The underlying molecular pathogenesis in hepatocellular carcinoma remains poorly understood. The transcription factor MEF2D promotes survival in various cell types and it seems to function as an oncogene in leukemia. However, its potential contributions to solid cancers have not been explored. In this study, we investigated MEF2D expression and function in hepatocellular carcinoma, finding that MEF2D elevation in hepatocellular carcinoma clinical specimens was associated with poor prognosis. MEF2D-positive primary hepatocellular carcinoma cells displayed a faster proliferation rate compared with MEF2D-negative cells, and silencing or promoting MEF2D expression in these settings limited or accelerated cell proliferation, respectively. Notably, MEF2D-silencing abolished hepatocellular carcinoma tumorigenicity in mouse xenograft models. Mechanistic investigations revealed that MEF2D-silencing triggered G2–M arrest in a manner associated with direct downregulation of the cell-cycle regulatory genes RPRM, GADD45A, GADD45B, and CDKN1A. Furthermore, we identified MEF2D as an authentic target of miR-122, the reduced expression of which in hepatocellular carcinoma may be responsible for MEF2D upregulation. Together, our results identify MEF2D as a candidate oncogene in hepatocellular carcinoma and a potential target for hepatocellular carcinoma therapy. Cancer Res; 74(5); 1452–62. ©2014 AACR.
Hepatocellular carcinoma is a highly lethal cancer, with increasing worldwide incidence (1). Lack of effective treatment of hepatocellular carcinoma is due to relatively poor understanding on molecular mechanisms underlying pathogenesis of hepatocellular carcinoma (2). Numerous studies have been focused on identification of hepatocellular carcinoma associated genes (3, 4). However, current knowledge about molecular pathogenesis of hepatocellular carcinoma is far from complete elucidation. Therefore, identification of new genes participating in hepatocarcinogenesis is critical for the development of novel targeted therapeutic strategies in hepatocellular carcinoma (5).
The MEF2 family of transcription factors comprises four members in mammals, MEF2A, 2B, 2C, and 2D. They were originally identified as major transcriptional activators for muscle differentiation (6, 7). Subsequently, MEF2 factors were found to participate in diverse gene regulatory programs, including muscle and neural differentiation, cardiac morphogenesis, blood vessel formation, and growth factor responsiveness (8–11). In addition to their effect on development, MEF2 family members behave as survival factors in different types of cells (12–15). Cyclic AMP–dependent protein kinase A signaling promotes apoptosis by regulating negatively MEF2D function in primary hippocampal neurons (16). And a small molecule, bis(3)-cognitin, acts as a potent neuroprotective agent in Parkinson disease neurons against toxic stress by upregulation of MEF2D (17).
However, expression and function of MEF2 is poorly understood in human tumors. Studies on leukemia showed that MEF2D/DAZAP1 and DAZAP1/MEF2D fusion proteins produced by t(1;19)(q23;p13.3) chromosome translocation maintained the malignant phenotype of acute lymphoblastic leukemia cells (ALL; refs. 18, 19). Integrated transcript and genome analysis demonstrated that ectopically activated MEF2C served as an oncogene in human ALL (20, 21). Consistent with this, large-scale retrovirus-mediated insertion mutagenesis identified the mouse MEF2D gene as a potential oncogene in the development of both myeloid and lymphoid tumors (22, 23). Therefore, MEF2 transcription factors may play an important role in progression of leukemia. It has been reported that MEF2D was overexpressed in nasopharyngeal carcinoma (24). However, the biologic function of MEF2 family members in solid cancers is not known.
In liver, expression of MEF2A, MEF2C, and MEF2D increased in the activated hepatic stellate cell (HSC) and enhancing MEF2 significantly increased the expression of α-smooth muscle actin (α-SMA), activated collagen I promoter activity, and stimulated HSC proliferation, suggesting that MEF2 plays a critical role in regulating multiple key aspects of HSC activation and fibrotic response (25, 26). It has been shown that retinoic acid could inhibit the expression of MEF2D in murine hepatocytes (27). Retinoic acid has a key function in the control of cell proliferation, differentiation, and apoptosis and retinoic acid may play a role in the prevention and treatment of tumors (28). The close association between liver fibrosis and hepatic cancer and regulation of MEF2D by retinoic acid suggest MEF2 family members might participate in hepatocarcinogenesis.
In this study, we investigated MEF2 expression and functions in hepatocellular carcinoma. Our data showed that MEF2D was overexpressed in hepatocellular carcinoma and high level of MEF2D expression was correlated with a poor prognosis in patients with hepatocellular carcinoma. MEF2D participated in tumorigenecity of hepatocellular carcinoma by transcriptional regulation of G2–M transition-retarding genes.
Materials and Methods
Human hepatocellular carcinoma cell lines Huh7, PLC/PRF/5, SMMC-7721, BEL-7404, MHCC97-H, MHCC97-L, Hep3B, and HepG2, human uterine cervix cancer line HeLa, were purchased from the Shanghai Cell Collection. HEK293 and HEK293FT cell lines were obtained from Microbix Biosystems. The cells were authenticated by short tandem repeat profiling and cultured according to the manufacturer's specifications for less than 6 months. The patient-derived primary hepatocellular carcinoma cultures of hepatocellular carcinoma cells were obtained from fresh tumor specimens from patients with hepatocellular carcinoma described previously (29). In brief, the single-cell suspension was obtained from tumors by mechanical manipulation. The primary culture was established initially in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 15% FBS and maintained in DMEM supplemented with 10% FBS. The cells were verified for expression of α-fetoprotein (AFP), albumin, and α-SMA by immunofluorescence staining. All cells expressed AFP and albumin, but did not express α-SMA. The primary cultures were named as T1216, T0127, T0408, T0420, and T0421.
The samples (n = 145) were randomly collected from patients with hepatocellular carcinoma who underwent curative resection in the Institute of Hepatobiliary Surgery in Southwest Hospital (Chongqing, China). No antitumor treatment was performed before hepatectomy. Tissue array blocks containing hepatocellular carcinoma tissues and their corresponding nonhepatocellular carcinoma tissues were generated with a tissue microarrayer (Leica).
The procedure of human sample collection and use of human samples for primary culture and gene expression were approved by the Ethical Committee of the Third Military Medical University (Chongqing, China).
Immunohistochemical staining was performed on tissue array slides and formalin-fixed, paraffin-embedded tissue sections using the streptavidin–biotin–peroxidase complex method. The antigen retrieval procedure was performed by heating the samples in Dako antigen retrieval solution containing 10 mmol/L EDTA (pH 8.0) with a pressure cooker. Rabbit anti-human MEF2D antibody (HPA004807; Sigma-Aldrich; 1:350) was used to detect MEF2D expression. Slides were counterstained with hematoxylin (Sigma). Expression of MEF2D was evaluated using graded semiquantitatively scoring system. The intensity of staining was classified into none (0), weak (1), strong (2), or very strong (3), and the staining patterns were classified into negative (0:≤10%), sporadic (1:11% to 25%), focal (2:26% to 50%), or diffuse(3:≥51%). An overall expression score was calculated by multiplying the intensity and positivity scores: 0 score (negative), 1 to 4 score (moderate), and 5 to 9 score (strong). In the analysis of clinical significance and prognosis, malignant samples with strongly and moderately positive MEF2D staining were merged as MEF2D+, whereas MEF2D− indicated no MEF2D expression.
All procedures for animal experiments were approved by the Committee on the Use and Care on Animals (The Third Military Medical University, Chongqing, China) and performed in accordance with the institution guidelines. After infection with indicated lentiviral vectors, Huh7 tumor xenografts were established by subcutaneously inoculating 5 × 105 cells into the both flanks of 6-week-old BALB/c nude mice (the Lv-scrambled–infected group, n = 9; the Lv-shMef2d-1–infected group, n = 9; and the Lv-shMef2d-2–infected group, n = 9). Twenty-one days later, animals were sacrificed to weight the established tumors. All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy.
The statistical significance of correlation between MEF2D expression and survival was estimated by the log-rank test. To study the relationship between MEF2D expression and other variables, we used either the independent sample t test or the nonparametric Mann–Whitney test for continuous variables. We used the Spearman rank test to analyze correlations between variables. The values of quantitative real-time PCR (qRT-PCR), cell growth rate, and colony formation were expressed as means ± SD, and compared at a given time point by a two-tailed independent sample t test. Data were considered to be statistically significant (*, P < 0.05; **, P < 0.01).
Other methodologies are detailed in Supplementary Data.
Aberrant expression of MEF2D in hepatocellular carcinoma samples
To investigate expression of MEF2 in hepatocellular carcinoma specimens, we measured mRNA levels of MEF2A, 2B, 2C, and 2D in eight fresh hepatocellular carcinoma samples and their corresponding nonhepatocellular carcinoma tissues. Our data showed that no significant differences in mRNA level of MEF2A and MEF2C were found between tumors and their corresponding noncancerous tissues (Fig. 1A and B) and there was no detectable MEF2B expression in these tissues (data not shown). However, mRNA abundance of MEF2D was significantly higher in hepatocellular carcinoma tissue than nonhepatocellular carcinoma tissues (Fig. 1C). We confirmed the elevated MEF2D expression in hepatocellular carcinoma samples at protein level by immunoblotting and MEF2D protein was not detectable in normal livers (Fig. 1D). Immunohistochemical staining showed an increased expression of MEF2D in cancerous tissue. MEF2D protein was mainly localized in the nuclei of cancer cells. No obvious staining was observed in noncancerous liver tissues nor in normal livers (Fig. 1E). These findings reveal a marked upregulation of MEF2D in hepatocellular carcinoma.
Overexpression of MEF2D in cancer cells correlates with poor prognosis of patients with hepatocellular carcinoma
Subsequently, we investigated whether aberrant expression of MEF2D predicts prognosis in patients with hepatocellular carcinoma. We generated a tissue array containing 145 hepatocellular carcinoma samples and their corresponding noncancerous liver tissues to determine MEF2D expression level by immunohistochemical staining. The representative images of hepatocellular carcinoma samples with strong, moderate, or no MEF2D expression and of nonhepatocellular carcinoma samples with moderate or no MEF2D expression were shown in Fig. 2A. Of note, 59% of hepatocellular carcinoma samples had strong MEF2D expression, whereas percentages of moderate and no MEF2D expression were 28% and 13%, respectively (Fig. 2B). In contrast, MEF2D expression was relatively low in noncancerous liver tissues. Percentages of moderate and no expression of MEF2D protein were 71% and 29%, respectively (Fig. 2C). Clinicopathologic analysis showed that overexpression of MEF2D in hepatocellular carcinoma was correlated with earlier hepatocellular carcinoma recurrence (P = 0.0158) and increased incidences of death (P = 0.0352; Supplementary Table S1 and S2). Importantly, MEF2D-positive patients had shorter total survival than MEF2D-negative patients (P = 0.033; Fig. 2D).
Expression of MEF2D in hepatocellular carcinoma cell lines and patient-derived primary hepatocellular carcinoma cultures of tumor cells
MEF2D expression pattern was evaluated in hepatocellular carcinoma cell lines and in patient-derived primary hepatocellular carcinoma cultures of tumor cells at mRNA and protein levels. HeLa cell line was used as a positive control because it has been validated to express MEF2D (30). High expression level of MEF2D mRNA was detected in Huh7, PLC/PRF/5, BEL-7404, SMMC-7721, and Hep3B cell lines as well as T1216, T0420, and T0421 patient-derived primary hepatocellular carcinoma cultures of tumor cells, but not in MHCC97-L, MHCC97-H, and HepG2 as well as T0127 and T0408 patient-derived primary hepatocellular carcinoma cultures of tumor cells (Supplementary Fig. S1A). Immunoblotting analysis showed that expression of MEF2D protein paralleled mRNA level in the tested cells (Supplementary Fig. S1D). The location of MEF2D protein was also studied by immunofluorescence in MEF2D-positive hepatocellular carcinoma cell lines. Similar to HeLa cells, nuclear accumulation of MEF2D was observed extensively in the majority of Huh7, PLC/PRF/5, and SMMC-7721 cells. No MEF2D staining was detected in the cytoplasm or on the membrane of hepatocellular carcinoma cells (Supplementary Fig. S1E).
MEF2D promotes the growth of hepatocellular carcinoma cells
Our data showed that the MEF2D-positive cells (T1216, T0420, and T0421) had increased proliferation rates than MEF2D-negative cells (T0127 and T0408; Fig. 3A). Correlation analysis showed an inverse correlation between MEF2D mRNA abundance and doubling time of proliferation in these primary hepatocellular carcinoma cells (Fig. 3A). These results suggested a proliferation-promoting role of MEF2D in hepatocellular carcinoma cells. To confirm this notion, we constructed a lentiviral vector carrying short hairpin RNA that specifically knocked down MEF2D expression (Lv-shMEF2D-1). Our data showed that Lv-shMEF2D-1 inhibited the expression of MEF2D, but not MEF2A and MEF2C (Fig. 3B and Supplementary Fig. S2A–S2C). We found that silencing MEF2D inhibited the growth of Huh7 and PLC/PRF/5 cell lines as well as T0420 primary hepatocellular carcinoma cells (Fig. 3C). Moreover, the growth-suppressing effect seemed to depend on the extent of MEF2D reduction, because infection of cells with Lv-shMEF2D-1 at multiplicity of infection (MOI) of 10 inhibited MEF2D expression and cell growth more efficiently, as compared with infection of cells with Lv-shMEF2D-1 at MOI of 1 (Fig. 3B and C). Similar data were obtained when a second MEF2D-silencing lentiviral vector (Lv-shMEF2D-2) was used to reduce MEF2D expression in hepatocellular carcinoma cells (Supplementary Fig. S2D). Thus, we used an MOI of 10 at the following experiments. In addition, we found that the efficiency of colony formation was also decreased when Huh7 cells were infected with Lv-shMEF2D-1 (Fig. 3D). Cell-cycle analysis revealed that silencing MEF2D expression in Huh7 and SMMC-7721 cells caused moderate G2–M arrest (Fig. 3E and Supplementary Fig. S5). However, MEF2D knockdown did not induce cell apoptosis (data not shown).
On the other hand, we infected MEF2D-negative MHCC97-H and HepG2 cells with lentiviral vector expressing MEF2D (Lv-MEF2D) to evaluate its growth-promoting effect on hepatocellular carcinoma cells. After infection of MHCC97-H and HepG2 cells with Lv-MEF2D, MHCC97-H, and HepG2 cells expressed high level of exogenous MEF2D (Fig. 4A and B). Lv-MEF2D–infected MHCC97-H and HepG2 cells exhibited higher proliferation rates, as compared with control vector Lv-GFP–infected cells (Fig. 4C). Consistently, Lv-MEF2D–infected MHCC97-H and HepG2 cells also displayed the increased colony forming capacity in comparison with Lv-GFP–infected counterparts (Fig. 4D). Overexpression of MEF2D in MHCC97-H and HepG2 cells resulted in the accelerated G2–M transition (Fig. 4E and Supplementary Fig. S5).
Downregulation of MEF2D abolished tumorigenecity of hepatocellular carcinoma cells
The role of MEF2D in tumor formation of hepatocellular carcinoma cells was also investigated in the animal model. Lv-shMEF2D-1- and Lv-shMEF2D-2–infected Huh7 cells formed small tumors in only 22% and 11% of nude mice, respectively. In contrast, Lv-scrambled–infected cells formed tumors in 89% of nude mice (Fig. 5A). The average weight of tumors was significantly lower in Lv-shMEF2D–infected groups than that in the Lv-scrambled–infected group (Fig. 5B). Immunohistochemical staining analysis revealed extensive expression of MEF2D in tumors from the Lv-scrambled–infected group, whereas MEF2D expression was not detected in the formed tumors from Lv-shMEF2D-1- and Lv-shMEF2D-2–infected groups (Fig. 5C). These data show that MEF2D targeting blocks tumor formation in vivo.
MEF2D regulates the G2–M transition of cell cycle in hepatocellular carcinoma cells
To elucidate the mechanisms by which MEF2D promotes proliferation of hepatocellular carcinoma cells, we examined global gene expression profiles in Huh7 cells after infection with Lv-shMEF2D-1 and Lv-scrambled as well as after transfection with siRNA against MEF2D and control siRNA by cDNA microarray. By an analysis of the combined data from Lv-shMEF2D-1 infection and siRNA MEF2D transfection, we found that there were 1,397 genes with 2-fold or higher change in their expression when MEF2D was knocked down. The Shanghai Biotechnology Corporation Analysis System analysis showed that these genes enriched in the categories of cell processes, including nicotinate and nicotinamide metabolism, mitogen-activated protein kinase (MAPK) signaling pathway, TGF-β signaling pathway, Janus-activated kinase–STAT signaling pathway, and cell cycle (Supplementary Fig. S3). Further analysis indicated a shift toward G2–M arrest in the cells with reduced MEF2D expression. The genes that inhibit G2–M transition were found to be expressed at higher levels in the MEF2D-downregulated group, as compared with the control group (Fig. 6A). Meanwhile, mRNA abundance of G2–M transition-promoting genes, except CDC2 and CDC25C, was reduced when MEF2D expression was depressed in Huh7 cells (Fig. 6A).
MEF2D suppresses the transcription of RPRM, CDKN1A, GADD45A, and GADD45B in hepatocellular carcinoma cells
To know whether MEF2D directly regulates the transcription of G2–M transition-related genes, we analyzed putative MEF2 recognition elements (MRE) in the upstream region of transcription starting points of these genes by bioinformatics approach. We found multiple putative MREs in regulatory regions of RPRM, CDKN1A, GADD45A, and GADD45B, providing grounds for the ability of MEF2D to modulate the transcription of these genes (Fig. 6B and Supplementary Table S3). Furthermore, our data confirmed that silencing MEF2D expression in Huh7 cells lead to increased expression of RPRM, CDKN1A, GADD45A, and GADD45B, whereas overexpression of MEF2D resulted in decreased expression of RPRM, CDKN1A, GADD45A, and GADD45B in MHCC97-H cells by RT-PCR (Fig. 6C and D).
To further confirm the binding of MEF2D to the putative MREs located in the upstream regions of RPRM, CDKN1A, GADD45A, and GADD45B promoters, we performed chromatin immunoprecipitation (ChIP) assay on Huh7 cells. The results showed that anti-MEF2D antibody coprecipitated all the DNA fragments containing the predicted MREs in the regulatory regions of the four genes (Fig. 6E), indicating that MEF2D directly binds the MREs in Huh7 cells. Subsequently, we generated a series of constructs in which luciferase expression was driven by regulatory regions of RPRM, CDKN1A, GADD45A, and GADD45B (Supplementary Fig. S4). We found that MEF2D overexpression significantly suppressed luciferase activity driven by the four gene promoters. Consistently, knocking down endogenous MEF2D levels resulted in increased promoter-driven luciferase activity (Fig. 6F). These data indicated that MEF2D directly suppressed transcription of RPRM, CDKN1A, GADD45A, and GADD45B genes, which promoted G2–M transition in hepatocellular carcinoma cells.
MiR-122 regulates MEF2D expression in hepatocellular carcinoma cells
Bioinformatic analysis using multiple algorithms showed that MEF2D is a predictive target of miR-122. Thus, we experimentally verified whether miR-122 can modulate MEF2D expression in hepatocellular carcinoma cells. In the same tumors from patients with hepatocellular carcinoma, which had increased expression level of MEF2D (Fig. 1C), miR-122 was found to be strongly downregulated (Fig. 7A). Expression levels of miR-122 and MEF2D were inversely correlated in these hepatocellular carcinoma samples (Fig. 7A). Also, there was an inverse correlation between MEF2D and miR-122 levels in hepatocellular carcinoma cell lines and patient-derived primary hepatocellular carcinoma cultures of tumor cells (Supplementary Fig. S1B and S1C). Next, we determined MEF2D expression in hepatocellular carcinoma cells by our previously constructed adenoviral vector expressing exogenous miR-122 (Ad-miR122; 31). Infection of hepatocellular carcinoma cells with Ad-miR122 resulted in the reduced MEF2D expression both at the mRNA and protein levels (Fig. 7B and C). With the help of a series of online databases, we predicted that miR-122–specific binding site was located within the 3′ untranslated region (UTR) of MEF2D mRNA (Fig. 7D). We then constructed a vector to investigate whether miR-122 could directly target MEF2D 3′UTR. We found that miR-122 markedly inhibited luciferase activity when MEF2D 3′UTR was inserted downstream of luciferase cDNA in our reporter vector (pMIR-MEF2D3UTR). In contrast, no significant suppressive effect on luciferase activity was observed in cells transfected with a control vector with mutant MEF2D 3′UTR (MIR-MEF2D3UTRm) when miR-122 expression was elevated (Fig. 7E). These data indicate that downregulation of miR-122 could be responsible for elevated expression of MEF2D in hepatocellular carcinoma cells.
In this study, we found elevated MEF2D expression in hepatocellular carcinoma tissues, compared with noncancerous tissue and normal liver. Importantly, we observed that overexpression of MEF2D in hepatocellular carcinoma was correlated with more frequent tumor recurrence and shorter survival, indicating that MEF2D expression level is of prognostic relevance. Our data also demonstrate that the expression of miR-122 and MEF2D correlate inversely and that miR-122 controls MEF2D by targeting MEF2D 3′UTR region. In addition to miR-122 repression, overexpression of MEF2D in hepatocellular carcinoma might also be due to gains of chromosome 1q12-q23 frequently detected in hepatocellular carcinoma cells, in which MEF2D gene is located (32, 33).
Interestingly, we observed that there was a correlation between MEF2D expression level and cell growth rate, suggesting that MEF2D may contribute to cancer cell proliferation. In fact, knocking down MEF2D expression in MEF2D-positive hepatocellular carcinoma cells suppressed cancer cell growth, whereas overexpression of MEF2D in MEF2D-negative hepatocellular carcinoma cells accelerated their proliferation. Most importantly, downregulation of MEF2D in hepatocellular carcinoma cells could abolish their tumorigenecity, when they were implanted into animals. In coincidence with our findings, MEF2D also plays an important role in cell growth in normal tissues. Zhao and colleagues demonstrated that MEF2D is required for p38- and BMK1 MAPKs-induced proliferation of vascular smooth muscle cells (34). This function of MEF2D was also reported during stress-dependent cardiac growth (35).
The critical role of MEF2D in proliferation of hepatocellular carcinoma cells was further supported by the finding that knocking down MEF2D expression in hepatocellular carcinoma cells blocked cell cycle at the G2–M checkpoints. Analysis of global expression microarray revealed that suppression of MEF2D resulted in downregulation of G2–M transition-promoting genes and upregulation of G2–M transition-inhibiting genes. Bioinformatics analysis identified multiply putative MREs in regulatory regions of some G2–M checkpoint genes, including RPRM, CDKN1A, GADD45A, and GADD45B, suggesting that MEF2D protein maybe bound these regions to modulate their transcription. Our data further confirmed that expression of RPRM, CDKN1A, GADD45A, and GADD45B was upregulated when MEF2D expression was decreased. Consistently, expression of RPRM, CDKN1A, GADD45A, and GADD45B was downregulated when MEF2D expression level was increased. Previous studies have shown that hypermethylation of RPRM promoter with reduced expression is a common event in many human cancers and that RPRM overexpression mediated by an adenoviral vector induced a strong G2–M arrest in HeLa cells (36, 37). CDKN1A (p21, Cip1) upregulation is also required for G2–M arrest induced by a variety of drugs (38). GADD45A is known to allow HepG2 cells to undergo G2–M arrest (39), whereas decreased GADD45B expression in human hepatocellular carcinoma tissues is significantly associated with histologic grading of tumors (40). Collectively, our data indicated that MEF2D promoted cell growth by downregulating G2–M transition-inhibiting genes. The pathways involved in MEF2D-mediated pathogenesis of hepatocellular carcinoma are outlined in Fig. 7F.
Our findings also confirmed that MEF2D protein directly binds to the putative MREs located in the upstream region from the transcription sites of RPRM, CDKN1A, GADD45A and GADD45B, and MEF2D regulated activity of these promoters. It has been demonstrated that MEF2D regulated the transcription of target genes by recruiting the necessary corepressors, such as histone modifiers SIRT1, HDAC4, and HDAC9. However, our preliminary data showed that some histone modifiers (SIRT1, HDAC4, and HDAC9) that are reported to form complexes with MEF2 family members did not interact with MEF2D in Huh7 cells (data not shown), suggesting that other corepressors may partner with MEF2D to inhibit the expression of G2–M checkpoint genes in hepatocellular carcinoma cells. The detailed mechanisms need further studies.
Finally, we confirmed that miR-122 inhibited MEF2D expression by targeting its mRNA 3′UTR. Gramantieri and colleagues have reported that miR-122 was able to inhibit cell-cycle progression in hepatocellular carcinoma cells (41). Our previous study also showed that overexpression of miR-122 mediated by adenoviral vector induced a G2–M arrest in hepatocellular carcinoma cells and rendered hepatocellular carcinoma cells sensitive to chemotherapy (31, 42). Therefore, we hypothesized that MEF2D suppression is responsible, at least in part, for the inhibitory effect of miR-122 on cell-cycle progression.
In conclusion, this study provides evidence identifying MEF2D as a tumor-promoting gene for human hepatocellular carcinoma. Our data also suggest that MEF2D may be a useful prognostic marker and a potential therapeutic target in patients with primary liver cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: L. Ma, J. Liu, P. Bie, X.-W. Bian, M.A. Avila, C. Qian
Development of methodology: L. Ma, J. Liu, J. Shan, J. Shen, Z. Yang
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Ma, J. Liu, L. Liu, G. Duan, Q. Wang, X.-W. Bian
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Ma, J. Liu, L. Liu, G. Duan, X.-W. Bian
Writing, review, and/or revision of the manuscript: L. Ma, J. Liu, Y. Cui, J. Prieto, M.A. Avila, C. Qian
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Ma, J. Liu, L. Liu, G. Duan, Q. Wang, Y. Xu, F. Xia, X.-W. Bian
Study supervision: F. Xia, P. Bie, X.-W. Bian, C. Qian
This work was supported by funds from National Natural Sciences Foundation of China (nos. 81090423 and 81020108026 to C. Qian; 81001104 to L. Ma; 81000966 to J. Shen; and 81101630 to J. Shan), National Basic Research Program of China (973 program, no. 2010CB529406; C. Qian), grant “UTE project CIMA,” and grants RTICC-RD06 00200061 and PI10/00038 from Instituto de Salud Carlos III, Spain (M.A. Avila).
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received July 30, 2013.
- Revision received December 2, 2013.
- Accepted December 2, 2013.
- ©2014 American Association for Cancer Research.