The Mechanism Of Drosha Gene Silencing Regulating The Development Of Gastric Cancer Cells

Part 1 The effect of Drosha gene silencing on Biological function of gastric cancer cellsObjective To explore the effect of Drosha gene silencing on Biological function of gastric cancer cells. Methods The gastric cancer cell with Drosha silencing and its control cells were established by lentivirus infection. After treatment with puromycin, the interference efficiency of Drosha in gastric cancer cells were approved by quantitative real-time PCR and Western blot analysis; The cell cycle and cell apoptosis were examined using flow cytometry; MTT assay were used to detect the affect of Drosha silencing to gastric cancer cell proliferation; Wound-Healing and transwell assay were detected cells migration; The sensitivity of MGC-803 with Drosha silencing and its control cells to epirubicin were detected through FCM. The protein expression of caspase3. caspase9. Bax and Bcl-2 were determined by Western blot. Results The gastric cancer cells with stable Drosha down-regulation and its control cells were established. Compared with NC-sh RNA cells, the proliferation of Drosha-sh RNA cells was not significantly different. And Drosha gene silencing could decrease themigration of gastric cancer cells. Meanwhile, the sensitivity of Drosha-sh RNA MGC-803 to epirubicin was significantly increased; the protein expressions of caspase3, caspase9 and Bax was significantly upregulated and Bcl-2 was downregulated after treatment with epirubicin when compared with the control cells. Conclusion We have successfully established a gastric cancer cell model with stable Drosha down-regulation, and certified that downregulated Drosha in Gastric cancer cells decreased migration and enhanced its sensitivity to epirubicin.Part 2 The molecular mechanism of Drosha gene silencing on gastric cancer cell migrationObjective To investigate The molecular mechanism of Drosha gene silencing on gastric cancer cell migration. Methods Mi RNA expression profiles of Drosha silencing cells compared to control analyzed with micro RNA microarray; Five of randomly selected mi RNA were analyzed by q RT-PCR; The potential target genes of the mi RNAs were predicted through Target Scan v6.2; KEGG pathways enriched by mi RNA target genes. Luciferase reporter assay and RT-PCR confirmed direct target of mi RNA-622 and mi RNA-197. Real time-PCR and Western blot were used to examine the expression of target genes; the prognostic value of target genes for gastric adenocarcinoma patients was assessed by Kaplan-Meier analysis; Target genes expression in gastric cancer tissues were detected by immunohistochemistry staining and RT-PCR. Western blot detected targetgene expression in the gastric cancer cell lines; silencing and over-expression target gene in MGC-803 gastric cancer cell; cell migration was detected by Transwell assay, the protein expressions of downstream signaling pathways were determined by Western blot. Results micro RNA microarray data showed 61 dysregulated mi RNAs(fold change≥2), which 47 up-regulated and 14 down-regulated; The Enriched KEGG pathways indicated that target genes involved in ECM, p53, m TOR signaling pathways; Luciferase reporter assay and RT-PCR confirmed LAMC2 and CD82 are respectively direct target of mi RNA-622 and mi RNA-197; RT-PCR and Western blot revealed that LAMC2 expression was decreased and CD82 increased after Drosha silencing; Kaplan survival curve analysis showed the survival rate in LAMC2-positive patients was significantly lower than the negative group, and in CD82-positive patients was significantly higher than the negative group; Immunohistochemistry staining and RT-PCR results confirmed that LAMC2 expression increased and CD82 expression decreased compared to normal adjacent tissues; Transwell experiment indicated that LAMC2 could enhance and CD82 inhibited gastric cancer cell migration, Western blot showed LAMC2 lowexpression and CD82 overexpression could suppress EGFR-ERK1/2 –MMP7 signaling pathway. Conclusion: Drosha silencing caused mi RNA-622 and mi RNA-197 dysregulated and respectively direct target LAMC2 and CD82 reduce gastric cancer cell migration via inhibitEGFR-ERK1/2-MMP7 signaling pathway.