| Traditional treatment for gastric cancer involves a combined approach of treatment types, including surgery chemotherapy and/or radiation therapy. Among these methods, Surgical resection is still the primary curative treatment for gastric cancer. Howerver, Conventional cancer treatments often have significant side effects. Thus, new therapeutic strategies are urgently needed. People have been exploring new methods of cancer treatment. Gene therapy has been recognized as a promising treatment method for cancer. With advances in molecular biology techniques and a greater understanding of the mechanisms in tumor pathogenesis,a variety of genes have been identified as molecular targets for gene therapy.RNA interference (RNAi) is the process of sequence-specific and post-transcriptional gene silencing(PTGS) initiated by double-stranded RNA (dsRNA). RNAi phenomenon is found in various organisms, such as plant, C.elegent , fungi and mammals. A number of technologies have been used in the study to mediate the down-regulation of gene expression. For example, anti-sense oligonucleotides and ribozymes. RNAi works much more efficiently than anti-sense oligonucleotides and ribozymes. As a highly specific gene knockout technology, RNAi not only provide a powerful tool in functional genomics ,but also show good prospects in gene therapy.In this work, Cyclin D1 highly expressed in gastric cacer was selected as the target gene. The RNAi technology was used to silence Cyclin D1 gene to observe its effect against gastric cancer.Objective: To evaluate the effect of small interferring RNA(siRNA) targetingCyclin D1 gene on the expression of Cyclin D1 and on the proliferation human gastric carcinoma SGC7901 cells.Methods:1.According to Cyclin D1 cDNA sequence in genebank ,two target sequences were designed and synthesized. After annealing, oligonucleotides were inserted into pGenesil-1 vector,the recombinant plasmid was named pGenesil-/C1 and pGenesil-1/C2, respectively.The recombinat plasmid was identifid by restriction endonuclease and DNA sequencing. At the same time,the eukaryotic expression vector pGenesil-1/HK was constructed as the negative control.2.Culture and transfection of SGC7901 cells. The cultured SGC7901 cells were divided into experiment groups(C1 group and C2 group),negative control group(HK group) and blank control group(NC group). The recombinant palsmids of pGenesil-1/C1,pGenesil-1/C2 and pGenesil-1/HK were transfected into cells in C1,C2,and HK group respectively.3. Following 48 hours, pGenesil-1/C1,pGenesil-1/C2 and pGenesil-1/HK were transfected into SGC7901 cells, and Western blot was carried out to detect the expression of the Cyclin D1 protein in groups.4.MTT was used to assay antiproliferative effects in vitro. At the time of 0h,12h,24h,36h,48h,72h,the enzyme marks instrument was used to evaluate absorbency (A) value at 490nm and the growth curves were drawn.Results1.By restriction endonuclease , eukaryotic expression plasmid of Cyclin D1 was proved to be successfully constructed. Sequence analysis of insertedfragment revealed the same sequence as synthesized oligonucleotides.2.Results of Western blot: The results showed no significant difference in Cyclin D1 expression in the blank control group and negative control group(The CyclinD1 / GAPDH protein western blot gray scale ratio was 1.01±0.04 and 0.98±0.01), but under the action of pGenesil-/C1, Cyclin D1 expression of SGC7901 cells significantly decreased (the CyclinD1 / GAPDH protein gray scale ratio was 0.58±0.19) compared with that in negative control group and blank control group(P<0.01).Under the action of pGenesil-/C2, Cyclin D1 expression also slightly decreased (the CyclinD1 / GAPDH protein gray scale ratio was 0.95±0.01), and there was no significant difference with that in negative control group, but there was difference with that in blank control group(P<0.05). There was no significant difference in GAPDH expression between groups, suggesting that the effect of pGenesil-/C1,pGenesil-/C2 on Cyclin D1 was specificly inhibited.3.The results of MTT: Compared with blank control group and negative control group , the cell absorbencies of C1 and C2 group were significantly decreased . The proliferation of transected SGC7901 cells in C1 group and C2 group was inhibited (P<0.01).Conclusions:1.The Cyclic D1-siRNA expression vector has been successfully constructed.2.Both pGenesil-/C1 and pGenesil-/C2 can inhibit Cyclic D1 gene expressing.3.After recombinant plasmid pGenesil-/C1 and pGenesil-/C2 were transfected into SGC7901 cells, the growth of the cells was inhibited significantly.4.These results suggested that a strategy based on RNAi targeting to CyclinD1 may build the foundation for the gene therapy of gastric cancer.
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