Study On Epigenetic Mechanism Of OAT2 Transcriptional Inhibition In Hepatocellular Carcinoma

Primary liver cancer(PLC)is one of the most common malignant tumors in the world.PLC can be divided into hepatocellular carcinoma(HCC),cholangiocellular carcinoma(CC).and mixed type liver cancer(HCC-CC)according to histological differences.Almost 90%of the PLC are HCC.Human organic anion transporter 2(OAT2)is mainly expressed in the liver and kidney,and is the second most abundant transporter in the liver.In our study,loss of SLC22A7 expression was found in HCC tissues compared with matched tumor-adjacent tissues.Transcriptional inactivity of SLC22A7 contributed to the lost of OAT2 expression in HCC tissues.Epigenetics played an important role in the genes transcriptional regulation.DNA methylation and histone modification were the two most intensely studied epigenetic pathways.Due to the absence of CpG islands at SLC22A7 locus,as well as that the expression of SLC22A 7 had no change after DNA methyltransferase inhibitor decitabine(DAC)treated on HCC cell lines,we found DNA methylation didn’t regulate the SLC22A7 expression in HCC.In the tumor-adjacent liver tissues,we found that H3K18ac and H3K4me3 were enriched at the promoter of SLC22A7,while the enrichment of H3K9ac and H3K27ac were low.Compared with the tumor-adjacent tissues.the enrichment H3K9ac and H3K18ac at the promoter of SLC22A7 in HCC were up-regulated,while H3K27ac was down-regulated and the enrichement of H3K4me3 did’t change significantly,suggesting that H3K9ac and H3K18ac may be involved in the transcriptional regulation of SLC22A7 repression in HCC,of which H3K18ac may play a major role.Comparing the enrichment of multiple histone modification at SLC22A7 promoter in 3 HCC cell lines HepG2,BEL-7402 and SMMC-7741.SLC22A7 was relatively high expressed in HepG2 cell line.Only H3K18ac was found enriched at SLC22A7 promoter in HepG2 cell line.Histone deacetylase inhibitor Vorinostat(Suberoylanilide hydroxamic acid,SAHA)could increase the enrichment of H3K18ac at SLC22A7 promoter and induce SLC22A7 expression in BEL-7402 and SMMC-7721 cell lines.The results from uptake of OAT2 classical substrate zidovudine demonstrated that the up-regulation of SLC22A7 caused an increase in OAT2 uptake function.Meanwhile,we found SAHA could inhibit the expression of histone deacetylase SirT7,and the silencing of SirT7 in the HCC cells could increase the enrichment of H3K18ac level at SLC22A 7 promoter,thereby inducing the transcription of SLC22A7.To investigate the expression of histone deacetylase HDAC1-10 in HCC tissues and their adjacent tissues,it was found that the expression of HDAC1 and HDAC7 was negatively correlated with the expression of SLC22A7,while the silencing of HDAC7 in HCC cell lines enricned H4K16ac at SLC22A7 promoter and induced SLC22A7 expression.Comparing the mRNA expression between HCC and the matchedtumor-adjacent-adjacent tissues;DMSO and SAHA-treated HCC cells,a histone acetyltransferase KAT8 was found to regulate the depression of SLC22A7 in HCC.SAHA decreased KAT8 expression at SLC22A7 promoter by inhibiting the HDAC7 expression,and enhanced the H4K16ac,thereby inducing the transcription of SLC22A7.In summary,two histone acetylation pathway was found to regulate the transcription depression of SLC22A7 in our study:1)SAHA inhibited the expression of histone deacetylase SirT7 and promoted the enrichment of H3K18ac at SLC22A7 promoter,thereby activating SLC22A7 transcription.2)SAHA inhibited the expression of HDAC7,which enriched KAT8 at SLC22A7 promoter region,and increased the acetylation level of H4K16,inducing the transcription of SLC22A7.Multiple-drug resistance(MDR)always causes the failure of cancer drug therapy.Several anti-cancer drugs,such as 5-fluorouracil(5-FU),bendamustine,irinotecan,paclitaxel,and methotrexate are all substrates of OAT2.The significant repression of OAT2 in HCC may influence the uptake of these anti-cancer drugs,thereby leading to MDR.In our study,we designed a combination regimen of SAHA and 5-FU on HCC cells.The results showed that SAHA can enhance the sensitivity of 5-FU on HCC cells.The synergistic effect of the SAHA and 5-FU increases with the increase of OAT2 induction level.It highlighted the potential of a novel combinational chemotherapy in clinical HCC therapy.