Inhibition And Molecular Mechanism Of SREBP1 On The Cellular Proliferation By Betulin In Hepatocellular Carcinoma

Background and aims:Liver cancer is one of the common malignant tumor.It is the largest number of the world’s highest incidence and death in our country.At present the clinical treatment have no effective targeted drugs,survival rate is lower than 30% because of recurrence and metastasis rates.It is an important issue needs to be solved that find effective targeted drugs.The study found sterol regulatory element binding proteins(SREBPs)is an important nuclear transcription factors that regulate lipid metabolism.SREBPs and lipid metabolism-related target genes are up-regulated in hepatocellular carcinoma.Inhibiting the lipid synthesis can inhibit hepatoma cell growth.So SREBPs is a promising targets to anti-cancer.Betulin is inhibitor of SREBPs and inhibit it’s activation.And several studies have shown that the anti-tumor activity of betulin has a broad spectrum,but the mechanism of action is not yet clear.This topic confirm that betuin can inhibit cell proliferation in liver cancer and SREBP1 can regulate the expression of apoptosis gene directly,so SREBP1 have a new regulatory mechanism in tumor cells.We will be taken SREBP1 to explanation the molecular mechanisms,provide theoretical basis and clinical support for the treatment of liver cancer.Methods:1.Determination proliferation,cycle and apoptosis in HepG2 cell: the different concentration: 0μg/m L,5μg/m L,8μg/m L,10μg/mL,20μg/mL,processing HepG2 cells 0h,24 h,48 h,72 h,96 h.Detect cell proliferation by MTT and cell apoptosis and cycle using flow cytometry instrument.qRT-PCR detecte the mRNA expression level of gene related cell apoptosis,protein kinase protein related cycle.2.Identification differentially expressed genes and functional analysis:RNA-seq technology screening expressed genes in HepG2 cells.The differentially expressed genes in the betulin and DMSO groups were analyzed and compared.3.The transcription factor pathway analysis and verify: In order to verify the results of RNA-seq analysis,pathway analysis was carried out and verify result by qRT-PCR and western blot.4.KEGG pathway enrichment analysis was performed on 993 DEGs to understand the biological significance of betulin regulation of gene expression in HepG2 cells.5.Predicte the gene near transcription factor binding sites: In order to predict the target gene downstream of SREBP1,we first predicted the proximity of its binding site: The ChIP-Seq and ChIP-seeker predict candidate target genes of the SREBP1 binding site in the promoter region.6.The prediction of target genes of transcription factors and functional analysis:In order to predict the target gene downstream of SREBP1,integrate ChIP-Seq data and DEGs,screened for genes of apoptosis related regulated by SREBP1;Luciferase detecte SREBP1 regulates this apoptotic gene and qRT-PCR detects the mRNA expression under betulin treatment.7.Analysis PIK3R3 overexpression and correlation of liver cancer: according to TCGA database、 GEO database and meta to analysis of PIK3R3 expression in HCC.Results:1.Betulin can significantly inhibit the proliferation of HepG2,and the concentration dependence.optimal concentration for 10μg/ml.2.qRT-PCR results showed that the mRNA levels of cyclin and cyclin kinases such as CDK1 and CDK6 were down-regulated in betulin treatment,while the levels of cycle-inhibitory proteins such as P21 and P27 were up-regulated;And pro-apoptotic genes such as BAX were up-regulated,while anti-apoptotic genes such as Bcl-xl,were down-regulated.And the m RNA levels of SREBP1 and FASN in the nuclear were down-regulated.Western blot results showed that the protein expression levels of SREBP1,FASN,CDK1,and CDC25 A in the nuclears under betulin treatment decreased.3.RNA-Seq results showed that 993 genes were differentially expressed among the 17339 genes detected in betulin group and DMSO group,accounting for 5.73% ofthe total number of expressed genes.These include up-regulation of 464 genes and down-regulation of 529 genes.4.Transcription factor pathway analysis confirmed that betulin is an inhibitor of SREBP1 by inhibiting the activation of karyotype to regulating the expression of downstream target genes.5.Pathway enrichment analysis indicate that betulin not only affects lipid metabolism but also affects the survival of cells.6.SREBP1 as a transcription factor that mainly binds to the promoter region of downstream genes.Betulin significantly inhibited the expression of apoptosis-related target gene PIK3R3.7.The expression of PIK3R3 in liver cancer tissues is higher than others,and it were higher in tumor tissues than adjacent to carcinoma tissue.Conclusion:1.Betulin can inhibit proliferation of HepG2 cells significantly,induce its apoptosis,and which are arrested in the G2/M phase.2.The mechanism of betulin inhibiting liver cancer is: SREBP1 can lower the expression of PIK3R3 directly which associated with apoptosis in HepG2 cell.