Inhibition CircPHKB/miR-1234-3p/CYP2W1 Pathway To Enhance The Sensitivity Of HCC Cells To Sorafenib

Research Background and Objective:One of the most common malignant tumors--liver cancer poses a great threat to health in clinic.More than 600,000 patients are diagnosed as liver cancer each year,and more than250,000 people die from it.Hepatocellular carcinoma(HCC)is the most common liver cancer type,and the incidence of HCC rate has been increased dramatically these years.According to the statistics in 2018,the incidence rate of HCC ranks the sixth and its mortality ranks the fourth in all kinds of cancer in the world.Sorafenib is an effective molecular targeted medcine and has been approved to the treatment of advanced liver cancer.Many studies have decleared that sorafenib can effectively improve the survival rate and curative effect of patients,but it is easy to develop drug resistance in HCC patients after treatment for several months.The emergence of primary and secondary drug resistance greatly reduces the effectiveness of the drug.Previous studies have shown that multiple mechanisms are involved in drug resistance,such as autophagy,tumor stem cells,tumor micro-environment,epigenetics,etc.However,the mechanisms of sorafenib resistance in HCC patients have not been fully clarified.circular RNA(circRNA)is an endogenous non-coding RNA that has been discovered recent years.Generally,it isn’t expressed or rarely expressed in normal tissues or cells,but is usually highly expressed in cancer tissues and cancer cells.Therefore,it is a good candidate of tumor marker and therapeutic target.Unlike linear mRNA with 5’end cap and 3’end tail,the 5’end and the 3’end of circRNA connect together and form a covalently closed loop structure.This special structure makes circRNA very conservative and stable.The circRNA plays an important role in specific cells or specific stages of cell growth.Natural circRNAs are mostly located in the cytoplasm and contain micro RNA binding sites,so circRNA can sponge miRNA.Some circRNAs are located in the nucleus,which regulates target genes through binding with RNA binding protein.Many studies have shown that circRNA participates in the regulation of cell function and varieties of diseases.micro RNA(miRNA)is a non-coding RNA that is very small and usually contains about20 bases.Generally,miRNA regulates target genes by partially or completely binding to the3’UTR region of its target mRNA.Most miRNAs can regulate multiple genes,while a small number of miRNAs only regulate a specific gene.The functions of miRNA have been extensively reported,but the interaction between miRNAs and circRNAs is still under exploring.Previous studies have shown that circRNA is closely related to sorafenib resistace.This study focused on exploring the effect of circPHKB on sorafenib resistance.Our results showed that over-expression of circPHKB promoted sorafenib resistance in HCC cells.Inhibition of circPHKB significantly enhanced sorafenib sensitivity in HCC cells.Therefore,circPHKB may be a potential target to sensitize sorafenib in HCC.Research Contents:Part UpⅠ-regulation of circPHKB decreased sorafenib sensitivity in HCC cells1.Screening of differential expressed circRNA in sorafenib treated HCC cells2.Analysis of the expression of circPHKB in sorafenib treated cells3.Analysis of circPHKB cyclization sites by DNA sequencing4.Analysis of circPHKB amplification by divergent and convergent primers5.Test the stability of circPHKB6.Investigation of circPHKB distribution in HCC cells7.Observing the effect of sorafenib on cell survival after silencing circPHKBPart Ⅱ Mechanisms of sorafenib triggered circPHKB over-expression in HCC cells1.Bio-informatics prediction of circPHKB binding proteins2.Verification the interaction between circPHKB and e IF4A33.Analysis the expression of e IF4A3 in sorafenib treated HCC cells4.Analysis circPHKB expression after silencing e IF4A3 in HCC cells5.Analysis circPHKB expression after silencing e IF4A3 in sorafenib treated HCC cellsPart Ⅲ circPHKB reduced sorafenib sensitivity in HCC cells by modulating the miR-1234-3p/ CYP2W1 pathway1.Bio-informatics prediction of circPHKB binding miRNAs2.Analysis of the expression of the candidate miRNAs in sorafenib treated HCC cells3.Investigating the mechanism of circPHKB as a molecular sponge of miR-1234-3p4.Observing the effect of sorafenib on cell survival after silencing or over-expression of miR-1234-3p in HCC cells5.Bio-informatics prediction and validation of miR-1234-3p targets6.Analysis of CYP2W1 expression in sorafenib treated HCC cells7.Investigating the regulating mechanism of miR-1234-3p on CYP2W18.Observing the effect of sorafenib on cell survival after silencing CYP2W19.Regulation of circPHKB on miR-1234-3p/CYP2W1 signaling pathwayResearch methods:1.Culture of HCC cells(Hep G2 and Huh7)2.Analysis of the protein expression level by Western blot3.Changing gene expression by transfection4.Analysis cell survival by CCK-8 assay5.Bio-informatics prediction through online databases and softwares6.Analysis the interaction between RNA and RNA by dual luciferase reporter gene assay7.Analysis the distribution of circRNA in cells by fluorescence in situ hybridization and nucleo-plasmic separation experiments8.Analysis circRNA cyclization sites by DNA sequencing9.Analysis circRNA amplication by agarosegel electrophoresis10.Analysis circRNA stability by RNase R and actinomycin D experiments11.Analysis RNA expression by Real-time PCR12.Analysis apoptosis by flow cytometryResearch results:Part Ⅰ The up-regulation of circPHKB decreased sorafenib sensitivity in HCC cells1.Three differential expressed circRNAs were screened in sorafenib treated HCC cells2.The expression level of circPHKB was significantly increased in sorafenib treated HCC cells3.circPHKB is formed by cyclization between exon 3 and exon 84.Only c DNA template can be amplified by divergent primers5.The stability of circPHKB was more stable than that of its linear gene PHKB6.circPHKB is mainly distributed in cytoplasm of HCC cells7.Silencing circPHKB dramatically increased sorafenib sensitivity in HCC cellsPart Ⅱ Mechanisms of sorafenib triggered circPHKB over-expression in HCC cells1.Bio-informatics analysis showed that e IF4A3 could interact with PHKB pre-mRNA2.There are three binding sites between e IF4A3 and PHKB pre-mRNA3.The expression of e IF4A3 was significantly increased in sorafenib treated HCC cells4.The expression of circPHKB was suppressed after silencing e IF4A3 in HCC cells5.Sorafenib induced circPHKB up-regulation was inhibited by e IF4A3 silencing in HCC cellsPart ⅢcircPHKB reduced sorafenib sensitivity in HCC cells by modulating miR-1234-3p/ CYP2W1 pathway1.Bio-informatics analysis showed that four miRNAs may combine with circPHKB2.The expression level of miR-1234-3p was significantly decreased in sorafenib treated HCC cells3.There are direct interaction between circPHKB and miR-1234-3p4.Silencing miR-1234-3p reduced sorafenib sensitivity while over-expression of miR-1234-3p enhanced it in HCC cells5.Bio-informatics analysis showed that miR-1234-3p can regulate the expression of CYP2W16.miR-1234-3p can bind with the 3’ UTR of CYP2W1 mRNA7.Silencing of miR-1234-3p up-regulated the level of CYP2W1 while over-expression of miR-1234-3p down-regulated it in HCC cells8.Over-expression of miR-1234-3p inhibited sorafenib induced CYP2W1 up-regulation9.Silencing CYP2W1 dramatically increased sorafenib sensitivity in HCC cells10.The down-regulation of CYP2W1 and sorafenib induced CYP2W1 up-regulation could be suppressed by circPHKB knock-down11.The increase of CYP2W1 and sorafenib induced CYP2W1 up-regulation could be triggered by circPHKB knockdown and miR-1234-3p inhibition.Conclusion1.Sorafenib up-regulated the expression of circPHKB,and the over-expression of circPHKB decreased sorafenib sensitivity in HCC cells.2.e IF4A3 promoted the formation of circPHKB in HCC cells.3.circPHKB reduced sorafenib sensitivity in HCC cells by regulating miR-1234-3p/CYP2W1 signaling pathway...