| Hepatocellular carcinoma is one of the most common malignant cancers in digestive system.The worldwide morbidity and mortality of this disease are ranked sixth and second respectively among all the malignant cancers.Hepatocellular carcinoma usually concealed onset without any symptom or with untypical symptoms at early stage.But the carcinoma develops and progresses quite fast.Most of the patients are diagnosed as hepatocellular carcinoma at late stage,when local invasion or distant metastasis have already happened.All these make it not easy to cure the disease.The lifetime of patients is short and the prognosis is poor.Recent years,more and more studies have been done on the pathogenetic mechanism of hepatocellular carcinoma,such as the regulation of cell cycle,epigenetic regulation,telomere maintenance,gene mutations and so on.Studies have showed that there exists P53 mutations and TERT promoter mutations in hepatocellular carcinoma.P53 is a classical and well-known tumor suppress gene,which plays an important role in progression of tumor.A great number of tumorigenesis is related to the mutation or inactivation of this protein.The most common mutation of P53 is the missense mutation of a specific base site,which not only destroy the anti-tumor function of normal protein,but also express a mutant P53 protein gaining a new function of carcinogenesis.So the missense mutation of P53 is also called the gain of function(GOF)mutation of P53.While in hepatocellular carcinoma the rate of GOF P53 mutations are reaching up to 32%.Telomerase reverse transcriptase(TERT)is the key catalytic subunit of telomerase and it is hyper-active in most malignant cancers leading the immortalization of cancer cells.Therefore,TERT is regarded as one of the hallmarks of cancer.TERT promoter mutation is one of the most studied mechanism of TERT activation in cancer cells.It has showed that the rate of TERT promoter mutations in hepatocellular carcinoma are also very high,which is more than 55%.GABPA is GA-binding protein subunit alpha,as a transcription factor,functions as a DNA-binding subunit.It is also likely involved in activation of cytochrome oxidase expression and nuclear control of mitochondrial function.It has been verified that GABPA can selectively bind to mutant TERT promoter through the ETS motif created by mutation and active TERT expression in urothelial carcinomas of the bladder,melanomas and glioblastomas.The rate of TERT promoter mutation is quite high in hepatocellular carcinoma,but the mechanism of regulation of TERT carrying promoter mutations is still unknown.Whether GABPA could regulate of TERT through the same way above in hepatocellular carcinoma need to be studied.What’s more,P53 protein mutation is common in hepatocellular carcinoma,whether there is relationship among mutant P53,GABPA and TERT in hepatocellular carcinoma is worth to be further explored.Purpose:Our study aims to explore whether typical tumor suppress gene P53 GOF mutation could regulate the transcriptional expression of TERT carrying specific promoter mutation mediating by GABPA or together with GABPA.We try to find a new way to regulate TERT transcriptional expression in hepatocellular cancer,and provide theoretical evidences for new molecular targets for treatment.Methods:(1)To sequence the promoter of the TERT in four hepatocellular cancer cell lines by extracting genome DNA from four different hepatocellular cancer cell lines including HepG2,Huh-7,Hep3B2.1.7,PCL/PRF/5 and amplifying prompter of TERT with specific primers by PCR.Then sent the products to Beijing Genomics Institute for the sequencing step.Base on the sequence results,we selected the cell lines with C228Tmutation in TERT promoter.(2)The level of P53 protein in HepG2 and Huh-7 were detected by extracting total cellular protein and performing western blotting.(3)The effects of specially downregulating P53 by siRNA or upregulating WT/mutant P53 by plasmids on TERT expression:After transfecting P53 siRNA into both HepG2 and Huh-7 cell lines for 72h,collect cells and extract cellular protein and mRNA to determine the lever of P53 protein by western blotting and the lever of TERT mRNA by q-PCR.What is more,transfect WT or R175H P53 vectors into HepG2 and determine the lever of P53 protein and TERT mRNA48h later.(4)The effects of specially downregulating GABPA by siRNA on TERT expression:After transfecting GABPA siRNA into both HepG2 and Huh-7 cell lines for 72h,collect cells and extract cellular protein and mRNA to determine the lever of GABPA protein by western blotting and the lever of TERT mRNA by q-PCR.(5)Co-immunoprecipitation to detect the relationship between P53 and GABPA:Extract total cellular protein from both HepG2 and Huh-7 cell lines and perform co-immunoprecipitation respectively.(6)Dual-luciferase report assay to explore the effect of P53 in regulation of TERT promoter in Huh-7 cell line:Transfect P53 siRNA or/and GABPA siRNA into Huh-7 cell lines for 24h,following by transfecting the cells with TK plasmid and mutant TERT promoter plasmid,and collect cells to conduct dual luciferase reporter assay.Results:(1)Among these four different hepatocellular cancer cell lines,HepG2 and Huh-7 had C228T mutation at promoter of TERT,while Hep3B2.1.7 and PCL/PRF/5 were wide-type(WT)promoter at this locus.(2)Hepatocellular carcinoma cell line HepG2 has WT P53 protein,while cell line Huh-7 has Y220C mutant P53 protein.Both the WT P53 protein in HepG2 and the Y220C mutant P53 protein were highly expressed respectively and the level of mutant P53 was much higher than WT p53.(3)P53 knockdown increased transcriptional expression of TERT in HepG2,on the other hand,upregulating WT P53 decreased TERT expression and upregulating R175H mutant P53 increased TERT expression in HepG2.But P53 knockdown decreased TERT expression in Huh-7.(4)GABPA knockdown decreased transcriptional expression of TERT in both HepG2 and Huh-7.(5)GABPA protein bind to P53 protein in Huh-7,but in HepG2 there was no binding between these two proteins.(6)P53 or/and GABPA knockdown downregulated the luciferase activity of TERT promoter in Huh-7,and P53 and GABPA knockdown at the same time had stronger effect than knockdown individually.Conclusions:(1)Different hepatocellular carcinoma cell lines have different phenotypes of P53 protein,HepG2 has WT P53 and Huh-7 has Y220C mutant P53.The proteins are highly expressed in these two cell lines respectively.(2)The roles of different P53 from different hepatocellular carcinoma cells lines in regulating TERT expression.WT P53 negatively regulates the expression of TERT,while mutant P53 regulates TERT expression in opposite direction in hepatocellular carcinoma.(3)GABPA positively regulates the expression of TERT in hepatocellular carcinoma.(4)Mutant p53 protein can bind to GABPA protein to active the mutant promoter of TERT and upregulate the expression of TERT together in hepatocellular carcinoma. |
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