| Gastric cancer is one of the most common malignant tumor of digestive system. The incidence of gastric cancer showed a downward trend in Europe and the United States, but in developing countries including China, the incidence has no obvious declining. Gastric cancer also have high mortality rates in developing countries, the 5-year survival rate is only 20%～25%, the median survival time was only 24 months.The major cause of extremely poor survival rate in gastric cancer patients is low of oearly diagnosis rate, high transfer rate, no treatment method is sensitive and effective, research on weak foundation. Therefore, an improved understanding of the molecular pathways involved in gastric cancer will be helpful in improving prevention, diagnosis and therapy of this disease. It can improve cancer patients’life quality and prolong survival time. MicroRNA(MiRNA) is a 22 nucleotide, single-stranded RNA molecules that play a crucial role in regulating gens expression including oncogene and tumor suppressor gene. Abberant expression of MiRNA was associated wih tumor proliferation, invasion and metastasis. Researcher showed that MiRNA has important value in prevention, diagnosis and therapy of gastric cancer, but the specific mechanism of miRNA in promoting occurrence and development of gastric carcinoma and its clinical significance remains to be further studied. We therefore for sought to systematically investigate the expression pattern of MiRNA in human gastric cancer, then study the function and molecular biology on the part of the key MiRNAs.Part I Investigate the expression pattern of MiRNA in gastric cancerObjective:Screening MiRNAs that are strongly associated with human gastric cancer, which will offer a new tool for the diagnosis of gastric cancer.Methords:We collected 162 different human gastric tumor entities and matched 62 non-cancerous human tissues. RT-PCR was uesd for the detection of differential MiRNAs expression between the two group. Analyed of the judgment of the tumor and non-cancerous tissues MiRNA expression profiling using biological information.Results:Twelve MiRNAs (hsa-miR-196a, hsa-miR-196b, hsa-miR-204, hsa-miR-133b, hsa-miR-148a, hsa-miR-375, hsa-miR-551b, hsa-miR-31#, hsa-miR-224, hsa-miR-31, hsa-miR-378d and hsa-miR-37) were identified significantly correlated with the incidence of gastric cancer, which can distinguish gastric cancer and normal mucosa, the effectiveness of 95.08%, specificity 97.56% (P< 0.005).Conclusion:Abnormal expression of MiRNAs were closely correlated with the occurrence of gastric cancer, suggesting that the MiRNAs can be used for the diagnosis of gastric cancer.Part II Research on hsa-miR-196a in gastric cancer cells functions and targetsObjective:To invstigate the function of hsa-miR-196a in promoting gastric cancer cell growth, invasion, and the possible molecular mechanisms.Methods:hsa-miR-196a-shRNA was constructed and transfected into SGC7901 and BGC823 gastric cancer cells., MTT, clone formation assay, scratch assay, invasion and metastasis assay were introduced to detect the down-regulation of miR-196a on gastric cancer cell functions. RT-PCR was used to detect the mRNAs levels of possible targets of miR-196a, including HOXA7, HOXC8, SLC9A6, ZMYND11, and CCDC47 after miR-196a was downregulated.Results:①RT-PCR results showed that hsa-miR-196a-shRNA can successfully interfere the expression of miR-196a in SGC7901 and BGC823;② When the expression of miR-196a in SGC7901 and BGC823 was inhibited, the ability of cell proliferation, clone formation, invasion, and metastasis were significantly decreased (P<0.05);③After downregulation of miR-196a in SGC7901 and BGC823,the HOXA7、HOXC8 and CCDC47 are decreased in the both cells, but the expression of SLC9A6、ZMYND11 in SGC7901 cells are increased, but decreased in BGC823 cells.Conclusion:MiR-196a may regulate the expression of HOXA7, HOXC8 and CCDC47, and then promote the gastric cancer cell proliferation, invasion and metastasis.Part Ⅲ The expression of HOXA7 and HOXC8 in human gastric carcinoma and its relationship with clinicopathological parameters and prognosis.Objective:To detect the expression of HOXA7 and HOXC8 in human gastric carcinoma and to analyze its clinical value.Methods:Using immunohistochemistry and tissue microarray, we detected the expression of HOXA7 and HOXC8 in human gastric carcinoma, and analysis the relationship between their expressions and clinicopathological parameters and prognosis.Results:①Compared with the adjacent normal mucosa tissue, the expression of HOXA7 and HOXC8 in gastric cancer tissue was significantly up-regulated, the expression rates were:55.6%(134/241) and 60.6%(146/241), respectively.② The high expression of HOXA7 was significantly correlated with the depth of invasion, and the high expression of HOXC8 obvious correlated with the tumor size, depth of invasion, lymph node metastasis, TNM staging. ③The relationship between the expression of HOXA7 and HOXC8 in human gastric carcinoma and prognosis: Univariate survival analysis showed that overall survival in gastric cancer patients with high expression of HOXA7 was 56 months,significantly lower than patients with low expression of HOXA7 whose overall survival was 75.6 months (P=0.003).The prognosis of gastric cancer patients with high expression of HOXC8 was also poorer than patients with low expression of HOXC8 (the median survival time 24.9 months V.S 90.0months, P<0.001). Multivariate COX regression analysis showed that the positive expression of HOXC8 is an important independent prognostic factor in gastric cancer patients.Conclusion:The overexpression of HOXA7 and HOXC8 play a certain role in the development of gastric cancer, and may become a potential target for molecular biological therapy of gastric cancer and a molecular markers for anticipating the prognosis. |
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