The Construction Of The Recombination Eukaryotic Cell Expression Plasmid Of Human FHIT And A Study On The Intervention Of That On The Proliferation, Apoptosis And Invasive Ability In Human Cholangiocarcinoma Cell Line

Introduction The construction and transfection of eukaryotic expression plasmid are commonly molecular biology method used in medical research. And the occurrence of tumor is closely related to oncogenes and anti-oncogenes. FHIT is an important tumor suppressor gene, in a variety of tumors expression was found missing. Cyclin D1 gene is a common oncogene. It can promote tumor cell through the cell-cycle restriction point like G1/S. The coadjustment of them, especially the intervention of FHIT on the proliferation, apoptosis and invasive ability in human cholangiocarcinoma cell become the focus of this study.Objective Through the construction of recombinant human fragile histidine triad (FHIT) gene eukaryotic expression plasmid, to discuss the effect of FHIT gene on the proliferation, apoptosis and invasive ability of cholangiocarcinoma cell line - QBC939. And the regulation functions of FHIT gene on Cyclin D1 was studied further.Methods Genomic RNA of the fresh specimens'tissue was extracted by Trizol for RT-PCR. The product above and the pcDNA 3.1 vector were purified and connected by double digestion with BamH I/Xho I. All of above, the recombinant eukaryotic expression plasmid was constructed and sequence identification. Then FHIT-pcDNA 3.1 was transfected into QBC939 by lipofectamine instant method and selected neomycin-resistant clones to identified by real-time RT-PCR. Experimental subject was randomly divided into three groups: natural control group (NCG), blank control group (BCG) and experimental group (EPG). Their situations of proliferation were observed using the MTT method and the apoptosis were detected by Flow Cytometer (FCM). The invasive ability was determined by Transwell chamber test. The expression of Cyclin D1 mRNA was detected by real-time RT-PCR and the protein was detected by Western Blot. All of data were analyzed by one-way ANOVA, using Bonfferoni test or rates by chi-square test.Results Human FHIT recombinant eukaryotic expression plasmid was constructed successfully and identified through sequencing. Real-time RT-PCR detection of transfected QBC939 showed that FHIT gene expression was restored limitedly. Experimental group compared with two control groups showed QBC939 cell proliferation was inhibited (P<0.05) and the apoptosis was promoted. The amount of cells which passed the filter was significantly less. At the same time, the expression of Cyclin D1 in gene and protein level was down-regulated (P<0.05).Conlusion The human FHIT recombinant eukaryotic expression plasmid was constructed successfully. FHIT gene can interfere proliferation and apoptosis of QBC939, weaken the invasive ability and reduced the expression level of Cyclin D1. Molecular biology tool and direction were provided for further fundamental and clinical research of cholangiocarcinoma.