MiR-26a Enhances Metastasis Potential Of Lung Cancer Cell Via JAK2/STAT3 Pathway By Targeting ITG?8

Objective: Lung cancer is a cancer with the highest number of new cases and deaths cases among malignant tumors in the world.It was easy to metastasis.The prognosis of lung cancer was poor,and the 5-year survival rate was less than 1/3.80% of lung cancer are non-small cell lung cancer(NSCLC).MicroRNAs(miRNAs)are a class of 20-22 nucleotides RNA which post-transcriptionally regulate gene expression in various cancers,including human lung cancer.Many studies have shown miRNA-26 a affect the metastasis and invasion of cancer cells,however,its role in lung cancer metastasis remains unclear.In the present study,we investigated the effect of miR-26 a on proliferation,migration and invasion of lung cancer cell,both in vitro and in vivo.We also investigated the underling mechanism.Our study provided useful information on miR-26 a as a novel theraputic target for lung cancer treatment.Methods: 1.Using transient transfection ways to achieve that miR-26 a were overexpressed or reduced expression in A549,H1299 and H661 lung cancer cell line,and then through the CCK-8,wound healing assay and Transwell assay study the effect of miR-26 a in proliferation and metastasis ability of lung cancer cells in vitro.2.We used bioinformatics software to analyze the target genes of miR-26 a,and chose ITG?8 as the target of miR-26 a.We constructe the 3'-UTR firefly fluorescence plasmid of ITG?8,then verify whether ITG?8 was the target gene of miR-26 a from the mRNA level and protein level using Dual-luciferase reporter gene assay and Western Blot assay.3.Overexpressed and knockdown the ITG?8 gene in A549,H1299 and H661 lung cancer cells to study the influence of ITG?8 on lung cancer cell metastasis by using wound healing assay and Transwell assay.4.The gene correlation between ITG?8 and miR-26 a in lung cancer tissues,and whether there was negative feedback regulation in A549,H1299 and H661 lung cancer cells on miR-26 a in ITG?8 was detected by RT-PCR.5.The effects of miR-26 a on metastatic genes(CD44 and MMP-9)and EMT-related proteins(E-cadherin and Snail)were detected by RT-PCR and Western Blot assay.We detected the influence of miR-26 a on JAK2/STAT3 pathway by Western blot.6.Using slow virus transfection miR Control and miR-26 a to A549 cell line,and using puro drug to screening A549-miR-Control-luc and A549-miR-26a-luc cell lines.Then we constructe subcutaneous transplantation tumor model in mice,using a Fluorescent imaging system for small animals to analysis tumors and metastases of fluorescence intensity,then evaluate the effect of miR-26 in growth and metastasis ability of lung cancer cells in vivo.Results: 1.miR-26 a has no effect on the proliferation of extracorporeal lung cancer cells,and increases the invasion and metastasis ability of lung cancer cells in vitro.2.ITG?8 was the target gene of miR-26 a.3.ITG?8 can inhibit the invasion and metastasis of A549,H1299 and H661 lung cancer cells.miR-26 a and ITG?8-overexpression plasmid co-transfection assay results show that ITG?8 can partially weaken the effect of miR-26 in metastasis ability on lung cancer cells.4.As the target of miR-26 a,the expression of miR-26 a was negatively correlated with the expression of ITG?8 in lung cancer tissue samples.In lung cancer cells,ITG?8 can negatively regulate the expression of miR-26 a.5.miR-26 a can promote the expression of CD44 and MMP-9 gene,and promote the EMT process of lung cancer cells.miR-26 a promotes the expression of p-JAK2,p-STAT3 and MMP-2 protein in JAK2/STAT3 pathway to promote the effect of lung cancer cell metastasis,while ITG?8 can reduce the expression level of these proteins to inhibit lung cancer cell metastasis.6.miR-26 a promote the growth and metastasis of lung cancer cell in vivo.Conclusion: 1.MiR-26 a enhanced the metastasis potential of lung cancer cells both in vitro and in vivo.2.ITG?8 was the direct target of miR-26 a,ITG?8 mediated the effect of miR-26 a on lung cancer cell metastasis.Furthermore,ITG?8 could negatively regulate the expression of miR-26 a.3.MiR-26 a increased the expression of metastasis-related genes,induced lung cancer EMT and activated JAK2/STAT3 pathway.4.This study indicated that miR-26 a could be a novel target for the treatment of metastasis of lung cancer.