Downregulation of BCL2 by miRNAs augments drug- induced ...

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Research Article

Downregulation of BCL2 by miRNAs augments druginduced apoptosis – a combined computational and experimental approach Richa Singh and Neeru Saini* Functional Genomics Unit, Institute of Genomics and Integrative Biology, Council of Scientific and Industrial Research (CSIR), Mall Road, Delhi-110007, India *Author for correspondence ([email protected])

Journal of Cell Science

Accepted 16 September 2011 Journal of Cell Science 125, 1568–1578 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.095976

Summary A number of anti-cancer strategies aim to target the mitochondrial apoptotic machinery to induce tumour cell death. Mitochondria play a key role as death amplifiers by releasing apoptogenic factors from the mitochondrial inter-membrane space into the cytosol. BCL2 proteins are known for their ability to regulate both mitochondrial physiology and cell death, and their deregulated expression often renders cancer cells insensitive to apoptosis-inducing anticancer drugs. Recently, a few microRNAs, a novel class of gene regulators, have been demonstrated to regulate expression of some members of the BCL2 family. Here, we have combined computational and experimental approaches to identify miRNAs that can regulate the anti-apoptotic protein BCL2. We report that miR-195, miR-24-2 and miR-365-2 act as negative regulators of BCL2 through direct binding to their respective binding sites in the 39-UTR of the human BCL2 gene. Ectopic expression of miR-195, miR-24-2 and miR-365-2 individually led to a significant reduction of the levels of BCL2 protein. Additionally, we found that overexpression of these miRNAs induced dissipation of the mitochondrial membrane potential and release of cytochrome c from mitochondria into the cytosol. Furthermore, we demonstrated that overexpression of these miRNAs not only caused an increase in apoptosis but also augmented the apoptotic effect of etoposide in breast cancer MCF7 cells. These data not only show the apoptotic nature of miR-195, miR-24-2 and miR-365-2 but also highlight the therapeutic potential of these miRNAs. Key words: Apoptosis, BCL2, Etoposide, MicroRNA

Introduction Most anticancer drugs work by induction of apoptosis (Danial and Korsmeyer, 2004). Alterations in susceptibility to apoptosis not only contribute to neoplastic development (Strasser et al., 1990) but also enhance resistance to conventional anticancer therapies, such as radiation and cytotoxic agents (Green and Reed, 1998). BCL2 (encoding the protein known as apoptosis regulator Bcl-2 or B cell lymphoma 2) is the founder member of the BCL2 family of apoptosis regulators and was identified as an oncogene that does not promote cell proliferation but the evasion of cell death (Cory and Adams, 2002; Youle and Strasser, 2008). BCL2 manifests its pro-survival effect by inhibiting dimerization of pro-apoptotic members at the mitochondrial membrane, thereby protecting the integrity of the mitochondrial membrane (Dlugosz et al., 2006; Zamzami et al., 1996). Overexpression of BCL2 protein has been reported in many types of cancers, including leukemia, lymphomas and carcinomas (Iqbal et al., 2006; Majid et al., 2008; Skirnisdottir et al., 2002; Yanai et al., 2010) and has been associated with chemotherapy resistance in various human cancers. Thus targeted inhibition of BCL2 can be used as a tool for the treatment of different cancers. Several classes of drugs have been found to regulate gene expression of anti-apoptotic BCL2 members, and several of these are in different phases of clinical trials (Andersen et al., 2005; Kausch et al., 2005; Oltersdorf et al., 2005). The prospects regarding the potential benefits of inhibiting the BCL2 members by drugs seem promising, but, as the tumours are

typically heterogeneous in nature, it is evident that the current strategies will not produce many cures and that resistance might emerge. Hence, there is an urgent need to look for other drugs or complementary therapies as well as establish a better understanding of the cellular mechanisms regulating BCL2 members to overcome chemoresistance and fight cancer. MicroRNAs (miRNAs), non-coding RNA molecules of length 22–26 nucleotides, have emerged as new regulators of gene expression (Bartel, 2004; Zamore and Haley, 2005). Recent studies have shown that miRNAs can be an additional class of cell death regulators (Cheng et al., 2005; Garzon et al., 2009; Vecchione and Croce, 2010). Aberrant miRNA expression has been frequently observed in various human tumours (Macfarlane and Murphy, 2010; Mayr et al., 2007). More than 50% of human miRNA genes are located in cancer-associated genomic regions or at fragile sites, which further suggests there exists an important role for miRNAs in tumorigenesis and cancer progression (Calin et al., 2004; Volinia et al., 2006). miRNAs pair with partially complementary sites in the 39-untranslated regions (UTRs) of target mRNAs, leading to translational repression and/or mRNA degradation. Computational and biological analyses estimate that ,30% of all genes and the majority of genetic pathways are subject to regulation by multiple miRNAs (Bartel, 2009). A number of studies have clearly shown that one miRNA can regulate several hundreds of target mRNAs, and, conversely, one mRNA can be targeted by multiple miRNAs (Lim et al., 2005).


miRNA-mediated downregulation of BCL2 By targeting multiple transcripts, miRNAs play important roles in a wide array of biological processes, including development, differentiation, cell proliferation, apoptosis and metabolism (Ambros, 2004; Brennecke et al., 2003; Reinhart et al., 2000). However, relatively few miRNA–target interactions have been experimentally validated, and the functions of a majority of miRNAs remain to be elucidated (Bentwich et al., 2005; Kloosterman and Plasterk, 2006). Recent findings have also suggested that miRNAs have important roles in development of chemosensitivity or chemoresistance in different cancers (Zhang et al., 2010; Ory et al., 2011; Hamano et al., 2011). Evidence for the involvement of miRNAs in the regulation of the antiapoptotic protein BCL2 came from the discovery of miR-15 and miR-16 as regulators of BCL2 in chronic lymphocytic leukemia (CLL) (Cimmino et al., 2005). Several lines of evidence show that miR-15b, miR-16 (Xia et al., 2008), miR-181b (Zhu et al., 2010) and miR-34a (Kojima et al., 2010) modulate multidrug resistance by targeting BCL2. In the current study, through computational approaches, we identified three miRNAs – miR-195, miR-24-2 and miR-365-2 – that could negatively regulate BCL2 expression by binding to its 39-UTR. The experiments presented here show that overexpression of miR-195, miR-24-2 and miR-365-2 individually impaired the pro-survival function of BCL2 in human embryonic kidney (HEK 293T) and breast cancer (MCF7) cells and augmented the apoptotic effect of etoposide in breast cancer cells. Our current data highlight the pro-apoptotic function miR-195, miR-24-2 and

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miR-365-2 and support the application of these miRNAs as therapeutic molecules. Results Identification of potential miRNAs targeting BCL2

MicroRNAs (miRNAs) are small non-coding RNAs that affect the expression of target genes by means of translational repression or mRNA degradation. Huang and colleagues showed that the simultaneous expression profiling of miRNAs and mRNAs is an effective strategy for miRNA target identification (Huang et al., 2007). With the increasing availability of mRNA and miRNA expression data, it might be possible to assess functional targets using the fact that a miRNA might downregulate its target mRNAs. In this work, we correlated gene expression data for 48 cell lines [from Gene Expression Omnibus (GEO), GSE5949] with miRNA expression data of the same cell lines from Cellminer (Blower et al., 2007) to identify miRNAs that could target BCL2. By using correlation coefficient values, we shortlisted 69 miRNAs (supplementary material Table S1) that showed significant negative correlation with BCL2 expression (R2#–0.287; P,0.05; Fig. 1A). Next, we compared this with a list of 320 miRNAs constituted from overlap of two online tools of miRNA target prediction, TargetScan (http://www.targetscan.org) and miRanda (http:// www.microrna.org), which resulted in a consensus list of 22 miRNAs. Comparative analysis showed six miRNAs – miR-365, miR-195, miR-15a, miR-24, miR-16 and miR-383 – to be

Fig. 1. Selection of candidate microRNAs targeting BCL2. (A) Graph showing correlation coefficient values between BCL2 mRNA and microRNA expression across 48 cell lines. Expression data of 319 miRNAs were available for the 48 cell lines. A total of 69 miRNAs show a significant negative correlation with BCL2 expression, with R2#20.287, P,0.05. (B) A list of 22 miRNAs was generated by consensus of the above 69 miRNAs and the 320 miRNAs predicted to target BCL2 by TargetScan or miRanda. Finally, six miRNAs were selected as potential miRNAs targeting BCL2, which were predicted by both software applications.

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Journal of Cell Science 125 (6)

potential miRNAs to target BCL2 as they were predicted by both the software tools and were significantly negatively correlated with BCL2 mRNA expression (Fig. 1B). Among the predicted miRNAs, hsa-miR-15 and hsa-miR-16 have already been reported to target BCL2 (Cimmino et al., 2005). We initiated our study with miR-195, miR-24 and miR-365. It should be noted here that miR-24 and miR-365 both are derived from two distinct hairpin loci, namely hsa-miR-24-1 and hsa-miR-24-2, and hsamiR-365-1 and hsa-miR-365-2, respectively. The nomenclature (‘1’ and ‘2’) is based on the difference in chromosomal origins of the hairpin precursors, but the mature miRNA sequence is identical. In the current study, we cloned the precursor sequences of miR-195, hsa-miR-24-2 and hsa-miR-365-2, designated them as p195, p24-2 and p365-2, respectively, and performed further experiments. hsa-miR-195, hsa-miR-24-2 and hsa-miR-365-2 negatively regulate BCL2 protein levels


Computational analysis suggested that the 2–7 base seed sequence regions of hsa-miR-195, hsa-miR-24-2 and hsa-miR365-2 have perfect complementarities to their respective putative binding sites on the 39-UTR of BCL2. The putative binding site of hsa-miR-195 is TGCTGCTA (2521–2527 bp from the start of the

39-UTR of human BCL2), of hsa-miR-24 is CTGAGCC (1011– 1017 bp from the start of the 39-UTR of human BCL2) and that of hsa-miR-365 is GGGCATT (1711–1717 bp; 1574–1581 bp from the start of the 39-UTR of human BCL2), as shown in Fig. 2A. To examine whether these three miRNAs target BCL2, pre-miRNA plasmids, namely p195, p24-2 and p365-2, were transfected into human embryonic kidney (HEK 293T) and breast cancer (MCF7) cells. These vectors express the precursor forms of miR-195, miR-24-2 and miR-365-2 in their native context, while preserving putative hairpin structures to ensure biologically relevant interactions with endogenous processing machinery and regulatory partners, which leads to properly cleaved miRNAs. A TaqMan-based real-time PCR assay showed that overexpression of p195, p24-2 and p365-2 plasmids led to a significant increase in the expression of the mature form of the respective miRNAs in HEK 293T and MCF7 cells (Fig. 2Bi,Ci). In HEK 293T cells, there was 3.3-fold (P50.05) increase in the mature form of miR-195, a 3-fold (P50.041) increase in the mature form of miR-24 and a 4.5-fold (P50.025) increase in the mature form of miR-365 expression in comparison with nontransfected HEK 293T cells. Similarly, there was 2.7fold (P50.024) increase in the mature form of miR-195, a 3.5fold (P50.001) increase in the mature form of miR-24 and a

Fig. 2. miR-195, miR-24-2 and miR-365-2 negatively regulate BCL2 protein levels in cells. (A) Schematic representation of BCL2 mRNA with predicted binding sites of hsa-miR-24, hsa-miR-365 and hsa-miR-195 in the 39-UTR. Hsa-miR-365 has two predicted binding sites in the BCL2 39-UTR. The 2–8 nucleotide seed region of the miRNAs is perfectly complementary to their respective binding sites (indicated in red) and numbers indicate respective binding positions. (B) Expression data for HEK 293T and (C) MCF7 cells. (i) Taqman assay for mature form of miR-195, miR-24 and miR-365 before and after transfection of p195, p24-2 and p365-2. (ii) Quantitative real-time RT-PCR for detection of BCL2 mRNA levels after overexpression of miR-195, miR-24-2 and miR-365-2. 18s rRNA was used for normalization of real-time data. (iii) Western blotting for BCL2 protein before and after transfection of p195, p24-2 and p365-2 in HEK 293T and MCF7 cells. The same blot was probed for b-actin for normalization. Graphs in the lower panel represent the fold change in the BCL2 protein levels (mean6s.d.) of three independent experiments. The bands were quantified by spot densitometry using AlphaEase Imager and normalized with b-actin. *P,0.05, **P,0.01 versus nontransfected controls.


miRNA-mediated downregulation of BCL2 2.9-fold (P50.002) increase in the mature form of miR-365 in MCF7 cells in comparison with nontransfected MCF7 cells Fig. 2Ci. As miRNAs can exert their role at either the transcriptional, post transcriptional or at both levels, the BCL2 mRNA and protein levels were determined at 24 hours posttransfection of p195, p24-2 and p365-2 in HEK 293T and MCF7 cells. As shown in Fig. 2B,C, the mRNA as well as protein levels of BCL2 were significantly reduced in both the cell lines. The BCL2 mRNA levels were decreased by 1.38-fold and 1.44-fold (P50.014) after overexpression of miR-195 in HEK 293T and MCF7 cells, respectively [Fig. 2B(ii); Fig. 2C(ii)]. Similarly, miR-24-2 overexpression led to a decrease in BCL2 mRNA levels by 1.47-fold and 1.7-fold (P50.049) in HEK 293T and MCF7 cells. Overexpression of miR-365-2 also led to a decrease in BCL2 mRNA levels by 2-fold and 1.74-fold (P50.09) in HEK 293T and MCF7 cells, respectively. We next wanted to check whether the decrease in the mRNA level of BCL2 correlates with the BCL2 protein levels. In miR-195-overexpressing HEK 293T and MCF7 cells, we detected a 2.6-fold (P,0.01) and 2-fold (P50.002) decrease in the levels of BCL2 protein in comparison with those of nontransfected cells, whereas in miR-24-2overexpressing HEK 293T and MCF7 cells we detected a 4fold (P,0.01) and 1.8-fold (P51.64E-6) decrease in the levels of BCL2 protein in comparison with those of nontransfected cells, respectively [Fig. 2B(iii); Fig. 2C(iii)]. Overexpression of miR365-2 also led to a decrease in BCL2 protein levels by 1.7-fold

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(P,0.01) in HEK 293T cells and 1.9-fold (P50.0013) in MCF7 cells, respectively. Taken together, these results demonstrate that miR-195, miR-24-2 and miR-365-2 could negatively regulate BCL2 at both the transcriptional as well as post-transcriptional levels. miR-195, miR-24-2 and miR-365-2 negatively regulate BCL2 through binding to the 39-UTR of human BCL2

Alignments of sequences showed that the binding sites of miR195, miR-24-2 and miR-365-2 on the 39-UTR of human BCL2 are highly conserved across different species (Fig. 3). To determine whether miR-195, miR-24-2 and miR-365-2 bind to the respective binding sites on the BCL2 39-UTR to repress BCL2 expression, we constructed a reporter vector consisting of luciferase cDNA followed by the BCL2 39-UTR. We transfected HEK 293T cells with BCL2 reporter construct in absence or in presence of p195, p24-2 or p365-2 and did a dual luciferase reporter assay 24 hours post transfection, as described in the Materials and Methods. Results in Fig. 3A(ii),B(ii),C(ii) show that the luciferase activities of the vectors containing the wild-type BCL2 39-UTR sequence was significantly decreased after overexpression of p195, p24-2 and p365-2 in HEK 293T cells by 2.8 fold (P,0.01), 2.5 fold (P,0.005) and 2 fold (P,0.05), respectively. To demonstrate further that miR-195, miR-24-2 and miR-365-2 interact with specific target sequences localized in the BCL2 39-UTR, additional reporter mutant

Fig. 3. BCL2 is a target of miR-195, miR-24-2 and miR-365-2. (A)(i), (B)(i), (C)(i) Comparison of the binding sites of miR-195, miR-24-2 and miR-365-2 in the BCL2 wild-type 39-UTR of different species shows that the target sites of the three miRNAs in the 39-UTR of BCL2 are highly conserved, as shown by the red font. The red broken line indicates the region of 39-UTR deleted for the respective mutant reporter construct. (A)(ii), (B)(ii), (C)(ii) Data from dual luciferase reporter assays performed in HEK 293T cells. Cells were co-transfected with BCL2 UTR construct (wild-type or mutant) and p195, p24-2 or p365-2. In the case of miR-365, three mutant constructs were designed, two (m3Del1, m3Del2) having individual binding sites deleted, and a third (m3Del 1+2) with both the binding sites deleted. Luminescence was measured at 24 hours post transfection. Luciferase counts were normalized with Renilla, and luciferase activity relative to a pMiR-Report vector was plotted. The bar diagram represents the means 6 s.d. for three independent experiments. **P,0.01 versus the BCL2 UTR.

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constructs were generated in which the predicted binding sites of the three miRNAs were deleted from the wild-type 39-UTR by site-directed mutagenesis. The resulting mutant reporter constructs were transfected with or without p195, p24-2 and p365-2 into HEK 293T cells, and luciferase activity was measured as described above. Interestingly, there was no significant change in the luciferase activity when mutant UTRs were co-transfected with p195, p24-2 or p365-2 [Fig. 3A(ii),B(ii),C(ii)], suggesting that repression of luciferase activity of the BCL2 39-UTR reporter construct shown by the miRNAs was because of binding of these miRNAs to the predicted binding sites on the BCL2 39-UTR that consequently led to negative regulation of BCL2 protein levels. Induction of apoptosis by hsa-miR-195, hsa-miR-24-2 and hsa-miR-365-2


As BCL2 is an anti-apoptotic protein, we next investigated whether downregulation of BCL2 by miR-195 or miR-24-2 or miR-365-2 overexpression leads to an induction of apoptosis in cells. As shown in Fig. 4A,B, there was an increase in annexin-V-PE- and TUNELpositive cells after transfection of p195, p24-2 and p365-2 in HEK 293T and MCF7 cells. The percentage of annexin-V-PE-positive cells increased from 3.7% in nontransfected cells to 17.763%

(P50.02), 16.661.5% (P50.01) and 18.261.7% (P50.003) after overexpression of p195, p24-2 and p365-2 in HEK 293T cells, respectively [Fig. 4A(i)]. We did not find a significant increase in the percentage of annexin-V-PE-positive cells (8.4%) when HEK 293T cells were transfected with a plasmid containing a scrambled sequence. Similar results were also observed in the case of MCF7 cells [Fig. 4B(i)]. The annexin-V-PE-positive cells increased from 3.15% in nontransfected MCF7 cells to 20.160.28% (P50.01), 20.860.28% (P50.009) and 2461.5% (P50.01) after overexpression of p195, p24-2 and p365-2, respectively. Transfection of a scrambled control also showed no significant apoptosis (6.9%). To confirm further the apoptotic nature of miR195, miR-24-2 or miR-365-2, we also performed a TUNEL assay and observed a significant increase in the number of TUNELpositive cells after ectopic expression of p195, p24-2 and p365-2 in HEK 293T as well as in MCF7 cells, respectively [Fig. 4A(ii),B(ii)]. As caspase activation plays a central role in execution of apoptosis (Budihardjo et al., 1999), we next checked for caspase-9 and caspase-3 activity by colorimetric assay. As shown in Fig. 4C, the caspase-9 and caspase-3 activities were significantly enhanced after overexpression of p195, p24-2 and p365-2 in HEK 293T cells. In comparisons with nontransfected cells, caspase-9 activity

Fig. 4. Induction of apoptosis in HEK 293T and MCF7 cells. Overexpression of miR-195, miR-24-2 and miR-365-2 induces apoptosis in (A) HEK 293T and (B) MCF7 cells. (A)(i), (B)(i) After transfection of p195, p24-2 and p365-2, an annexin V assay was performed as described in the Materials and Methods section. The percentage plotted indicates the percentage of annexin V-PE-positive or apoptotic cells. The data are representative of three independent experiments with similar results (P,0.01). (A)(ii), (B)(ii) A TUNEL assay was performed in HEK 293T and MCF7 cells to detect apoptotic cells after overexpression of miR-195, miR-24-2 or miR-365-2. Brown cells are TUNEL positive and are indicated by arrows. Pictures are representative of three independent experiments. (C) As described in Materials and Methods, (i) caspase 9 and caspase 3 enzyme activities were measured in HEK 293T cells, and (ii) caspase 9 activity was measured in MCF7 cells. After transfection with either p195 or p24-2 or p365-2 or scrambled control, the enzyme activity was measured by determining the extent of cleavage of the caspase substrates N-acetyl-Leu-Glu-His-Asp-p-nitroanilide (Ac-LEHD-pNA) and N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA), respectively. Bar graphs represent the means 6 s.d. of the fold increase in enzyme activity versus nontransfected control of three independent experiments performed in duplicate. *P,0.05, **P,0.01.

miRNA-mediated downregulation of BCL2 was increased by 2.2-fold (P50.041), 1.9-fold (P50.0321) and 2fold (P50.027), and caspase-3 activity was found to be increased by 1.6-fold (P50.014), 1.75-fold (P50.02) and 1.8-fold (P50.046), in p195-, p24-2- and p365-2-transfected HEK 293T cells. Similar results were obtained in the case of MCF7 cells, where ectopic expression of miR-195, miR-24-2 and miR-365-2 resulted in caspase-9 activity increased by 2-fold (P50.021), 1.75fold (P50.028) and 1.86-fold (P50.034), respectively. The negative control showed no significant change in caspase-9 or caspase-3 activity in comparison with that of nontransfected cells. As induction of apoptosis is often preceded by changes in the cell cycle, we therefore next looked for cell cycle changes assessed by flow cytometry before and after expression of p195, p24-2 and p365-2. Overexpression of p195, p24-2 and p365-2 in HEK 293T and MCF7 cells led to a significant increase in the percentage of the sub-G1 or G0 population in comparison with that of nontransfected cells (supplementary material Fig. S1), suggesting an increase in the apoptotic process. Taken together, the above experiments confirmed the apoptotic potential of miR-195, miR24-2 and miR-365-2.


Disruption of mitochondrial membrane potential (DYm) and release of cytochrome c

As an anti-apoptotic protein, BCL2 is known to guard the mitochondrial outer membrane permeability (MOMP) and

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thereby prevent diffusion of soluble pro-apoptotic proteins such as cytochrome c from the intermembrane space into the cytosol (Chipuk et al., 2010). To investigate the effect of BCL2 repression by the three miRNAs on mitochondrial membrane integrity, we examined the mitochondrial membrane potential (DYm) using the fluorescent probe DiOC6 (3) in cells before and after overexpression of p195, p24-2 and p365-2. The decrease in green fluorescence is a marker of mitochondrial membrane potential dissipation and is measured as the percentage of cells shifting towards the left. As depicted in Fig. 5A(i),A(ii), p195-, p24-2- and p365-2-transfected HEK 293T and MCF7 cells showed a marked shift towards the left in comparison with nontransfected cells, indicating that the overexpression of these miRNAs causes disruption of the mitochondrial membrane potential. The cells transfected with the scrambled negative control did not show a substantial shift in comparison with the miRNA-transfected cells. As the dissipation of mitochondrial membrane potential results in diffusion of cytochrome c from the mitochondrial intermembrane space into the cytosol, we next checked the levels of cytochrome c protein in mitochondria and cytosol by western blotting. As shown in Fig. 5B(i), the levels of cytochrome c protein in the cytosol were increased by 1.8-fold (P50.035), 2.4-fold (P50.022) and 2.2-fold (P50.03) by overexpression of p195, p24-2 and p365-2 in HEK 293T cells

Fig. 5. Disruption of mitochondrial membrane potential and release of cytochrome c. (A) The change in mitochondrial membrane potential (Dym) was estimated using DiOC6 (3). 20 minutes prior to harvesting, cells were incubated with 40 nM DiOC6 (3). After incubation, (i) HEK 293T and (ii) MCF7 cells were harvested, and the change in fluorescence was measured using flow cytometry. The X-axis represents green fluorescence, and the Y-axis represents the count scale. The illustrated histograms are representative of three independent experiments with similar results (P,0.05). (B) Release of cytochrome c from mitochondria into the cytosol after overexpression of miR-195, miR-24-2 and miR-365-2 in (i) HEK 293T and (ii) MCF7 cells. Mitochondrial (‘M’) and cytosolic (‘C’) fractions were separated as described in the Materials and Methods section. The purity of the fractions was determined by expression of the mitochondria-specific COX IV protein. GAPDH was used as a loading control. The protein bands were quantified and normalized to GAPDH intensities. Bars represent the means 6 s.d. of the fold change in cytochrome c protein levels versus those of the nontransfected control of three independent experiments. *P,0.05.

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in comparison with those of nontransfected cells. Similar results were obtained in the case of MCF7 cells also where cytosolic cytochrome c levels increased by 1.7-fold (P50.041), 1.76-fold (P50.031) and 1.5-fold (P50.024), respectively, after overexpression of p195, p24-2 and p365-2 [Fig. 5B(ii)]. The purity of the mitochondrial fraction was checked by use of an antibody against cytochrome c oxidase IV subunit (Cox IV). Taken together, these results suggested that miR-195, miR-24-2 and miR-365-2 disrupt mitochondria and cause release of cytochrome c into the cytosol.


miR-195, miR-24-2 and miR-365-2 augments the apoptotic effect of anti-tumour drug etoposide

Overexpression of anti-apoptotic proteins such as BCL2 has been a major cause of evasion of drug-induced cell death by cancer cells (Reed, 2006). Considering the well-characterized role of BCL2 in apoptosis and chemoresistance, we hypothesized that miR-195, miR-24-2 and miR-365-2, by repressing expression of BCL2, might play role in modulating the drug resistance of cancer cells. To confirm this hypothesis, we next examined whether overexpression of p195, p24-2 and p365-2 would have any effect on the sensitivity of breast cancer MCF7 cells to the standard chemotherapeutic agent etoposide. As shown in Fig. 6A, etoposide by itself showed 16.2% annexin-positive cells in comparison with 35.8%, 40.2% and 37.8% (P,0.005) annexinpositive cells when cells were treated with etoposide after

overexpression of p195, p24-2 and p365-2, respectively, in MCF7 cells. Considering the fact that miRNAs can target several genes simultaneously, we further sought to establish that the apoptosispromoting effect of these miRNAs is mainly through targeting BCL2 expression. To confirm that it is really the regulation of BCL2 that is crucial, we performed an annexin assay in the presence or absence of a plasmid construct harbouring BCL2 cDNA (without the 39-UTR) in MCF7 cells. At 24 hours post transfection, the percentages of apoptotic cells after p195, p24-2 and p365-2 overexpression were reduced from 19.6% to 11.5%; from 19% to 11.6% and from 16.8% to 9.6% (P,0.05), respectively, which was similar to the percentage of apoptotic cells found after BCL2 overexpression (11.4%). Our current data thus show that the overexpression of BCL2 rescued the apoptotic effect of miR-195, miR-24-2 as well as miR-365-2 in MCF7 cells (Fig. 6B). Transfection of this construct into MCF7 cells also significantly increased BCL2 protein levels (data not shown) and, as it lacked the 39-UTR region, the levels of BCL2 were found not to be downregulated by these miRNAs. Taken together, these results confirmed that miR-195, miR-24-2 and miR-365-2 directly regulate expression of the anti-apoptotic protein BCL2 and induce apoptosis. Discussion BCL2 is an important anti-apoptotic protein that has been studied intensively for the past decade owing to its role in the regulation

Fig. 6. Induction of apoptosis in MCF7 cells with or without etoposide or BCL2 cDNA. (A) Overexpression of either miR-195 or miR-24-2 or miR-365-2 significantly increased etoposide-induced cell death in MCF7 cells. Cells were transfected with p195 or p24-2 or p365-2. After 4 hours, etoposide (100 mg ml–1) was added in fresh media and the cells were incubated for a further 24 hours. After incubation, apoptosis was measured using flow cytometry as described in Materials and Methods. (i) Histograms are representative of three independent experiments. (ii) Bar graphs show the percentage of apoptotic cells. The data presented are the means 6 s.d. of three independent experiments. **P,0.01. (B) Overexpression of BCL2 rescues apoptosis induced by miR-195, miR-24-2 and miR-365-2. p195, p24-2 or p365-2 was co-transfected with BCL2-overexpressing cDNA clone (pBCL2) into MCF7 cells. Apoptosis was measured by flow cytometry at 24 hours after transfection, as described in Materials and Methods. (i) The histograms are representative of three independent experiments. (ii) Bar graphs showing the percentage of apoptotic cells. The data presented are the means 6 s.d. of three independent experiments. *P,0.05.

miRNA-mediated downregulation of BCL2


of apoptosis, tumorigenesis and the cellular response to anticancer therapies. Several observations in our study suggest that ectopic expression of the pre-miRNA clones p195, p24-2 and p365-2 increased the mature forms of miR-195, miR-24-2 and miR-365-2 and significantly reduced the expression of BCL2 protein through direct binding of miRNAs to their respective binding sites within the 39-UTR of the human BCL2 gene in HEK 293T and MCF7 cells. In apoptosis, coincident with the permeabilization of the outer mitochondrial membrane, there is typically a rapid reduction in the mitochondrial membrane potential (Desagher and Martinou, 2000). Furthermore, BCL2, being a crucial regulator of the mitochondrial pathway of apoptosis, exhibits its anti-apoptotic function by prohibiting release of cytochrome c from the mitochondrial intermembrane space into the cytosol (Yang et al., 1997). In the current study, we observed that overexpression of p195, p24-2 and p365-2 led to a significant decrease in the mitochondrial membrane potential, increase in the release of cytochrome c into the cytosol, increase in the activity of caspase-9 and the induction of apoptosis (see Fig. 7 for a model). We have observed that the increased expression of miRNAs is sufficient to initiate the apoptotic process. As protection by BCL2 against cell death induced by various

Fig. 7. Diagram showing a mechanism of cellular apoptosis induction by miRNAs miR-195, miR-24-2 and miR-365-2. By downregulating expression of the anti-apoptotic protein BCL2, miR-195, miR-24-2 and miR-365-2 reduce mitochondrial membrane potential and cause release of apoptotic molecules such as cytochrome c into the cytosol. Cytosolic cytochrome c leads to activation of a caspase cascade and ultimately results in apoptosis.

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anticancer drugs has been proposed as an important mechanism of drug resistance, our current results are of considerable therapeutic significance because miR-195, miR-24-2 and miR365-2 have been identified as naturally occurring antisense interactors with BCL2 mRNA that could be used for therapy in tumours overexpressing BCL2. In addition to targeting BCL2, miR-195 can regulate apoptosis by modulating other targets also. Recently, miR-195 has been reported to promote apoptosis by downregulating SIRT1, which encodes the NAD-dependent deacetylase sirtuin-1 (Zhu et al., 2011). Another report showed miR-195 can suppress tumorigenicity and regulate the cell cycle by targeting several G1–S transition-related molecules (Xu et al., 2009). Interestingly, miR-195 has been implicated in acquired resistance to temozolamide (TMZ) in glioblastoma multiforme cells (Ujifuku et al., 2010) and has also been identified as a novel biomarker in the prognosis of colorectal cancer (Wang et al., 2011). Recent work by another group has also identified BCL2 as a direct target of miR-24-2, consistent with our findings (Srivastava et al., 2011). Several lines of evidence suggest that there is a role of miR-24 in the regulation of apoptosis by targeting molecules other than BCL2, for example repression of Bcl2l11 (Bim) (Qian et al., 2011) and FAF1 (encoding Fas-associated factor 1) (Qin et al., 2010) in cancer cells. Other regulatory functions of miR-24 have been identified in haematopoietic differentiation (Wang et al., 2008) and tumorigenesis (Lal et al., 2009). For miR-365, there are very few reports that characterize its functions. Recently, parallel to our study, Qin and colleagues have shown that miR-365 induces apoptosis and targets BCL2 in HUVEC cells (Qin et al., 2011). miR-365 has also been identified as a direct negative regulator of the expression of IL-6 (Xu et al., 2011). Furthermore, Papetti and Augenlicht recently showed that miR-365 suppresses Mybl2 protein expression in proliferating Caco-2 cells (Papetti and Augenlicht, 2011). Acquired or innate resistance to chemotherapy is a crucial determinant of the outcome of cancer treatment (Pommier et al., 2004). The development of drug resistance in various cancer cells has been linked to reduced susceptibility to drug-induced cell death. Cancer cells evade apoptosis induced by these stimuli by increasing expression of anti-apoptotic proteins such as BCL2, BCL-xL and MCL1. In the present study, we observed augmentation of etoposide-induced apoptosis of breast cancer (MCF7) cells after overexpression of the individual miRNAs studied. Etoposide is an important chemotherapeutic drug that is used to treat a wide range of human cancers by targeting topoisomerase II (Baldwin and Osheroff, 2005). BCL2 is an important target for cancer therapy, and its regulation by miRNAs is an interesting and crucial approach for such therapies. Furthermore, miRNAs might be used as biomarkers to predict the response to chemotherapy (Ranade et al., 2010). In addition, miRNAs combined with traditional chemotherapy agents might provide a new strategy to treat malignant tumours in the future. Exploring the miRNA-mediated regulation of proteins involved in the development of resistance or sensitivity towards anti-neoplastic drugs can provide mechanistic insights into the relatively poorly understood process of chemosensitivity in cancer cells. Investigating the combinatorial effects of the miRNAs in the present study, as well as identifying other cellular targets of these miRNAs, will be interesting and crucial areas of future work. The findings of such studies will be important for

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estimating the therapeutic potential of these miRNAs as well as characterizing their biological functions in the cells. Materials and Methods Computational approach to identify candidate miRNAs targeting BCL2

The gene expression data for 48 cell lines were downloaded from the Gene Expression Omnibus (GEO) series GSE5949. For miRNA expression data of different cell lines, Cellminer (http://discover.nci.nih.gov/cellminer/) was used. The expression values were normalized by Z-transformation (Cheadle et al., 2003). Correlation coefficients were computed between expression of BCL2 and each miRNA. A freely available online tool (http://danielsoper.com/statcalc3/calc. aspx?id544) was used for calculating the significance of correlations. This tool provides the significance (both one-tailed and two-tailed probability values) of a Pearson correlation coefficient, given the correlation value and the sample size. The selection of miRNAs was done on the basis of a significant (P,0.05, twotailed) negative correlation.


Plasmid constructs

The sequences of the 39-UTR of BCL2 and pre-miRNAs were retrieved from ENSEMBL. For primer design, PrimerSelect (DNASTAR) and Primer3 (http:// frodo.wi.mit.edu) applications were used. To generate the 39-UTR reporter construct, a 2.26 kb region of the 39-UTR of BCL2 mRNA (ENST00000333681, 557-2805) containing the predicted binding sites for the three miRNAs was cloned into pMIR-REPORT Luciferase Vector (Ambion, Austin, TX) between HindIII and MluI restriction sites and designated BCL2 39-UTR. For construction of the mutant UTR, site-directed mutagenesis was performed using the QuickChange II kit (Stratagene, USA) and designated as m1del for miR-195; m2Del for miR-24; m3Del1, m3Del2 and m3Del 1+2 for miR-365. Plasmids expressing miR-195, miR-24-2 and miR-365-2 were constructed by amplifying sequences by PCR from human genomic DNA. These amplified sequences were then cloned into the pSilencer 4.1 CMV expression vector (Ambion) between HindIII and BamHI restriction sites and designated as p195, p24-2 and p365-2, respectively. All the resulting plasmids were sequenced to ensure accuracy. The sequences of the primers used for different cloning experiments are mentioned in supplementary material Table S2. A plasmid construct harbouring human BCL2 in pcDNA3 and Addgene plasmid 8768, for overexpression of BCL2, was purchased from Addgene (addgene.org) (Yamamoto et al., 1999). We refer it as pBCL2 in the present study. Cell culture and transfection

HEK 293T (human embryonic kidney) and MCF7 (Breast cancer) cell lines were procured from the National Centre for Cell Sciences (NCCS), Pune, India and maintained in DMEM containing 10% (v/v) fetal calf serum, 100 units ml–1 penicillin, 100 mg ml–1 streptomycin, 0.25 mg ml–1 amphotericin at 37 ˚C in a humidified atmosphere at 5% CO2. For overexpression studies, ,2.5–36105 cells of HEK 293T or MCF7 cells were seeded into six-well plates (for westerns, realtime PCRs, caspase activity assays and cell cycle analysis) and 1–1.26105 cells were seeded into 12-well plates (for luciferase assays, apoptosis assays and measurements of mitochondrial membrane potential). Transfections were performed by using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA) reagent according to the manufacturer’s protocol. For overexpression of miRNA, 3 mg of the pre-miRNA clones were transfected in six-well plates, and 1.5 mg of plasmids were transfected in 12-well plates. A pSilencer 4.1 plasmid containing a scrambled sequence was used as negative control in the experiments as it does not target any known sequence in human cells. The cells were harvested by trypsinization 24 hours post transfection and used for all the experiments. Real-time RT PCR

Total RNA was extracted with the Trizol reagent as per the manufacturer’s instructions (Invitrogen). TaqMan microRNA assays (Applied Biosystems) that include specific RT primers and TaqMan probes were used to quantify the expression of mature miR-195 (AB Assay ID 000494), miR-24-2 (AB Assay ID 000402) and miR-365-2 (AB Assay ID 001020), as described by the manufacturer. The primers used for detection of BCL2 mRNA levels were as follows: forward primer – 59-GGGGAGGATTGTGGCCTTC-39 and reverse primer – 59CAGGGCGATGTTGTCCAC-39. 18S rRNA (AB Assay ID 4333760F) was used for normalization. The reaction was incubated in a 7500 Real-Time PCR System (Applied Biosystems) in 96-well plates at 95 ˚C for 10 minutes, followed by 40 cycles of the following steps: 95 ˚C for 15 seconds and 60 ˚C for 1 minute. The real-time PCR data were analyzed using Pfaffl’s method (Pfaffl, 2001). Western blotting

Cells were trypsinized and lysed with modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate, 1 mM EDTA) containing protease inhibitors (1 mg ml–1 aprotinin, 1 mg ml–1 leupeptin, 1 mg ml–1 pepstatin, 1 mM PMSF, 1 mM sodium orthovanadate and 1 mM sodium

fluoride) for 30 minutes on ice. The lysates were centrifuged at 16,000 g for 30 minutes at 4 ˚C, and the supernatant was then collected. Protein concentration was determined by the BCA (Sigma, St Louis, MO) method. Equal amounts of proteins (30–50 mg) were separated by 12–15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Mdi; Advanced Microdevices, India). Membrane was blocked using 3% skimmed milk for 1 hour at room temperature and incubated with the appropriate antibodies in 1% skimmed milk for 2–3 hours, followed by incubation with a secondary antibody for 1 hour. Primary antibodies against the BCL2, cytochrome c, COX4, GAPDH and b-actin proteins were sourced from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibodies were ALP-linked, and blots were developed using NBT-BCIP (Sigma). Integrated density values were calculated using an AlphaImager 3400 (Alpha InnoTech, San Leandro, CA). These values were then normalized to the values for b-actin or GAPDH. All experiments were repeated at least three times; representative results are presented. Luciferase reporter assay

HEK 293T cells were co-transfected with 750 ng of pMIR-REPORT luciferase reporter vector containing the BCL2 39-UTR and 750 ng of p195 or p24-2 or p3652 using Lipofectamine 2000 (Invitrogen), as described by the manufacturer. To monitor the transfection efficiency, the samples were also co-transfected with 20 ng of the pRL-CMV plasmid that expresses the Renilla luciferase. For studies of mutants, cells were co-transfected with 750 ng of the BCL2 39-UTR reporter (wild-type or mutated) and 750 ng of p195, p24-2 or p365-2, as appropriate. Luciferase activity was measured by using the dual luciferase reporter assay system (Promega, Madison, WI). Readings were taken on a luminometer (Berthold Autolumat). The firefly luciferase activity was normalized by dividing by the Renilla luciferase activity. Normalized readings were then compared against those of a pMIR control vector. A pcDNA3.1 plasmid was used to normalize the total amounts of DNA transfected. Mitochondrial membrane potential (DYm) measurement

The mitochondrial membrane potential (DYm) was measured with DiOC6 (3) (3,39-dihexyloxacarbocyanine iodide; Sigma), a fluorochrome that is incorporated into cells depending upon the DYm (Su et al., 2005). Loss of DiOC6 (3) fluorescence indicates a reduction in the mitochondrial inner transmembrane potential. Briefly, cells were stained with DiOC6 (3) at a final concentration of 40 nM for 20 minutes at 37 ˚C in the dark. Cells were washed, and the fluorescence intensity was analyzed by a flow cytometer (Guava Technologies). A minimum of 5000 events were counted. Subcellular fractionation

The mitochondrial fractions from nontransfected and transfected HEK 293T and MCF7 cells were isolated by using a Mitochondrial/cytosol fractionation kit (Biovision, USA). Briefly, cells were washed and resuspended in 16 Cytosol Extraction Buffer. Cells were then homogenized and centrifuged first at 700 g to collect the cytosolic fraction, followed by 12,000 g for 30 minutes at 4 ˚C to obtain the mitochondrial pellet. The mitochondrial pellet was lysed with 16 mitochondrial extraction buffer. Annexin assay

Apoptosis was assessed by using the Guava Nexin kit and the Guava PCA system (Guava Technologies, Hayward, CA). The assay utilises Annexin V-PE to detect the translocation of phosphatidylserine to the external surface of the membrane of apoptotic cells, an early indication of commitment to apoptosis. The cellimpermeable dye 7-amino actinomycin D (7-AAD) is included in the Guava Nexin Reagent as an indicator of membrane structural integrity. 7-AAD is excluded from live healthy cells and early apoptotic cells, but permeates late-stage apoptotic and dead cells. Annexin V-PE fluorescence was analyzed by cytosoft software (Guava Technologies). A minimum of 2000 events were counted. For assay of etoposide sensitivity, MCF7 cells were transfected with 1.5 mg of individual miRNAs using Lipofectamine 2000 reagent. After, 4 hours, 100 mg ml–1 etoposide was added to all the wells, and apoptosis was measured at 24 hours post transfection. For detection of apoptosis before and after BCL2 overexpression, MCF7 cells were transfected with 750 ng of pBCL2 in the presence or absence of 750 ng p195, p242 or p365-2. pcDNA 3.1 was used to normalize the total amount of DNA transfected. TUNEL assay

Apoptotic cells were visualised by the terminal deoxynucleotidyl transferasemediated dUTP end-labeling (TUNEL) technique using the DeadEnd Colorimetric TUNEL system (Promega) 24 hours post transfection, as described previously (Goel et al., 2007). Cell cycle analysis

For analysis of cell cycle distribution, cells were fixed overnight with ice-cold 70% ethanol and treated with 1 mg ml–1 RNase for 30 minutes at 37 ˚C. Intracellular

miRNA-mediated downregulation of BCL2 DNA was labelled with 50 mg ml–1 propidium iodide (Sigma) at 4 ˚C for 30 minutes in the dark and analysed using a flow cytometer (Guava Technologies). A minimum of 5000 events were counted. Caspase activity measurement

The activities of caspase-3 and caspase-9 were determined using the respective colorimetric substrates (Calbiochem, Germany). Briefly, transfected and nontransfected HEK 293T and MCF7 cells were homogenized in lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM DTT, 0.1 mM EDTA) supplemented with protease inhibitor cocktail (Sigma). Then, 100 mg of total protein was incubated with colorimetric caspase-3 substrate Ac-DEVD-pNA or colorimetric caspase-9 substrate Ac-LEHD-pNA in an assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 0.1 mM EDTA, 10% glycerol), at 37 ˚C for 1 hour in the dark. The assay was based on the ability of the active enzyme to cleave the chromophore from the enzyme substrates Ac-DEVDpNA and Ac-LEHD-pNA, respectively. pNA released upon caspase cleavage produces a yellow colour, which is measured by a spectrophotometer at 405 nm. The amount of yellow colour produced upon cleavage is proportional to the amount of caspase activity present in the sample. One unit is defined as the amount of enzyme that will cleave 1 picomole of the substrate per minute at 37 ˚C and pH 7.4. Results are presented as the fold change of the activity, in comparison with the nontransfected control. Statistical significance analysis

Results are given as means of three independent experiments 6 s.d. An independent Student’s two-tailed t test was performed using replicate values. Values of P,0.05 were considered statistically significant.


Acknowledgements

We especially thank Vinod Kumar Yadav for statistical analysis and his contributions to Fig. 1. We acknowledge Stanley Korsmeyer for the Addgene plasmid 8768 used for overexpression of BCL2. Funding

This work was supported by the Council of Scientific and Industrial Research (CSIR) [grant number NWP0036]. R.S. was supported by a senior research fellowship from CSIR. Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.095976/-/DC1

References Ambros, V. (2004). The functions of animal microRNAs. Nature 431, 350-355. Andersen, M. H., Svane, I. M., Kvistborg, P., Nielsen, O. J., Balslev, E., Reker, S., Becker, J. C. and Straten, P. T. (2005). Immunogenicity of Bcl-2 in patients with cancer. Blood 105, 728-734. Baldwin, E. L. and Osheroff, N. (2005). Etoposide, topoisomerase II and cancer. Curr. Med. Chem. Anticancer Agents 5, 363-372. Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-297. Bartel, D. P. (2009). MicroRNAs: target recognition and regulatory functions. Cell 136, 215-233. Bentwich, I., Avniel, A., Karov, Y., Aharonov, R., Gilad, S., Barad, O., Barzilai, A., Einat, P., Einav, U., Meiri, E. et al. (2005). Identification of hundreds of conserved and nonconserved human microRNAs. Nat. Genet. 37, 766-770. Blower, P. E. et al. (2007). MicroRNA expression profiles for the NCI-60 cancer cell panel. Mol. Cancer Ther. 6, 1483-1491. Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B. and Cohen, S. M. (2003). bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113, 25-36. Budihardjo, I., Oliver, H., Lutter, M., Luo, X. and Wang, X. (1999). Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 15, 269290. Calin, G. A., Sevignani, C., Dumitru, C. D., Hyslop, T., Noch, E., Yendamuri, S., Shimizu, M., Rattan, S., Bullrich, F., Negrini, M. et al. (2004). Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. USA 101, 2999-3004. Cheadle, C., Vawter, M. P., Freed, W. J. and Becker, K. G. (2003). Analysis of microarray data using Z score transformation. J. Mol. Diagn. 5, 73-81. Cheng, A. M., Byrom, M. W., Shelton, J. and Ford, L. P. (2005). Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res. 33, 1290-1297. Chipuk, J. E., Moldoveanu, T., Llambi, F., Parsons, M. J. and Green, D. R. (2010). The BCL-2 family reunion. Mol. Cell 37, 299-310. Cimmino, A., Calin, G. A., Fabbri, M., Iorio, M. V., Ferracin, M., Shimizu, M., Wojcik, S. E., Aqeilan, R. I., Zupo, S., Dono, M. et al. (2005). miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc. Natl. Acad. Sci. USA 102, 13944-13949.

1577

Cory, S. and Adams, J. M. (2002). The Bcl2 family: regulators of the cellular life-ordeath switch. Nat. Rev. Cancer 2, 647-656. Danial, N. N. and Korsmeyer, S. J. (2004). Cell death: critical control points. Cell 116, 205-219. Desagher, S. and Martinou, J. C. (2000). Mitochondria as the central control point of apoptosis. Trends Cell Biol. 10, 369-377. Dlugosz, P. J., Billen, L. P., Annis, M. G., Zhu, W., Zhang, Z., Lin, J., Leber, B. and Andrews, D. W. (2006). Bcl-2 changes conformation to inhibit Bax oligomerization. EMBO J. 25, 2287-2296. Garzon, R., Calin, G. A. and Croce, C. M. (2009). MicroRNAs in Cancer. Annu. Rev. Med. 60, 167-179. Goel, A., Prasad, A. K., Parmar, V. S., Ghosh, B. and Saini, N. (2007). 7,8Dihydroxy-4-methylcoumarin induces apoptosis of human lung adenocarcinoma cells by ROS-independent mitochondrial pathway through partial inhibition of ERK/ MAPK signaling. FEBS Lett. 581, 2447-2454. Green, D. R. and Reed, J. C. (1998). Mitochondria and apoptosis. Science 281, 13091312. Hamano, R., Miyata, H., Yamasaki, M., Kurokawa, Y., Hara, J., Moon, J. H., Nakajima, K., Takiguchi, S., Fujiwara, Y., Mori, M. et al. (2011). Overexpression of miR-200c induces chemoresistance in esophageal cancers mediated through activation of the Akt signaling pathway. Clin. Cancer Res. 17, 3029-3038. Huang, J. C., Babak, T., Corson, T. W., Chua, G., Khan, S., Gallie, B. L., Hughes, T. R., Blencowe, B. J., Frey, B. J. and Morris, Q. D. (2007). Using expression profiling data to identify human microRNA targets. Nat. Methods 4, 1045-1049. Iqbal, J., Neppalli, V. T., Wright, G., Dave, B. J., Horsman, D. E., Rosenwald, A., Lynch, J., Hans, C. P., Weisenburger, D. D., Greiner, T. C. et al. (2006). BCL2 expression is a prognostic marker for the activated B-cell-like type of diffuse large Bcell lymphoma. J. Clin. Oncol. 24, 961-968. Kausch, I., Jiang, H., Thode, B., Doehn, C., Kruger, S. and Jocham, D. (2005). Inhibition of bcl-2 enhances the efficacy of chemotherapy in renal cell carcinoma. Eur. Urol. 47, 703-709. Kloosterman, W. P. and Plasterk, R. H. (2006). The diverse functions of microRNAs in animal development and disease. Dev. Cell 11, 441-450. Kojima, K., Fujita, Y., Nozawa, Y., Deguchi, T. and Ito, M. (2010). MiR-34a attenuates paclitaxel-resistance of hormone-refractory prostate cancer PC3 cells through direct and indirect mechanisms. Prostate 70, 1501-1512. Lal, A., Navarro, F., Maher, C. A., Maliszewski, L. E., Yan, N., O’Day, E., Chowdhury, D., Dykxhoorn, D. M., Tsai, P., Hofmann, O. et al. (2009). miR-24 Inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to ‘‘seedless’’ 39UTR microRNA recognition elements. Mol. Cell 35, 610625. Lim, L. P., Lau, N. C., Garrett-Engele, P., Grimson, A., Schelter, J. M., Castle, J., Bartel, D. P., Linsley, P. S. and Johnson, J. M. (2005). Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769-773. Macfarlane, L. A. and Murphy, P. R. (2010). MicroRNA: biogenesis, function and role in cancer. Curr. Genomics 11, 537-561. Majid, A., Tsoulakis, O., Walewska, R., Gesk, S., Siebert, R., Kennedy, D. B. and Dyer, M. J. (2008). BCL2 expression in chronic lymphocytic leukemia: lack of association with the BCL2 938A.C promoter single nucleotide polymorphism. Blood 111, 874-877. Mayr, C., Hemann, M. T. and Bartel, D. P. (2007). Disrupting the pairing between let7 and Hmga2 enhances oncogenic transformation. Science 315, 1576-1579. Oltersdorf, T., Elmore, S. W., Shoemaker, A. R., Armstrong, R. C., Augeri, D. J., Belli, B. A., Bruncko, M., Deckwerth, T. L., Dinges, J., Hajduk, P. J. et al. (2005). An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677-681. Ory, B., Ramsey, M. R., Wilson, C., Vadysirisack, D. D., Forster, N., Rocco, J. W., Rothenberg, S. M. and Ellisen, L. W. (2011). A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma. J. Clin. Invest. 121, 809-820. Papetti, M. and Augenlicht, L. H. (2011). Mybl2, downregulated during colon epithelial cell maturation, is suppressed by miR-365. Am. J. Physiol. Gastrointest. Liver Physiol. 3, G508-G518. Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45. Pommier, Y., Sordet, O., Antony, S., Hayward, R. L. and Kohn, K. W. (2004). Apoptosis defects and chemotherapy resistance: molecular interaction maps and networks. Oncogene 23, 2934-2949. Qian, L., Van Laake, L. W., Huang, Y., Liu, S., Wendland, M. F. and Srivastava, D. (2011). miR-24 inhibits apoptosis and represses Bim in mouse cardiomyocytes. J. Exp. Med. 208, 549-560. Qin, B., Xiao, B., Liang, D., Xia, J., Li, Y. and Yang, H. (2011). MicroRNAs expression in ox-LDL treated HUVECs: MiR-365 modulates apoptosis and Bcl-2 expression. Biochem. Biophys. Res. Commun. 410, 127-133. Qin, W., Shi, Y., Zhao, B., Yao, C., Jin, L., Ma, J. and Jin, Y. (2010). miR-24 regulates apoptosis by targeting the open reading frame (ORF) region of FAF1 in cancer cells. PLoS. ONE. 5, e9429. Ranade, A. R., Cherba, D., Sridhar, S., Richardson, P., Webb, C., Paripati, A., Bowles, B. and Weiss, G. J. (2010). MicroRNA 92a-2*: a biomarker predictive for chemoresistance and prognostic for survival in patients with small cell lung cancer. J. Thorac. Oncol. 5, 1273-1278.


1578


Reed, J. C. (2006). Drug insight: cancer therapy strategies based on restoration of endogenous cell death mechanisms. Nat. Clin. Pract. Oncol. 3, 388-398. Reinhart, B. J., Slack, F. J., Basson, M., Pasquinelli, A. E., Bettinger, J. C., Rougvie, A. E., Horvitz, H. R. and Ruvkun, G. (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901-906. Skirnisdottir, I., Seidal, T., Gerdin, E. and Sorbe, B. (2002). The prognostic importance of p53, bcl-2, and bax in early stage epithelial ovarian carcinoma treated with adjuvant chemotherapy. Int. J. Gynecol. Cancer 12, 265-276. Srivastava, N., Manvati, S., Srivastava, A., Pal, R., Kalaiarasan, P., Chattopadhyay, S., Gochhait, S., Dua, R. and Bamezai, R. N. (2011). miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention. Breast Cancer Res. 13, R39. Strasser, A., Harris, A. W., Bath, M. L. and Cory, S. (1990). Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2. Nature 348, 331-333. Su, Y. T., Chang, H. L., Shyue, S. K. and Hsu, S. L. (2005). Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway. Biochem. Pharmacol. 70, 229-241. Ujifuku, K., Mitsutake, N., Takakura, S., Matsuse, M., Saenko, V., Suzuki, K., Hayashi, K., Matsuo, T., Kamada, K., Nagata, I. et al. (2010). miR-195, miR-4553p and miR-10a(*) are implicated in acquired temozolomide resistance in glioblastoma multiforme cells. Cancer Lett. 296, 241-248. Vecchione, A. and Croce, C. M. (2010). Apoptomirs: small molecules have gained the license to kill. Endocr. Relat. Cancer 17, F37-F50. Volinia, S., Calin, G. A., Liu, C. G., Ambs, S., Cimmino, A., Petrocca, F., Visone, R., Iorio, M., Roldo, C., Ferracin, M. et al. (2006). A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl. Acad. Sci. USA 103, 2257-2261. Wang, Q., Huang, Z., Xue, H., Jin, C., Ju, X. L., Han, J. D. and Chen, Y. G. (2008). MicroRNA miR-24 inhibits erythropoiesis by targeting activin type I receptor ALK4. Blood 111, 588-595. Wang, X., Wang, J., Ma, H., Zhang, J. and Zhou, X. (2011). Downregulation of miR195 correlates with lymph node metastasis and poor prognosis in colorectal cancer. Med. Oncol. [Epub ahead of print].

Xia, L., Zhang, D., Du, R., Pan, Y., Zhao, L., Sun, S., Hong, L., Liu, J. and Fan, D. (2008). miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cells. Int. J. Cancer 123, 372-379. Xu, T., Zhu, Y., Xiong, Y., Ge, Y. Y., Yun, J. P. and Zhuang, S. M. (2009). MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. Hepatology 50, 113-121. Xu, Z., Xiao, S. B., Xu, P., Xie, Q., Cao, L., Wang, D., Luo, R., Zhong, Y., Chen, H. C. and Fang, L. R. (2011). miR-365, a novel negative regulator of interleukin-6 gene expression, is cooperatively regulated by Sp1 and NF-kappaB. J. Biol. Chem. 286, 21401-21412. Yamamoto, K., Ichijo, H. and Korsmeyer, S. J. (1999). BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol. Cell. Biol. 19, 8469-8478. Yanai, S., Nakamura, S., Takeshita, M., Fujita, K., Hirahashi, M., Kawasaki, K., Kurahara, K., Sakai, Y. and Matsumoto, T. (2010). Translocation t(14;18)/IGHBCL2 in gastrointestinal follicular lymphoma: correlation with clinicopathologic features in 48 patients. Cancer 117, 2467-2477. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. I., Jones, D. P. and Wang, X. (1997). Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129-1132. Youle, R. J. and Strasser, A. (2008). The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47-59. Zamore, P. D. and Haley, B. (2005). Ribo-gnome: the big world of small RNAs. Science 309, 1519-1524. Zamzami, N., Susin, S. A., Marchetti, P., Hirsch, T., Gomez-Monterrey, I., Castedo, M. and Kroemer, G. (1996). Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 1533-1544. Zhang, J., Du, Y., Wu, C., Ren, X., Ti, X., Shi, J., Zhao, F. and Yin, H. (2010). Curcumin promotes apoptosis in human lung adenocarcinoma cells through miR-186* signaling pathway. Oncol. Rep. 24, 1217-1223. Zhu, W., Shan, X., Wang, T., Shu, Y. and Liu, P. (2010). miR-181b modulates multidrug resistance by targeting BCL2 in human cancer cell lines. Int. J. Cancer 127, 2520-2529. Zhu, H., Yang, Y., Wang, Y., Li, J., Schiller, P. W. and Peng, T. (2011). MicroRNA195 promotes palmitate-induced apoptosis in cardiomyocytes by down-regulating Sirt1. Cardiovasc. Res. 92, 75-84.