The Effect Of DNA Methylation On The Regulation Of HNF4α Expression

【Background and Objective】Hepatocellular carcinoma（HCC） is one of the most common malignant tumorsworldwide. HCC occurs mostly in patients with known risk factors, such as livercirrhosis,hepatitis virus infection, alcohol consumption, aflatoxin exposure and so on.However, the molecular biological mechanism of hepatocarinogensis process is stillunclear. Recent studies have shown that DNA methylation plays an important role in thehepatocarinogenesis.HNF4α (hepatocyte nuclear factor-4α) is a transcription factor of steroid hormonereceptor superfamily and is enriched in liver. HNF4α is important in maintaining albuminsynthesis of hepatocytes, drug detoxification, energy metabolism, bile acid synthesis andfat metabolism. The nine isoforms of HNF4α have been cloned and characterized with thealternative promoter (P1and P2). Exon1A corresponds to the first exon originallydescribed for the HNF4α1promoter（P1promoter），which can generate six differentisoforms (HNF4α1-6). The P2promoter-driven isoforms HNF4α7-9are characterized byan alternative first exon (1D), resulting in a short amino-terminal sequence.Transforming growth factor-β1(TGF-β1) is a pluripotent cytokine and plays animportant biological role in the development of tissues and organs, cell proliferation,differentiation, survival, apoptosis and fibrosis. TGF-β1has a dual role in tumor growthand acts as a tumor suppressor in early-stage tumors but becomes an oncogenic factor inadvanced tumors, which causes tumor invasione and the formation of the EMT. Studieshave indicated that TGF-β1plays an important role in the differentiation of liver cells andliver at different stages of liver development.The current study aims to investigate the effect of methylation in the promoter ofHNF4α on the expression of HNF4α and its role in the regulation of HNF4α by TGF-β1.【Methods】1. The expression of HNF4αP1and HNF4αP2in the clinical HCC samples andthe hepatocellular carcinoma cell linesRealtime RT-PCR was performed to detect the expressions of HNF4αP1、HNF4αP2in the20pairs of clinical HCC in compared with their adjacent tissues and in differenthepatocellular carcinoma cell lines, such as Hep3B、HepG2、Huh-7and YY.2. The methylation status of the promoter region of P1and P2in differenthepatoma cell linesBSP method was used to examine the methylation status of the promoter region ofHNF4αP1. methylation specific PCR MSP was carried out to detect the methylation statusof the promoter region of HNF4αP2.3. The expression of HNF4αP1and HNF4αP2in hepatoma cell line FOCUStreated with5-AZA-CdRThe hepatoma cell line FOCUS were inoculated in6-well plates as2×105/plate anddifferent concentrations of5-AZA-CdR (0.1μM,1μM,2.5μM) were added into culturesin10%FBS DMEM. The medium was changed every24h in37℃5%CO2incubators andcells were collected72h later. BSP was performed method to examine the methylationlevel of the promoter region of P1in compared with blank control. PCR was used to detectthe expression of HNF4αP1and HNF4αP2in comparision with blank control.4. The regulation of HNF4α expression in hepatoma cell lines by TGF-β1The human liver tumor cell lines of Hep3B, HepG2, Huh-7, SMMC-7721,BEL-7405and FOCUS were inoculated in6-well plates as7×105/plate, then cultured in mediumcontaining1%fetal bovine serum containing TGF-β1in2ng/ml concentration. Themedium was replaced every24hours with a medium containing the same concentration ofTGF-β1. RT-PCR was used to detect the expression of HNF4α.5. The role of DNA methylation in the regulation of HNF4α by TGF-β1The human hepatoma cell lines FOCUS were inoculated in a6-well plates at5×105cells/plate and divided into A, B, C, D four groups. Group A as control group, group Bstimulated with1μM5-AZA-CdR for6days, Group C without any treatment in the first3days and then given2ng/ml TGF-β1on the fourth day for3days, Group D treated with1μM5-AZA-CdR for3days and then added2ng/mlTGF-β1for the next3days. The cellswere cultured in37℃5%CO2in cubators. The medium was changed every24h and thecells were collected6later. RT-PCR was carried out to detect the expression ofHNF4αP1.6. Statistical analysisThe arithmetic mean and standard deviation (s.d.) were calculated for the data, and statistically evaluated using2-tailed unpaired t-test. P <0.05was considered statisticallysignificant.【Results】1. Expression of HNF4αP1was reduced and HNF4αP2was increased in humanHCC specimensReal time RT-PCR was used to detected the expression of HNF4αP1in20pairs ofhuman liver cancer The results revealed that HNF4αP1expression is reduced in cancertussues than that in non-cancer tissues in13pairs of clinical samples (P<0.05). Three pairsof liver cancer clinical specimens showed no significant difference of HNF4αP1expression in comparision with the adjacent normal tissue and cancer tissue. In contrast,the expression of HNF4αP1was increased in cancer tissues than that in the adjacentnoncancerous tissues in the rest4pairs of human samples (P<0.05).Inversely, The HNF4αP2expression was higher in cancer tissues than that innon-cancer tissues from13of pairs of human samples (P<0.05). Two pairs of specimensshowed no difference and the expression of HNF4αP2was repressed in cancer tissues thanthat in the adjacent noncancerous tissues in the rest5pairs of human samples (P<0.05).The expression of HNF4αP1and HNF4αP2on different Hepatoma cell lines usingRT-PCR method to detect Hep3B, HepG2and Huh-7, YY, MHCC-H, MHCC-L,SMMC-7721,BEL-7405and FOCUS, the results showed the expression of HNF4αP1andHNF4αP2is markedly higher in Hep3B, HepG2, Huh-7compared with that inSMMC-7721, BEL-7405and FOCUS. The HNF4αP1average expression level is900times higher in Hep3B, HepG2and Huh-7than that in SMMC-7721,BEL-7405andFOCUS. While HNF4αP2average expression in Hep3B, HepG2, Huh-7is300timerhigher than that in SMMC-7721, BEL-7405, and FOCUS.2. The methylation status of promoter region of HNF4αP1and HNF4αP2isinversely correlated with the expression of HNF4αP1and HNF4αP2BSP results indicated that the degree of P1methylation in Hep3B, HepG2, Huh-7is3.6%,3%, and2%, respectively, whereas the degree of P1methylation in SMMC-7721、BEL-7405and FOCUS was88%、42%and64%, respectively. In addition, MSP dectectiondisplayed that the degree of P2methylation in Hep3B,HepG2and Huh-7is also lower thanthat in the SMMC-7721, BEL-7405and FOCUS. 3.5-AZA-CdR treatment increased the expression of HNF4αP1and HNF4αP2Demethylation drug5-AZA-CdR was used to treat hepatoma cells FOCUS for72h.BSP examination showed that the degree of P1methylation in FOCUS cells wassignificantly suppressed by5-AZA-CdR in a dose-dependent manner. Nevertheless, theexpression of HNF4αP1and HNF4αP2was induced in a dose-dependent manner.4. TGF-β1inhibites the expression of HNF4αP1Hep3B, HepG2, Huh-7, SMMC-7721,BEL-7405and FOCUS cells were treated withTGF-β1for72h, the expression of HNF4αP1was detected by RT-PCR. The resultsindicated that HNF4αP1mRNA leves were downregulated with TGF-β1treatment inHep3B, HepG2, Huh-7which have low methylation on promoter of P1(P<0.05). However,TGF-β1treatment did not result in significant change of HNF4αP1mRNA in SMMC-7721,BEL-7405and FOCUS which have high level of methylation on promoter of P1.5. DNA methylation impedes the regulation of HNF4αP1by TGF-β1HNF4αP1expression was significantly upregulated wth the treatment of5-AZA-CdRcompared with control (P <0.05), while no obvious change of HNF4αP1expression inFOCUS cells was detected with the addition of TGF-β1. Nevertheless, pretreatment ofFOCUS cells with5-AZA-CdR led to the reduction of HNF4αP1induced by TGF-β1（P<0.05）.【Conclusion】1. The expression of HNF4αP1is elevated in liver cancer tissues, whereas HNF4αP2expression is repressed.2. DNA methylation may regulate the expression of HNF4αP1and HNF4αP2inhepatoma cells.3. The methylation in HNF4αP1promoter region may prevent the regulatory role ofTGF-β1for HNF4αP1.