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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Based on microarray data, we have previously shown a significant down-regulation of miR-29 in hepatocellular carcinoma (HCC) tissues. To date, the role of miR-29 deregulation in hepatocarcinogenesis and the signaling pathways by which miR-29 exerts its function and modulates the malignant phenotypes of HCC cells remain largely unknown. In this study, we confirmed that reduced expression of miR-29 was a frequent event in HCC tissues using both Northern blot and real-time quantitative reverse-transcription polymerase chain reaction. More interestingly, we found that miR-29 down-regulation was significantly associated with worse disease-free survival of HCC patients. Both gain- and loss-of-function studies revealed that miR-29 could sensitize HCC cells to apoptosis that was triggered by either serum starvation and hypoxia or chemotherapeutic drugs, which mimicked the tumor growth environment in vivo and the clinical treatment. Moreover, introduction of miR-29 dramatically repressed the ability of HCC cells to form tumor in nude mice. Subsequent investigation characterized two antiapoptotic molecules, Bcl-2 and Mcl-1, as direct targets of miR-29. Furthermore, silencing of Bcl-2 and Mcl-1 phenocopied the proapoptotic effect of miR-29, whereas overexpression of these proteins attenuated the effect of miR-29. In addition, enhanced expression of miR-29 resulted in the loss of mitochondrial potential and the release of cytochrome c to cytoplasm, suggesting that miR-29 may promote apoptosis through a mitochondrial pathway that involves Mcl-1 and Bcl-2. Conclusion: Our data highlight an important role of miR-29 in the regulation of apoptosis and in the molecular etiology of HCC, and implicate the potential application of miR-29 in prognosis prediction and in cancer therapy. (HEPATOLOGY 2010.)

MicroRNAs (miRNAs) are a class of phylogenetically conserved short RNAs that suppress protein expression through base-pairing with the 3'-untranslated region (3'-UTR) of target mRNA.1 Growing evidence suggests that miRNAs play important roles in diverse biological processes1-3 and the dysfunction of miRNAs is involved in the development of cancer.4, 5

Hepatocellular carcinoma (HCC) is one of the most common cancers and the leading cause of cancer-related death globally.6, 7 Altered miRNA expression is observed in HCCs that have been collected from different study cohorts.8-16 Furthermore, several deregulated miRNAs (e.g., miR-21, miR-101, miR-195, miR-122, miR-221, miR-223, and miR-224) have been shown to regulate cell growth, apoptosis, migration, or invasion.9, 11, 13, 15-18 These findings suggest that deregulation of miRNA may be associated with hepatocarcinogenesis. More extensive investigations are required to elucidate the role of miRNAs in the development of HCC, to identify those miRNAs that may be employed as novel prognosis predictor or as therapeutic targets for HCC.

We have previously shown a significant down-regulation of miR-29 family members, including miR-29a, miR-29b, and miR-29c (miR-29a/b/c), in HCC tissues,16 which is in accordance with previous observations in other types of human neoplasms.19-23 It has been shown that ectopic expression of miR-29b inhibits cell growth and promotes tumor necrosis factor–related apoptosis-inducing ligand–triggered apoptosis.22, 24, 25 In addition, miR-29a/b/c significantly repressed the ability of lung cancer and rhabdomyosarcoma cell lines to form tumor in vivo.24, 25 These data suggest a potential tumor suppressive function of the miR-29 family. To date, several oncogenes, including apoptosis-related molecules, such as T cell leukemia/lymphoma 1 (TCL1), myeloid cell leukemia sequence 1 (Mcl-1), cell division cycle 42 (CDC42) and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), have been characterized as targets of the miR-29 family.20, 22, 26 However, the role of miR-29 deregulation in hepatocarcinogenesis and the signaling pathways by which miR-29 exerts its function and modulates the malignant phenotypes of HCC cells remain largely unknown.

In this study, we showed that miR-29 expression was obviously reduced in the majority of examined HCC tissues, and its down-regulation was significantly associated with worse disease-free survival (DFS) of HCC patients. Furthermore, enhanced miR-29 expression dramatically sensitized HCC cells to various apoptotic signals and suppressed the ability of HCC cells to form tumor in vivo. Moreover, we found that both Bcl-2 and Mcl-1 were direct targets of miR-29 and the mitochondrial pathway was activated in miR-29–promoted apoptosis. Our findings will help to elucidate the functions of miRNAs and their roles in tumorigenesis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Tissue Specimens and Cell Lines.

Information about HCC cell lines and tissue specimens is given in the Supporting Materials and Methods. The relevant characteristics of the studied subjects are shown in Supporting Table 1.

Northern Blotting, Semiquantitative Reverse-Transcription Polymerase Chain Reaction, and Real-Time Quantitative Reverse-Transcription Polymerase Chain Reaction for miRNA.

Northern blot and semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) were performed as described.27 Quantitative RT-PCR (qPCR) was performed using kits for qPCR of U6 and mature miR-29b (Genpharma, Shanghai, China), according to the manufacturer's instructions. Detailed information is provided in the Supporting Materials and Methods.

RNA Oligoribonucleotides and Plasmids.

All RNA oligoribonucleotides were purchased from Genepharma. miRNA duplexes corresponding to mature miR-29a, miR-29b, and miR-29c were designed as described.28 The small interfering RNAs that targeted human Bcl-2 (GenBank accession no. NM_000633) and Mcl-1 (GenBank accession no. NM_021960) mRNAs were designated siBCL2 and siMCL1, respectively. The negative control RNA duplex (named NC) for both miRNA mimic and small interfering RNA was nonhomologous to any human genome sequences. For the in vivo tumorigenicity assay, all pyrimidine nucleotides in NC or miR-29 duplex were substituted by their 2'-O-methyl analogs to improve RNA stability. The anti–miR-29a, anti–miR-29b, and anti–miR-29c, with sequences that were complementary to the mature miR-29a, miR-29b, and miR-29c, respectively, were 2'-O-methyl-modified oligoribonucleotides designed as inhibitors of miR-29a, miR-29b, and miR-29c individually. The anti–miR-C was used as a negative control in the antagonism experiments.

A wild-type 3'-UTR segment of human Bcl-2 (618 bp) or Mcl-1 (721 bp) mRNA that contained putative binding site for miR-29 was PCR-amplified and inserted into the EcoRI/XbaI or ApaI/XbaI sites downstream of the stop codon of firefly luciferase in pGL3cm,16 which was created based upon the pGL3-control (Promega). The resulting plasmids were denoted pGL3cm-BCL2-3'-UTR-WT and pGL3cm-MCL1-3'-UTR-WT, respectively. The plasmids pGL3cm- BCL2-3'-UTR-MUT and pGL3cm-MCL1-3'-UTR-MUT, which carried mutated sequence in the complementary site for the seed region of miR-29 (Supporting Fig. 1A), were generated by site-specific mutagenesis based on pGL3cm-BCL2-3'-UTR-WT and pGL3cm-MCL1-3'-UTR-WT, respectively.

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Figure 1. Expression analysis of mature miR-29 in paired HCC and adjacent nontumor tissues by Northern blotting. The same membrane was hybridized sequentially with miR-29b and U6 probes at 42°C. Hybridization with miR-29b probe revealed a single band that migrated between xylene cyanol FF and bromophenol blue, the loading dyes which comigrate with ∼30 bp and ∼10 bp RNA fragments in 15% denaturing polyacrylamide gel, respectively. The intensity for each band, representing gene level, was densitometrically quantified. miR-29 expression in each sample was normalized by U6, the control for RNA loading. The value under each pair of samples (T/N), which is represented by the ratio of normalized miR-29 expression between HCC tissue and its corresponding adjacent nontumor tissue, indicates fold change of miR-29 level in HCC. N, adjacent noncancerous tissue; T, HCC tissue.

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The coding sequence of Bcl-2 and Mcl-1 were cloned into the EcoRI/XhoI and BamHI/EcoRI sites of pc3-gab, respectively. The pc3-gab was produced based upon pcDNA3.0 (Invitrogen, Carlsbad, CA) by replacing the neomycin open reading frame with an expression cassette of the enhanced green fluorescent protein (EGFP) gene.16 The generated expression vectors were named pc3-gab-BCL2 and pc3-gab-MCL1, respectively.

All oligo sequences are provided in Supporting Table 2.

Cell Transfections.

Reverse transfection of RNA oligoribonucleotides was performed using Lipofectamine-RNAiMAX (Invitrogen). Transfection of plasmid DNA or cotransfection of RNA duplex with plasmid DNA was performed using Lipofectamine 2000 (Invitrogen). Unless otherwise indicated, 50 nM of RNA duplex and 200 nM of miRNA inhibitor were used for each transfection.

Cell Viability and Apoptosis Analysis.

Cell viability was analyzed by the Alamar Blue assay (AbD Serotec, Oxford, UK) as reported.16 Apoptosis was evaluated by morphological examination and the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assay, as described in the Supporting Materials and Methods.

Tumorigenicity Assays in Nude Mice.

Analysis for tumorigenicity was performed as described in the Supporting Materials and Methods.

Luciferase Reporter Assay.

The luciferase assay was performed in HEK293T or HepG2 cells, as described in the Supporting Materials and Methods.

Western Blot.

Cytosolic protein fractions were prepared using the cell mitochondria isolation kit (Beyotime, Shanghai, China). The antibodies used for western blotting are described in the Supporting Materials and Methods.

Immunohistochemisty.

Paraffin-embedded, formalin-fixed tissues were immunostained for Bcl-2 and Mcl-1, as described in the Supporting Materials and Methods.

Measurement of Mitochondrial Membrane Potential.

Cells were stained with both MitoTracker Deep Red FM (MTRed, invitrogen) and MitoTracker Green FM (MTGreen, Invitrogen) at 37°C for 20 minutes in the dark and washed in Ca2+-free phosphate-buffered saline, followed by flow cytometry analysis.

Statistical Analysis.

Data are expressed as the mean ± standard error of the mean from at least three independent experiments. Unless otherwise noted, the differences between groups were analyzed using Student t test when only two groups, or by one-way analysis of variance when more than two groups were compared. Association between expression levels of miR-29 and its target genes in HCC tissues was explored using Pearson's correlation coefficient. The DFS was calculated from the date of tumor resection to the time of first recurrence or death. Patients who were lost to follow-up or died from causes unrelated to HCC were treated as censored events. Kaplan-Meier survival curves were constructed, and the differences between groups were analyzed using a log rank test. Association between the molecular changes or clinical characteristics of patients and the DFS was first analyzed by univariate Cox proportional hazards regression analysis. Significant prognostic factors found by univariate analysis were further evaluated by multivariate Cox regression analysis. All statistical tests were two-sided, and P values less than 0.05 were considered as statistically significant. All analyses were performed using SPSS software (version 13.0, SPSS Inc., Chicago, IL).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Down-Regulation of miR-29 Is a Frequent Event in HCC Tissues and Is Associated with Worse Prognosis.

In a previous study, we observed that miR-29a/b/c (miR-29), especially miR-29b and miR-29c, were significantly down-regulated in HCC compared with normal liver tissues.16 Here, miR-29 expression was further analyzed in 17 paired HCC and adjacent nontumor liver tissues by way of Northern blotting. Like other miRNA family members, miR-29a/b/c display high sequence similarity (Supporting Fig. 1B) and share common seed sequences for target recognition. We determined 42°C as the proper hybridization temperature to detect all three members of the miR-29 family, whereas 58°C was the temperature that allowed miR-29b to be distinguished from miR-29a and miR-29c (Supporting Fig. 2A). However, miR-29a and miR-29c, whose mature sequences differ only by one base, were indistinguishable even at 60°C. Notably, miR-29 was down-regulated in the majority of examined HCC tissues and cell lines (Fig. 1, Supporting Fig. 2B), with 10 out of 17 (58.8%) HCC tissues displayed a more than 50% reduction. Consistent with the results from Northern blotting, qPCR analysis revealed a similar trend of miR-29b decrease in HCC tissues (Supporting Fig. 2C, Supporting Table 3, Fig. 1).

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Figure 2. Kaplan-Meier DFS analysis for HCC patients according to miR-29b level. The mature miR-29b level was analyzed by real-time qPCR and its median value of all 127 cases was chosen as the cut-off point for separating miR-29b low-expression tumors (n = 64) from miR-29b high-expression cases (n = 63).

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We next investigated whether miR-29 down-regulation was correlated with clinical features or prognosis of HCC patients. Since the expression pattern of miR-29b resembled that of miR-29 in HCC, qPCR was applied to analyze the level of miR-29b on 127 HCC tissues, including those 17 samples used above. An association between the decreased miR-29b expression and the increased AFP level was observed (Supporting Table 1). Furthermore, the Kaplan-Meier method revealed that lower miR-29b level was associated with shorter disease-free survival (P = 0.002) (Fig. 2). To exclude the confounder effect, we further performed Cox proportional hazards regression analysis. Univariate analysis was first conducted to identify those factors that affected the DFS, followed by multivariate analysis, which controlled for potential confounders (Table 1). Strikingly, multivariate analysis further confirmed that reduced miR-29 level is an independent predictor for shorter DFS of HCC patients (P = 0.008) (Table 1).

Table 1. Univariate and Multivariate Analysis of Factors Associated with Disease-Free Survival
Clinical VariablesCase NumberHR (95% CI)P Value
  • Analysis was conducted on 127 cases, including those 17 cases shown in Fig. 1. Hazard ratios (95% confidence interval) and P values were calculated using univariate or multivariate Cox proportional hazards regression.

  • AFP, alpha-fetoprotein; ALT, alanine aminotransferase, HBV, hepatitis B virus, TNM, tumor-node-metastasis.

  • *

    The mature miR-29b level was examined by real-time qPCR and normalized to U6 level. The median value of all 127 samples was chosen as the cut-off point for separating miR-29b low tumors from miR-29b high cases.

  • −, none or incomplete capsule; +, complete capsule.

  • Boldface P values indicate statistical significant.

Univariate analysis   
miR-29b (low versus high)*64/632.0 (1.3–3.3)0.003
Sex (M versus F)108/190.9 (0.5–1.8)0.751
Age (>50 versus ≤50 years)54/731.0 (0.8–1.3)0.860
HBV (positive versus negative)111/161.0 (0.5–2.1)0.885
Cirrhosis (yes versus no)110/131.6 (0.7–3.6)0.299
Ascites (yes versus no)14/1122.2 (1.2–4.0)0.011
AFP (≥400 versus <400 ng/mL)67/601.0 (0.8–1.2)0.701
ALT (≥50 versus <50 U/L)68/591.0 (0.8–1.3)0.996
Tumor size (>5 cm versus ≤5 cm)94/331.5 (1.1–2.0)0.014
Tumor number (>1 versus 1)29/942.4 (1.5–4.0)0.001
Tumor capsule (− versus +)72/531.7 (1.0–2.7)0.036
Portal vein tumor thrombus (yes versus no)18/1074.0 (2.2–7.3)<0.001
TNM stage (II/III versus I)57/702.9 (1.8–4.7)<0.001
Edmondson grade (>II versus I-II)64/631.7 (1.1–2.8)0.019
Multivariate analysis   
miR-29b (low versus high)*64/631.9 (1.2–3.2)0.008
Ascites (yes versus no)14/1121.8 (1.0–3.4)0.058
Tumor capsule (− versus +)72/531.4 (0.8–2.4)0.257
TNM stage (II/III versus I)57/702.7 (1.5–4.6)<0.001
Edmondson grade (>II versus I-II)64/631.4 (0.8–2.2)0.226

Collectively, these data suggest that deregulation of miR-29 may contribute to the development of HCC.

miR-29 Promotes Apoptosis and Represses Tumorigenicity.

The above findings prompted us to explore the biological significance of miR-29 in tumorigenesis. Rapid growth of malignancy causes insufficient blood supply and, thus, solid cancer cells should evolve to tolerate hypoxia and nutritional starvation.29 We therefore analyzed the effect of miR-29 on the phenotype of HCC cells that were undergoing hypoxia (1% O2) and serum starvation. Interestingly, the Alamar Blue assay revealed that introduction of miR-29 resulted in significantly decreased viability of serum-deprived and hypoxia-exposed HepG2 cells (Supporting Fig. 3A). Furthermore, both morphological examination and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining demonstrated that transfection with miR-29a/b/c obviously increased the apoptotic rates of HepG2 cells upon the treatment of hypoxia and serum deprivation (Fig. 3A, Supporting Fig. 3B,C). The apoptosis-promoting effect of miR-29 was reproducible in two other HCC cells, MHCC97H and QGY-7703 (Fig. 3B). To verify the findings from gain-of-function study, loss-of-function analysis was performed using inhibitors of miR-29a/b/c (anti–miR-29, a mixture of anti–miR-29a/b/c). Dramatically, anti–miR-29 attenuated the apoptosis-promoting effect of exogenous miR-29 (Supporting Fig. 3D), suggesting the efficiency of anti–miR-29. Then we analyzed the effect of anti–miR-29 on endogenous miR-29. Consistently, compared with negative controls, anti–miR-29 transfectants displayed obviously lower apoptotic rate, in response to hypoxia and serum deprivation (Fig. 3C).

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Figure 3. The impact of miR-29 family members on apoptosis. (A,B) Ectopic expression of miR-29 sensitizes HCC cells to serum deprivation and hypoxia-induced apoptosis. HepG2 (A) and MHCC97H and QGY-7703 (B) cells without transfection or transfected with NC or miR-29a/b/c were cultured in 1% O2 and serum-deprived medium for indicated times before DAPI staining. In figure B, cells underwent serum starvation and hypoxia for 72 hours. (C) Antagonism of miR-29 desensitizes HepG2 cells to serum deprivation and hypoxia-induced apoptosis. Cells without transfection or transfected with anti–miR-C or anti–miR-29 were deprived of serum and cultured in 1% O2for 96 hours before DAPI staining. (D) miR-29 sensitizes HepG2 cells to doxorubicin-induced apoptosis. Cells without transfection or transfected with NC or miR-29a/b/c were treated with doxorubicin (0.1 μg/mL) for indicated times before morphological examination. (E) Antagonism of miR-29 desensitizes HepG2 cells to doxorubicin-induced apoptosis. Cells without transfection or transfected with anti–miR-C or anti–miR-29 were treated with doxorubicin (0.2 μg/mL) for 72 hours before DAPI staining. (F) miR-29 sensitizes HepG2 cells to curcumin- and etoposide-induced apoptosis. Cells without transfection or transfected with NC or miR-29a/b/c were exposed to curcumin (12.5 μM) or etoposide (1.5 μg/mL) for 48 hours before DAPI staining. Cells without transfection or transfected with NC or anti–miR-C were used as negative controls. *P < 0.05, **P < 0.01, ***P < 0.001 compared with NC or anti–miR-C transfectants.

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We next investigated whether miR-29 could also enhance the chemosensitivity of HCC cells. Compared with negative controls, enhanced expression of miR-29 significantly increased the apoptotic rates of HepG2 cells that were exposed to doxorubicin, which is one of the major drugs used for the chemotherapy of HCC (Fig. 3D, Supporting Fig. 4A). This result was reproducible in QGY-7703 cells (Supporting Fig. 4B) and was also confirmed by loss-of-function analysis, as shown by anti–miR-29 significantly repressing doxorubicin-triggered apoptosis (Fig. 3E). Furthermore, miR-29 transfectants were also much more sensitive to curcumin or etoposide-induced apoptosis compared with negative controls (Fig. 3F). Taken together, both gain- and loss-of function studies imply that miR-29 family members may increase the sensitivity of HCC cells to different apoptotic stimuli, including hypoxia, serum starvation, and chemotherapeutic agents.

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Figure 4. Effect of miR-29b on tumorigenicity in nude mice xenograft model. Photographs of dissected tumors from nude mice (A) and the curve of tumor growth (B) are shown. Wilcoxon signed-ranks test was used for the comparison of tumor volumes. *P < 0.05 versus NC transfectants.

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An in vivo model was subsequently applied to evaluate the effect of miR-29 on tumorigenicity. Because miR-29a/b/c displayed a similar apoptosis-promoting effect, we used miR-29b to represent the miR-29 family in tumorigenicity assay. Compared with NC transfectants, miR-29b–transfected cells revealed a delayed tumor formation time (4/6 versus 1/6 on day 10) and a significant reduction in the tumor size (Fig. 4), suggesting a potential tumor suppressive effect of miR-29.

Bcl-2 and Mcl-1 Are Direct Targets of miR-29.

Next, we explored the molecular mechanisms responsible for the function of miR-29 that were observed above. Predicted target genes of miR-29a/b/c were retrieved using publicly available databases (TargetScan and miRanda). Bcl-2 and Mcl-1 were chosen for further analysis, because they are antiapoptotic members of the Bcl-2 family and have displayed frequent overexpression in HCC tissues.30, 31 To verify whether Bcl-2 and Mcl-1 are direct targets of miR-29, a dual-luciferase reporter system was first employed. Cotransfection of miR-29a/b/c significantly suppressed the firefly luciferase activity of the reporter with wild-type 3'-UTR but not that of the mutant reporter (Fig. 5A). These results were also reproducible in HepG2 cells (data not shown). In addition, inhibition of endogenous miR-29a/b/c by anti-miR-29 led to increased firefly luciferase activity of the wild-type reporter but not that of the mutant one (Fig. 5B).

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Figure 5. Bcl-2 and Mcl-1 are direct targets of miR-29. (A,B) Analysis of luciferase activity. In figure A, cells were cotransfected with indicated RNA duplex, pRL-TK and firefly luciferase reporter plasmid containing wild-type or mutant 3'-UTR of putative target gene. In figure B, cells were first transfected with anti–miR-29 or anti–miR-C and then with pRL-TK and the firefly luciferase reporter comprising the wild-type or mutant 3'-UTR of the putative target gene. pRL-TK expressing Renilla luciferase was cotransfected as an internal control to correct the differences in both transfection and harvest efficiencies. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. The normalized luciferase activity of NC or anti–miR-C transfectant was set as relative luciferase activity 1, therefore, no error bar was shown for NC and anti–miR-C transfectant. Values represent the mean ± standard error of the mean of at least three independent experiments performed in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001 compared with NC or anti–miR-C transfectants. (C,D) Effects of miR-29 overexpression (C) or suppression (D) on the endogenous Bcl-2 and Mcl-1 levels. Forty-eight hours after transfection with the indicated RNA oligoribonucleotides, HepG2 cells were analyzed by Western blotting and RT-PCR. The intensity for each band was densitometrically quantified. The value under each lane indicates the relative expression level of the putative target gene, which is represented by the intensity ratio between Bcl-2 or Mcl-1 and β-actin bands in each lane (β-actin, internal control). (E) Immunohistochemistry staining of Bcl-2 and Mcl-1 in HCC. Representative immunohistochemical stainings in HCC tissues with low (730T) or normal (714T) miR-29b expression are shown (magnification ×400). (F) Inverse correlation between miR-29b and Bcl-2/Mcl-1 levels in HCC tissues. Expression of Bcl-2 and Mcl-1 analyzed by immunohistochemical staining was quantified using modified Histo-score as described in the Supporting Materials and Methods. The mature miR-29b level was examined by real-time qPCR analysis and normalized to U6 expression. Statistical analysis was performed using Pearson's correlation coefficient.

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Further investigation showed that transfection with miR-29a/b/c diminished the endogenous expression of both Bcl-2 and Mcl-1 proteins, under conditions of conventional culture (Fig. 5C) or serum deprivation and hypoxia (Supporting Fig. 5). In addition, antagonism of endogenous miR-29 resulted in the up-regulation of Bcl-2 and Mcl-1 proteins (Fig. 5D).

Correlation between the miR-29b level and the expression of Bcl-2 and Mcl-1 was further examined in HCC tissues. Bcl-2 and Mcl-1 were analyzed by immunohistochemistry, and miR-29b was determined by qPCR in the same set of specimens shown in Fig. 1. Markedly, miR-29b level was inversely correlated with Bcl-2 and Mcl-1 expression (Fig. 5E, F).

These data suggest that miR-29 may negatively regulate the expression of Bcl-2 and Mcl-1 by directly targeting the 3'-UTR of their mRNAs.

miR-29 May Promote Apoptosis Through a Mitochondrial Pathway That Involves Mcl-1 and Bcl-2.

To evaluate whether Bcl-2 and Mcl-1 are involved in miR-29–promoted apoptosis, cells were transfected with siBCL2 and/or siMCL1, which resulted in greatly reduced mRNA and protein levels of the respective genes (Fig. 6A). Silencing of either target gene significantly increased the apoptotic rate in serum-deprived and hypoxia-exposed HepG2 cells (Fig. 6B), which phenocopied the consequence of enhanced miR-29 expression. Furthermore, the apoptosis-promoting effect of double-knockdown was more pronounced than that of single knockdown of either Bcl-2 or Mcl-1 (Fig. 6B). We subsequently investigated whether Bcl-2 and Mcl-1 could counteract the pro-apoptotic function of miR-29. The expression vector pc3-gab-BCL2 or pc3-gab-MCL1, which encoded the entire coding sequence of Bcl-2 or Mcl-1 but lacked the 3'-UTR (Fig. 6C), was cotransfected with miR-29b into HepG2 cells. Notably, overexpression of Bcl-2 and Mcl-1 abrogated miR-29b–promoted apoptosis (Fig. 6D). These observations suggest that Bcl-2 and Mcl-1 are potentially involved in miR-29-regulated apoptosis.

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Figure 6. Bcl-2 and Mcl-1 are involved in miR-29-promoted apoptosis. (A) Endogenous Bcl-2 and Mcl-1 expression is efficiently repressed by small interfering RNA. HepG2 cells were transfected with siBCL2 or siMCL1 for 48 hours and then analyzed by RT-PCR or Western blotting (β-actin, internal control). (B) Knockdown of either or both Bcl-2 and Mcl-1 increases the cellular sensitivity to apoptosis. Twenty-four hours after transfection with indicated RNA duplex, HepG2 cells were deprived of serum and cultured in 1% O2 for 72 hours before DAPI staining. (C) Ectopic expression of Bcl-2 and Mcl-1 in HepG2 cells. Cells were transfected with indicated expression vector for 48 hours and then analyzed by Western blotting (β-actin, internal control). (D) Ectopic expression of either or both of Bcl-2 and Mcl-1 abrogates the proapoptotic effect of miR-29. Twenty-four hours after cotransfection with indicated expression vector and RNA duplex, HepG2 cells were serum-starved and cultured in 1% O2 for 48 hours before DAPI staining. Vec, pc3-gab expressing EGFP only; Bcl-2, pc3-gab-BCL2 expressing both Bcl-2 and EGFP under the control of different promoters; Mcl-1, pc3-gab-MCL1 expressing both Mcl-1 and EGFP under the control of different promoters. Cells with green fluorescence were analyzed. HepG2, cells without transfection. **P < 0.01, ***P < 0.001; comparison between two groups as indicated.

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Given that both Bcl-2 and Mcl-1 play crucial roles in the mitochondrial apoptosis pathway, we examined whether introduction of miR-29 triggered a mitochondrial pathway, based on assays of mitochondrial membrane potential (ΔΨm) and cytochrome c. The ΔΨm was analyzed by double-staining of MTRed and MTGreen. The intensity of MTRed staining depends on ΔΨm, while the intensity of MTGreen remains the same regardless of ΔΨm and was thus used as an internal staining control. As shown in Fig. 7A,B, the negative controls and the miR-29a/b/c transfectants displayed similar fluorescent intensity of MTGreen. In contrast, an obvious increase in the propotion of cells with loss of ΔΨm, as shown by substantially decreased fluorescent intensity of MTRed, was observed after transfections with miR-29a/b/c. Furthermore, miR-29a/b/c-transfectants displayed a much higher level of cytochrome c in the cytoplasmic fraction but an equivalent level of total cytochrome c (Fig. 7C), which indicated the release of cytochrome c from the mitochondria to the cytoplasm.

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Figure 7. miR-29 may promote apoptosis through a mitochondrial pathway. (A,B) Introduction of miR-29 results in depolarization of mitochondrial membrane. Cells without transfection or transfected with NC or miR-29a/b/c were serum-starved and cultured in 1% O2 for 48 hours, then stained with MTRed and MTGreen, followed by flow cytometry analysis. Both dot plot (A) and fluorescent histograms (B) are shown. R1, cell population with intact mitochondria; R2, population with loss of mitochondrial membrane potential (ΔΨm). ***P < 0.001 compared with NC transfectants. (C) Ectopic expression of miR-29 promotes mitochondrial release of cytochrome c. HepG2 cells without transfection or transfected with indicated RNA duplex were serum-starved and cultured in 1% O2 for 48 hours before Western blot analysis. Cyt c, cytochrome c; β-actin, internal control.

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Taken together, our data suggest that miR-29 may promote apoptosis by suppressing the expression of Mcl-1 and Bcl-2, and in turn triggering the mitochondrial pathway.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Although misexpression of miRNAs has been observed in various types of cancers,32, 33 the molecular mechanisms by which miRNAs modulate the process of carcinogenesis and the behavior of cancer cells are still largely unknown. In the present report, we demonstrated that down-regulation of miR-29 was a frequent event in HCC tissues and an independent prognosis predictor for HCC patients. Furthermore, reintroduction of miR-29 dramatically repressed the tumorigenicity of HCC cells and also sensitized HCC cells to apoptosis triggered by different stimuli. We further characterized Bcl-2 and Mcl-1 as functional targets of miR-29 and proved the involvement of the mitochondrial pathway in miR-29–promoted apoptosis. Our findings, together with those from other groups,20, 22-26, 34, 35 suggest a fundamental role of miR-29 in tumorigenesis as well as in the phenotypes of cancer cells, and implicate the potential application of miR-29 in prognosis prediction and cancer therapy.

Apoptosis is a major barrier that must be circumvented during malignant transformation and tumor progression. Cancer cells evolve to evade apoptosis so that they can escape from the surveillance system and survive in the crucial tumor growth environment, such as low nutrition and hypoxia.29 We showed that miR-29 could sensitize cancer cells to hypoxia and serum starvation-induced apoptosis. Therefore, down-regulation of miR-29 may facilitate the adaptation of cancer cells to the crucial growth environment and, in turn, facilitate the development of HCC. It is noteworthy that HCC is poorly responsive to chemotherapy. Interestingly, miR-29 could also sensitize HCC cells to chemotherapeutic drug-induced apoptosis. Therefore, reintroduction of miR-29 into HCC cells may not only limit cancer growth but also sensitize HCC cells to anticancer therapy.

Recently, it has been shown that miR-29a/b/c up-regulates p53 by targeting p85α and CDC42, and induces apoptosis in a p53-dependent manner in breast and colorectal cancer cell lines.26 miR-29b is also shown to target Mcl-1 and sensitize cholangiocarcinoma cells to tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis.22 In the present study, we employed a new research model, HCC cells, to study the apoptosis pathways by which miR-29 exerts its function and modulates the malignant phenotypes of cancer cells. We not only confirmed that Mcl-1 is a target of miR-29, but also provided new evidence to support Bcl-2 as another target of miR-29 and defined the activation of mitochondrial pathway as an important event in miR-29–promoted apoptosis. This study, together with the work of other groups, demonstrates that miR-29 may target multiple proteins that function spatiotemporally or in cooperation in different cellular processes including cell growth, death, and differentiation.20, 22, 24-26 It is intriguing when an miRNA can suppress multiple genes that favor the process of tumorigenesis, because introduction of a single miRNA may modulate complex downstream signals and inhibit tumor growth, and thus, is more likely effective as anticancer drugs.

Both Bcl-2 and Mcl-1 exert an antiapoptotic function through the mitochondrial signaling pathway. It has been shown that Mcl-1 and/or Bcl-2 are up-regulated in different types of cancers30, 31, 36 and their overexpression is correlated with tumor progression and poor prognosis,37, 38 which is consistent with our and other's findings that down-regulation of miR-29 is associated with poorer survival in HCC and in chronic lymphocytic leukemia.23 These data suggest that functional loss of the miR-29 family may result in the enhanced expression of Mcl-1 and Bcl-2 and, in turn, the resistance of cells to apoptosis, which consequently favors tumor progression. We are aware that other miR-29 target genes may also be involved in miR-29–promoted apoptosis. However, the observations that silencing of Bcl-2 and Mcl-1 can largely mimic the apoptosis-promoting effect of miR-29 overexpression and that Bcl-2 and Mcl-1 expression can dramatically reverse the effect of miR-29 implicates Bcl-2 and Mcl-1 as predominant mediators of miR-29–promoted apoptosis in HCC cells. Interestingly, exogenous introduction of Bcl-2 and Mcl-1 could significantly reduce the apoptotic rates in miR-29 transfectants but not that in NC transfectants treated with serum starvation and hypoxia (Fig. 6D). This is likely due to high basal levels of Bcl-2 and Mcl-1 in HepG2 cells. We speculate that miR-29 transfection, which results in a decrease in endogenous Bcl-2 and Mcl-1 proteins, may provide a more proper condition for observing the antiapoptotic effect of exogenous Bcl-2 and Mcl-1. In addition, cells that were cotransfected with control vector and RNA duplex (Fig. 6D) displayed higher apoptotic rates than those transfected with RNA duplex only (Fig. 3A) upon the same treatment, which may be explained by the enhanced cytotoxicity that results from double-transfection as well as the use of Lipofectamine 2000 instead of Lipofectamine-RNAiMAX.

We have previously shown that the RNA transfection efficiency by Lipofectamine-RNAiMAX is around 70% to 80% in different HCC cells, including HepG2.16, 18 In this study, we examined the stability of exogenous miR-29b and found that overexpression of miR-29b may be maintained for at least 4 days but maximally 7 days in HepG2 cells (Supporting Fig. 6). Clearly, 4 weeks after inoculation in nude mice, no difference should be expected between NC and miR-29 transfectants in the cellular concentration of exogenous miR-29b, although we were unable to examine the levels of miR-29 and its target genes in xenografts because of limited tumor materials derived from miR-29b transfectants (Fig. 4). We presume that the apoptosis-promoting effect of miR-29b mainly occurs in the first week after inoculation, which in turn results in the observed suppressed tumorigenicity of miR-29b transfectants.

In summary, we investigated the potential role of the miR-29 family in tumorigenesis and its underlying mechanisms. Our data suggest that down-regulation of miR-29 may play important role in the development of cancer, such as HCC, and that miR-29 may be employed as prognosis marker and therapeutic target for HCC.

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  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_23380_sm_suppfig1.tif1142KSupplementary Fig. 1. Sequences of mature miR-29a/b/c and their putative binding sites. (A) Predicted miR-29b-binding sequences in the 3′-UTR of Bcl-2 and Mcl-1 mRNAs. Mutation was generated in the complementary site for the seed region of miR-29 family members, as indicated. (B) Sequence alignment of the miR-29 family members.
HEP_23380_sm_suppfig2.tif113KSupplementary Fig. 2. Analysis of miR-29 expression. (A) Optimal hybridization temperature for specific detection of miR-29b and miR-29a/c by Northern blot. Five pmol of synthetic RNA duplexes corresponding to miR-29a (lane 1), miR-29b (lane 2) and miR-29c (lane 3) were separated on a 15% denaturing polyacrylamide gel. Hybridizations were performed at 58°C. The same membrane was hybridized sequentially with probes for miR-29a, miR-29b and miR-29c, as indicated on the left. (B) Expression analysis of mature miR-29 in HEK 293T and four human HCC cell lines by Northern blot. The same membrane was hybridized sequentially with miR-29b and U6 probe at 42°C. Hybridization with miR-29b probe revealed a single band that migrated between xylene cyanol FF and bromophenol blue, the loading dyes which co-migrates with ∼30 bp and ∼10bp RNA fragments in 15% denaturing polyacrylamide gel, respectively. U6, internal control for RNA loading. Normal liver, RNA from normal liver tissue. (C) Real-time quantitative RT-PCR analysis of mature miR-29b expression in 17 paired HCC (T) and adjacent non-tumor liver tissues (N). miR-29b level was normalized to U6 expression in each sample. The median values of miR-29b in each group are indicated by a solid horizontal line. The difference between two groups was analyzed by paired Student t test.
HEP_23380_sm_suppfig3.tif1809KSupplementary Fig. 3. miR-29 sensitizes HCC cells to serum deprivation and hypoxia-induced apoptosis. (A) Analysis of cell viability by Alamar Blue. HepG2 cells were first transfected with NC or miR-29a/b/c duplex in a 24-well plate for 24 h, and then replated into 96-well plate at about 20% confluence. Twenty-four hours after splitting, cells were deprived of serum and cultured in 1% O2. Cell viability was evaluated by Alamar Blue assay at the indicated time points. (B–C) Analysis of apoptosis in HepG2 cells by TUNEL staining. Images were captured at 100× magnification. Cells without transfection or transfected with NC or miR-29a/b/c were deprived of serum and cultured in 1% O2 for 24 h, and then subjected to TUNEL staining followed by counterstaining with DAPI. Representative photographs (B) and the apoptotic rates (C) are shown. (D) Effect of anti-miR-29 on exogenous miR-29-promoted apoptosis. HepG2 cells were transfected with indicated RNA dulplex for 24 h, followed by culture in serum-deprived medium and 1% O2 for 72 h before DAPI staining. ***, P < 0.001, comparison between two groups as indicated.
HEP_23380_sm_suppfig4.tif428KSupplementary Fig. 4. miR-29 sensitizes HCC cells to doxorubicin-induced apoptosis. (A) miR-29 sensitizes HepG2 cells to doxorubicin-induced apoptosis in a dose-dependent manner. Cells without transfection or transfected with NC or miR-29a/b/c were treated with indicated concentration of doxorubicin for 48 h before DAPI staining. (B) miR-29 sensitizes QGY-7703 cells to doxorubicin-induced apoptosis. Cells without transfection or transfected with NC or miR-29a/b/c were treated with doxorubicin (0.2 μg /ml) for 72 h before DAPI staining. ***, P < 0.001, compared with NC-transfectants.
HEP_23380_sm_suppfig5.tif88KSupplementary Fig. 5. Enhanced expression of miR-29 decreases the level of endogenous Bcl-2 and Mcl-1 under conditions of serum deprivation and hypoxia. HepG2 cells without transfection or transfected with indicated RNA duplex were deprived of serum and cultured in 1% O2 for 72 h before Western blot analysis. The value under each lane indicates the relative expression level of the putative target gene, which is represented by the intensity ratio between Bcl-2 or Mcl-1and β-actin bands in each lane. β-actin, internal control.
HEP_23380_sm_suppfig6.tif42KSupplementary Fig. 6. Analysis of miR-29b level in miR-29b-transfected HepG2 cells. Cells were first transfected with NC or miR-29b duplex (all pyrimidine nucleotides in the NC or miR-29b duplex were substituted by their 2'-O-methyl analogs) and then maintained in 24-well plate at 37°C with 5% CO2. At every indicated time point, cells were trypsinized and split 1/3 into 24-well plate for sustained culture, and the rest 2/3 cells were applied to RNA isolation and expression analysis of mature miR-29b. The miR-29b level was evaluated by real-time quantitative RT-PCR and normalized by the expression of U6.
HEP_23380_sm_supptabs.doc99KSupplementary Tables
HEP_23380_sm_supptext.doc47KSupplementary Materials and Methods

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