| Objective(s):Hepatocellular carcinoma is the most important type of liver cancer,and the most important related factor of liver cancer is chronic infection of hepatitis B and C.In 2011,liver cancer became the fourth most common cancer and the second leading cause of cancer death in China.In 2014,China added 364,800 new cases of liver cancer,about 318,800 deaths from liver cancer,and the Chinese population accounted for about 19%of the world.Liver cancer cases account for more than 50%.Methods for the treatment of liver cancer Clinically,liver resection and liver transplantation are often used.Some studies have shown that the 5-year survival rate of liver cancer patients is related to the timing of surgical treatment.Early intervention is beneficial to patients.The early symptoms of liver cancer are atypical and lack of early diagnostic indicators,so that the disease has progressed at the time of diagnosis and accompanied by distant metastasis of the tumor.At this time,the patient has lost the best opportunity for treatment.An experiment with survival time as the research object showed that the age-standardized five-year relative survival rate of liver cancer patients in China is only 12.1%.This is a major health problem with a very low survival rate.This is not only true in China,but also urgently needed to be solved globally.Furthermore,surgical treatment may also recur.Therefore,for the prevention and treatment of liver cancer,it is particularly important to find sensitive early diagnosis and treatment targets.The purpose of this study was to perform whole transcriptome sequencing of hepatocellular carcinoma and adjacent cancer tissues,and then identify the target circRNA molecules by quantitative screening,and quantitatively detect and compare the RNA expression levels of target circRNAs in HCC and adjacent cancer tissues.Experimental verification in four dimensions:tissue,cell,molecule and animal,looking for changes and determining its diagnostic value for HCC,and exploring related mechanisms involved in its impact on liver cancer metastasis.Methods:1.Clinical data section:Three paired whole-transcriptome sequencings of hepatocellular carcinoma and paracancerous tissues were collected,and differentially exoressed circRNAs were analyzed by bioinformatics for GO and KEGG enrichment.Finally,circ-SIRT1 was selected and 19 paired tissues were selected.Liver cancer and paracancerous tissues were verified by qPCR,and 71 paired tissues were used for fluorescence in situ hybridization verification of liver cancer and adjacent tissues.The obtained fluorescence signal information and the clinical data of the patients were analyzed by single and multiple factors.2.In vitro experiments:?First,detect the expression of HCC cell lines and normal liver cells by qPCR.Then recombinantly express the plasmid pcDNA 3.1(+)-circ-SIRT1 of circ-SIRT1,and transfect HCC cell line HepG2;and synthesize si-circ-SIRT1 to transfect cells Huh7 cells.After transfection,perform qPCR to verify the transfection efficiency.?CCK-8 test to detect cell proliferation,flow cytometry to detect apoptosis and cycle,scratch and Transwell test without matrigel to detect cell migration ability,Transwell test with matrigel to detect cell invasion ability,and The effects of circ-SIRT1 overexpression/knockdown on E-cadherin,Fibronectin,a-SMA,Snail 1,FAK,Src,Tks5,and Girdin proteins in HCC cells were studied using Westemblot experiments.?Determine the subcellular distribution of circ-SIRT1 in the cells through nuclear separation,and then combine the three databases of targetscan,miRanda and RNAhybrid to pre-target the circ-SIRT1 miRNA and ceRNA by bioinformatics,and verify the cells co-localization of circ-SIRT1 and SIRT1 with FISH.The wild-type LUC-circ-SIRT1 vector was recombined,The dual-luciferase reporting experiment was used to verify the direct binding relationship between miR-132/miR-212 and circ-SIRTl/SIRT1.In addition,circ-SIRT1 was knocked down to study its effect on the expression of SIRT1 downstream of the target miRNA,while SIRT1 was knocked down to study its effect on the expression of circ-SIRT1.?ChIP experiments after knocking down circ-SIRT1 verified the condition of SOX2 promoters H3K4Me3 and H3K27Me3,clarified the changes in DNMT3A,and the effects on the expression of SOX2 after knocking down circ-SIRT1 and DNMT3A simultaneously.Subsequently,a Co-IP study was performed after knocking down circ-SIRT1 to determine the effect of circ-SIRT1 on the binding of SIRT1 to SOX2.The effects of dual-operation circ-SIRT1 and SOX2 on invasion and E-cadherin,Fibronectin,a-SMA,Snail 1,FAK,Src,Tks5,and Girdin proteins were studied in rescue experiments.?In the exploration of the upstream mechanism,we performed dual operations on TGF?1 and circ-SIRT1 and observed the effects on invasion and Tks5 and Girdin.3.In vivo experiments:Establish subcutaneous tumor models on nude mice,record tumor growth and volume,and detect the expression of circ-SIRT1 in tumors and verify the mechanism of in vivo experiments to explore the effects of circ-SIRT1 on tumor growth.Results:1.Of the 2874 circRNAs obtained by sequencing,148 circRNAs were significantly overexpressed in tumors,180 circRNAs were significantly downregulated in tumors,1269 were tumor-specific differential circRNAs,and 860 were adjacent tumors-specific differential circRNAs.There are 745 different circRNAs shared by tumors and adjacent tumors.2.The expression levels of circ-SIRT1 in HCC tissues were significantly higher than those in adjacent tissues.The expressions of p65 and SOX2 in HCC cancer tissues were higher than those in adjacent tissues(P<0.05),and the expression of circ-SIRT1 was related to the invasion of patients.3.The expression levels of circ-SIRT1 of HEP3B2.1-7,HepG2,Huh-7;and SK-Hepl were commonly higher than those of HL-7702 and HepaRG cells(p<0.05).4.Huh-7 and HepG2 cells were successfully established after knocking down and overexpressing circ-SIRT1,respectively.SIRT1,miR-132/miR-212 also changed after knocking down circ-SIRT1.5.Cell biological function experiments show that over-expression of circ-SIRT1 can promote the cycle progression,proliferation,epithelial-mesenchymal transition(EMT),invasion and migration of HCC cells,and inhibit cell apoptosis.However,getting the opposite result after knocking down circ-SIRT1.6.The potential target of circ-SIRT1 was predicted by bioinformatics,and miR-132/miR-212 was selected as the molecule that can be adsorbed.The miR-132/miR-212 was proved by the double luciferase report experiment.Can directly combine circ-SIRT1 and SIRT1?In addition,we found that knockdown of circ-SIRT1 in Huh7 cell lines can inhibit SIRT1 expression and vice versa.Knocking down circ-SIRT1 can inhibit the expression of SOX2 and reduce the binding of SIRT1 to SOX2.7.After knocking down circ-SIRT1 in Huh7 cell line,it was found that the methylation level of SOX2 promoter was changed,H3K4Me3 was decreased and H3K27Me3 was increased,and the expression level of DNMT3 A was changed after circ-SIRT1 was knocked down.In Huh7,The co-transfected with circ-SIRT1 and DNMT3A knockdown plasmids in cell lines could partially reverse the expression of SOX2.8.Co-transfection of circ-SIRT1 knockdown plasmid and SOX2 overexpression plasmid can partially reverse HCC cell invasion and E-cadherin,Fibronectin,a-SMA,Snail1,FAK,Src,Tks5,and Girdin proteins.9.Subcutaneous tumor models in nude mice showed that knockdown of circ-SIRT1 can inhibit tumor growth.Conclusion(s):circ-SIRT1 is highly expressed in liver cancer tissues relative to adjacent tissues,and its high expression is related to the clinical metastasis characteristics of patients,and has a protmoting effect on the proliferation,invasion and migration of liver cancer cells,which may be promoted by adsorption of miR-132/miR-212,then promoting the expression of SIRT1,thereby reducing the DNMT3A protein level,increases the H3K4Me3 and H3K27Me3 of the SOX2 promoter,further promotes the expression of SOX2.promoting the Invadopodia function of hepatocellular carcinoma,and ultimately regulates the invasion and metastasis of liver cancer. |
PDF Full Text Request