The Influence On Cell Proliferation By Small Interfering RNA Of RegIα Expression In Gastric Cancer Cell Lines

Gastric cancer is a very common carcinoma in the world, and remains the leading cause of cancer incidence and mortality in china. Because there are lack of special clinical symptoms in early staged patients, which may result in delayed diagnosis and treatment. The five-year survival rate of gastric cancer is nearly thirty percents. Chemotherapeutic Resistance and drug caused toxicity remain the major causes of failure in chemotherapy in middle or late staged patients. The regenerating gene (RegⅠα) is a new growth factor, which is mainly isolated in pancreatic islet and gastric mucosa in human being..RegⅠαprotein in Human contains of 166 amino acids. Studies showed that RegⅠαhad effect on cell proliferation and anti-apoptosis in gastric mucosa. It was reported overexpressed in gastric cancer,which was related with TMN stage. differentiation,lymph node metastasis and survival rate. Hence, Dysregulation of RegⅠαgene is closely related to the occurrence and progress in gastric cancer. It is a potential marker of target-gene therapy. Small RNA interfering (RNAi), which was described as the "scientific breakthrough of the year" in 2002,is a process involving post-transcriptional gene silencing triggered by double-stranded RNA(dsRNA). RNAi is now widely used in cancer studies, but few reports focused on the effect and mechanism of RegⅠαgene in gastric cancer. We therefore designed present study to discuss the possibility function in gastric cancer therapy by RNAi targeting the RegⅠαgene,which mainly in vitro study on the inhibitory effect of RegⅠαRNAi in high expression gastric cancer cells.Firstly, Real-time RT-PCR was used to detect the mRNA expression of RegⅠαin 6 gastric cancer cell line. Result showed that MKN45 and AGS had high expression of RegⅠαmRNA. On the basis of RNAi design discipline, We designed two strips target sequence, and synthesized DNA template single-stranded of short hairpin(shRNA), including the positive-sense strand and antisense strand of siRNA. GFP (green fluorescence protein) were successfully inserted into as vector, which had BamHI and ALF-II enzyme restricted sites.After constructing RNAi plasmid expression vector in vitro, Escherchia coli DH5αcells which contain the plasmid DNA and the negative control were planted on LB medium. Ampicillin-resistant colonies were cultured at 37℃over-night in a rocking bed, then the recombinant RegⅠαRNAi plasmid was prepared after 18hs. UA spectrop-hotometer tested the OD value,λ-HindⅢenzyme sites were observed to confirm plasmid DNA. The recombinant plasmid was proved by agarose gel electrophoresis and nucleotide sequence testing.MKN45 and AGS with a high expression of RegⅠαmRNA was then stably transfected with RegⅠαRNAi and negative recombination plasmids. In short, MKN45 and AGS cells (1×105) were inoculated in a 6-well plate and transfected when the confluence was about 80%. After 24 hours, the efficiency of transfection was analysed by fluorescence microscope.Twenty-four hours after transfetion, cells were diluted to 1:100 for 96-wells re-plate, cultured for three weeks in medium with G418(MKN45:600μg/ml and AGS: 400μg/ml). After three weeks, we selected the survived growth cell with monoclones,To further verify the stable transfection of RegⅠαRNAi plasmid, Regla mRNA and protein expression was measured by RT-PCR and Western blot respectively in stable transfected cell lines. RT-PCR results indicated that Regla mRNA expression was significantly inhibited by RNAi in both cell lines, when compared with empty-vector. Western blot results showed that Regla protein was downregulated to (44±4)% and (25±4)% respectively in MKN45 and AGS cells, when compared with empty-vector.To observe the influence on gastric cancer cells growth by RNAi RegⅠα, we used MTT assay for 4 days continuously after cell seeding. Results showed that cell growth was significantly inhibited in MKN45 and AGS cells with RNAi. Cells were observed to grow slowly in day 2, and the shape was mostly rod or spindle, partly triangular or irregular. It demonstrated that RNAi of Regla gene could inhibit cell growth. The cell apoptosis were detected with Annexin V-FITC/PI double labeling flow cytometry assay. The apoptosis rate was (12.96±0.5)% and (11.59±1.1)% respectively in MKN45 and AGS cells, when compared with which of (3.99±0.3)% and (4.22±0.4)% in empty-vector, had remarkably increased (P<0.05), It demonstrated that RNAi of RegⅠαgene could induce cell apoptosis.Summary:(1) Gastric cancer cell lines MKN45 and AGS have high Regla mRNA expression.(2) Small interfering RNA of Regla gene could efficiently downregulate Regla mRNA expression and protein expression in MKN45 and AGS gastric cell lines. (3) Small interfering RNA of Regla gene could inhibit cell growth and induce cell apoptosis in MKN45 and AGS cells.