| Background:Hepatocellular carcinoma(HCC)is a common malignant gastrointestinal tumor with high mortality rate all over the world.Because of the difficulty of early detection,80%of HCC patients are already in the advanced stage of cancer when they first visit.And there is no effective treatment to maintain the long-term survival time of patients due to the metastasis of HCC or poor liver function.Alpha-fetoprotein is currently used as an early screening indicator for hepatocellular carcinoma in clinical work,but its sensitivity and accuracy are relatively limited.Liver tissue biopsy has good accuracy in the diagnosis of hepatocellular carcinoma,but it is a invasive operation which can lead to tumor metastasis.Therefore,liver tissue biopsy is not suitable used as a routine clinical detection method especially in the diagnosis of early hepatocellular carcinoma.Hence,it is necessary to discover a new biomarker to improve the detection,the treatment and the prognosis of HCC patients.Circular RNA is a special type of non-coding RNA,which is a research hotspot in the field of RNA in recent years.Circular RNA plays an important role in the development of tumors.Many studies reported that there is a close relationship between the expression of circular RNAs and the clinical manifestations of cancer patients.In addition,circular RNA could regulate many tumor phenotypes including cell cycle,apoptosis,invasion and metastasis,etc.Therefore,circular RNAs have the potential to become a molecular markers or therapeutic targets for tumors.It is of great significance to further explore the relationship between circular RNA and the hepatocellular carcinoma.Based on the above reasons,we designed this subject which mainly to discuss the function and the molecular mechanism of circular RNA(circBACH1)in hepatocellular carcinoma.First,we detected the expression of circBACHl in hepatocellular carcinoma and analyzed the relationship between circBACH1 and clinical characteristics of patients(gender,age,AFP,HBsAg,tumor volume,number,MVI,Child-Pugh classification,degree of differentiation,TNM stage and survival time).Then,we verified the effect of circBACH1 on the cell cycle and proliferation of hepatocellular carcinoma cells through cell and animal experiments(3)Finally,we discussed the molecular mechanism of circBACHl in regulating the cell cycle and proliferation of hepatocellular carcinoma cells.Part I:The expression of circBACHl in HCC tissues and the relationship between circBACHl and clinical characteristicsObjectiveTo detect the expression of circBACH1 in hepatocellular carcinoma cells and tissues,and analyze the relationship between circBACH1 and clinical characteristics of patients(gender,age,AFP,HBsAg,tumor volume,number,MVI,Child-Pugh classification,degree of differentiation,TNM stage and survival time).Methods1.In order to verify the circular structure of circBACH1,we extracted the total RNA and genomic DNA(gDNA)from the hepatocellular carcinoma cell lines(HepG2 and Hep3B),And the total RNAs were reverse-transcripted to cDNAs which were then amplified to target fragments.Finally,we analyzed the sequence of the product by using the sanger sequencing.Two pairs of primers for GAPDH and circBACH1 were designed for the next experiments.One pair are divergent primers and the other pair are convergent primers.Two sets of primers were used for amplifition with the templates of cDNA and gDNA.At last,the amplified products were subjected to agarose gel electrophoresis experiments.2.Human normal hepatocyte cell line(L-02)and human hepatocellular carcinoma cell lines(HepG2,Huh7,Hep3B and SNU475)are selected for cell experiments to certify the functions of circBACH1.We collected the cancer and adjacent tissues of HCC which were confirmed by pathological test.Then clinical datas(gender,age,AFP,HBsAg,tumor volume,number,MVI,Child-Pugh classification,degree of differentiation,TNM staging and survival time)of the patients were collected and sorted.The expression of circBACH1 in cells and tissues was detected by qRT-PCR technology,and the relationship between circBACH1 and clinical manifestations of hepatocellular carcinoma was analyzed by statistical methods such as chi-square test and survival analysis.Results1.Sanger sequencing results showed that the amplifed sequences by RT-PCR contains the head and tail splice region of circBACH1.The results of agarose gel electrophoresis assay showed that the sequence of circBACH1 could only be amplified divergent primers.And the sequence of gapdh could only be amplified by convergent primers.Moreover,when we performed PCR assay by using gDNA as a template,we could not achieve the sequence of circBACH1.2.The expressions of circBACH1 in hepatocellular carcinoma cell lines were 2-6 times that of normal liver cell lines.And the expression of circBACH1 in cancer tissues is about 2 times that of adjacent cancer cells.The difference is statistically significant.The analysis results of clinical datas show that the expression of circBACH1 was closely related with the tumor volume(p=0.0164),the tumor differentiation(p=0.0019)and survival time(p=0.0283)in HCC.The results of ROC analysis showed that the AUC=0.8506.Conclusion1.CircBACH1 is a closed-loop RNA molecule.The circular sequence of circBACH1 can be obtained by RT-PCR.2.CircBACHl is significantly overexpressed in hepatocellular carcinoma cell lines and cancer tissues.The expression of circBACH1 is closely related to the tumor volume and differentiation of hepatocellular carcinoma.And circBACH1 has the potential to be a diagnostic and prognostic indicator for hepatocellular carcinoma.Part II:CircBACH1 regulate the cell cycle and proliferation of hepatocellular carcinomaObjectiveTo clarify the function of circBACH1 on regulating the cell cycle and proliferation activity of hepatocellular carcinoma cells through cell and animal experiments.Methods1.First,we constructed lentivirus with the sequence of circBACH1,sh-circBACH1,vector or negative control.Then,the lentivirus were transfected into HepG2 and Hep3B cell lines and the expressions of circBACH1 in cell lines were detected.Cell Counting Kit-8(CCK-8)experiments,clony formation experiments,Ethynyldeoxyuridine(EdU)experiments and Flow cytometry experiment were used to detect the proliferation ability of hepatocellular carcinoma cell after knockouting or overexpressing circBACHl.2.The HepG2 cell line was used to construct a subcutaneous implanted tumor model in nude mice,which was divided into five groups(blank control group,high expression circBACHl group and vector group,low expression circBACH1 group and negative control group).Regularly detecting and recording the tumor volume and weight every three days and draw the growth curves.After 4 weeks,the subcutaneous transplanted tumor was removed and the expression levels of Ki-67 and p27 were detected by Immunohistochemistry(IHC)assay.Results1.The results of cell experiments showed that circBACH1 has a close relationship with the proliferation ability of HCC cells.Silencing the expression of circBACH1 leaded to the decrease of absorbance value in 450nm by CCK-8 assay.The clony formation experiment results shows that the cell cloning ability was weakened by knocking down circBACH1's expression.Moreover,when knockouting circBACH1,the percentage of EdU positive cells was decreased.The cell cycle assay showed that the reduction of circBACH1 increased percentage of GO/G1 phase cells,and decreased proportion of S phase cells.2.The results of animal experiments showed that the volume and weight of hepatocellular carcinoma in the overexpressing circBACH1 group were increased,and knocking out circBACH1 inhibited the tumor growth rate.Immunohistochemical assay results showed that Ki-67 expression was increased and p27 expression was downregulated in the circBACH1 overexpression group,while Ki-67 expression was decreased and p27 expression was increased in the circBACH1 knockout group.Conclusion1.circBACH1 can enhance the transformation of G1 phase to S phase in hepatocellular carcinoma cells which accelerate the cell cycle progression.2.circBACH1 can promote the proliferation of HCC cells3.CircBACH1 can enhance the replication of DNA in HCC cells4.circBACH1 can improve the cloning ability of hepatocellular carcinoma5.In the animal experiments,circBACH1 can promote the proliferation and growth of hepatocellular carcinoma.Part III:The molecular mechanism of circBACH1 in hepatocellular carcinomaObjectiveTo discuss the molecular mechanism of circBACH1 in regulating the cell cycle and proliferation activity of hepatocellular carcinoma cellsMethods1.To certify the relationship of circBACH1 and cell cycle,we extracted the total protein of circBACH1 in HCC cells and detcted the key cyclins(cyclinDl,cyclinE,CDK2,CDK4,etc.)in cell cycle by Western blot assay.And we found that expression of p27 was regulated by circBACH1.Moreover,silencing the expression of p27 could abolished the function of circBACH1 in HCC proliferation through the CCK-8 experiment,clony formation experiment and flow cytometry experiment2.To further study the mechanism of circBACH1 in HCC,we first analyzed the circRNA-protein interaction databases(RBPDB,RBPmap,and Circlnteractome)and found out the RNA-binding proteins which can interact with circBACH1.Then we used RNA-binding protein immunoprecipitation(RIP)assay,magnetic bead RNA pull-down experiment,electrophoretic mobility shift assay(EMSA)to verify the above results.3.Total proteins were extracted from circBACH1 overexpressed and knockdowned HCC cells,and the expression of RNA binding protein(HuR)was detected by Western blotting.Fluorescence in situ hybridization(FISH)assay and RNA nuclear separation techniques were used to detect the location of circBACH1 in HCC cells under normal conditions.After overexpressing or knockdowning the circBACHI's expression,the expression of circBACH1 and HUR were detected by FISH and Western blot assays.Moreover,we detected the distribution of HuR in the cytoplasm and nucleus.4.In order to verify the circBACH1's function in cell cycle was through HuR which regulating the translation of p27.First,we knocked down HuR and detected the expression changes in p27 protein and RNA levels.Then we performed rescue experiments to verify HuR' functions in regulation pathway of circBACH1.While overexpressing circBACHl's level in HCC,we knocked down HuR and observed the cell proliferation abilities by CCK-8,and cell flow cytometry.Results1.Western blot results showed that p27 protein levels in the over-expressed circBACH1 group were significantly reduced,and p27 protein levels in the low-expressed circBACH1 group were significantly increased.The results of cell phenotypic experiments showed that inhibiting p27's expression could rescue the regulation function of circBACH1 in HCC.2.Through RNA-protein binding databases,we found that RNA binding protein(HuR)could bind to circBACH1.And the results of RNA binding protein immunoprecipitation experiment,RNA pull-down experiment,and EMSA experiment confirmed results of the database analysis.3.There was no significant change in HuR expression in total protein after overexpressing and knockdowning the expression of circBACH1.FISH assay and nuclear and cytoplasmic separation experiments showed that circBACH1 was distributed both in the cytoplasm and nucleus,while HuR was mainly distributed in the nucleus.Moreover,FISH and IF assays showed that distribution of HUR in the cytoplasm increased and the distribution in the nucleus decreased when increasing the expression of circBACH1.4.Western blot and qRT-PCR results showed that there was no significant change in the RNA level of p27 after knocking down HUR' expression,but the protein level was significantly reduced.In addition,knockdowning of HUR'expression can reverse the decreasion of p27 protein levels and cell phenotype changes caused by overexpression of circBACH1(increased cell proliferation activity,enhanced colony forming ability,and accelerated cell cycle progression).Conclusion1.circBACH1 accelerates the cell cycle transition from GO/G1 phase to S phase by inhibiting the translation of p27,and promotes the proliferation of hepatocellular carcinoma.2.CircBACHl can directly bind to HUR and promote the transfer of HUR from the nucleus to the cytoplasm,while it can't affect the expression of HUR.3.In hepatocellular carcinoma,circBACH1 can strengthen the binding of HUR and the intracellular ribosome entry site(IRES)of p27 mRNA to inhibit the translation of p27and affecting cell's proliferation activity... |
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