Construct Of Gastrin-shRNA Vector And Its Effects On Gastric Cancer Cell Line BGC-823

Gastric cancer is one of the leading causes of cancer death in the world. Although the incidence and mortality of gastric carcinoma are decreasing in many countries, gastric cancer still represents the most frequent malignancy in the world. As a kind of polypeptide hormone, the major site of gastrin synthesis and secretion is the gastrin-containing cell (G cell) in the antropyloric mucosa. Gastrin is now firmly established as the physiological regulator of gastric acid secretion and an important regulator of gastric mucosal cell proliferation. Gastrin has been shown to be a growth factor for gastric cancer in established BGC-823 cell lines. Gastrin has been found to potentialize the movement of gastric cancer BGC-823 cell lines and to facilitate the metastasis of cancer cell. In addition, gastrin promotes cellular multiplication and suppresses cellular apoptosis by its receptor mediated the signal transduction. Therefore, gastrin gene may provide a potential target for genetic therapy of gastric cancer.RNAi is an innate cellular process activated when a double-stranded RNA (dsRNA) molecule enters the cell, causing the degradation of not only the invading dsRNA molecule, but also single-stranded (ssRNAs) RNAs of identical sequences, including endogenous mRNAs. The Double-stranded RNA in cell is diced-up a class of 20-25 nucleotide long RNA (siRNA) by the enzyme Dicer. The siRNA degrades its homologous mRNA to interfere with the gene expression by mixing with and activating multicomponent nucleases. RNAi technology is currently being evaluated not only as an extremely powerful technology for functional genomic analysis, but also as a potentially useful method to develop highly specific dsRNA based cancer therapeutics. RNAi has been successfully used for many human tumors study, including colon cancer, liver cancer, ovary cancer, and et al. Little research in the field of gastric cancer using this technique has been reported yet. Because of the time limits of synthetic siRNA, construction of shRNA expression vector and establishment of gene knok-down cell line has been a tendency in this field.Based on the previous study, the gastrin-shRNA vector was constructed. The vector contains neo resistance gene and GFP gene. After transfection, the transfected BGC-823 cell was screened by G418, and established gastrin-shRNA vector stable transfected gastric cancer cell line. The suppressing efficiency of gastrin gene on mRNA level was detected by RT-PCR. The effects of gastrin-shRNA vector on BGC-823 cell were demonstrated by Flow Cytometry and MTT. The results of this study might provide an experimental basis for further study of the molecular mechanism of the effects of gastrin on the development of gastric cancer.Material and methods1. Reconstruction plasmid pSUPER-EGFP-NRG Since the Bgl II wasdestroyed in the pSUPER-EGFP-NRG, the vector was reconstructed in which contained Bgl II site and no NRG coding sequence. Based on the sequence of pSUPER-EGFP-NRG, a pair of primers containing Bgl II site was designed. The pSUPER-EGFP-NRG DNA was used as template for PCR, and the product was subcloned into pSUPER-EGFP-NRG to construct vector pSUPER-EGFP-I. The reconstructed vector was identified by enzyme digestion and sequencing.2. Construction gastrin-shRNA expression vector A pair of complementary oligonucleotide fragments coding gastrin-shRNA were designed and synthesized. The annealed fragment containing Bgl II and HindIII sticky end was judged by 2.0% agarose gel electrophoresis and subcloned into pSUPER-EGFP to construct gastrin-shRNA vector pSUPER-EGFP-G. The constructed vector was identified by enzyme digestion and sequencing.3. Cell culture and transfection BGC-823 human gastric cancer cell line was cultured in RPMI—1640 supplemented with 10% heat-inactivated FBS, 100μ/ml penicillin and 100μg/ml streptomycin in a 37℃, 5 % CO₂ incubator. pSUPER-EGFP-G and pSUPER-EGFP-I were purified by non-endotoxin group plasmid purification kit and transfected with Lipofectamine^? 2000 Transfaction Reagent. BGC-823 cells were randomly divided into experimental group (pSUPER-EGFP-G transfection), control group (pSUPER-EGFP-I transfection) and blank group (no plasmid transfection).4. Screening and proliferation The transfected cells were screened with RPMI-1640 supplement with 400μg/ml G418. About two weeks, the formed positive clones were inoculated into 24 culture plate. When the cells was 80%-90% confluence, the cells were inoculated into culture bottles.5. Effects of gastrin-shRNA vector on gastric cancer cell line BGC-823 The gastrin expression on mRNA level was detected by using RT-PCR. The alterations of cellular proliferation and cell cycle were demonstrated by using MTT and FCM.6. Statistical analysis SPSS10.0 statistical software was used to analyze experimental data. a= 0.05 was selected as test level.Results1. Construction of gastrin-shRNA expression vector The results of HindIII/EcoR Irestriction map and sequencing of inserted sequence of pSUPER-EGFP-G were same to anticipation.2. The results of RT-PCR The length of amplified products of gastrin andβ-actin was 207bp and 300bp respectively. The ratio of gastrin/β-actin in three groups was 1.09±0.011 (blank group), 1.074±0.016 (control group) and 0.155±0.012 (experimental group) respectively. Significant difference was found between experimental group and blank group (P < 0.01) . There was no obvious difference between control group and blank group (P>0.05) . The results indicated that gastrin-shRNA vector could effectively inhibit expression of gastrin in gastric cancer cell line BGC-823. 3. Results of MTT Survival rate of three groups was 0.988±0.042 in blank group,0.958±0.033 in control group and 0.734±0.026 in experimental group. Compared with blank group, the cellular proliferation in experimental group was significantly inhibited (q=15.426, P<0.05). The results indicated that the proliferation of gastric cancer cell line BGC-823 was obviously inhibited by gastrin-shRNA vector.4. FCM Detection In experimental group, the percentage of G₀/G₁ cells wasincreased from 59.5%in control group to 62.7%, G₂/M cells from 10.1% to 17.9%. The percentage of S cells was decreased from 30.4% in control group to 19.4%.Conclusion1. The gastrin-shRNA expression vector pSUPER-EGFP-G is successfully constructed.2. The gastrin gene knock-down gastric cancer cell line is successfully established.3. The gastrin-shRNA expression vector can inhibit the proliferation of gastric cancercell line BGC-823 via silence the gastrin gene.