Accepted Manuscript Synthetic artificial "long non-coding RNAs" targeting oncogenic microRNAs and transcriptional factors inhibit malignant phenotypes of bladder cancer cells Haibiao Xie, Hengji Zhan, Qunjun Gao, Jianfa Li, Qun Zhou, Zhicong Chen, Yuhan Liu, Mengting Ding, Huizhong Xiao, Yuchen Liu, Weiren Huang, Zhiming Cai PII:
S0304-3835(18)30179-4
DOI:
10.1016/j.canlet.2018.02.038
Reference:
CAN 13788
To appear in:
Cancer Letters
Received Date: 20 December 2017 Revised Date:
13 February 2018
Accepted Date: 26 February 2018
Please cite this article as: H. Xie, H. Zhan, Q. Gao, Jianfa Li, Q. Zhou, Z. Chen, Y. Liu, M. Ding, H. Xiao, Y. Liu, W. Huang, Z. Cai, Synthetic artificial "long non-coding RNAs" targeting oncogenic microRNAs and transcriptional factors inhibit malignant phenotypes of bladder cancer cells, Cancer Letters (2018), doi: 10.1016/j.canlet.2018.02.038. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Abstract Both oncogenic transcription factors (TFs) and microRNAs (miRNAs) play important roles in human cancers, acting as transcriptional and post-transcriptional regulators, respectively. These phenomena raise questions about the ability of an artificial device to simultaneously regulate miRNAs
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and TFs. In this study, we aimed to construct artificial long non-coding RNAs, “alncRNAs”, and to investigate their therapeutic effects on bladder cancer cell lines. Based on engineering principles of synthetic biology, we combined tandem arrayed aptamer cDNA sequences for TFs with tandem
arrayed cDNA copies of binding sites for the miRNAs to construct alncRNAs. In order to prove the
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utility of this platform, we chose β-catenin and the miR-183-182-96 cluster as the functional targets and used the bladder cancer cell lines 5637 and SW780 as the test models. Dual-luciferase reporter
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assay, real-time quantitative PCR (qRT-PCR) and related phenotypic experiments were used to test the expression of related genes and the therapeutic effects of our devices. The result of dual-luciferase reporter assay and qRT-PCR showed that alncRNAs could inhibit transcriptional activity of TFs and expression of corresponding microRNAs. Using functional experiments, we observed decreased cell proliferation, increased apoptosis, and motility inhibition in alncRNA-infected bladder cancer cells.
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What’s more, follow-up mechanism experiments further confirmed the anti-tumor effect of our devices. In summary, our synthetic devices indeed function as anti-tumor regulators, which synchronously accomplish transcriptional and post-transcriptional regulation in bladder cancer cells. Most importantly,
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anti-cancer effects were induced by the synthetic alncRNAs in the bladder cancer lines. Our devices, all in all, provided a novel strategy and methodology for cancer studies, and might show a great potential
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for cancer therapy if the challenges of in vivo DNA delivery are overcome.
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Synthetic artificial "long non-coding RNAs" targeting oncogenic microRNAs
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and transcriptional factors inhibit malignant phenotypes of bladder cancer cells
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Haibiao Xie1, 2, 3*, Hengji Zhan1,3*, Qunjun Gao1, 3, 4*,Jianfa Li1, 2, 5*,Qun Zhou1,3, Zhicong Chen1,3,
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Yuhan Liu1,3, Mengting Ding1,3,Huizhong Xiao1,3, Yuchen Liu1,2Ψ, Weiren Huang1Ψ, Zhiming Cai1, 2, 3Ψ
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5 1.
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Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital,
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First Affiliated Hospital of Shenzhen University, Shenzhen 518039, China 2.
Shantou University Medical College, Shantou 515041, Guangdong Province, China;
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3.
Department of Urology, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen
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University, Shenzhen 518039, China
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Guangzhou Medical University, Guangzhou, China,
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5.
Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics, Institute
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of Urology, Peking University Shenzhen Hospital, Peking University, Shenzhen 518036, China
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Ψ
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Zhiming Cai, Weiren Huang &Yuchen Liu
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Emails:
[email protected] (ZC) or
[email protected](WH) &
[email protected]
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(YL)
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*
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Authors for correspondence:
The three authors contributed this work equally.
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Abstract Both oncogenic transcription factors (TFs) and microRNAs (miRNAs) play important roles in human cancers, acting as transcriptional and post-transcriptional regulators, respectively. These
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phenomena raise questions about the ability of an artificial device to simultaneously regulate miRNAs
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and TFs. In this study, we aimed to construct artificial long non-coding RNAs, “alncRNAs”, and to
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investigate their therapeutic effects on bladder cancer cell lines. Based on engineering principles of
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synthetic biology, we combined tandem arrayed aptamer cDNA sequences for TFs with tandem
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arrayed cDNA copies of binding sites for the miRNAs to construct alncRNAs. In order to prove the
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utility of this platform, we chose β-catenin and the miR-183-182-96 cluster as the functional targets
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and used the bladder cancer cell lines 5637 and SW780 as the test models. Dual-luciferase reporter
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assay, real-time quantitative PCR (qRT-PCR) and related phenotypic experiments were used to test the
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expression of related genes and the therapeutic effects of our devices. The result of dual-luciferase
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reporter assay and qRT-PCR showed that alncRNAs could inhibit transcriptional activity of TFs and
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expression of corresponding microRNAs. Using functional experiments, we observed decreased cell
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proliferation, increased apoptosis, and motility inhibition in alncRNA-infected bladder cancer cells.
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What’s more, follow-up mechanism experiments further confirmed the anti-tumor effect of our devices.
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In summary, our synthetic devices indeed function as anti-tumor regulators, which synchronously
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accomplish transcriptional and post-transcriptional regulation in bladder cancer cells. Most importantly,
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anti-cancer effects were induced by the synthetic alncRNAs in the bladder cancer lines. Our devices, all
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in all, provided a novel strategy and methodology for cancer studies, and might show a great potential
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for cancer therapy if the challenges of in vivo DNA delivery are overcome.
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Key words
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Bladder cancer, synthetic biology, aptamer, miRNA, TFs.
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Introduction Bladder cancer (BCa) is one of the most common urologic malignancies worldwide, and its high prevalence and recurrence rates have captured great attention in the past decades1-3. Although there
ACCEPTED MANUSCRIPT have been great advances in the therapy of BCa, such as chemotherapy, radiotherapy and surgery, the
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five-year survival rate still remains unsatisfactory4-6. Although several novel antineoplastic drugs have
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been developed and subjected to clinical trials to assess their use as cancer therapy, no major
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breakthroughs have been achieved with these strategies7. Thus, there is an increasing need for the
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development of new and efficient strategies to treat BCa.
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With the completion of the ENCODE project, more than three-quarters of the genome has been found to be transcribed into RNA, but only 1–2% of this is translated into proteins8. Such RNAs
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without protein-coding function are classified as non-coding RNAs (ncRNAs). Among them,
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microRNAs (miRNAs), a class of small non-coding RNAs, regulate gene expression by binding to
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partially complementary sequences in the 3'UTR of mRNAs, participate in cellular differentiation and
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homeostasis, and are consequently involved in the development of various diseases, including cancers9.
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In our previous work, we used deep sequencing of genome-wide miRNA profiles in human BCa and
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demonstrated that the miR-183-96-182 cluster and miR-17-92 are strongly up-regulated in BCa10. Thus,
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miRNAs could be a potential target for BCa therapy11. To enhance the effect of inhibiting tumor
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progression, concurrently knocking down or knocking out various miRNAs are common strategies.
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However, accomplishing multiple miRNA knockouts simultaneously is difficult. Therefore, a device
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for inhibiting these functional classes of paralogous miRNAs is needed.
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Aptamers are small single-stranded oligonucleotides that can fold into unique tertiary structures and
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bind to target molecules such as proteins and RNAs with high affinity and specificity. Therefore, they
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are considered “chemical antibodies”12. Using a technique known as systematic evolution of ligands by
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exponential enrichment (SELEX), aptamers binding to various proteins have been routinely selected
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from synthetic combinatorial libraries13. Compared with monoclonal antibodies, aptamers can be
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synthesized quickly and are structurally stable under a wide range of temperatures and storage
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conditions. Most significantly, they exhibit low toxicity and non-immunogenicity in contrast to
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conventional antibodies12. To date, various studies have demonstrated that RNA aptamers can inhibit
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tumorigenesis by targeting oncogenic transcription factors (TFs) such as β-catenin14 and NF-κB15.
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Synthetic biology is a discipline that enables multiple signals to be cascaded and even to alter the
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signal flow by designing and linking various elements16. It is flexible, interesting and programmable so
ACCEPTED MANUSCRIPT that scientists can use a variety of elements to verify their own ideas such as a logic gate17, signal
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conductor18 or signal-connectors19. In this study, we constructed “artificial long non-coding RNAs”
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(alncRNAs) based on the principles in the field of synthetic biology, by combining an RNA aptamer
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for TFs with miRNA sponges targeting oncogenic miRNAs, and tested the utility of this device in BCa
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cells. The results showed that alncRNA could inhibit the transcription of TF target genes and the
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expression of targeted miRNAs, as expected. Importantly, our devices partly inhibited the malignant
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phenotypes of BCa cells. These results showed that our tools could simultaneously accomplish both
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transcriptional and posttranscriptional regulation of genes of interest, thus providing a novel strategy
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for treating human BCa cells.
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Materials and Methods
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Cell lines and cell culture
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Human bladder transitional cell carcinoma cell lines (SW780, 5637) and normal human cell lines (SV-40-immortalized human uroepithelial cell line, SV-HUC1) were purchased from the American
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Type Culture Collection (ATCC, Manassas, VA, USA). The 5637 cells were grown in RPMI-1640
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Medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine
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serum (Gibco). The SW780 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM;
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Gibco), supplemented with 10% fetal bovine serum (Gibco). The SV-HUC-1 cells were cultured in
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F12K (Gibco) plus 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin sulfate), with 10%
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fetal bovine serum. All cells were cultured at 37°C, in a humidified atmosphere of 5% CO2.
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Creation of the artificial lncRNAs and lentivirus infection
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Bulged miRNA binding sites for the miR-183, miR-182, miR96 and miR-17-5p and linkers
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between them were designed and chemically synthesized. In addition, RNA-aptamers specifically
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modulating β-catenin and NF-κB were applied from previous studies14,15. The combination DNA parts
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for the miR-183-182-96 cluster-β-catenin and miR-17-5p-NF-κB were driven by CMV or U6. These
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sequences and untargeted-control binding sites (negative control; NC) were cloned into a lentiviral
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vector (PLV-EGFP-N). Detailed descriptions of the related sequences are presented in Table 1, and
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detailed maps of alncRNA vectors are shown in supplementary Figure 1A.
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To produce lentivirus, lentivirus expression vectors (PLV-EGFP-N) were transfected into 293T cells. Supernatants were collected 48 and 72 hours after transfection, and cell debris was removed by
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centrifugation at 1500 × g for 30 minutes then passing through a syringe filter. Titers of the
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concentrated viruses were determined by infecting 293T cells with seral dilutions. The titers of the
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virus stocks were calculated to be 5 × 107–1 × 108 transduction units (TU)/mL based on the formula:
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(%GFP + cells) × (1/dilution). To effectively construct a stable cell line, three parts of the viral
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supernatant were mixed with one part fresh medium and 10 µg/mL of polybrene, and the mixture was
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applied to freshly-seeded cells. MOI (multiplicities of infection) of 10 pfu/cell were used, as shown in
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supplementary Figure 2. Stable cell lines were selected by flow cytometric assay by observing GFP
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expression.
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Enzyme-linked immunosorbent assay (ELISA)
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The concentration of β-catenin and NF-κB was measured by ELISA, which was carried out according to the manufacturer’s instructions. Briefly, 106 sample cells (SW780, 5637 and SW780) were
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harvested and resuspended in 200 µL of lysis buffer. The supernatants of lysates were collected by
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centrifugation and used for the subsequent procedures. The OD values were then measured using an
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automatic microplate reader (Bio-Rad, Hercules, CA, USA) and converted into protein concentrations
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using standard calibration curves.
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Dual-luciferase reporter assay
To measure the transcriptional activity of β-catenin and NF-κB, a dual-luciferase reporter assay was
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performed using β-catenin or NF-κB reporter constructs (Supplementary Figure 1B). The β-catenin
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reporter contained copies of Tcf-binding elements (responsive elements), minimal promoter and
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dual-reporter vector (Beijing Syngenetech CO., Ltd, China). In addition, the NF-κB reporter contained
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multiple binding sites for NF-κB, minimal promoter and dual-reporter vector (Beijing Syngenetech Co.,
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Ltd, China). cDNAs of the binding sites of β-catenin and NF-κB are shown in Table 1. Cells were
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seeded into six-well plates (5 × 105 per well) and transfected with the β-catenin or NF-κB
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dual-luciferase reporter vectors. Luciferase activity was measured using the dual luciferase assay
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system (Promega, Madison, WI, USA) as per the manufacturer’s instructions at 48 hours after
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transfection. Firefly luciferase activities were normalized to Renilla luciferase activities. All assays
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were performed in duplicate, and all data shown are representative of at least two independent
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experiments.
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RNA extraction and real-time quantitative PCR
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Total RNAs were isolated from SW780, 5637 and SV-HUC1 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration and purity of total
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RNA were measured using UV spectrophotometric analysis at 260 nm. cDNAs of coding RNA and
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microRNA were synthesized using a Revertra Ace qPCR RT Kit (Toyobo, Osaka, Japan) and a
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miScript II RT Kit (Qiagen, Hilden, Germany). Real time PCR was carried out using real time PCR
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Master Mix (Toyobo) and the miScript® SYBR® Green PCR Kit (Qiagen). GAPDH or U6 small
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nuclear RNA (snRNA) was selected as the endogenous control. The miRNA qRT-PCR primers were
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ordered from GeneCopoeia, Inc. (Rockville, MD, USA). The qPCR primers are listed in Table 2. The
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PCR mixtures were prepared according to the manufacturer’s protocols and amplification was
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performed under PCR conditions of 40 cycles of 15 sec at 95°C, 20 sec at 55°C, and 30 sec at 70°C on
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a ABI PRISM 7300 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA,
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USA). Expression fold changes were calculated using the 2-△△ct method.
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Cell proliferation assay
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The CCK-8 assay (TransGen, Beijing, China) was used to examine cell proliferation. Aliquots of 5 × 103 cells were seeded into 96-well plates and cultured at 37°C. After 24, 48, or 72 h, 10 µL CCK-8
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was added to each well and incubated at 37°C for 1 h. The absorbance at 450 nm was read by an
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automatic microplate reader (Bio-Rad). To directly observe the proliferating cells, Edu incorporation
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assay was performed using an Edu assay kit (Ribobio, Guangzhou, China) according to the
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manufacturer’s instructions. Each experiment was repeated at least three times.
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Apoptosis assay and cell cycle analysis
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Apoptosis was determined by caspase 3 and cleaved-PARP ELISA assay. Aliquots of 1 × 106 cells
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were counted to measure the activity of caspase 3 and PARP ELISA using caspase 3 and PARP ELISA
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assay kits respectively (Biovision, Milpitas, CA, USA and American). A microplate reader was used to
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measure the OD values at 405 nm. Each experiment was repeated at least three times.
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phosphate-buffered saline (PBS) and fixed in 75% cold alcohol overnight at 4°C. After washing in cold
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PBS three times, cells were incubated with 1 × PI/RNase staining buffer for 15 min in the dark at room
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temperature. Samples were then analyzed for their DNA content using flow cytometry. All assays were
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performed in duplicate, and all data shown are representative of at least two independent experiments.
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Cell motility assay
Cell motility was determined by wound-healing assay. A wound field was created using a sterile 200 µL pipette tip when cells reached approximately 90% confluence. The migration of cells was
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monitored with a digital camera system. The cell migration distance (µm) was calculated using the
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software program HMIAS-2000, at 24 hours after the wound was created. These experiments were
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repeated at least three times.
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Additionally, the cell motility assay was also performed using a transwell insert (8 µm, Dow Corning
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Corp., Midland, MI, USA). After 24 h, 5 × 10 4 SW780 cells and 15 × 105 5637 cells were first starved
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in 200 uL serum-free medium and then placed in the uncoated dishes. Concurrently, the lower
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chambers were filled with 500 µL medium containing 10% fetal bovine serum. Cells were cultured at
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37°C in a 5% CO2 atmosphere for 24 hours. Cells under the surface of the lower chamber were washed
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with 1 × PBS, fixed with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet for 25 min,
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and then washed three times. Invaded cells were observed under the inverted microscope and imaged.
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Afterwards, each chamber with the invaded cells was soaked in 1 mL 33% acetic acid for 10 min to
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wash out the crystal violet. Then 100 µL of 33% acetic acid was added into each well of a 96-well plate,
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and the absorbance was measured at a wavelength of 570 nm using a microplate reader (Bio-Rad).
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Experiments were performed in triplicate.
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Statistical analysis
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Each experiment was performed in triplicate. All quantitative data are expressed as mean ± standard
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deviation (SD). Statistical analyses were performed using SPSS8.0 software (IBM Corp., Armonk, NY,
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USA). Statistical significance was tested by Student’s t-test, Chi square test or ANOVA. P < 0.05 was
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considered to be statistically significant.
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Results
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Design and construction of the alncRNAs.
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Inspired by two observations that some RNA molecules could regulate miRNAs by complementary base pairing in eukaryotic cells20, and that some RNA aptamers are capable of capturing proteins and
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consequently inhibiting their function by protein–RNA interactions15,21,22, we set out to construct an
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RNA tool, “alncRNA”, which could inhibit the malignant phenotype of BCa by regulating oncogenic
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signal pathways at transcriptional and post-transcriptional levels simultaneously. This proposed tool
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contains two modules, encoded on plasmids (Figure 1A). The first module, the RNA aptamer, can
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capture proteins including transcription factors (TFs). The second module, the microRNA sponge,
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causes miRNA loss-of-function by partially-complementary binding to target miRNAs. We connected
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these modules by a linker of a few nucleotides to allow correct folding of aptamers and sponges23. To
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ensure effective expression of alncRNAs, we used a powerful promoter (CMV) or U6 promoter, which
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expresses nuclear localized RNAs24, to drive expression of alncRNAs.
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To test the utility of the designed device, we combined two tandem arrayed cDNA sequences of
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aptamers for β-catenin with six tandem arrayed cDNA copies of binding sites for the miR-183-96-182
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cluster (two copies for each miRNA of the cluster) to construct alncRNA1. As previously reported,
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β-catenin is a well-known multifunctional protein, which always activates the transcription of several
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oncogenic TFs25. To demonstrate the modularity of the circuit design, we also used another two cDNA
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copies of the RNA aptamer for NF-κB, and six cDNA copies of binding sites for miR-17-5p to replace
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the corresponding locations in alncRNA1, producing alncRNA2. Afterwards, we inserted these
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alncRNAs and corresponding control cDNA, a repeated binding element not targeting any protein or
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microRNA, into lentivirus vectors and used lentivirus to deliver the devices into the target cells for use
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in subsequent experiments. As shown in Supplementary Figure 2, we used a MOI of 10 pfu/cell to
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construct the stable cell lines. The mechanism diagram is shown in Figure 1B.
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Repression of dual-luciferase reporters by alncRNA in BCa cell lines
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NF-κB transcriptional activity by specifically subcloning the β-catenin and NF-κB responsive promoter
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sequences into a dual-luciferase vector, named β-catenin and NF-κB reporter (Figure 2A). In order to
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verify reporter detection efficiency, we first measured the protein expression of β-catenin and NF-κB in
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SW780, 5637 and SV-HUC1 cells by ELISA. The results showed that both β-catenin and NF-κB were
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overexpressed in BCa cell lines (SW780 and 5637) compared with the normal human uroepithelial cell
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line SV-HUC1 (Figure 2B). Next, we respectively transfected our β-catenin or NF-κB reporter into
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BCa cell lines and SV-HUC1, and measured activity by luciferase assay. As expected, the ratio of
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firefly luciferase versus Renilla luciferase expression was consistent with the ELISA results, indicating
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that our reporter did indeed sense the transcriptional activity of β-catenin and NF- κB (Figure 2C).
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Next, in an effort to verify the effect of alncRNA on TF transcription, we introduced the β-catenin
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and NF-κB reporter into BCa cell lines stably expressing alncRNA or NC, respectively (SW780, 5637).
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After 48 hours, the results of the dual-fluorescein assay confirmed that our alncRNA did inhibit
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transcription of β-catenin and NF-κB reporter, and that the CMV and the U6 promoter-driven effects
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were similar (Figure 2D). In addition, we found that alncRNA did not conduct crosstalk between the
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β-catenin and NF-κB reporter pathways. These results confirmed that alncRNA could directly inhibit
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transcriptional activity of both β-catenin and NF-κB without crosstalk. Since we previously verified the
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effect of microRNA sponges by the luciferase assay, we did not repeat it here26.
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The alncRNA achieved effective suppression of oncogenic signals and target miRNA levels in BCa cell lines.
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To further investigate the functionality of alncRNAs, we examined the relative expression levels of
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the target miRNAs and the downstream genes of target TFs in the human BCa cell lines SW780 and
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5637 treated with our device. The qRT-PCR results demonstrated that expression of the corresponding
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alncRNAs induced a remarkable decrease in expression levels of the corresponding downstream genes
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and the targeted miRNAs in the two BCa cell lines (Figure 3). The expression levels of the downstream
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genes of NF-κB (Bcl-XL, TRAF1) and miR-17-5p could not be suppressed by alncRNA1. At the same
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time, the expression level of the miR-183-182-96 cluster also could not be changed by alncRNA2.
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Interestingly, we found that the expression levels of two well-known downstream genes (c-myc, cyclin
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D1) of β-catenin could be decreased by alncRNA2 (Figure 3A, B, C and D). After searching the
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literature, we found that c-myc and cyclin D1 can also be transcriptionally activated by NF-κB27.
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Inhibition of cell proliferation induced by alncRNAs in BCa cell lines Stable BCa cell lines (SW780 and 5637) expressing alncRNAs or corresponding NC vector were
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seeded into 96-well plates to test the cancer cell proliferation ability. Using the CCK-8 assay, we
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demonstrated that the alncRNAs could effectively reduce cell growth compared with the NC group in
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both BCa cell lines (Figure 4A, B, C, and D). The Edu incorporation assay was then used to determine
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the effects of alncRNAs on proliferation of BCa cell lines (Figure 4E, F and Supplementary Figure3).
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The results showed that alncRNAs could markedly inhibit cancer cell growth.
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Since alncRNA could induce the down-regulation of myc and cyclin D1, we hypothesized that
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alncRNA acted to inhibit tumor proliferation by affecting the cell cycle. To verify our hypothesis, we
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performed cell cycle analysis of SW780 cells stably expressing alncRNA and NC. The results showed
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that BCa cells expressing alncRNAs in G0 / G1 phase increased significantly, while those in G2 phase
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decreased significantly (Figure 5A). The result was consistent with the down-regulation of myc and
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cyclinD1. These data suggest that alncRNA may cause cell cycle arrest by affecting the transcriptional
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activity of TFs, thus inhibiting the expression of cell cycle regulators.
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Induction of apoptosis by alncRNAs in BCa cell lines.
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According to cell cycle experiments, we found that alncRNA can increase the sub-G1 population, indicating the induction of apoptosis (Figure 5A). To verify and explore the mechanism by which
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alncRNAs induce apoptosis, BCa cell lines (SW780, 5637) stably expressing alncRNAs or the
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corresponding NC vectors were seeded into 6-well plates to investigate the extent of apoptosis and the
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underlying mechanisms. We used ELISA to analyze the expression of caspase-3 protein and
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cleaved-PARP-apoptotic proteins. We found an obvious increase in the expression of caspase-3 and
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cleaved-PARP in the cells expressing alncRNA2, but only the expression of caspase3 was increased in
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the cells expressing alncRNA1 (Figure 5B and Supplementary Figure 4). Previous studies suggested
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that NF-κB exerts anti-apoptotic effects by inhibiting caspase3 and cleaved-PARP, whereas β-catenin
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acts only to inhibit caspase3, rarely affecting the expression of PARP in BCa28,29. These results indicate
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that alncRNA can induce apoptosis, with alncRNA2 eliciting a higher rate of apoptosis than alncRNA1
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(Figure 5A), which is consistent with the expression of apoptosis-related proteins.
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Inhibition of cell migration induced by alncRNAs in BCa cell lines. Finally, we further investigated whether alncRNAs could inhibit cell migration in BCa. Stable BCa
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cell lines (SW780 and 5637) expressing alncRNAs and corresponding NC vector were seeded into
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24-well plates to test their cell migration ability using the wound healing assay. Cell migration arrest
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was observed in SW780 (Figure 6A) and 5637 cells (Supplementary Figure 5A) as expected. Similarly,
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the transwell assay was used to further confirm our results. Decreased cell motility was observed in
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stable cell lines expressing alncRNAs compared with NC cell groups (Figure 6B, Supplementary
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Figure 5B). These results confirmed that alncRNAs suppress cell migration in BCa.
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To elucidate the mechanism of inhibitory effects of alncRNAs on cell migration, we conducted a
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literature search, and found that β-catenin and NF-κB, as well as the targeted microRNAs, could induce
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epithelial-mesenchymal transition (EMT) and sequentially induce cancer cell migration and
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invasion30-33. We hypothesized that our alncRNAs might reverse EMT and resulted in decreased cell
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migration. To confirm our hypothesis, we used qRT-PCR to analyze the expression of several common
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epithelial markers (E-cadherin) and mesenchymal markers (vimentin, slug) in SW780 cells stably
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expressing alncRNAs and NC. Compared with control group, the expression levels of slug and
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vimentin in SW780 cells stably expressing alncRNAs were significantly decreased, and the expression
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of E-cadherin was significantly increased(Figure 4D). Additionally, we found that alncRNA-infected
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cells underwent a phenotypic change from a spindle-shaped morphology to a cuboidal, epithelial-like
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morphology (Figure 4C). These data suggested that our alncRNAs were likely to suppress migration by
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reversing the EMT process.
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Discussion Medical synthetic biology is an interdisciplinary branch of medical biology and engineering34. One
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of the major purposes of medical synthetic biology research is to connect synthetic genetic devices
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together to control complex biological phenotypes16. In our present study, we connected the cDNA of
ACCEPTED MANUSCRIPT RNA aptamers and miRNA sponges to construct an artificial anti-tumor device, named alncRNA. The
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reason for this name is that this device can accomplish simultaneous transcriptional and
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post-transcriptional regulation, functioning just like a natural lncRNA35. To confirm the utility of our
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device, we chose oncogenic transcription factors (TFs) and miRNAs as our targets. Based on the results
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of previous studies36-40, we chose two tandem copies of aptamers and six binding sites of microRNAs
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to construct our device. The results of qRT-PCR showed that the BCa cell lines (5637, SW780) stably
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expressing alncRNA indeed decreased expression of the TF target genes and the corresponding
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miRNAs. Lastly, phenotypic experiments revealed that alncRNA could inhibit cell proliferation,
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suppress cell motility and induce apoptosis at the same time. These results demonstrate that our device
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can effectively inhibit oncogenic signaling transduction to suppress the malignant phenotypes of BCa
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cells.
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It is widely acknowledged that miRNAs degrade mRNA and non-coding RNA by complementary base pairing with 3’UTR of these RNAs, achieving post-transcriptional regulation41. Interestingly,
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recent studies have revealed that mRNA–miRNAs interaction can also block the function of
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miRNAs20,42. For instance, the pseudogene PTENP1 was found to regulate cellular levels of PTEN by
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interacting with miRNAs targeting PTEN. A similar result was shown in the reciprocal relationship
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between MALAT1 and miR-125b. MALAT1 is an lncRNA which inhibits miR-125b by functioning as
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a sponge43. Based on these findings, we previously constructed a microRNA mower that can inhibit a
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miRNA of interest by integrating the specific recognition sites into a mower RNA sequence26. The
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mower indeed performed as expected. However, a single protein can be involved in more than one step
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of gene expression, such as transcription, post-transcriptional processing, and translation. Our previous
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device focused only on transcription and did not show an absolute effect. Therefore, we aimed to
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construct a more effective device which could achieve both transcriptional and post-transcriptional
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regulation.
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An interesting tool–an RNA aptamer specifically targeting proteins–came to our attention. Its high
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specificity and high-level expression in cells make it possible to specifically recognize proteins and
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even modulate their activity. Previous studies revealed that DNA or RNA molecules can compete with
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genes for TFs, thus affecting the transcription of these genes44,45. For example, nucleophosmin (NPM)
ACCEPTED MANUSCRIPT is a multi-functional protein that plays a pivotal role in cell proliferation and apoptosis46. Jian et al.
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took advantage of an RNA aptamer to target the central acidic region of NPM and inhibit its
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oligomerization resulting in disruption of the downstream signaling pathway47. Based on these
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observations, we, first constructed alncRNA by connecting the sequences of miRNA sponges and RNA
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aptamers to accomplish simultaneous transcriptional and post-transcriptional regulation. For maximum
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expression of alncRNA, we used the strong promoters CMV or U6 to drive our devices and inserted
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them into cells using a lentivirus to achieve stable and highly-efficient expression. Based on these
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designs, we hypothesized that our devices would target oncogenic signals and subsequently inhibit
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malignant phenotypes of BCa cells.
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To test our hypothesis, we first chose the TFs β-catenin and the miR-183-182-96 cluster as targets.
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β-catenin is a multifunctional TF involved in cell adhesion and the Wnt pathway. It is also well known
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to activate transcription of various oncogenes, such as c-myc, cyclinD1 and Cox-225,48. Another factor,
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the miR-183-182-96 cluster, has been considered to play an important role in tumor apoptosis and the
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AKT–mTOR signaling pathway 49. The results of qRT-PCR confirmed that our tool also successfully
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inhibited the expression of the miRNA of interest and the function of β-catenin. The next step was to
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conduct a functional experiment to demonstrate that our design strongly suppressed bladder urothelial
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carcinoma cell proliferation, increased the apoptosis rate and decreased cell motility by modulating the
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corresponding genes.
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miRNA that could in theory be replaced by other similar elements. Here, we replaced the binding sites
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of β-catenin and the mir-183-182-96 cluster with sequences of NF-κB and mir-17-5p to test the
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modularity. NF-κB is also a cancer-associated TF involved in transcription of multiple oncogenes, such
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as Bcl-xl, vascular endothelial growth factor (VEGF) and HIF-1α50. MiR-17-5p has been shown to
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regulate basic cellular processes such as proliferation and apoptosis. In a wide variety of cancers,
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miR-17-5p is highly expressed and always associates with cancer development and aggressiveness51.
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As we expected, alncRNA2 also played an anti-tumor role in BCa.
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The highlight of our work is the simultaneous regulation of transcription and post-transcriptional
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regulation by designing an RNA-based device. Compared with traditional single elements, our device
ACCEPTED MANUSCRIPT exerts a more significant effect in the regulation of signal flow. Due to our tandem and programmable
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design, we can change the TFs we want to target as well as the miRNAs. However, there remain a few
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limitations that need to be addressed. Firstly, as an anti-tumor devices, alncRNAs really need to be
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effectively controlled to avoid damage to normal cells. There are a variety of ways previously shown to
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control expression, such as riboswitches52, logic gates 17 and light-induced switches53. However the
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availability of these tools for alncRNA will need verification. Secondly, one notable limitation to the
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application of this methodology is that functional aptamers still need to be screened from randomized
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libraries, which currently limits the widespread application of alncRNAs. Finally, before any such
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anti-tumor device can be used in the clinic, it will need to be tested in vivo. Our group used lentivirus to
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transport alncRNA into cancer cells. Thus, in vivo experiments would be needed to address the key
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question of identifying the best concentration (MOI) of alncRNA that will inhibit tumor cells but not
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normal cells. Even so, we still believe that our novel device will be widely applied in biology and
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medicine in future. Here, we show a future scheme for application of alncRNA (Figure 7).
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In summary, our team constructed anti-tumor devices, alncRNAs, which had the ability to capture oncogenic TFs and miRNAs of interest and to effectively inhibit malignant phenotypes of BCa cells.
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Although there is a long way to go before our tool can be used for clinical diagnosis and therapy,
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alncRNAs still provide a novel strategy for cancer therapy and could be useful “weapons” against
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cancer cells when the challenges of in vivo DNA delivery are overcome.
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Author contributions
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H.X., Q.G, H.Z., J.L. and Y.L. performed experiments and data analysis. Y.L. and H.X. designed the
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project and wrote the paper. Z.C., W.H. and Y.L. supervised the project. W.H. and Z.C. provided
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financial support for the project.
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Acknowledgments
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We are indebted to the donors whose names were not included in the author list, but who participated in this program. This work was supported by the National Key Basic Research Program of China (973 Program) (2014CB745201), the Chinese High-Tech (863) Program (2014AA020607), National Natural Science Foundation of China (81773257), International S&T Cooperation program of China (ISTCP)
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(2014DFA31050),
The
National
Science
Foundation
Projects
of
Guangdong
Province
(2014A030313717), the Shenzhen Municipal Government of China (ZDSYS201504301722174, JCYJ20150330102720130, GJHZ20150316154912494), and Special Support Funds of Shenzhen for Introduced High-Level Medical Team. What’s more, we thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.
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Figure legends Figure 1. Design and construction of the alncRNAs in cancer cells. (A) General compositional
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framework and design strategy for constructing alncRNAs. (B) Thorough description of the engineered
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alncRNAs for inducing cancer cell inhibition.
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Figure 2. Repression of dual-luciferase reporters by alncRNA in bladder cancer (BCa) cell lines. (A) Description of the working mechanism of TF reporters. (B) The expression of β-catenin and NF-κB
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protein were detected by ELISA in SW780, 5637 and SV-HUC1 cells. (C) Luciferase expression levels
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of several cell lines transfected with β-catenin or NF-κB reporters. (D) Luciferase expression levels of
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BCa cell lines (SW780 and 5637) expressing alncRNA and NC transfected with β-catenin or NF-κB
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reporters. Results are shown as mean ± SD. ***P < 0.001 compared with control. ** P < 0.01
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compared with control. * P < 0.05 compared with control.
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Figure 3. The artificial lncRNAs simultaneously reduced the expression levels of mRNA of various
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downstream genes of the targeted TFs and corresponding miRNAs. alncRNA1 reduced the expression
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of myc and cyclinD1 mRNAs (A, B, C, and D) and miR-183-182-96 (E, F, G, H) in BCa cell lines
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(SW780, 5637) whether it was driven by CMV or U6 promoters. alncRNA2 reduced the expression of
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myc, cyclinD1, Bcl-XL, and TRAF1 mRNAs (A, B, C, D) and miR-17-5p (E, F, G, H) in BCa cell
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lines (SW780, 5637) whether it was driven by CMV or U6 promoters. Results are shown as mean ± SD.
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***P < 0.001 compared with control. ** P < 0.01 compared with control. * P < 0.05 compared with
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control.
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Figure 4. The effects of artificial lncRNAs on cell proliferation in BCa cells. The growth curves of
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SW780 or 5637 cells infected with alncRNA driven by CMV (A, B) and U6 (C, D) promoters were
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determined using CCK-8 assay. Data are shown as mean ± SD. The proliferation of 5637 (E) and
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SW780 (F) cells infected with alncRNA were also determined using the EDU incorporation assay.
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Figure 5. The effects of artificial lncRNAs on apoptosis in BCa cells. (A) AlncRNA induce
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apoptosis in SW780 cell. (B) The relative activity of caspase 3 was calculated in SW780 and 5637 cells
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infected with alncRNA driven by CMV, and U6 promoters using ELISA.
432 Figure 6. The effects of artificial lncRNAs on migration of BCa cells. (A) The relative rate of cell
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migration was calculated in SW780 cells infected with alncRNAs using the wound-healing assay. (B)
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Cell migration was determined by the transwell assay. Inhibition of cell migration following infection
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with alncRNAs was observed in SW780 cells. Data are shown as mean ± SD. (C) Cell morphology of
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SW780 cells expressing alncRNAs and NC. (D) The expression of E-cadherin, vimentin and slug in
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SW780 expressing alncRNA and NC.
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Figure 7. The future optimization and application of alncRNA.
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Supplementary Figure 1. The detailed profile of the vectors. A. Map of alncRNA lentivirus vectors. B. Map of NF-κB and β-catenin responsive-reporter vector.
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Supplementary Figure 2. Cell viability and transduction efficiency of SW780 cells after infection
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with various MOI of lentivirus. A. Cell viability of SW780 cells infected with alncRNA and NC driven
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by CMV was determined by CCK8 assay. B. GFP positive SW780 cells infected with alncRNA and
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NC driven by CMV were evaluated by manual counting after 72 hours.
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Supplementary Figure 3. The effects of artificial lncRNAs on proliferation of BCa cells. The
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Supplementary Figure 4. The effects of artificial lncRNAs on apoptosis in BCa cells. The relative
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proliferation of 5637 (A) and SW780 (B) cells infected with alncRNA were also determined using the
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EDU incorporation assay.
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activity of cleaved-PARP was calculated in SW780 cells infected with alncRNA driven by CMV (A)
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and U6 (B) promoters using ELISA. Results are shown as mean ± SD. ***P < 0.001 compared with
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control. ** P < 0.01 compared with control. * P < 0.05 compared with control.
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Supplementary Figure 5. The effects of artificial lncRNAs on cell migration in BCa cells. (A) The relative rate of cell migration was calculated in 5637 cells infected with alncRNAs using the
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wound-healing assay. (B) Cell migration was determined by transwell assay. Inhibition of cell
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migration following transfection with alncRNAs was observed in 5637 cells. Data are shown as mean ±
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SD.
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Table 1. cDNA sequences of the protein-binding elements used in the vectors.
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Table 2. Relative primers used in this research.
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ACCEPTED MANUSCRIPT Table1. cDNA sequences of vectors. Synthetic Sequences
CMV-alncRNA1
AGGCCGATCTATGGACGCTATAGGCACACCGGATACTTTAACGATTG GCTATATTCAGGCCGATCTATGGACGCTATAGGCACACCGGATACTTT AACGATTGGCTATATTCAGTGTGAGTTCTGAAGTTGCCAAACTTCAG TGAATTCTCAACGTGCCATACTTCAGCAAAAATGTTAGCGTGCCAAA CTTCAGTGTGAGTTCTGAAGTTGCCAAACTTCAGTGAATTCTCAACG TGCCATACTTCAGCAAAAATGTTAGCGTGCCAAA
CMV-alncRNA2
GATCTTGAAACTGTTTTAAGGTTGGCCGATCATATTCGATCTTGAAA CTGTTTTAAGGTTGGCCGATCATATTCCTACCTGCACTACGTGCACTT GCTTCCTACCTGCACTACGTGCACTTGCTTCCTACCTGCACTACGTG CACTTGCTTCCTACCTGCACTACGTGCACTTGCTTCCTACCTGCACT ACGTGCACTTGCTTCCTACCTGCACTACGTGCACTTG
CMV-NC
TTCTCCGAACGTGTCACGTCACGTTTCAAGAGAACGTGACACGTTC GGAGAAATATTCTTCTCCGAACGTGTCACGTCACGTTTCAAGAGAA CGTGACACGTTCGGAGAAATATTCAAGTTTTCAGAAAGCTAACAAA GTTTTCAGAAAGCTAACAAAGTTTTCAGAAAGCTAACAAAGTTTTC AGAAAGCTAACAAAGTTTTCAGAAAGCTAACAAAGTTTTCAGAAA GCTAACA
U6-alncRNA1
GAGGCCGATCTATGGACGCTATAGGCACACCGGATACTTTAACGATT GGCTATATTCAGGCCGATCTATGGACGCTATAGGCACACCGGATACTT TAACGATTGGCTATATTCAGTGTGAGTTCTGAAGTTGCCAAACTTCA GTGAATTCTCAACGTGCCATACTTCAGCAAAAATGTTAGCGTGCCAA ACTTCAGTGTGAGTTCTGAAGTTGCCAAACTTCAGTGAATTCTCAAC GTGCCATACTTCAGCAAAAATGTTAGCGTGCCAAATTTTTT
U6-alncRNA2
GATCTTGAAACTGTTTTAAGGTTGGCCGATCATATTCGATCTTGAAA CTGTTTTAAGGTTGGCCGATCATATTCCTACCTGCACTACGTGCACTT GCTTCCTACCTGCACTACGTGCACTTGCTTCCTACCTGCACTACGTG CACTTGCTTCCTACCTGCACTACGTGCACTTGCTTCCTACCTGCACT ACGTGCACTTGCTTCCTACCTGCACTACGTGCACTTGTTTTTT
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Vector Names
U6-NC
GTTCTCCGAACGTGTCACGTCACGTTTCAAGAGAACGTGACACGTT CGGAGAAATATTCTTCTCCGAACGTGTCACGTCACGTTTCAAGAGA ACGTGACACGTTCGGAGAAATATTCAAGTTTTCAGAAAGCTAACAA AGTTTTCAGAAAGCTAACAAAGTTTTCAGAAAGCTAACAAAGTTTT CAGAAAGCTAACAAAGTTTTCAGAAAGCTAACAAAGTTTTCAGAAA GCTAACATTTTTT
Beta-catenin luciferase responsive elements
AAAGGGGGTAAGATCAAAGGGGGTAAGATCAAAGGGGCGCGAGAT CAAAGGGGGTAAGATCAAAGGGGGTAAGATCAAAGGGGGTAAGAT C
NF-KB luciferase
GGGAATTTCCGGGGACTTTCCGGGAATTTCCGGGGACTTTCCGGGA ATTTCC
ACCEPTED MANUSCRIPT responsive elements Note: Yellow and green parts are the cDNA sequences of RNA aptamers for β-catenin and NF-κB p50 respectively. Pink and blue parts are the cDNA sequences of miRNAs sponges for miR-183/96/182 cluster and miR-17-5p.
Sequences
GAPDH-F
CGCTCTCTGCTCCTCCTGTTC
GAPDH-R
ATCCGTTGACTCCGACCTTCAC
MYC-F
GGCTCCTGGCAAAAGGTCA
MYC-R
CTGCGTAGTTGTGCTGATGT
Cyclin D1-F
GCTGCGAAGTGGAAACCATC
Cyclin D1-R
CCTCCTTCTGCACACATTTGAA
BCL-XL-F
GAGCTGGTGGTTGACTTTCTC
BCL-XL-R
TCCATCTCCGATTCAGTCCCT
Vimentin-F
GACGCCATCAACACCGAGTT
Vimentin-R
CTTTGTCGTTGGTTAGCTGGT
E-cadherin-F
CGAGAGCTACACGTTCACGG
E-cadherin-R
GGGTGTCGAGGGAAAAATAGG
Slug-F
CGAACTGGACACACATACAGTG
Slug-R
CTGAGGATCTCTGGTTGTGGT
TRAF1-F
TCCTGTGGAAGATCACCAATGT
TE D
M AN U
SC
Name
RI PT
Table2. Relative primers used in this research.
TRAF1-R
GCAGGCACAACTTGTAGCC
Has-miR-96~HmiRQP0852
Has-miR-182~HmiRQP0239
The company did not provide sequence information of these Primers.
EP
Has-miR-183~HmiRQP0244 Hsa-miR-17-5P~ HmiRQP0230
AC C
SnRNA U6~HmiRQP9001
Note: F, forward primer; R, reverse primer.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Highlights
RI PT SC M AN U TE D
EP
An anti-tumor device, artificial lncRNA (alncRNA), was constructed by connecting microRNA sponge and TFs aptamer. The alncRNA could synchronously accomplish transcriptional and post-transcriptional regulations in bladder cancer cells. Anti-cancer effects were induced by the synthetic alncRNAs in the bladder cancer lines.
AC C
