Screening Of MEN1 Mutants In Primary Hepatocellular Carcinoma And Its Protein Stability Studies

Primary hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the clinic,with 90%of it being hepatocellular carcinoma(HCC).Globally,its incidence is on the rise.Epidemiological statistics show that the incidence and mortality of hepatocellular carcinoma in China are among the highest in the world.Genetic mutations can lead to the change of protein structure and function,further studies have found increases in expression of MEN1 gene in HCC,and the expression of MEN1 associated with tumor size.A previous research of our lab,preliminary illustrates epigenetic role of the MEN1 gene in HCC and its expression level increased and the occurrence,development,invasion and metastasis of HCC is closely related to it.However,the complete role of MEN1 in HCC,has not been fully elucidated and remains to be further studied.We sequenced the genome of MEN1 gene of cancer tissue samples from 49 patients with hepatocellular carcinoma(HCC)using the second generation sequencing,found that compared MEN1 sequence with wild type,there are several kinds of single-nucleotide-change in exon 9 and exon 10 including SNPS,synonymous mutations and missense mutations.Through bioinformatics mining,we preliminarily studied the effect of three missense-point mutations on protein structure and stability,and predicted its correlation.We further constructed MEN1 wild type and point mutation plasmid on the slow virus vector,and stable transfect MEN1 knockout mice fibroblast cells and then stable express wild type and mutant menin,and then study the stability of the mutants.We collected tissue from hepatocellular carcinoma patients and healthy blood samples to extract blood DNA and then sequence and carry on the statistics.The characteristics of the mutants,inner connections of them,interrelationship of mutations and clinical symptoms were analyzed,hoping better for the prevention,diagnosis,treatment and prognosis of hepatocellular carcinoma(HCC).Through the study,we found that:1.The three MEN1 point mutations we screened were not reported in liver cancer previously.2.According to the sequencing results,it is shown that MEN1 mutations in hepatocellular carcinoma is concentrated in exon 9 and exon 10,which may be the hotspots of MEN1 mutation in hepatocellular carcinoma.3,by building a stable transfection cell line,we found that correlation between mutant protein levels has little correlation with copy number and transcription.Mutant protein levels vary,but are higher than the wild type.4,According to the protein structure prediction,the protein stability experiment(CHX-chase)and MG 132 experiment,we found that three mutant compared with wild type:G503D stability is similar to that of the wild type,L551P has poor stability,and the C-terminal and N-terminal stability of P595L is different and the incidence of mutations in liver cancer tissue and blood and normal blood differs a lot.In summary,there may be hot spots in MEN1 mutation in hepatocellular carcinoma;MEN1 missense mutation affects the expression level and stability of protein,the menin C-terminal plays an important role in protein stability.The relationship between missense mutation and clinical manifestation,and the distribution of certain point mutations in different parts and populations may become potential target of early diagnosis and treatment of tumors.