Overexpression of the Transcription Factor MEF2D in Hepatocellular Carcinoma Sustains Malignant Character by Suppressing G 2 – M Transition Genes

, Abstract 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, ﬁ nding 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 G 2 – M arrest in a manner associated with direct downregulation of the cell-cycle regulatory genes RPRM , GADD45A , GADD45B , and CDKN1A . Furthermore, we identi ﬁ ed 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); 1 – 11. (cid:1) 2014 AACR.


Introduction
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)(9)(10)(11).In addition to their effect on development, MEF2 family members behave as survival factors in different types of cells (12)(13)(14)(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 a-smooth muscle actin (a-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 G 2 -M transition-retarding genes.

Cell culture
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 a-fetoprotein (AFP), albumin, and a-SMA by immunofluorescence staining.All cells expressed AFP and albumin, but did not express a-SMA.The primary cultures were named as T1216, T0127, T0408, T0420, and T0421.

Tissue arrays
The samples (n ¼ 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).

Animal experiments
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 Â 10 5 cells into the both flanks of 6-week-old BALB/c nude mice (the Lv-scrambled-infected group, n ¼ 9; the Lv-shMef2d-1-infected group, n ¼ 9; and the Lv-shMef2d-2-infected group, n ¼ 9).Twentyone 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.

Statistical analysis
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 AE SD, and compared at a given time point by a two-tailed independent sample t test.Data were considered to be statistically significant ( Ã , P < 0.05; ÃÃ , 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 sam-ples 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  A-C, mRNA levels of MEF2A, 2B, 2C, and 2D were quantified in hepatocellular carcinoma samples and their corresponding noncancerous liver tissues as well as normal liver tissues by qRT-PCR (n ¼ 8).MEF2 expression was normalized by glyceraldehyde-3phosphate dehydrogenase (GAPDH) expression and each noncancerous liver tissue was used as a control.Data, mean AE SD.T, tumors; N, noncancerous liver tissues.D, protein level of MEF2D was determined by immunoblotting.GAPDH expression was used as endogenous reference.E, immunohistochemical analysis on MEF2D expression was performed in hepatocellular carcinoma samples and their corresponding noncancerous liver tissues, as well as in normal liver tissues.The representative microphotographs of MEF2D expression are displayed.
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 ¼ 0.0158) and increased incidences of death (P ¼ 0.0352; Supplementary Table S1 and  S2).Importantly, MEF2D-positive patients had shorter total survival than MEF2D-negative patients (P ¼ 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 MEF2Dnegative 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 G 2 -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-GFPinfected 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 G 2 -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 G 2 -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-b signaling pathway, Janus-activated kinase-STAT signaling pathway, and cell cycle (Supplementary Fig. S3).Further analysis indicated a shift toward G 2 -M arrest in the cells with reduced MEF2D expression.The genes that inhibit G 2 -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 G 2 -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 G 2 -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 G 2 -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-122specific binding site was located within the 3 0 untranslated region (UTR) of MEF2D mRNA (Fig. 7D).We then constructed a vector to investigate whether miR-122 could directly target MEF2D 3 0 UTR.We found that miR-122 markedly inhibited luciferase activity when MEF2D 3 0 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 0 UTR (MIR-MEF2-D3UTRm) 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.

Discussion
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 Figure 6.MEF2D regulates the G2-M cell-cycle transition in hepatocellular carcinoma cells.A, Huh7 cells were infected with Lv-scrambled or Lv-shMEF2D-1 lentiviruses, or transfected with MEF2D-specific siRNAs and control siRNAs.Forty-eight hours later global gene expression profiles of these cells were determined using expression microarrays.The changes in the expression of G2-M transition involved genes of both lentivirus-infected and siRNA-transfected cells and are shown as means AE SD.B, bioinformatic analysis for putative MEF2D-binding sites in the regulatory regions of RPRM, CDKN1A, GADD45A, and GADD45B genes are shown.C and D, expression of RPRM, CDKN1A, GADD45A, and GADD45B at the mRNA level in both Lv-shMEF2D-1-infected Huh7 cells (C) and Lv-MEF2D-infected MHCC97H cells (D) were determined by RT-PCR.mRNA levels were normalized by GAPDH expression and control vector-infected cells were used as a controls.Data, means AE SD from three independent experiments ( Ã , P < 0.05; ÃÃ , P < 0.01).E, ChIP assays were performed to detect the binding of MEF2D to the potential MREs identified in the promoter regions mentioned above.The IgG-incubated and blank groups were considered as negative controls, whereas the input fraction was the positive control.F, luciferase expression driven by the regulatory regions of RPRM, CDKN1A, GADD45A, and GADD45B was tested in Lv-MEF2D-infected MHCC97-H cells and Lv-shMEF2D-1-infected Huh7 cells.The fold changes of relative luciferase activity in Lv-MEF2D-and Lv-shMEF2D-1-infected cells were normalized to Lv-GFP-and Lv-scrambled-infected cells, respectively.Data, means AE SD from three independent experiments ( Ã , P < 0.05; ÃÃ , P < 0.01).
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 0 UTR region.In addition to miR-122 repression, overexpression of MEF2D in hepatocellular carcinoma might also be due to gains of chromosome Figure 7. miR-122 regulates MEF2D expression in hepatocellular carcinoma (HCC) cells.A, miR-122 level was quantified in HCC samples and noncancerous liver tissues by qRT-PCR.miR-122 expression was normalized by U6 and each noncancerous liver tissue was used as a control.MEF2D mRNA levels of individual tumors relative to their corresponding nontumor tissue samples are also shown.A statistically significant negative correlation was found between miR-122 and MEF2D expression levels in HCC samples.B, MEF2D mRNA level was determined in noninfected HCC cells and after infection with control adenovirus Ad-NC or Ad-miR122 adenovirus.GAPDH expression was used as control.Data, means AE SD from three independent experiments.C, MEF2D protein levels were assessed in the same cells.b-Actin was used as control.D, a putative binding site targeted by miR-122 was predicted to be located in 3 0 UTR of MEF2D mRNA.This site is framed in the solid line box, the miR-122 seed sequence is framed by a dotted line box.E, HeLa cells were cotransfected with miR-122 mimics or control RNA (negative control, NC) with luciferase reporter plasmids containing either wild-type (pMIR-MEF2D3UTR) or mutant 3 0 UTR (pMIR-MEF2D3UTRm) of MEF2D gene or control plasmid without 3 0 UTR of MEF2D gene (pMIR-REPORT).Luciferase expression was measured.The fold changes of relative luciferase activity in miR-122 mimics with indicated plasmids transfected cells were normalized to negative control (NC) with corresponding indicated plasmids transtected cells, respectively.Data, means AE SD from three independent experiments ( Ã , P < 0.05).F, schematic summary of the findings presented in this study on the role of MEF2D in the regulation of HCC cell growth.Downregulation of miR-122 in HCC cells leads to MEF2D overexpression.In turn, MEF2D overexpression suppresses that of G2-M transition inhibition genes such as RPRM, CDKN1A, GADD45A, and GADD45B, resulting in the dysregulation of HCC cell cycle and promotion of HCC growth.
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 MEF2Dpositive hepatocellular carcinoma cells suppressed cancer cell growth, whereas overexpression of MEF2D in MEF2Dnegative 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 G 2 -M checkpoints.Analysis of global expression microarray revealed that suppression of MEF2D resulted in downregulation of G 2 -M transition-promoting genes and upregulation of G 2 -M transition-inhibiting genes.Bioinformatics analysis identified multiply putative MREs in regulatory regions of some G 2 -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 G 2 -M arrest in HeLa cells (36,37).CDKN1A (p21, Cip1) upregulation is also required for G 2 -M arrest induced by a variety of drugs (38).GADD45A is known to allow HepG2 cells to undergo G 2 -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 G 2 -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 G 2 -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 0 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 G 2 -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.

Figure 1 .
Figure 1.Aberrant expression profile of MEF2D in hepatocellular carcinoma tissue samples.A-C, mRNA levels of MEF2A, 2B, 2C, and 2D were quantified in hepatocellular carcinoma samples and their corresponding noncancerous liver tissues as well as normal liver tissues by qRT-PCR (n ¼ 8).MEF2 expression was normalized by glyceraldehyde-3phosphate dehydrogenase (GAPDH) expression and each noncancerous liver tissue was used as a control.Data, mean AE SD.T, tumors; N, noncancerous liver tissues.D, protein level of MEF2D was determined by immunoblotting.GAPDH expression was used as endogenous reference.E, immunohistochemical analysis on MEF2D expression was performed in hepatocellular carcinoma samples and their corresponding noncancerous liver tissues, as well as in normal liver tissues.The representative microphotographs of MEF2D expression are displayed.

Figure
Figure 2. MEF2D overexpression in cancer cells correlates with poor prognosis of patients with hepatocellular carcinoma.A, the representative microphotographs showed strong, moderate, and no MEF2D expression in hepatocellular carcinoma tissues and their corresponding noncancerous liver tissues by immunohistochemistry in tissue array.B and C, percentages of strong, moderate, and no MEF2D expression in hepatocellular carcinoma samples (B) and corresponding noncancerous liver tissues (C) are shown in the pie charts.D, Kaplan-Meier analysis of overall survival of patients with hepatocellular carcinoma according to the expression level of MEF2D protein in hepatocellular carcinoma tissues [MEF2D À (n ¼ 19) and

Figure 3 .
Figure3.Downregulation of MEF2D expression results in the inhibition of hepatocellular carcinoma cell growth.A, growth rates of patient-derived primary hepatocellular carcinoma cultures of tumor cells with various levels of MEF2D expression as determined by cell proliferation assay.Data, means AE SD from three independent experiments.A highly significant inverse correlation is shown between MEF2D mRNA levels and cell doubling time for these primary hepatocellular carcinoma cells.B, hepatocellular carcinoma cell lines were infected with lentiviral vectors (Lv-scrambled and Lv-shMEF2D-1) at MOI of 1 or 10.MEF2D protein was determined by immunoblotting at 72 hours of infection.GAPDH served as endogenous reference protein.C, growth rates of hepatocellular carcinoma cell lines and primary hepatocellular carcinoma cells in which MEF2D expression was suppressed were determined.Data, means AE SD from three independent experiments.D, colony formation ability was performed on Huh7 cells at 72 hours after infection with Lv-scrambled and Lv-shMEF2D-1 at MOI of 10.The number of Huh7 cells colonies is shown as means AE SD from three independent experiments ( ÃÃ , P < 0.01).E, cell-cycle analysis was performed on Huh7 and SMMC-7721 cells at 72 hours after infection with Lv-scrambled and Lv-shMEF2D-1 at MOI of 10.The average values of population percentages at G0-G1, S, and G2-M phases are shown as mean AE SD from three independent experiments ( ÃÃ , P < 0.01).

Figure 4 .
Figure 4. Overexpression of MEF2Dresults in increased growth of hepatocellular carcinoma cells.A, MHCC97-H and HepG2 cells were infected with Lv-GFP or Lv-MEF2D lentiviral vectors at MOI of 10.MEF2D protein level was determined by immunoblotting at 72 hours of infection.GAPDH served as endogenous reference.B, MHCC97-H and HepG2 cells were infected with Lv-GFP and Lv-MEF2D at MOI of 10.Seventy-two hours later MEF2D protein was determined by immunofluorescent staining (original magnification, Â200).C, growth rates of MEF2Dexpressing MHCC97-H and HepG2 cells and control GFPexpressing cells were determined.Data, means AE SD from three independent experiments.D, colony formation ability of control and MEF2D-expressing MHCC97-H and HepG2 cells.The numbers of colonies are shown as means AE SD from three independent experiments ( Ã , P < 0.05).E, cellcycle analysis was performed on MHCC97-H and HepG2 cells 72 hours after infection with Lv-GFP and Lv-MEF2D.The average values of population percentages at G0-G1, S, and G2-M phases are shown as mean AE SD from three independent experiments ( Ã , P < 0.05).

Figure 5 .
Figure 5. Downregulation of MEF2D expression abolishes the in vivo tumorigenic capacity of hepatocellular carcinoma cells.A, Huh7 cells were subcutaneously inoculated in BALB/c nude mice after infection with Lv-scrambled, Lv-shMEF2D-1, or Lv-shMEF2D-2 at MOI of 10 (n ¼ 9 for each group).Twenty-one days later tumors were removed.B, the weight of established tumors was measured and is shown in a scatter plot.Horizontal lines, average values.C, immunohistochemical analysis of MEF2D expression was performed on Huh7 tumor xenografts.The representative images are shown (original magnification, Â200).