| Objective Micro RNAis closely related to the inflammation induced by Helicobacter pylori infection, and Helicobacter pylori infection is the very important cause of gastric cancer. Micro RNAcan regulate the expression of inflammatory gene through the combination of the 3 ’-UTR regionof target inflammatory gene, and then influence the activation of inflammatory pathway and the expression of inflammatory factors, thus control and participatemany pathological processes in the occurrence and development stage of tumor. The IL-1 family is a very important kind of inflammatory cytokines, but at present, the research on IL-1F5 is relatively less, especially associated with gastric cancer. Acorrding to the target gene prediction, we found that the target binding sites of mi R-197 may exist in the 3 ’-UTR region of IL-1F5. This research mainly about the preliminary verification of the functions and relations between the mi R-197 and IL-1F5, the chronic inflammatory gene related to gastric cancer.Methods According to the resullts of target gene prediction and the research on the correlation between the SNP in 3 ’-UTR region of IL-1F5 and the genetic susceptibility of gastric cancer, an over expressed vectorp Genesil-1-mi R-197 was conctructedusingartificial cloning method; and then transfect the cell lines of human gastric cancer SGC-7901 and BGC-823, with the p Genesil-1, mi R-197 inhibitor and mi RNA inhibitor N.C together. After transfection, the total RNA and protein was extracted from the cells of each group. Then, q RT-PCR was used to detect the expression of mi R-197, U6, IL-1F5 and GAPDHin each group, and Western Blot was used to detect the protein expression of IL-1F5 and GAPDHin each group. The t-test was used to analysis the difference of the relative expression in each group, and the test standardα was 0.05. All experiments were independently repeated 3 times, and the results were showed use mean±standard deviation. The statistical software used was SPSS 21.0.Results(1) The over expressed vector of mi R-197, p Genesil-1-mi R-197, was identified use the single and double enzyme digestion method, and was found that the sizes of enzyme fragments were completly consistent with theoretical prediction; the concordance rate of sequence comparison analysis about the sequencing resultswas 100%. Thus, the vector was constructed successfully.(2) In the human gastric cancer cell line SGC-7901 and BGC-823,the relative expression of mi R-197 of thep Genesil-1-mi R-197 group and p Genesil-1 group was 3.55±0.032 and 0.77±0.072(P< 0.001), 3.07±0.056 and 0.94±0.030(P< 0.001), respectively; the relative expression of mi R-197 of the mi R-197 inhibitor group and mi RNA inhibitor N.C group was 0.33±0.025 and 0.59±0.069(P = 0.003),0.62±0.035 and 0.84±0.015(P = 0.001), respectively; all the differences were statistically significant. The relative expression of IL-1F5 of the p Genesil-1-mi R-197 group and p Genesil-1 group was 0.26±0.051 and 0.39±0.042(P = 0.033), 1.01±0.087 and 1.37±0.10(P = 0.01), respectively; the relative expression of IL-1F5 of the mi R-197 inhibitor group and mi RNA inhibitor N.C group was 3.23±0.098 and 0.72±0.063(P< 0.001),2.94±0.17 and 1.97±0.061(P = 0.001), respectively; all the differences were statistically significant.(3) In the human gastric cancer cell line SGC-7901 and BGC-823,the relative expression of protein IL-1F5 of the p Genesil-1-mi R-197 group and p Genesil-1 group was 0.09±0.025 and 0.30±0.103(P< 0.001), 0.18±0.004 and 0.48±0.013(P< 0.001), respectively; the relative expression of protein IL-1F5 of the mi R-197 inhibitor group and mi RNA inhibitor N.C group was 0.46±0.025 and 0.38±0.013(P= 0.006),0.65±0.021 and 0.38±0.027(P< 0.001), respectively; all the differences were statistically significant.Conclusions The over expressed vector of mi R-197, p Genesil-1-mi R-197, was constructed successfully. The vector also transfected the human gastric cancer cell line SGC-7901 and BGC-823 successfully, and after transfection, the vector upregalated the expression of mi R-197 in the gastric cancer cells.The recombinant plasmid, p Genesil-1-mi R-197, down regalated the expression of IL-1F5 after transfected the human gastric cancer cell line SGC-7901 and BGC-823. The mi R-197 inhibitor upregalated the expression of IL-1F5 after transfected the human gastric cancer cell line SGC-7901 and BGC-823. The research indicates that the IL-1F5, inflammatory gene, may be the target gene of mi R-197, and mi R-197 may negative regulate the expression of IL-1F5. |
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