Aberrant Methylation Of ACTL6B CpG Island Mediated By ACTL6A/c-Myc Interaction Promot Hepatocellular Carcinoma Metastasis Via EMT

Background:The treatment of Hepatocellular Carcinoma is diverse,but the prognosis is still not ideal.Distant metastasis,postoperative recurrence has seriously affected the patient's long-term prognosis.At present,the most important cellular biological mechanism leading to recurrence of tumor metastasis is Epithelial-Mesenchymal Transition(EMT).EMT has been shown to be involved in the formation of circulating tumor cells,it is a key factor in triggering tumor metastasis,and tumor stem cell formation,it can make tumor cells for chemotherapy tolerance and immunotherapy tolerance.At the same time in the process of tumor cell EMT,epigenetic changes,especially the inactivation of tumor suppressor mediated by methylation was more extensively studied.There is growing evidence that methylation of tumor suppressor genes is involved in the regulation of tumor progression,including EMT,and that abnormal methylation of these promoter regions can also be used as diagnostic markers.There exists abnormal ACTL6B methylation in HCC tissue and cell lines,but the regulation of methylation,and whether methylation inactivation involved in tumor metastasis is still unknown.Chapter 1 The CpG island of ACTL6B gene promoter region in HCC tissues and cell lines is abnormally methylated and leads to low expression of ACTL6BObjectives:The methylation status of CpG island in the promoter region of ACTL6B gene in HCC was preliminarily studied.The tumor-specific CpG islands were screened,and the impact of promoter methylation on gene expression was investigated.Methods:The CpG island of ACTL6B promoter was predicted online.The methylation status of ACTL6B promoter was investigated by Direct Bisulfite sequencing PCR(DBSP).Tumor-specific CpG sites were determined by comparing the methylation status of ACTL6B in HCC Tissue and Para-Cancerous Histological Normal Tissue.Methylation specific polymerase chain reaction(MSP)primers were designed for tumor-specific CpG sites.The expression levels of ACTL6B in HCC tissues and cell lines were detected by Real Time Polymerase Chain Reaction(RT-PCR)and Western Blot(WB).Results:The high frequency of abnormal methylation was detected in HCC by BSP technique.The results were compared with the sequencing results of Para-Cancerous Histological Normal Tissue.The tumor-specific CpG sites were as follows:-622,-610,-590,-568,-550,-519,-33,-27,-22,-13,-8,+6,+12.Methylation of ACTL6B in tissues and cell lines were detected by MSP primers designed for tumor specific CpG sites.The results were consistent with the sequencing results.ACTL6B gene methylation was inhibited via TSA,5-Aza combined treatment of HCC cell lines,and the mRNA,protein expression was restored.The mRNA and protein expression of ACTL6B gene in 24 HCC tissues were negatively correlated with the methylation level of ACTL6B.ConclusionIn high-frequency abnormal methylation in HCC tissues and common HCC cell lines.HCC abnormal methylation is mainly reflected in a series of tumor-specific CpG sites.Abnormal methylation of the ACTL6B promoter region leads to low expression of ACTL6B mRNA and protein.TSA,5-Aza can reverse the methylation of ACTL6B,and then restore the expression of ACTL6B.Chapter 2 ACTL6B promoter methylation was mediated by ACTL6A/c-Myc interaction induced DNMT3a targeting to the promoter of ACTL6BObjectives:To study the molecular mechanism of ACTL6B methylation in HCC.Methods:The expression of ACTL6A in 24 cases of HCC was detected by immunohistochemistry(IHC),and the correlation between ACTL6A expression and ACTL6B methylation was analyzed.RT-PCR and WB were used to detect the mRNA and protein expression of ACTL6B,DNMT3a,DNMT3b and DNMT1 in cell lines with suppressed expressing of ACTL6A by siRNA,and the metylation of ACTL6B promoter was detected by BSP and MSP.The relative methyltransferase(DNMT)activity was examined in siRNA ACTL6A-expressing cell lines.BSP and MSP were used to detect the level of methylation of ACTL6B promoter in cells with repressed expressing DNMTs by siRNA.Luciferase reporter ssay was used to detect the inhibitory activity of ACTL6A cotransfecting with DNMT3a wild type(DNMT3a wt)or DNMT3a methyltransferase-deficient(DNMT3a mut)on the activity of ACTL6B gene promoter.ChIP technique was used to identify the ACTL6B promoter region binding of ACTL6A,and the mechanism of ACTL6A and c-Myc mediating DNMT3a binding to ACTL6B promoter regio.Co-IP technology verifies the physical binding between ACTL6A,c-Myc and DNMT3a.Results:The expression level of ACTL6A in 24 cases of HCC tissues was detected by IHC.The methylation data of ACTL6B showed that the expression of ACTL6A was positively correlated with the methylation level of ACTL6B.Cell function validation showed that ACTL6A overexpression could promote ACTL6B methylation.RT-PCR,WB and methyltransferase activity showed that overexpression of ACTL6A did not affect the expression of DNMTs and the activity of total methyltransferase.BSP and MSP showed that the methylation of ACTL6B gene was reversed only after inhibition of DNMT3a expression,and the expression of mRNA and protein was restored.The luciferase reporter assay confirmed that DNMT3a induced methylation of ACTL6B-by ACTL6A.Screening results of ChIP-PCR showed that ACTL6A might target the-120 to-50 segments and bind to the ACTL6B promoter region.ChIP showed that ACTL6A,c-Myc and DNMT3a could be enriched in the promoter region of ACTL6B gene.After c-Myc inhibition the enrichment of ACTL6A was not changed,and the enrichment of DNMT3a was inhibited.BSP assay showed that methylation of ACTL6B gene was inhibited while c-Myc expression was inhibited.Co-IP technique showed that ACTL6B,DNMT3a,c-Myc had an interacting effect,and the direct binding of ACTL6A and DNMT3a was inhibited after c-Myc inhibition.Conclusion:In HCC,ACTL6A promotes the methylation of ACTL6B and inhibits the expression of ACTL6B.This process is achieved by ACTL6A/c-Myc interaction mediated DNMT3a binding to ACTL6B promoter.Chapter 3 ACTL6B inhibits the growth and invasion and migration of HCC cell lines by inhibiting EMTObjectives:To study the effect of methylation of ACTL6B promoter on the biological function of HCC cell line in HCCMethods:The ACTL6B gene was inhibited or overexpressed in HCC cell lines.Cell viability was measured by CCK8,and the growth curve was plotted.WB technique was used to detect the expression of pERK,ERK,p-AKT and AKT.Nude mice were implanted subcutaneously,and the growth of transplanted tumor was detected by small animal in vivo fluorescence imaging.Scrap healing experiment and transwell migration assay were used to detect the effect of ACTL6B expression on migration and invasion.WB technique was used to detect the expression levels of ZO-1,E-cadherin,Zeb-1 and Snail.Results:The expression of p-ERK and p-AKT was decreased after the expression of ACTL6B in SMMA-7721,SKHep-1,MHCC97-H and MHCC-LM3.The results of transplanted tumorigenesis also indicated that overexpression of ACTL6B could inhibit the growth of HCC cell line.Scrap healing experiment,transwell migration test results showed that migration and invasion ability were inhibited in ACTL6B overexpressed HCC cell lines.WB assay showed that the expression of EMT-related markers changed after ACTL6B overexpression,including the expression of E-cadherin and ZO-1,and the expression of Zeb-1 and Snail was inhibited.Conclusion:ACTL6B inhibits the growth,migration and invasion of HCC by inhibiting EMTChapter 4 ACLP6B gene methylation is associated with HCC progression,and has potential diagnostic and prognostic valueObjectives:To study the relationship between the methylation of ACTL6B gene promoter and the clinicopathological features in HCC,and to investigate the diagnostic value and prognostic value of ACTL6B gene methylation in HCC tissues and preoperative peripheral blood serum.Methods:MSP technique was used to detect 155 cases of HCC tissues and adjacent normal tissues,as well as peripheral blood serum free DNA(cell DNA,cfDNA)obtained from 155 cases of HCC,60 cases of chronic hepatitis cirrhosis(cirrhosis)patients,60 cases of healthy volunteers.The relationship between ACTL6B methylation in HCC tissues and tumor progression and between ACTL6B methylation and prognosis was analyzed.The consistency of ACTL6B gene methylation in serum cfDNA and tissue-derived genomes was validated.The diagnostic and prognostic value of ACTL6B gene in serum cfDNA was compared with the diagnostic value of AFP.Results:The positive rate of ACTL6B methylation in HCC tissues was 74.2%,and the positive rate of methylation in adjacent normal tissues was 9.0%.Methylation of ACTL6B in HCC tissue was associated with gender,tumor size,portal vein tumor thrombus,TNM staging.Methylation of ACTL6B in peripheral blood serum cfDNA has a good consistency with methylation of ACTL6B in tissues.The methylation of ACTL6B in HCC tissue and serum cfDNA is an independent prognostic factor for the overall survival and disease free survival of patients with HCC.The overall diagnostic efficacy of ACTL6B methylation positive as a diagnostic marker in serum cfDNA was similar to that of serum AFP,and the combination of the two as diagnostic criteria could significantly improve the overall diagnostic efficacy(AUC:0.913,95%CI:0.865-0.961,P:5.914E-21).ACTL6B methylation single index can not be used as a diagnostic index to distinguish between HCC and-CHC.AFP still has the diagnostic efficacy of distinguishing HCC and CHC.The combination of the two can improve the diagnostic performance(AUC:0.797,95%CI:0.693-0.901,P:1.929E-06).The diagnostic efficacy of ACTL6B to distinguish stage III HCC was significantly higher than that of AFP,both of which had the greatest diagnostic efficacy as a diagnostic index(AUC:0.958,95%CI:0.917-1.000,P:669E-20).Conclusion:The methylation level of ACTL6B gene in HCC tissue is related to the progression of tumor.It is an independent prognostic factor for total survival and disease-free survival after HCC hepatectomy.There was a good consistency between the methylation level of ACTL6B in the HCC tissue and the methylation level of peripheral blood serum cfDNA.Methylation of ACTL6B in peripheral serum cfDNA has potential diagnostic and prognostic value.