Solute Carrier Family 27 Member 5 Regulates Proliferation And Migration Of Hepatoma Cells And The Molecular Mechanisms

Objective:Hepatocellular carcinoma(HCC)is a malignant tumor with a high incidence rate.In 2015,more than 850 thousand new cases have been diagnosed worldwide,with the similar mortality rates.And it is the fourth leading cause of cancer death in China.Typically,patients tend to eventually develop advanced HCC because of its characteristic of metastasis and limited treatment.Therefore,exploring the pathogenesis of HCC and searching for the prognosis of molecular markers are important for the identification of novel therapeutic targets in HCC prevention and treatment.Lipid metabolism plays central role in the intracellular environmental homeostasis,and it is one of the most significant metabolic pathway in the organism.Fatty acids which are constituents for biosynthesis of membranes and signaling molecules are important substrates for energy metabolism.Cellular proliferation is an important feature of malignant tumor.In cancer cells,Fatty acids must be more required for biosynthesis of membranes andgeneration of signaling molecules.The diversification of lipid metabolism reveals that cancerous cells may through enhancing de novo FA synthesis to obtain their required fatty acid,thus providing energy for tumor cells rapid proliferation.The proteins encoded by SLC27(solute carrier family 27)are named fatty acid transporters(FATPs),which belong to solute carrier proteins family.They are integral transmembrane proteins that enhance the uptake of long-chain and very long chain fatty acids into cells,and important in the fatty acids and lipids metabolism.SLC27A5(solute carrier family 27 member 5)is exclusively expressed in the liver,suggesting that it is very important in fatty acid metabolism of liver.The literature reported that hepatocytes from FATP5 knockout mice show an approximately 50%decrease in fatty acid uptake and in free fatty acids and triglyceride,but the other FATP family members has no compensatory increase.It indicated that SLC27A5 is major in the uptake of long-chain fatty acids to maintain the hepatic lipid balance.However,there have been no reports that the biological effects of SLC27A5 on HCC occurrence and development and its mechanisms research.Our previous dates suggested that SLC27A5 gene expressed low level in HCC by GEO database.And we further confirmed SLC27A5 is low in HCC tissues by TCGA database analysis.In this study,we mainly studied the effects of SLC27A5 on the HCC cells proliferation and migration and the related mechanisms.Methods:1.SLC27A5 expression in HCC samples was determined by GEO database analysis.The expression of SLC27A5 in tumor tissues was analyzed by bioinformatics methods in TCGA database.The relationship between the expression of SLC27A5 and the prognosis of patients was analyzed.2.The expression of SLC27A5 in fourty pairs of clinic HCC tissues was measured by Western blot,qRT-PCR and immunohistochemistry(IHC).The endogenerous levels of SLC27A5 gene in hepatocyte cell lines and hepatoma cell lines were examined by Western blot.3.Recombinant adenovirus plasmid pAdTrack-SLC27A5 was transfected into HEK-293 cells to package recombinant adenoviruses encoding SLC27A5 which was transfected into hepatoma cells.The effect of SLC27A5 on liver cancer cell lines proliferation was detected by MTS,Edu and direct clonoy formation assays.The effect of SLC27A5 on liver cancer cell lines migration was measured by transwell and wound healing assays.4.Through transcriptome sequencing and reviewing literatures to select target genes.To verify the expression of target genes at the RNA and protein levels by qRT-PCR and Western blot.5.SLC27A5 and its downstream target gene TXNRD1 in HCC and adjacent tissues were measured by Western blot,q RT-PCR and IHC.TheNrf2 nuclear translocation was detected by immunofluorescence(IF)and Western blot assays.6.The effect of SLC27A5 suppressed tumor gowth was detected in xenograft implantation models.The correlation between SLC27A5 and its downstream target gene TXNRD1 was further verified in vivo by Western blot,qRT-PCR and IHC analysis.Results:1.The expression of SLC27A5 in forty pairs of HCC tissues was detected by Western blot and q-RT PCR.And there were twenty-seven pairs were down-regulated by Western blot,accounting for 67.5%.The significant decrease of SLC27A5 was detected by qRT-PCR,and the difference was statistically significant(P<0.01).SLC27A5 down-regulated in eighteen pairs HCC tissues was detected by IHC,and the intracellular localization of SLC27A5 was mainly located in the cytoplasm.It showed that the expression of SLC27A5 in HCC tissues was significantly lower than that in paracancerous tissues.2.SLC27A5 recombinant adenoviral plasmid was constructed and packaged into the recombinant adenovirus.SLC27A5 overexpression in AdSLC27A5 infected cells was identified by Western blot.The effects of AdSLC27A5 on cell proliferation were detected by MTS,EdU and direct clonoy formation assays.The results of MTS showed that compared with Mock and AdGFP group,the growth of the cells in the AdSLC27A5 groupstarted to get slower after 96 h of normal culture,and it was most obvious at120h;the results of EdU showed that compared with Mock and AdGFP group,the newly synthesized DNA of AdSLC27A5 group was significantly reduced.Direct colony formation assays showed that the number of colony formation in the AdSLC27A5 group was significantly less than Mock and AdGFP group.Wound healing assay and transwell chamber assay were used to investigate cell migration and invasion.The results of wound healing showed that compared with Mock and AdGFP group,the cell migration rate of AdSLC27A5 group was significantly decreased;the results of transwell chamber showed that AdSLC27A5 group cell migration and invasion numbers were lower than Mock and AdGFP group.It indicated that SLC27A5 had the ability to inhibit the proliferation and migration of hepatoma cells in vitro.3.The target gene TXNRD1 related to SLC27A5 was seeked by RNA-Seq;the mRNA and protein expressions were measured by Real-time PCR and Western blot.We found that the expression of TXNRD1 was significantly influenced by SLC27A5,while SLC27A5 overexpression reduced the level of TXNRD1.Nrf2 is an important upstream signaling molecule that can regulate TXNRD1.The expression of Nrf2 was detected by Western blot and IF(immunofluorescence).It was found that when SLC27A5 overexpressed,the protein level and fluorescence intensity of Nrf2 in the nucleus was decreased.It indicated that the reduction ofTXNRD1 was mediated by SLC27A5 through the Keap1/Nrf2 signaling pathway.4.MHCC-97 H cells,MHCC-97 H cells infected with AdGFP and MHCC-97 H cells infected AdSLC27A5 were injected subcutaneously into the nude mice to observe tumor growth,and mices were killed after four to six weeks.These results showed that the weight and volume of subcutaneous tumors of AdSLC27A5 group were significantly lower than control groups.The negative correlation between SLC27A5 and TXNRD1 was verified in vivo by q PCR,Western blot,and IHC.It demonstrated that SLC27A5 can inhibit tumor formation in nude mice model.Conclusion:SLC27A5 down-regulates TXNRD1 through the Keap1/Nrf2 signaling pathway to inhibit hepatoma cells proliferation.In this study,we firstly detected the expression of SLC27A5 in hepatocellular carcinoma,and explored its effects on the proliferation and migration of hepatocellular carcinoma cells in vitro and vivo,in order to reveal its possible mechanism of inhibiting the proliferation of hepatocellular carcinoma and provide new ideas for clinical diagnosis and treatment of HCC.