The Role And Mechanism Of MicroRNA-9/eIF5A2 Pathway In Invasion,Metastasis And Cisplatin Resistance Of Non-small Cell Lung Cancer

Aim:The purpose of this study is to validate the effect and mechanism of eIF5A2 gene in invasion,metastasis and cisplatin resistance of non-small cell lung cancer,and further explore the effect and mechanism of MicroRNA-9/eIF5A2 pathway in invasion,metastasis and cisplatin resistance of non-small cell lung cancer.Methods and method:1.Four NSCLC cell lines,A549、HCC827、NCI-H358、NCI-H1299,were selected and cultured.Using Western-blot and RT-PCR to detect the expression of eIF5A2,we selected NCI-H1299 cells of high expression of eIF5A2 and A549 cells of low expression of eIF5A2,inhibit the expression of eIF5A2 by RNAi interfering,to observe the changes of migration and invasion.2.We choose TGF-β1 to induced the EMT in A549 cells,inhibit the expression of eIF5A2 by RNAi interfering,and then detect the expression level of EMT related proteins and the ability of invasion and migration.3.We use GC7(eIF5A2 inhibitor)to inhibit the expression of in A549 and NCI-11299 cells,detect the changes of cisplatin sensitivity to the lung cancer cell,and then inhibit the expression of eIF5A2 in lung cancer cell lines by RNAi interfering,and detected the expression of EMT markers by Western-blot and immunofluorescence technique.4.We examined the expression of miR-9 by RT-PCR and transfected with miR-9 mimic and miR-9 inhibitor to determine the ability of migration and invasion by wound-healing assay and Transwell-Matrigel invasion assay in A549 and NCI-41299 cells.And we used Western Blot to detect E-cadherin and Vimentin expression and RT-PCR to detect the expression of eIF5A2.5.We transfected the lung cancer cells(A549、NCI-H1299、NCI-H358)with miR-9 mimic and miR-9 inhibitor,and then examined the expression of miR-9 by RT-PCR.The viability,proliferation and apoptosis level of NSCLC cells were detected by CCK-8 detection kit,EDU assay and flow cytometry after chemotherapy with cisplatin.Results:1.The expression of eIF5A2 in A549 cells was low,and the expression of eIF5A2 in NCI-H1299 cells was high.eIF5A2 siRNA plasmid transfection can significantly down regulate the expression of eIF5A2 which was positively related with the migration,invasion ability of lung cancer cells.2.We can reduce the migration and invasion ability of lung cancer cells by inhibiting the expression of eIF5A2 and inhibiting the EMT process induced by TGF-β1 in A549 cells3.We found that the expression of eIF5A2 in A549 and NCI-H1299 lung cancer cells is negatively correlated with the chemosensitivity of cisplatin,while eIF5A2 regulates the resistance of lung cancer cells to cisplatin by regulating the EMT process.4.We found that the high expression of miR-9 in A549 and NCI-H1299 lung cancer cells can down regulate the expression of eIF5A2 and reduce the migration and invasion ability of lung cancer cells by reversing the process of EMT.5.We found that the expression of miR-9 in A549,NCI-H1299 and NCI-H358 lung cancer cells was positively correlated with the chemosensitivity of cisplatin,and miR-9 could increase the sensitivity of lung cancer cells to cisplatin chemotherapy by down regulating the expression of eIF5A2.Conclusions:The results of this study showed that the expression level of eIF5A2 was positively correlated with invasion and migration of non-small cell lung cancer cells,and the cisplatin resistance was induced by upregulating the expression of eIF5A2 in lung cancer cells,which was closely related to the mechanism of EMT process.Further studies have shown that MicroRNA-9/eIF5A2 pathway can regulate the ability of migration and invasion and the cisplatin resistance in non-small cell lung cancer cells,and its mechanism may be closely related to the EMT process.