MicroRNA-125b Induces Cancer Cell Apoptosis Through Suppression of Bcl-2 Expression

Available online at www.sciencedirect.com

Journal of Genetics and Genomics 39 (2012) 29e35

JGG ORIGINAL RESEARCH

MicroRNA-125b Induces Cancer Cell Apoptosis Through Suppression of Bcl-2 Expression Aihua Zhao a,b,c,1, Quan Zeng b,1, Xiaoyan Xie b, Junnian Zhou b, Wen Yue b,*, Yali Li a,*, Xuetao Pei b,* b

a Department of Obstetrics and Gynecology, General Hospital of PLA, Beijing 100853, China Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China c Department of Obstetrics and Gynecology, The 309th Hospital of PLA, Beijing 100091, China

Received 25 April 2011; revised 25 November 2011; accepted 23 December 2011 Available online 28 December 2011

ABSTRACT MicroRNAs (miRNAs) are small, noncoding RNAs which can often act as an oncogene or a tumor suppressor. Several miRNAs are associated with the development of hepatocellular carcinoma (HCC). We demonstrated that miR-125b significantly suppresses HCC cell proliferation and promotes apoptosis by inhibiting the gene expression of the anti-apoptotic protein, Bcl-2. Bioinformatic analysis indicated that the 30 UTR of Bcl-2 has binding sites for miR-125b. Luciferase reporter assay confirmed the ability of miR-125b to dramatically suppress Bcl-2 transcription, suggesting that Bcl-2 is a target gene for miR-125b. We concluded that miR-125b acts as a tumor suppressor in hepatic tumor development by targeting Bcl-2 and inducing cancer cell apoptosis. KEYWORDS: miR-125b; Bcl-2; Apoptosis; Proliferation

1. INTRODUCTION MicroRNAs (miRNAs) belong to a large family of noncoding single-stranded RNAs, which are approximately 22 nucleotides in length (Bartel, 2004). Growing evidence indicates that miRNAs, by pairing with the 30 -untranslated region (30 UTR) of specific target messenger RNA (mRNA), can regulate the expression of protein-coding genes, either at the level of a mRNA degradation or translation. miRNAs are involved in many biological processes including cell cycle regulation, development, differentiation, metabolism, and aging (Spizzo et al., 2009). Calin et al. (2004) reported that * Corresponding authors. Tel: þ86 10 6693 1949, fax: þ86 10 6816 4807 (W. Yue; X. Pei); Tel: þ86 10 6693 8044, fax: þ86 10 6828 8056 (Y. Li). E-mail addresses: [email protected] (W. Yue), [email protected] (Y. Li), [email protected] (X. Pei). 1 These authors contributed equally to this work.

more than 50% of miRNA genes are located in genomic regions associated with cancer in fragile sitesdhot spots for mutations, deletions and amplification. miRNA dysregulation has been observed in many human malignancies and there is evidence for miRNA involvement in tumor progression as either an oncogene or a tumor suppressor. Hepatocellular carcinoma (HCC) is one of the most prevalent cancers and the leading cause of cancer-related deaths worldwide (Aravalli et al., 2008). Several miRNAs are upregulated or down-regulated in tumors and can modulate cell growth, apoptosis, migration and invasion (Li et al., 2008; Su et al., 2009; Xu et al., 2009; Liang et al., 2010; Xiong et al., 2010). However, their exact biological role in HCC remains unclear. miR-125b was recently identified as a tumor-associated miRNA. miR-125b dysregulation is involved in tumorigenesis and the progression of many types of cancer including HCC (Iorio et al., 2005; Visone et al., 2007; Ichimi et al.,

1673-8527/$ - see front matter Copyright Ó 2012, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. doi:10.1016/j.jgg.2011.12.003

30

A. Zhao et al. / Journal of Genetics and Genomics 39 (2012) 29e35

2009). It was also reported that miR-125b suppressed liver cancer proliferation and metastasis by targeting the oncogene LIN28B (Liang et al., 2010). Here, we demonstrate that Bcl-2, an anti-apoptotic gene which has essential roles in tumor progression, is a direct target for miR-125b and that ectopic miR-125b expression can suppress tumor cell proliferation by inhibiting Bcl-2 expression. 2. MATERIALS AND METHODS 2.1. Cell culture, tissues and mice We used the following cell lines: HEK 293T, immortalized liver cell line L-02 and six human hepatoma cell lines (MHCC97H, MHCC97L, PLC, SNU-449, HepG2 and SMMC7721). All the cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 C with 5% CO2. HCC and adjacent normal tissue specimens were obtained, with informed consent, from nine patients at the General Hospital of PLA (Beijing, China). Male Nu/Nu nude mice, aged 5e6 weeks, were purchased from the Vital River Laboratories (Beijing, China). All experimental procedures were performed in accordance with the internationally accepted ethical guidelines. 2.2. RNA isolation, reverse-transcription and quantitative real-time PCR Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) and the first strand cDNA was synthesized using miScript Reverse Transcription Kit (Qiagen, Hilden, Germany). The expression of mature miR-125b was quantified by real-time PCR using the miScript SYBR Green PCR Kit which contained 10 miScript Universal Primer (Qiagen) and was performed according to the manufacturer’s protocol. Quantization of U6 was used to normalize miRNA expression level. Real-time PCR was carried out in the Bio-Rad IQ5 amplification system (Bio-Rad, USA) and the results were calculated using deltaedelta CT method. Primer sequences were: U6 forward, 50 -CGCTTCGGCAGCACATATACTA-30 ; miR-125b forward, 50 -TCCCTGAGACCCTAACTTGTGA-30 . 2.3. Plasmid construction We used the lentiviral vector pHRS-1cla-EGFP to construct the plasmid for stable expression of miR-125b. The CMV promoter from the pcDNA3.1()-myc-his vector was amplified by PCR and was inserted into the pHRS-1cla-EGFP vector to obtain pHRS-1cla-CMV-EGFP, using the following primers: CMV forward/SpeI, 50 -GCCAGATATACGCGTTG ACA-30 ; CMV reverse/Mlu 50 -ACTAGTGGATGGGTCATGG TGAAAAC-30 . Pri-miR-125b was amplified from human genomic DNA from whole blood and then inserted into the pHRS-1cla-CMV-EGFP vector to generate the pHRS-1cla-miR125b-CMV-EGFP vector, using the following primers: pri-miR-125b forward/ BamHI, 50 -GGATCCGGGTCCCATTAACTGGCATA-30 ; pri-

miR-125b reverse/SpeI, 50 -ACTAGTGGATGGGTCATGGTG AAAAC-30 . To construct pGL3-Bcl-2 30 UTR-WT plasmid, a wild-type 0 3 UTR segment of human Bcl-2 mRNA (4681e5821 nt, GenBank accession No. NM_000633) containing the putative miR-125b binding sequence was amplified by PCR using the following primers: forward: 50 -GTAGCTCTGGCCCAGTG GGAAA-30 ; reverse: 50 -TTATTTTTCTGGGGCAGTCCAG ATGA-30 , and then subcloned into the Xba I site downstream of the stop codon of firefly luciferase in pGL3-control (Promega, Madison, WI, USA). pGL3-Bcl-2 30 UTR-MUT, which carried the mutated sequence in the complementary site for the seed region of miR-125b, was generated by sitespecific mutagenesis based on the wild-type plasmid. 2.4. Lentivirus packaging and cell transfection HEK 293T cells were seeded into a 10 cm dish. Either pHRS-1cla-miR-125b-CMV-EGFP or pHRS-1cla-CMV-EGFP vector, together with lentiviral packaging plasmid (PLP1, PLP2, PLP/VSVG; Invitrogen) was co-transfected using Lipofectamine 2000 (Invitrogen), following manufacturer’s instruction. The supernatant was harvested 48 h after transfection, and was concentrated with PEG 8000. MHCC97H and PLC cells were infected with the virus at a multiplicity of infection (MOI) of 8. After 6 days of culture, the EGFP-positive cells were collected by fluorescence activated cell sorting as miR-125b overexpression stable cells and vector control cells. The anti-miR-125b, with the sequence of 50 -UCACAA GUUAGGGUCUCAGGGA-30 , is a 20 -O-methyl-modified oligoribonucleotide designed as an inhibitor of miR-125b. The sequence of 50 -CAGUACUUUUGUGUAGUACAA-30 was used as a negative control for anti-miR-125b (anti-miRNANC) in the antagonism experiment. They were obtained from Genepharma (Shanghai, China). The small interference RNA (siRNA) targeting human Bcl-2 (si-Bcl-2, identification number 000633 and catalog number 3755) and the negative control siRNA (si-NC, catalog number 1079) were purchased from Sigma (Louis, MO, USA). Cells were transfected with miR125b inhibitor or siRNA against Bcl-2 using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Unless otherwise indicated, 50 nmol/L miRNA inhibitor and 50 nmol/L RNA duplex were used for each transfection. 2.5. Cell proliferation and colony formation assays Three thousand cells (MHCC97H or PLC) with a stable overexpression of miR-125b or control vector were seeded into 96-well plates and cultured for 1e6 days. MTT (20 mL, 5 mg/ mL; Sigma) was added to each well and incubated for 4 h. The optical density at 490 nm was measured using a Microplate Reader (Bio-Rad 550, USA). The proliferation index was calculated as an experimental OD value/control OD value. Five hundred transfected cells were seeded in a six-well plate and maintained in DMEM containing 10% FBS for 2 weeks. Colonies were fixed and stained with 0.1% crystal violet in 20% methanol for 15 min.

A. Zhao et al. / Journal of Genetics and Genomics 39 (2012) 29e35

2.6. Tumor formation in nude mice Stably transfected MHCC97H cells (1  107) with either miR-125b or control vector were suspended in 200 mL PBS and injected subcutaneously into either side of the same male Nu/Nu nude mouse. Six mice were used and tumor growth was examined every other day over the course of 37 days. Tumor volume (V) was monitored by measuring its length (L) and width (W ) with calipers and calculated using the formula (L  W2)  0.5. 2.7. Luciferase reporter assay HEK293T cells (6  104) were incubated in 24-well plates. The cells were co-transfected with 800 ng of either miR-125b expression plasmid (pHRS-1cla-miR125b-CMV-EGFP) or control plasmid (pHRS-1cla-CMV-EGFP) with 100 ng of either pGL3-Bcl-2 30 UTR-WT or pGL3-Bcl-2 30 UTR-MUT plasmid, and 8 ng of pRL-CMV (Promega). Cells were collected 48 h after transfection and analyzed using the dual-luciferase assay kit (Promega), according to the manufacturer’s instruction. 2.8. Western blot analysis Protein extracts were subjected to 10% SDS-PAGE and transferred onto PVDF membranes. This was followed by probing with mouse primary antibodies against human Bcl-2 (sc-7382; Santa Cruz, CA, USA) and incubation with the secondary HRP-conjugated anti-mouse antibody (Santa Cruz). After washing, the proteins were visualized with an ECL kit (Santa Cruz).

31

Differences were assessed by two tailed Student t-test using SPSS version 17.0. P < 0.05 was considered to be statistically significant. 3. RESULTS 3.1. Down-regulation of miR-125b in human HCC Using quantitative RT-PCR analysis, we found that the expression of miR-125b is substantially lower in five out of the six HCC cell lines than in L-02 cells (Fig. 1A). We examined the expression of miR-125b in nine paired samples of clinical HCC tumor and adjacent normal liver tissues. Among them, miR-125b was significantly down-regulated in seven of the tumor samples (Fig. 1B). 3.2. miR-125b inhibits HCC cell proliferation in vitro and in vivo To explore the effect of miR-125b on cancer cell proliferation, MHCC97H and PLC cells, which have low basal miR-125b levels, were used to establish stable miR-125bexpressing cell lines via lentivirus infection. Overexpression of miR-125b in these two cell lines was confirmed by real-time

2.9. Hematoxylineeosin (HeE) staining and immunohistochemistry (IHC) HeE staining was performed in a usual manner. In the IHC assay, we employed Bcl-2 mouse monoclonal antibody (sc7382; Santa Cruz) as the primary. The immunostaining was performed using the PV-9000 two-step method plus the PolyHRP Anti-Mouse/Rabbit IgG Detection System (Zhongshan Golden Bridge Biotechnology Co., Ltd, China). 2.10. Apoptosis assay Apoptosis was evaluated by the apoptotic morphology and Annexin V expression. For morphologic examination, cells were stained with Hoechst 33342 (Sigma, St. Louis, MO, USA) and those with fragmented or condensed nuclei were counted as apoptotic cells. At least 500 cells were counted for each sample. Annexin V expression was measured with the Annexin V-PE kits from KeyGEN (Nanjing, China) and performed according to manufacturer’s protocols. The Annexin V expression was quantified by flow cytometry. 2.11. Statistical analysis Data are presented as mean  standard error from at least three separate experiments.

Fig. 1. Analysis of miR-125b expression in cancer cell lines and human HCC tissues by quantitative RT-PCR. A: miR-125b expression in L-02 and various HCC cell lines. B: miR-125b expression in 9 paired HCC and adjacent non-tumor tissue samples. N, adjacent normal tissue; T, HCC tissue. U6dendogenous control.

32

A. Zhao et al. / Journal of Genetics and Genomics 39 (2012) 29e35

PCR (Fig. 2A). As shown in Fig. 2B, the proliferation of miR125b-expressing MHCC97H and PLC cells was significantly lower than control cells. A significant suppression of long-term cell growth was also observed by a colony formation assay (Fig. 2C). To examine the effect of miR-125b in vivo, 1  107 miR125b-expressing MHCC97H cells or control cells were subcutaneously injected into six nude mice. Thirty-seven days later, all the control mice developed tumors with mean volume of up to 815.6 mm3. However, only one out of six mice injected with miR-125b-expressing MHCC97H cells developed a tumor (16.9 mm3) (Fig. 2D and E).

48 h and 72 h after their normal passage with Annexin V assays using flow cytometry. We found that apoptotic rate for miR-125b-expressing cells is much higher than that for the control cells (Fig. 3A and B). Next, by using Hoechst 33342, we investigated whether miR-125b expression could sensitize tumor cells to chemotherapeutic drug-induced apoptosis. Compared with the control cells, the enhanced expression of miR-125b caused a strong increase in apoptosis of MHCC97H cells exposed to doxorubicin (P < 0.01), a drug commonly used in HCC chemotherapy. But when miR-125b-expressing MHCC97H cells were co-transfected with the inhibitor of miR-125b, the apoptotic rate was significantly decreased (P < 0.01) (Fig. 3C).

3.3. miR-125b induces HCC cell apoptosis Tumorigenicity inhibition by miR-125b suggests that it might promote cell apoptosis or inhibit the cell cycle. To examine its effect on apoptosis, we analyzed cell apoptosis of miR-125b-expressing MHCC97H cells and control at 24 h,

3.4. miR-125b promotes apoptosis by directly targeting anti-apoptotic Bcl-2 gene To determine how miR-125b promotes cancer cell apoptosis, we used publicly available databases and found

Fig. 2. Overexpression of miR-125b inhibited HCC cell proliferation in vitro and in vivo. A: miR-125b expression in transfected MHCC97H and PLC cell lines examined by quantitative RT-PCR. *: P < 0.05. B: cell growth determined by an MTT assay. C: miR-125b’s impact on colony formation of transfected MHCC97H and PLC cell lines. D: effect of miR-125b on xenograft tumor growth. E: photographs of dissected tumors from nude mouse xenografts.

A. Zhao et al. / Journal of Genetics and Genomics 39 (2012) 29e35

33

Fig. 3. miR-125b sensitized cancer cells to undergo apoptosis. A and B: apoptotic rate of normal cultured miR-125b-expressing MHCC97H cells and control cells at different time points using Annexin V-PE apoptosis assay. C: miR-125b sensitized MHCC97H cells to undergo drug-induced apoptosis. But, when miR-125b-expressing MHCC97H cells were transfected with a miR-125b inhibitor, the apoptotic rate was significantly decreased. Doxorubicin (0.1 mg/mL) was added 24 h after passage or transfection and incubated for 24 h or 48 h followed by apoptosis analysis using Hoechst 33342 staining. **: P < 0.01.

several predicted miR-125b target genes, including Lin28, BAK1, CBX7 and Bcl-2. For further study, we chose to focus on Bcl-2, an important anti-apoptotic protein that is well characterized in many tumors. We hypothesized that Bcl-2 inhibition by miR-125b promotes HCC cell apoptosis and inhibits HCC cell proliferation. To validate whether Bcl-2 is an actual target of miR-125b, a human Bcl-2 30 UTR fragment containing either a wild-type or mutant miR-125b binding sequence (Fig. 4A) was cloned downstream of a firefly luciferase reporter gene. The relative luciferase activity of the wild-type reporter was significantly suppressed by pHRS-1cla-miR125b (Fig. 4B). In contrast, the luciferase activity of the mutant reporter in the presence of pHRS-1cla-miR125b was almost unaffected (Fig. 4B), indicating that miR-125b may suppress Bcl-2 gene expression through its binding sequence at the 30 UTR of Bcl-2. Decreased Bcl-2 protein level was observed by Western blot analysis in MHCC97H cells transfected with miR-125b compared with control cells. Endogenous Bcl-2 protein level

was increased in L-02 cells that were transfected with the inhibitor, anti-miR-125b (Fig. 4C). These data were confirmed by IHC staining of xenograft tumor tissue, which showed that Bcl-2 protein level was inversely correlated with miR-125b expression (Fig. 4D). To further confirm whether Bcl-2 is an important miR-125b target during HCC apoptosis, we designed a “rescue” experiment by co-knocking down miR-125b and Bcl-2 and examined doxorubicin-induced apoptosis. Because SMMC-7721 cell line has a higher basic level of miR-125b (Fig. 1A), we chose it for this “rescue” experiment. SMMC-7721 cells were co-transfected with the following different combinations: (i) si-Bcl-2/anti-miRNA-NC; (ii) anti-miR-125b/si-NC; (iii) siNC/anti-miRNA-NC; and (iv) anti-miR-125b/si-Bcl-2 and two wells with the same treatment in six-well plates. After 48 h, the expression of Bcl-2 proteins was checked by Western blot. Also, half of the cells were treated for another 48 h with doxorubicin (0.1 mg/mL) followed by apoptosis analysis using Hoechst 33342 staining. We found that the expression of

34

A. Zhao et al. / Journal of Genetics and Genomics 39 (2012) 29e35

Fig. 4. Bcl-2 is a direct target of miR-125b and contributes to HCC cell apoptosis. A: putative miR-125b binding sequence in the 30 UTR of Bcl-2 mRNA. Mutation was generated on the Bcl-2 30 UTR sequence in the complementary site for the seed region of miR-125b, as indicated. B: analysis of luciferase activity, normalized to Renilla luciferase. C: Western blot analysis of Bcl-2 in miR-125b overexpressed MHCC97H cells or suppressed L-02 cells. b-actin was used as an internal control. D: IHC staining of Bcl-2, in xenograft tumors from mice injected with MHCC97H cells overexpressing either miR-125b or a control vector. Bar, 50 um. E: the expression of Bcl-2 protein in SMMC-7721 cells which were co-transfected with different combinations 48 h later. F: the apoptotic rate of SMMC-7721 cells in E which were treated for another 48 h with doxorubicin (0.1 mg/mL) using Hoechst 33342 staining. *: P < 0.05; **: P < 0.01. (i) si-Bcl-2/anti-miRNA-NC; (ii) anti-miR-125b/si-NC; (iii) si-NC/anti-miRNA-NC and (iv) anti-miR-125b/si-Bcl-2.

Bcl-2 protein in cells transfected with (ii) anti-miR-125b/ si-NC was increased, the expression of Bcl-2 protein in cells transfected with (i) si-Bcl-2/anti-miRNA-NC was decreased, and the expression of Bcl-2 protein in cells transfected with (iv) anti-miR-125b/si-Bcl-2 was little changed compared with cells transfected with (iii) si-NC/anti-miRNA-NC (Fig. 4E). Furthermore, their apoptotic rates were approximately consistent with the expression of Bcl-2 protein (Fig. 4F). Taken together, these results suggest that miR-125b can promote cancer cells apoptosis by directly inhibiting the expression of Bcl-2. 4. DISCUSSION Apoptosis is essential for normal development and homeostasis (Degterev et al., 2003; Danial, 2007). Deregulation of

apoptosis plays an essential role in tumor initiation, progression, and acquired chemotherapy resistance. The ability to bypass apoptosis is a hallmark of cancer and is a key factor in the development of neoplastic cells (Hanahan and Weinberg, 2000). Apoptosis induced by anticancer agents is largely mediated through the pathway regulated by the Bcl-2 family proteins, with the anti-apoptotic protein Bcl-2 playing an important role in this process. Overexpression of Bcl-2 has been reported in many cancers including HCC, which is implicated in chemoresistance of cancer cells (Raffo et al., 1995; Abou El Hassan et al., 2004; Yip and Reed, 2008). In recent years, many aberrant miRNAs were found in both clinical tumors and cancer cell lines. Emerging evidence suggests that miRNAs are important in cancer cell apoptosis and chemoresistance, although their exact function is not fully

A. Zhao et al. / Journal of Genetics and Genomics 39 (2012) 29e35

understood. Aberrantly expressed miRNAs can contribute to the pathogenesis of human diseases by targeting Bcl-2 family members. miR-15 and miR-16, which are down-regulated in chronic lymphocytic leukemia, inhibit Bcl-2 expression (Cimmino et al., 2005). miR-29 can sensitize HCC cells to undergo apoptosis as well as suppress HCC tumorigenicity by targeting Bcl-2 and Mcl-1 (Xiong et al., 2010). In this study, we found that reduced miR-125b expression is a frequent event in human HCC, which is consistent with other published studies (Li et al., 2008; Su et al., 2009; Liang et al., 2010). Then, we demonstrated for the first time that Bcl-2 is a target gene of miR-125b. It is likely that in HCC, downregulation of miR-125b and subsequent up-regulation of Bcl-2 expression affect tumorigenesis and chemoresistance. Liang et al. (2010) reported that miR-125b suppresses human liver cancer cell proliferation and metastasis by directly targeting the oncogene LIN28B. Here, we also showed that miR-125b significantly inhibited HCC cell growth in vitro and in vivo. Moreover, we demonstrated that miR-125b suppressed cancer growth by promoting cell apoptosis through the down-regulation of Bcl-2 expression. This is consistent with the current view that a single miRNA can regulate gene expression by targeting multiple mRNAs, which is collectively named a “targetome” (Selbach et al., 2008). In summary, we investigated the altered expression pattern of miR-125b in HCC and its indication of cancer cell apoptosis through suppression of Bcl-2 expression. Our data demonstrated an important role for miR-125b in the molecular etiology of cancer and suggested its potential application in cancer therapy. ACKNOWLEDGEMENTS This work was supported by the National High Technology Research and Development Program of China (No. 2006AA02A107), the Major State Basic Research Program of China (Nos. 2009CB521704 and 2010CB945504), and the National Nature Science Foundation of China (No. 30873031). REFERENCES Abou El Hassan, M.A., Mastenbroek, D.C., Gerritsen, W.R., Giaccone, G., Kruyt, F.A., 2004. Overexpression of Bcl2 abrogates chemo- and radiotherapy-induced sensitisation of NCI-H460 non-small-cell lung cancer cells to adenovirus-mediated expression of full-length TRAIL. Br. J. Cancer 91, 171e177. Aravalli, R.N., Steer, C.J., Cressman, E.N., 2008. Molecular mechanisms of hepatocellular carcinoma. Hepatology 48, 2047e2063. Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281e297.

35

Calin, G.A., Sevignani, C., Dumitru, C.D., Hyslop, T., Noch, E., Yendamuri, S., Shimizu, M., Rattan, S., Bullrich, F., Negrini, M., Croce, C.M., 2004. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. USA 101, 2999e3004. Cimmino, A., Calin, G.A., Fabbri, M., Iorio, M.V., Ferracin, M., Shimizu, M., Wojcik, S.E., Aqeilan, R.I., Zupo, S., Dono, M., Rassenti, L., Alder, H., Volinia, S., Liu, C.G., Kipps, T.J., Negrini, M., Croce, C.M., 2005. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc. Natl. Acad. Sci. USA 102, 13944e13949. Danial, N.N., 2007. BCL-2 family proteins: critical checkpoints of apoptotic cell death. Clin. Cancer Res. 13, 7254e7263. Degterev, A., Boyce, M., Yuan, J., 2003. A decade of caspases. Oncogene 22, 8543e8567. Hanahan, D., Weinberg, R.A., 2000. The hallmarks of cancer. Cell 100, 57e70. Ichimi, T., Enokida, H., Okuno, Y., Kunimoto, R., Chiyomaru, T., Kawamoto, K., Kawahara, K., Toki, K., Kawakami, K., Nishiyama, K., Tsujimoto, G., Nakagawa, M., Seki, N., 2009. Identification of novel microRNA targets based on microRNA signatures in bladder cancer. Int. J. Cancer 125, 345e352. Iorio, M.V., Ferracin, M., Liu, C.G., Veronese, A., Spizzo, R., Sabbioni, S., Magri, E., Pedriali, M., Fabbri, M., Campiglio, M., Menard, S., Palazzo, J.P., Rosenberg, A., Musiani, P., Volinia, S., Nenci, I., Calin, G.A., Querzoli, P., Negrini, M., Croce, C.M., 2005. MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 65, 7065e7070. Li, W., Xie, L., He, X., Li, J., Tu, K., Wei, L., Wu, J., Guo, Y., Ma, X., Zhang, P., Pan, Z., Hu, X., Zhao, Y., Xie, H., Jiang, G., Chen, T., Wang, J., Zheng, S., Cheng, J., Wan, D., Yang, S., Li, Y., Gu, J., 2008. Diagnostic and prognostic implications of microRNAs in human hepatocellular carcinoma. Int. J. Cancer 123, 1616e1622. Liang, L., Wong, C.M., Ying, Q., Fan, D.N., Huang, S., Ding, J., Yao, J., Yan, M., Li, J., Yao, M., Ng, I.O., He, X., 2010. MicroRNA-125b suppressed human liver cancer cell proliferation and metastasis by directly targeting oncogene LIN28B2. Hepatology 52, 1731e1740. Raffo, A.J., Perlman, H., Chen, M.W., Day, M.L., Streitman, J.S., Buttyan, R., 1995. Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res. 55, 4438e4445. Selbach, M., Schwanhausser, B., Thierfelder, N., Fang, Z., Khanin, R., Rajewsky, N., 2008. Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58e63. Spizzo, R., Nicoloso, M.S., Croce, C.M., Calin, G.A., 2009. SnapShot: microRNAs in cancer. Cell 137 586e586 e581. Su, H., Yang, J.R., Xu, T., Huang, J., Xu, L., Yuan, Y., Zhuang, S.M., 2009. MicroRNA-101, down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity. Cancer Res. 69, 1135e1142. Visone, R., Pallante, P., Vecchione, A., Cirombella, R., Ferracin, M., Ferraro, A., Volinia, S., Coluzzi, S., Leone, V., Borbone, E., Liu, C.G., Petrocca, F., Troncone, G., Calin, G.A., Scarpa, A., Colato, C., Tallini, G., Santoro, M., Croce, C.M., Fusco, A., 2007. Specific microRNAs are downregulated in human thyroid anaplastic carcinomas. Oncogene 26, 7590e7595. Xiong, Y., Fang, J.H., Yun, J.P., Yang, J., Zhang, Y., Jia, W.H., Zhuang, S.M., 2010. Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma. Hepatology 51, 836e845. Xu, T., Zhu, Y., Xiong, Y., Ge, Y.Y., Yun, J.P., Zhuang, S.M., 2009. MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. Hepatology 50, 113e121. Yip, K.W., Reed, J.C., 2008. Bcl-2 family proteins and cancer. Oncogene 27, 6398e6406.