| BackgroundGastric cancer is one of the most common malignant tumors of digestive tract in the world. Asia, Japan,South Korea and China is high incidence area of gastric cancer, in China each year about 400 thousand new cases, accounting for 42% of the worldwide total number of the cases. The 5 year survival rate of early gastric cancer patients after operation is more than 90%, but the vast majority of gastric cancer belongs to advanced stage when diagnosed, or has undergone tumor metastasis, at this period the 5 year survival rate is less than 30%. To reduce the death rate of gastric cancer, the most effective measure is early detection and early treatment. The development of molecular biology provides advanced technical means and methodology for the study on the mechanism of gastric cancer, and has important guiding significance for treatment of gastric cancer.The miRNA is a conserved non coding small molecule RNA about 18-22 nucleotides long, miRNA at the transcriptional or post transcriptional level can negatively regulate the expression of the protein coding gene: through a combination of incomplete complementary or approximately complete complementary of m RNA with its target gene, cause m RNA degradation or inhibit its translation. Since miRNA was discovered, many studies have reported the abnormal expression of miRNA in tumor tissues, and it is confirmed that miRNA has a significant correlation with some tumor suppressor factors and tumor promoting factors. PurposeIn this study,By detecting the differential expression of miRNA in gastric cancer tissue and adjacent tissues, the correlation between the expression level and tumor size, TNM stage and metastasis was discussed.The bioinformatics methods were used to do the prediction, the target gene which has the highest correlation with gastric cancer invasion and metastasis has been selected out, and the topic about how it affects the proliferation and invasion of gastric cancer cells through the target gene has been discussed. Method(1) In this study, three pair samples of gastric cancer and adjacent tissues were detected, the expression level of 27 miRNA screened by previous step in gastric cancer tissues and adjacent tissues was verified by q RT-PCR method.(2) In order to explore the relationship between miRNA and gastric cancer occurrence and development, the miRNA expression situation of miR-223, miR-20 a and miR-150 in metastatic and non metastatic gastric cancer samples has been analyzed, and the patient survival situation has been counted. The expression of miR-223,-20 a and-150 in 84 primary metastatic gastric cancer samples and 48 non metastatic gastric cancer samples has been done fluorescence quantitative PCR detection in real time.(3) In order to detect the effect of miR-223 on the proliferation of gastric cancer cells, GT3 TKB and MKN45 were selected as the research objects. First, the original expression levels of miR-223 in GT3 TKB and MKN45 were measured. Next, in GT3 TKB and MKN45 cells chemically synthesized miR-223 mimics and inhibitor RNA were respectively transfected. Secondly, GT3 TKB and MKN45 cell lines were transfected with mimics miR-223 and inhibitor, respectively. Finally, the wound healing experiment of transfected cells was done.(4) In order to search for the target gene of miRNA-223, using bioinformatics tools to predict and according to the experience rules and published research results, the most relevant target gene associated with the invasion and metastasis of gastric cancer PAX6 has been screened out. The miR-223 mimics and miR-223 inhibitor were transfected in GT3 TKB and MKN45 cell lines respectively, after the transfection, the total RNA of GT3 TKB and MKN45 as well as the corresponding control group cells were extracted, the transcription level of PAX6 was detected by RT-PCR method.The expression level of PAX6 was detected by RT-PCR method, and the expression of PAX6 was detected by blot Western method. The expression levels of miR-223 and PAX6 were detected by q RT-PCR method and blot Western method in 132 cases of gastric cancer, and which correlation was analyzed. Results(1) The differentially expressed miRNA in gastric cancer cells screening study has been done, the result showed that 13 expression of miRNA in gastric carcinoma was significantly up-regulated, 14 miRNA expression was down regulated in gastric cancer. the further validation results showed that there were 21 miRNA were consistent with the chip experimental results, 10 of them were highly expressed in gastric cancer, and 11 were low expressed in gastric cancer. There was no statistical significance of the difference in the expression of 6 miRNA in gastric cancer and adjacent tissues. Among them, 3 miRNA with the most significant differences were miR-223, miR-20 a and miR-150, respectively.(2) The abnormal expression situation of miRNA in metastatic gastric cancer and the patient survival analysis has been done. And the result showed that the expression of miR-223 was significantly correlated with tumor size and TNM stage(P<0.05),the correlation between miR-223 expression and metastasis was very significant(P<0.01), Mi R-20 a was significantly correlated with TNM stage(P<0.05), the correlation between miR-20 a and metastasis was very significant(P<0.05). Then the expression of miR-223 in gastric cancer samples in 76 patients with severe histological characters was analyzed, the correlation between miR-223 in these samples and survival rate was analyzed by Kaplan-Meier survival analysis, the result showed that the disease free survival rate of miR-223 low expression group was significantly higher than that of miR-223 high expression group. These results indicate that the expression of miR-223 is related to the development of gastric cancer.(3) The biological behavior study of miR-223 to migration and invasion of gastric cancer cells has been done. The experiment showed that the expression level of miR-223 of high invasive ability MKN45 was significantly higher than that of MKN45(p<0.01). GT3 TKB proliferation activity was significantly increased at the third day after being transfected with miR-223 mimics, and on the contrary, MKN45 proliferation activity was decreased at the third day after being transfected with miR-223 inhibitor. By transfection of miR-223 mimics, up regulate endogenous miR-223 expression of GT3 TKB, under the same multiple microscope, it can be observed that GT3 TKB cells transfected by mimics through the matrigel invasion chamber was more intensive than the GT3 TKB cell of control group, and quantity also increased significantly(150 : 100; P<0.01),which indicated the increased invasion ability. On the contrary, using miR-223 inhibitor down regulate the miR-223 expression of MKN45, the number of cells through the matrix glue was less than that of the control group(150 : 200; P<0.05), which indicated the decreased invasion ability. GT3 TKB that transfected with miR-223 mimics was cultured for one day, the wound healing situation was significantly better than that of the control group, which showed that miR-223 improved the healing rate of GT3 TKB, that also called transfer capability. On the contrary, MKN45 that transfected with miR-223 inhibitor was cultured for one day, the wound healing situation was significantly worse than that of the control group, which indicated that miR-223 played an important role in the wound healing of MKN45.(4) The miR-223 promotes the molecular mechanism research of migration and invasion of gastric cancer cells. The result showed that the PAX6 transcription level of GT3 TKB cell line transfected with miR-223 mimics was significantly lower than that of the non transfected GT3 TKB control group, and the PAX6 transcription level of MKN45 cell line transfected with miR-223 inhibitor was higher than that of the control group MKN45. Western blot experiment also showed the consistent result: expression amount of PAX6 in gastric cancer cells with upregulated miR-223 decreased. Conclusion(1) There are a total of 21 differentially expressed miRNAs in gastric cancer tissues, miRNA with most significant differences is mir-223, miR-20 a and miR-150.(2) The miR-223 expression level was significantly correlated with tumor size, TNM stage, and significantly correlated with metastasis status; miR-20 a was significantly correlated with TNM stage, and significantly correlated with metastasis status; miR-150 only significantly correlated with TNM stage. Further analysis showed that the high expression of miR-223 was related to the severity of the histological characters and the low survival rate.(3) The miR-223 can promote the proliferation of gastric cancer cells, and promote the invasion and metastasis of gastric cancer cells.(4) PAX6 the target gene of miR-223 in gastric cancer cells.(5) The miR-223 can enhance the proliferation and invasion ability of gastric cancer cells by down regulating PAX6. Therefore, miR-223 and PAX6 may be the target of gastric cancer treatment. |
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