miR-129 regulates cell proliferation by downregulating Cdk6 expression

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Cell Cycle 9:9, 1809-1818; May 1, 2010; © 2010 Landes Bioscience

miR-129 regulates cell proliferation by downregulating Cdk6 expression Junjie Wu,1 Jun Qian,1 Chun Li,1 Letty Kwok,1 Feng Cheng,1 Peijun Liu,2 Catalina Perdomo,1 Darrell Kotton,1 Cyrus Vaziri,2 Christina Anderlind,1 Avrum Spira,1 Wellington V. Cardoso1 and Jining Lü1,* Pulmonary Center; Department of Medicine; 2Department of Pathology and Laboratory Medicine; Boston University School of Medicine; Boston, MA USA

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Key words: miR-129, G1 phase arrest, Cdk6, post-transcriptional suppression, cell proliferation

Reduced expression of miR-129 has been reported in multiple tumor cell lines and in primary tumors including medulloblastoma, undifferentiated gastric cancers, lung adenocarcinoma, endometrial cancer and colorectal carcinoma. There is also recent evidence of an anti-proliferative activity of miR-129 in tumor cell lines. Still, little is known about how miR-129 regulates cell proliferation. Here we found that lentiviral-mediated overexpression of miR-129 in mouse lung epithelial cells (E10 cells) results in significant G1 phase arrest that eventually leads to cell death. miR-129 induce G1 phase arrest in multiple human lung adenocarcinoma cell lines, suggesting miR-129 targeting of G1/S phase-specific regulators. Interestingly, we show that Cdk6, a kinase involved in G1-S transition, is a direct target of miR-129. We also found the downregulation of three other cell cycle-related novel targets of miR-129, including Erk1, Erk2 and protein kinase C epsilon (Prkce). We further show that among these targets, only Cdk6 is functionally relevant. Restoring expression of Cdk6, but not Prkce partially rescues the cell growth arrest and cell death phenotype that results from miR-129 overexpression. Together, our data indicate that miR-129 plays an important role in regulating cell proliferation by downregulation of Cdk6.

Introduction miRNAs are endogenous small RNAs averaging 20 to 24 nucleotides, transcribed from non-protein-coding genes or introns, which mediate translational suppression or cleavage of their target mRNAs by binding to complementary sites in their 3'UTR.1-3 miRNAs play an essential role in cell cycle regulation, apoptosis and tumorigenesis. Aberrant miRNA expression profiles have been identified in tumors, as compared to normal tissues.4,5 Moreover, a large number of miRNAs are located inside or close to chromosomal fragile sites that are frequently lost or amplified in cancers.6 miRNAs have been characterized as oncogenes, tumor suppressors or as components of regulatory pathways critical for tumorigenesis. For example, the oncogenic activities of miR-17-92 have been implicated in B-cell lymphoma,7 miR-372 and miR-373 in testicular cancer.8 By contrast, let-7 behaves as a tumor suppressor in lung cancer cells by downregulating the expression of oncogenes, such as Ras9 and HMGA2 (high mobility group AT-hook 2).10 miR-129 is among the candidate miRNAs with potential tumor suppressor activity. Evidence that miR-129 may suppress cell growth comes from studies showing that miR-129 levels are much lower in tumor cell lines or primary tumors derived from neural, gastric, or colorectal tissue than in their respective normal tissues.11-14 A recent study showed that miR-129 is significantly downregulated in lung adenocarcinoma.15 Furthermore, levels of miR-129 appear to be associated with the differentiation status of

tumors. For example, miR-129 expression is lower in nonfunctioning pituitary tumors than in the more differentiated growth hormone-secreting pituitary adenomas.16 Human miR-129-1 is among the seven miRNAs identified in genomic regions near FRA7H, one of the fragile sites in chromosome 7q32 which is frequently deleted in several solid tumors.6 There is recent evidence that overexpression of miR-129-5p using mimics greatly reduces the proliferation activity of endometrial tumor cells and bladder cancer cells.17,18 Although the anti-proliferation activity of miR-129 is strongly suggested by the studies above, it is still unclear how its effects are achieved. Here we address this issue by investigating miR-129-mediated events and targets in lung epithelial derived cells. We show that miR-129 expression levels are very low in lung adenocarcinoma cells compared to normal lung tissue. Introduction of miR-129 expression in these cells using a lentivirus resulted in significant G1 phase arrest. We provide novel evidence that miR-129 targets directly cell proliferation regulators, such as Cdk6, Erk1, Erk2 and Prkce. Moreover, we show that among these target genes, Cdk6 is the only target that is critically involved in miR-129mediated growth arrest. Results Expression of miR-129 in lung tissue and cell lines. We asked whether the downregulation of miR-129 reported in primary

*Correspondence to: Jining Lü; Email: [email protected] Submitted: 12/07/09; Revised: 02/11/10; Accepted: 02/15/10 Previously published online: www.landesbioscience.com/journals/cc/article/11535 www.landesbioscience.com

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Figure 1. Expression of miR-129. (A) Expression of both miR-129-5p and -3p become detectable at E16 (embryonic day 16), and increased during lung development. miR-129 expression in brain and lung is higher than in other organs. (B) Levels of miR-129-5p and -3p in human lung adenocarcinoma cell lines (H1299, H460, H596 and A549) and in mouse lung epithelial cell line (E10) are significantly lower than those of mouse or human adult lung.

tumors and in tumor cell lines from different organs11-15,17 could be also seen in lung derived cell lines compared to normal lung tissue. We examined miR-129 levels in the adult and developing mouse lungs and in other adult tissues by northern blot analysis. miR-129 is conserved among vertebrates (miRBase); in most organisms, two loci encode miR-129 and two mature miRNAs are processed from both 5' prime (miR-129-5p) and 3'prime (miR-129-3p) of its pre-miRNA precursor.19 Expression of both miR-129-5p and miR-129-3p in mouse adult lung and brain was higher than in the kidney and heart. Interestingly, miR-129 levels in adult murine lungs were much higher than in embryonic lungs (Fig. 1A). This correlates well with the overall advanced differentiation and decreased proliferation status of the adult lung. Analysis of miR-129 in the murine E10 cell line,20 a nontransformed lung epithelial cell line and in different human lung adenocarcinoma cell lines, including H1299, H596, A549 and H460, showed very low expression levels, compared to normal adult lungs (Fig. 1B). This raised the possibility that downregulation of miR-129 could presumably result in increased cell proliferation. Since the expression of miR-129 in these cells is extremely low, we used a gain of function approach to address this issue. Overexpression of miR-129 in lung epithelial cells results in G1 phase arrest and cell death. To investigate the impact of 1810

miR-129 in cell growth, we increased miR-129 levels in the E10 lung epithelial cell line using lentiviral-mediated transduction. E10 cells have been previously reported as a line established from normal adult mouse lung; they are non-tumorigenic, exhibit contact inhibition, and display features typical of non-transformed cell lines.20 E10 cells grow rapidly, with similar doubling time comparable to A549 and other lung adenocarcinoma cell lines used in this study. Infection of E10 cells with pHAGE-CMV-eGFP-miR-129 virus or control virus (pHAGE-CMV-eGFP) resulted in efficient transduction (>95% of cells were GFP positive) and robust expression of both mature miR-129-5p and -3p within 48 hrs (Fig. 2A). Cell counting at different time points after infection revealed a significant decline in the number of miR-129 overexpressing cells three days after infection and thereafter, suggestive of growth arrest (Fig. 2D). Cell cycle analysis showed a greater than 10% increase in the G1 population of miR-129-expressing cells relative to controls at day 3, consistent with a G1 phase arrest (Fig. 2E and F). At day 4, a sub-G0 population became evident (about 40%), coincident with the appearance of apoptotic miR129 expressing cells (Fig. 2G and H). Thus, overexpression of miR-129 resulted in G1 phase arrest that eventually led to cell death. A similar effect was observed in E10 cells infected with different doses of miR-129 lentivirus (Fig. S1). This suggested that the

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Figure 2. Overexpression of miR-129 in E10 cells results in G1 arrest and cell death. (A) Expression of both mature miR-129-5p and -3p was detected in the E10 cells two days after infection. (B and C) miR-129 overexpressing cells were larger than those cells infected with control lentivirus. (D) Expression of miR-129 significantly arrested the growth of E10 cells (p < 0.05 for all time points). (E and F) G1 phase arrest was observed three days after infection. (G and H) Expression of miR-129 resulted in cell death four days after infection. *p = 0.015; **p = 0.004.

growth arrest and cell death phenotype was unlikely the result of lentiviral-mediated toxicity. The specificity of these effects was further demonstrated by overexpressing several other miRNAs in E10 cells using lentiviral vectors, including miR-29, miR-126, miR-223 and miR-451, known to be present in embryonic or adult lungs, but at extremely low levels.21,22 Highly efficient transduction was observed for all these lentiviral vectors, as indicated by GFP expression (Fig. S2A). The levels of mature miRNAs generated by these lentiviral vectors were different, likely due to the different efficiency in miRNA processing and in miRNA stability (Fig. 2SB). We found similar levels of overexpression of miR-29 and miR-126, as compared to those of miR-129, while levels of miR-223 and miR-451 were much lower (Fig. S2B). Among them, we only observed the sub-G0 population in miR-129 overexpressing cells (Fig. S2A). We also observed that although the levels of overexpressed miR-129 in E10 cells was higher than in adult lung, they were similar to those of adult brain (Fig. S2C). This suggested that the levels of overexpressed miR-129 were comparable to the physiological levels in cells of adult brain. Thus, the cell death and growth arrest phenotype was unlikely of the results from non-specific effects caused by overwhelming expression of mature miR-129. To gain initial insights into molecular phenotype of miR-129mediate cell growth arrest, we performed a genome-wide mRNA expression profiling of E10 cells infected with miR-129 or a GFPcontrol virus. Total RNA was isolated 48 hours after infection

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and processed for Affymetrix array (Mouse 430 2.0) hybridization and analysis. As expected, cell cycle-related genes were significantly over-represented in the list of genes downregulated in E10 cells with high miR-129 (EASE score, 7.10E-26, supporting information, Table S1). These include cyclins, Mcm (minichromosome maintenance deficient), DNA polymerase and replication factors (Table S2). By contrast, cell growth arrest- and cell death-related genes were over-represented among those upregulated by miR-129 (EASE score, 1.32E-2), including positive regulators of cell death, such as Cdkn1a or p21, Inhba, Gadd45a, Pdcd4, Braf, Tnfrsf10b, Sulf1, Bnip3, Apaf1, casp7 and Bok (Table S2). These results were consistent with the growth arrest phenotype observed. Anti-proliferation activity of miR-129 in multiple tumor cell lines. Next, we tested whether miR-129 could influence the growth behavior of other cell types, including lung cancer cells. Thus, we transduced miR-129 into lung adenocarcinoma cell lines A549, H596 and H1299, which express endogenous miR129 at extremely low levels (Fig. 1B). Remarkably, overexpression of miR-129 in all three lines resulted in a significant increased G1 phase population (more than 10%) and growth arrest (Fig. 3A and B). We did not observe the increase in sub-G0 population seen in the E10 cells even after 5 days post-infection. We also tested two brain glioblastoma cell lines (SNB75 and SF295) and one colorectal cancer cell line (HCT116), in which expression of endogenous miR-129 was nearly undetectable.12 In all cases,

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miR-410 expression construct, an unrelated miRNA with no complementary site in these luciferase reporters, respectively. Luciferase analysis revealed that the activities of three wild type 3'UTR reporters were significantly suppressed by miR-129, but not by miR-410 (Fig. 4B). Suppression by miR-129 depends on the wild type miR-129 complementary sites, and was no longer observed in reporters in which miR-129 complementary sites were mutated (Fig. 4B). The collective effect of these multiple complementary sites in the 3'UTR of CDK6 was likely the cause for the dramatic reduction of endogenous CDK6 protein levels by miR-129. Since these binding sites are scattered in a 11 kb 3'UTR, it is a challenge to analyze their synergistic effect in its natural context. Nevertheless, our data strongly suggested that CDK6 is a direct target of miR-129. Overexpression of Cdk6 partially rescues the miR129-mediated cell growth arrest and death phenotype. To investigate how critical is Cdk6 for the miR-129mediated effects, we rescued the Cdk6 expression in miR-129 overexpressing E10 cells. For this we generated a pLVTHM lentiviral vector containing the coding region of Cdk6. Co-transfection of a miR-129 and a Cdk6 lentiviral vector resulted in a four-fold increase in the cell numFigure 3. Expression of miR-129 resulted in reduced growth of human lung adenober, compared to cells co-transfected with miR-129 plus carcinoma cells. (A) The growth curve of A549 cells infected with miR-129 overexcontrol vector (Fig. 5A). We also observed a significant pressing lentivirus or with control lentivirus (p < 0.05 for D3, D4 and D5). reduction (31%) in the sub-G0 population in cells co(B) Significant G1 phase arrest was observed in A549, H1299 and H596 cells five days transfected with Cdk6 and miR-129 vectors (Fig. 5B). after infection. Thus, our data indicated that downregulation of Cdk6 by miR-129 is at least partially responsible for the E10 overexpression of miR-129 resulted in cell growth arrest and cell cell growth arrest and death phenotype observed. death (data not shown). These results confirmed a general role of Downregulation of cell cycle-related targets Prkce or Erk1/ miR-129 in regulating cell proliferation. Erk2 is not responsible for cell growth arrest phenotype. By Cdk6 is a direct target of miR-129. Next, we asked what tar- examining the expression levels of miR-129 predicted targets in gets of miR-129 were responsible for the G1 phase arrest pheno- our array data, we have identified that mRNA levels of 81 pretype. First, we searched multiple databases for predicted targets dicted targets of miR-129-5p (Targetscan) and 105 predicted tarof miR-129-5p or -3p involved in cell cycle regulation. We found gets of miR-129-3p (Targetscan) are significantly downregulated that human Cdk6 is one of the top predicted targets of miR- by miR-129 (Tables S3 and S4). Among these, 17 have known 129-5p with four complementary sites in its 3'UTR (microRNA. function in cell proliferation (Table 1). We asked what other tarorg). CDK6 plays an important role in G1/S phase transition by gets could be potentially involved in miR-129-mediated cell cycle associating with the D-type cyclins and by targeting the retino- regulation. blastoma protein (Rb).23 We reasoned that Cdk6 could be a canWe first examined the protein levels of additional genes from didate target in miR-129-mediated cell growth arrest and death. Table 1 in miR-129-expressing cells. We found that in both A549 By western blot, we found a three-fold reduction in Cdk6 and H1299 cells, protein levels of Prkce were significantly reduced protein level in both E10 and H1299 cells in which miR-129 is by miR-129 (Fig. 6A). 3'UTR luciferase reporter with wild type overexpressed (Fig. 4A). However, our array analysis showed no 3'UTR of Prkce was significantly suppressed by miR-129, but not difference in the mRNA levels of Cdk6 between miR-129 over- by miR-410 (Fig. 6B). This indicated that Prkce is a direct target expressing and control E10 cells (Fig. 4A, FC = 1.06, p = 0.24). of miR-129. To examine the impact of reduced Prkce expression This raised the possibility that miR-129 suppresses Cdk6 protein on the cell growth. We performed a similar rescue experiment as synthesis by a post-transcriptional repression mechanism, via its we did for Cdk6. Unlike Cdk6, we found that restoring Prkce expression by co3'UTR complementary sites. To test this possibility, fragments of 3'UTR of human CDK6 infection of Phage-CMV-GFP-miR-129 with LVTHM-Prkce did containing wild type or mutated miR-129 complementary sites not result in significant changes in cell number, as compared to were cloned into psiCHECK2 dual luciferase reporter plas- the combination of Phage-CMV-GFP-miR-129 with LVTHMmid. Luciferase reporters were co-transfected with miR-129 or GFP (Fig. 6C). This indicates that of the downregulation of

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Figure 4. Downregulation of Cdk6 by miR-129 is mediated by 3'UTR complementary sites. (A) Cdk6 protein levels in H1229 and E10 cells are significantly suppressed by miR-129. (B) FG293 cells were co-transfected with Cdk6 3'-UTR luciferase reporters, along with expression plasmids of miR-129 and miR-410. Firefly luciferase activity from the same construct were used to normalize and to generate relative activity of Renilla luciferase which was subject to the regulation of cloned Cdk6 3'UTR. These reporter constructs were named after the nucleotide position of complimentary site of miR-129 in the 3'UTR of Cdk6.

Prkce is not responsible for the miR-129-mediated growth arrest phenotype. Erk1 (Mapk3) is a predicted target of miR-129-3p, while Erk2 (Mapk1) a predicted target of both miR-129-5p and -3p. The total protein and mRNA levels of Erk1/2 were significantly downregulated by miR-129 in E10 cells (Fig. 7A) and lung adenocarcinoma cells. However, we detected significantly increased levels of the phosphorylated form of Erk1/2 in miR-129 overexpressing cells (Fig. 7A). Activation of Erk1/2 is known to promote cell proliferation and survival.24 Thus it is unlikely that the increased activity of Erk1/Erk2 in miR-129-expressing cells could be involved in miR-129-mediated growth arrest. Several studies indicate that prolonged activation of Erk1/ Erk2 results in neuronal and tumor cell death.25,26 This led us to investigate whether inhibiting activity of the Erk1/Erk2 could prevent the growth arrest or cell death of miR-129 overexpressing

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cells. Interestingly, treatment with U120 or PD98059, two different inhibitors of Erk1/Erk2 pathway resulted in significantly reduced growth of GFP control cells, but had no effect in the growth of miR-129 overexpressing cells (Fig. 7B and C). In contrast, inhibition of Akt kinase, which is also important for cell growth and survival resulted in dramatic growth arrest of both miR-129 overexpressing and GFP control cells (Fig. 7B and C). Thus, we concluded that activated Erk1/Erk2 was not relevant for the miR-129-mediated growth arrest. Discussion Little is known about the function of miR-129 in cell proliferation and cell death. Here, we report that miR-129 expression is significantly reduced in multiple cell lines, including mouse lung epithelial cells (E10) and human lung adenocarcinoma cell lines.

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Figure 5. Restoring the expression of Cdk6 partially rescues the cell growth arrest and cell death phenotype. (A) Expression of Cdk6 partially rescues cell growth arrest phenotype, cells were counted five days after infection. (B) Expression of Cdk6 significantly reduced the sub-G0 population cells of miR-129 overexpressing cells.

miR-129 gain of function resulted in G1 phase arrest. We show that this effect is, at least in part, due to downregulation of Cdk6. Cdk6 plays an important role in G1/S phase transition. Several studies have shown that amplification, overexpression or activation of Cdk6 is associated with human glioblastoma, medulloblastoma and lung adenocarcinomas.27-29 Cdk6 has been shown to play an essential role in F-box protein 7 (Fbxo7)-mediated transformation of murine fibroblasts.30 We showed that posttranscriptional downregulation of Cdk6, but not Prkce or Erk1/Erk2 by miR-129 results in reduced cell growth, suggesting an important role of miR-129-Cdk6 axis in tumor cell growth. There are predicted binding sites for a large number of miRNAs within the 11 kb sequence of 3'UTR of human Cdk6, including four complementary sites for miR-129-5p. Experimentally, Cdk6 has been shown to be the target of miR-34a in pancreatic

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carcinoma cells, and the target of miR-124 in medulloblastoma cells.31,32 In lung adenocarcinoma A549 cells, Cdk6 is under the control of let-7.33 Here, we showed that in mouse lung epithelial cells, and lung adenocarcinoma cells, Cdk6 is under the control of miR-129. Cdk6 may be regulated collectively by multiple miRNAs in the same cell. However, conversely a particular cell type may express only one of these miRNAs that target Cdk6. We have evidence that miR-129 expression is restricted to specific cell types in developing and adult organs (Wu et al., unpublished results). This suggests Cdk6 may be a critical mediator of the miR-129 biological effects in these cells. We observed downregulation of a large number of predicted targets of both miR-129-5p and miR-129-3p in miR-129 overexpressing cells, indicating that both mature miRNAs are functional. miR-129-5p is at least partially responsible for cell growth

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Table 1. miR-129 predicted targets that are related to cell proliferation Gene symbol

Gene title

miR-129-5p/3p

FC (miR-129/Gfp)

p-value

Mapre1

microtubule-associated protein, RP/EB family, member 1

5p

0.24

1.95E-04

Mapk3

mitogen activated protein kinase 3

3p

0.29

1.06E-03

Dedd

death effector domain-containing

3p

0.32

5.28E-03

Pura

purine rich element binding protein A

5p/3p

0.41

5.85E-04

Yes1

Yamaguchi sarcoma viral (v-yes) oncogene homolog 1

3p

0.43

4.90E-02

Hmgb1

high mobility group box 1

5p

0.46

3.62E-05

Apc

adenomatosis polyposis coli

5p

0.48

8.90E-03

Unc84b

unc-84 homolog B (C. elegans)

5p

0.5

2.14E-04

Fmr1

fragile X mental retardation syndrome 1 homolog

5p

0.5

3.82E-02 4.04E-04

Nek1

NIMA-related expressed kinase 1

3p

0.58

Mapk1

mitogen activated protein kinase 1

5p/3p

0.59

1.42E-03

Birc6

Baculoviral IAP repeat-containing 6

3p

0.61

1.20E-03

Rap1b

RAS related protein 1b

3p

0.65

3.25E-03

Prkce

protein kinase C, epsilon

3p

0.65

1.08E-02

Son

Son cell proliferation protein

3p

0.72

1.60E-02

E2f3

E2F transcription factor 3

3p

0.83

9.53E-03

arrest, since Cdk6 is targeted by miR-129-5p, but not by miR129-3p. We showed that three miR-129-3p targets including Erk1/Erk2 and Prkce are downregulated by miR-129, however, they are not involved in growth arrest. This is consistent with a recent finding that transfection of miR-129-5p, but not a miR129-3p mimic significantly reduces the proliferation of endometrial cancer cells.17,18 The role of miR-129-3p in cell growth arrest, if any, is currently unknown. Whether there are synergistic roles of these two miRNAs in cell proliferation is still an important issue to be addressed in future studies. Restoring the expression of Cdk6 only partially rescue the growth arrest and cell death phenotype, suggesting the involvement of other genes downstream of miR-129. Expression of more than 100 predicted targets of miR-129-5p was significantly reduced in miR-129 overexpressing cells. These included several genes with known indirect role in cell proliferation, such as poly (ADP-ribose) polymerase 1 (Parp1),34,35 SRY (sex determining region Y)-box 4 (Sox4)17 (Table S3). We attempted to analyze the role of endogenous miR-129 in cell proliferation. Extensive search for cell lines that express high level of miR-129 led us to choose L1236 cells (Hodgkin’s lymphoma cell line) as a potential line to explore. We detected relatively high levels of miR-129-3p in L1236 cells, but very low levels of miR-129-5p which is responsible for cell growth arrest.17,18 Thus, addressing the role of endogenous miR-129 will require a mouse genetic model, beyond the scope of this study. In summary, we have characterized the role of miR-129 in cell cycle regulation in mouse lung epithelial cells and human adenocarcinoma cells. We showed that miR-129 arrests cell growth at least in part by downregulation of Cdk6. Since downregulation of miR-129 has been reported in medulloblastoma, gastric cancer, lung adenocarcinoma, endometrial tumors and colon cancer, it will be interesting to examine the role of miR-129 in the pathogenesis of these primary tumors.

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Materials and Methods Northern blot. Total RNA was isolated from mouse or human tissues or cell lines using Trizol (Invitrogen). Timed pregnant CD1 mice (Charles River Laboratory International) were used for collection of lungs at different developmental stages. 20 ug total RNA was separated on 15% polyacrylamide gel with 7 M urea and 20 mM MOPS buffer (pH 7.0) and transferred to Hybond Nx membrane (Amersham Biosciences). RNA was cross-linked to the membrane using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).22 Ultrahyb-Ultrasensitive Hybridization buffer (Ambion) was used for pre-hybridization and hybridization at 40°C with 0.1 nM DIG-labeled LNA probe (Exiqon). After three washes in 2X SSC, 0.1% SDS, blots were rinsed in washing buffer (0.1 M maleic acid, 0.15 M NaCl, 0.1% Tween, pH 7.5) and blocked with 1% blocking reagent (Roche). Subsequently, blots were incubated for 1 hour at room temperature with antiDIG-AP antibody (Roche) in blocking buffer, washed 3 times for 15 min in washing buffer and twice for 5 min with AP-buffer (0.1 M Tris-HCl pH 9.5, 50 mM MgCl2, 0.1 M NaCl, 0.1% Tween). Signal was developed and detected by using CDP-star chemiluminescent substrate (Roche). Antibodies and western blot. Total protein samples were prepared by lysing cells with 1.5X SDS Lane Marker Sample Buffers (PIERCE, Cat# 39000). Western blot analysis was carried out following the manufacturer’s protocol. We used antibodies against Cdk6, Prkce and Erk1/Erk2 (Cell Signalling Technology), Gapdh (R&D Systems). HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (Millipore) were used as secondary antibodies. Immune-star HRP Developer System (Bio-Rad) was used for detection. Overexpression of miR-129-2, Cdk6, Prkce using a lentiviral system. Overexpression of miRNAs under the CMV promoter was achieved using the third generation, replication incompetent,

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Figure 6. Prkce is another novel target of miR-129-3p. (A) Protein level of Prkce is significantly reduced in A549 or H1299 cells by miR-129; (B) FG293 cells were co-transfected with Prkce 3'-UTR luciferase reporters, along with expression plasmids of miR-129 and miR-410. Firefly luciferase activity from the same construct were used to normalize and to generate relative activity of Renilla luciferase which was subject to the regulation of cloned Prkce 3'UTR. (C) Restoring Prkce expression did not rescue cell growth arrest phenotype, cells were counted five days after infection.

VSV-G pseudotyped lentiviral vector backbone (generous gift of Dr. Richard C. Mulligan, Harvard Medical School) previously published as pHAGE.36,37 The DNA sequence encoding the indicated miRNA together with surrounding miRNA precursor sequence (approximately 500 bp in total) was amplified

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Figure 7. Predicted targets Erk1/Erk2 are downregulated by miR-129. (A) Levels of total Erk1/Erk2 protein and mRNAs are reduced by miR-129 in E10 cells, while phosphorylated Erk1/Erk2 are increased. (B) Inhibition of Akt and Erk1/Erk2 results in cell growth arrest and cell death in Gfp control cells. (C) Inhibition of Erk1/Erk2 has no effect on the growth of miR-129 overexpressing cells, while blocking Akt activity significantly reduces the cell number.

by conventional PCR. This sequence was then ligated into the BamH1 site of pHAGE-CMV-eGFP-W38 immediately following the eGFP TAA stop site. The resulting integrating lentiviral vector results in constitutive transcription of an RNA encoding the eGFP mRNA as well as the relevant miRNA. This approach allows translation of the eGFP tracking reporter gene as well as

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processing and maturation of the indicated miRNA precursor from the same RNA transcript.39 For overexpression of human Cdk6 and Prkce, the coding region of each gene were cloned into the pLVTHM lentiviral vector, under the control of a constitutively active EF1-alfa promoter (generous gift of Dr. Didier Trono, University of Geneva; available from addgene.org) at PmeI and NdeI sites.40 Packaging of all lentiviral vectors was performed as published36,37 by co-transfection of the 293T cell line with 5 plasmids encoding the lentiviral backbone as well as tat, rev, gag/pol and vsv-g viral genes. Viral particles in the resulting supernatants were collected and concentrated by ultracentrifugation. Titers used in all experiments ranged from 0.5–1.5 x 109 TU/mL. Sequences of PCR primers used for these cloning are included in Supplementary Information (Table S5). Cell cycle analysis. Murine non-transformed lung epithelial cells (E10) or human lung adenocarcinoma cells (A540, H1299 and H596, from ATCC) were grown in DMEM with 10% FBS and antibiotics in 24 or 6 well plates. Lentiviral infections were performed when cells reached 80% –90% confluency. For cell cycle analysis, cells were harvested at different time points, washed with PBS and fixed in 35% ethanol at 4°C for 4 hrs. After fixation, cells were washed twice with PBS before re-suspension in propidium iodide/RNase A solution (50 µg/ml propidium iodide and 100 µg/ml RNase A). Cells were incubated with propidium iodide at room temperature in the dark for 1 hour. Stained cells were analyzed by flow cytometry for light-scattering properties and for DNA content using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). These data were analyzed by using Flowjo (Flowjo). 3'-UTR luciferase reporter assay. DNA fragments from 3'UTR of Cdk6 and Prkce that host the predicted complementary sites of miR-129 or the mutated sites were cloned downstream of the Renilla luciferase reporter gene in psiCHECK2 dual luciferase reporter plasmid (Promega). FG293 cells were seeded in 96 well plates, and co-transfected with luciferase reporters together with either miR-129 or miR-410 expression plasmid. Cells were lysed and luciferase activities were measured 48 hrs after transfection, according to manufacturer’s instruction using the Dual-GloTM Luciferase Assay System and GloMax Multi-Detection System (Promega). All experiments were carried out in six replicates and data were normalized to the activity of the firefly luciferase expressed from the same psiCHECK-2 vector as an internal control. Sequences of PCR primers used for these cloning are included in Supplementary Information (Table S5). References 1. 2. 3.

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Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 2009; 10:1-3. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009; 136:215-33. Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev 2006; 20:515-24. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, et al. MicroRNA expression profiles classify human cancers. Nature 2005; 435:834-8. Mi S, Lu J, Sun M, Li Z, Zhang H, Neilly MB, et al. MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia. Proc Natl Acad Sci USA 2007; 104:19971-6.

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8.

9.

Affymetrix oligonucleotide array analysis. E10 cells infected with miR-129 lentivirus or the control lentivirus were cultured for 48 hours. More than 80% of cells were infected. Total RNA samples were isolated using Trizol (invitrogen), and subjected to labeling, fragmentation and hybridization, according to manufacturer’s protocols. The Mouse Genome 430 2.0 Array (Affymetrix) was used for mRNA expression profiling. Experiments were performed in triplicates for each condition. Expression levels for probe sets on the array along with detection p values (indicating whether the transcript was reliably detected) were derived using Affymetrix Microarray Suite 5.0 software. All microarrays passed the quality control criteria, 70% of genes are present and 3':5' ratio for Actin range from 3.0 to 6.9. Data from each array were normalized for inter-array comparisons by scaling the target intensity to 500. Genes were filtered out from further analysis if their detection p-values were greater than 0.05 in all six chips. A two-sample t-test was performed in order to identify differential gene expression between comparative groups (p < 0.05 considered significant). To explore the functional significance of differentially expressed transcripts, DAVID (david.abcc.ncifcrf.gov), Targetscan (www.targetscan.org) databases were used for data analysis. The array data has been deposited into GEO database in NCBI (GSE15121). Human lung tissue acquisition. Subjects undergoing thoracotomy at Boston Medical Center for early stage lung cancer were recruited into this study. Lung tissue samples from the tumor and adjacent histologically normal lung were collected intraoperatively and flash frozen in liquid nitrogen. The tissue samples were stored at -80°C until RNA isolation was performed. This study was approved by the Institutional Review Board at Boston Medical Center. All subjects provided written informed consent. Total RNA samples from adjacent histologically normal lung were used in this study. Acknowledgements

This work is supported by Grants from NIHBL (R01HL081800, P01 HL47049). We are all grateful for the comments and suggestions provided by Jerome Brody, Mary Williams, Xingbin Ai, Zhi-Xiong Xiao, Yujun Zhang, Arjun Guha and also technical supports provided by Yanhui Deng. Note

Supplementary materials can be found at: www.landesbioscience.com/supplement/WuCC9-9-Sup.pdf

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