| Gastric cancer is one of the most common malignant tumors worldwide. Up tillnow, the5-year survival rates for patients with advanced gastric cancer or withmetastases is stll very poor. The dead rate is at the head of the list of various cancer inprovinces near sea-side such as Fujian, Shangdong, Zhejiang and etc in China. Basedon a large number of literature and the previous work of our laboratory, in this study,immunohistochemistry, siRNA, gene transfection, RT-PCR and MTT assays wereapplied to determine the expression of MEK5in gastric cancer tissue and topreliminarily investigate the effect of MEK5on the biological behaviors of gastriccancer cells with the purpose to provide a foundation for understanding the potentialmechanism of MEK5’s action and discovering the possible therapeutic target of drug.In the first part of this study, whether MEK5expression in human gastricadenocarcinoma tissue exists difference between non-neoplastic and neoplastic hasbeen investigated. The paraffin embedded blocks for105cases of gastricadenocarcinoma tissue with different TNM stage and different differentiation grade,and the paired non-neoplastic tissue were collected, the expression of MEK5in thesesamples had been determined by immunohistochemistry with anti-MEK5antibody,and the relationship between MEK5expression and the clinical pathologicalcharacteristic of patients with gastric adenocarcinoma was then analyzed. The resultsshowed that MEK5was down-expression in71of105cases (low expression rate was67.6%) of cancer tissues, compared to the levels in non-neoplastic tissues (P=0.01)(Wilcoxon Signed Ranks Test). Down-expression of MEK5was not correlated withpatients’ age, nor with tumor size, TNM stage and lymph node metastasis, but relatedwith differentiation grade (p=0.047) and5-years survival of patients (p=0.012).In the second part of this study, three different siRNA template DNA sequencetargeting in MEK5gene were designed by the application of Genscript siRNA design software, and the corresponding DNA fragments were synthesized in vitro, were thencloned into the pRNAT-H1.1/Adeno shuttle vector with its two restriction enzymeMluI and XhoI sites to construct three targeted MEK5gene siRNA expression shuttlevector: pRNAT-H1.1/Adeno-MEK5siRNA, then transformed into Escherichia coliBJ5183carrying backbone plasmid pADEasy-1to obtain adenovirus plasmidpAD-MEK5siRNA through homologous recombination.The adenovirus plasmid wastransfected into293A cells to form adenovirus particle. The RT-PCR results showedthat pAD-MEK5siRNA could effectively suppress MEK5expression in gastricadenocarcinoma AGS cells with a suppression ratio which was up to more than90%.In the third part of this study, the effect of MEK5on the biological behaviors ofAGS cells was preliminarily investigated. The activity of cell proliferation before andafter transducted MEK5siRNA adenovirus was detected by MTT assay. The resultsshowed that the proliferation of AGS cells was increased after the expression ofMEK5had been silenced with siRNA. The analystical results of statistics indicatedthe difference was significant between AGS cells transduced with pAD-MEK5siRNAadenovirus and pAd-scramble siRNA adenovirus or wild type cells (P＜0.05). Thepreliminary results indicate knock-down MEK5might cause the changes of cellproliferation. The molecular mechanism of its action is still unclear, which needs tobe further investigated. |
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