MiR-9-5p Inhibits Metastasis Of Non-small Cell Lung Cancer By Targeting TGFBR2

Objectives:Lung cancer is a kind of malignant tumor which present the greatest harm on people’s lives and health in the world including China.The incidence and mortality of lung cancer have risen rapidly over the past half century.Lung cancer has became the first cause of death of malignant tumors.In recent years,great progress has been made in the treatment of advanced lung cancer by chemotherapy,radiotherapy and target therapy.However,resistance and disease progression continue to occur in this group of patients.The metastasis of lung cancer is the main reason for the failure of tumor treatment and the death of lung cancer patients.Therefore,it is important to elucidate the metastasis mechanism of lung cancer.Recent studies have shown that aberrant expression of miRNA including miR-9-5p can promote the expression of proto oncogenes or the expression of tumor suppressor genes.In the past few years,there have been very few reports of miR-9-5P in lung cancer,especially in non-small cell lung cancer.Transforming growth factor-β(TGF-β)is a cytokine with many functions.The TGF-β signaling pathway regulates cell growth,differentiation,apoptosis,migration,furthermore,it also plays an important role in the formation of extracellular matrix,and has tumor suppressor and oncogenic activity.lt plays a very important role in the development of cancer.But the biological role of TGF-β must be combined with TGFBR to play,it has a high affinity with TGFBR2,combined with TGFBR1 in the two dimer form ISO four dimer complexes,TGFBR2 through phosphorylation of TGFBR1 glycine serine phosphorylation,configuration changes of TGFBRl after being activated,Smad MAPK pathway is activated downstream.Therefore,abnormal expression of TGFBR2 protein leads to disorder of TGF-beta signal transduction,losing control of cell growth and differentiation,and promoting the occurrence and development of cancer.MiR-9-5p has been reported to directly regulate the expression of TGFBR2 in lung epithelial cells,but the mechanism is unknown.In view of this,in this study,we will investigate the expression of miR-9-5p in lung cancer tissues and cell lines,and its relationship with the growth and metastasis of lung cancer.Methods:1.To investigate the expression of miR-9-5p RNA in human non-small cell lung cancer tissues and surrounding normal lung tissues,and to analyze the relationship between miR-9-5p and clinicopathological features of non-small cell lung cancer,then to further investigate the clinical significance of miR-9-5p in non-small cell lung cancer(NSCLC).2.To investigate the effects of MiR-9-5p expression on the proliferation and apoptosis of human lung adenocarcinoma cell carcinoma A549 cell line and human lung squamous cell line SK-MES-1 through injecting MiR-9-5p plasmid vector into human lung adenocarcinoma cell carcinoma A549 cell line and human lung squamous cell line SK-MES-1 by liposome 2000.3.To investigate the mechanism of high expression of miR-9-5p in inhibiting migration and metastasis of lung cancer cells in A549 and SK-MES-1 cells.Results:1.The results of RT-PCR detected in 30 cases of lung cancer tissues and normal tissues confirmed by HE staining indicated that compared with normal tissue specimens,miR-9-5p levels in lung cancer samples decreased significantly(P<0.05),the difference was statistically significant(P<0.05).MiR-9-5p levels in these with lymph node metastasis were significantly lower than those without lymph node metastasis(P<0.05).2.RT-PCR results of 4 cell lines before treatment indicated that MiR-9-5p RNA level in A549,SK-MES-1 were signi-ficantly lower than that in BEAS-2B cell lines.After transfection of MiR-9-5p mimics,cell scratch test,MTT assay and Transwell chamber method showed that the cell invasion ability was significantly decreased.3.Targetscan software is used to predict that TGFBR2 3’UTR is a potential binding site for miR-9-5p and TGFBR1 and TGFBR2mRNA.Putative wild-type and mutant 3’UTR clones were cloned onto luciferase reporter vectors,MiR-9-5p was then transfected into these two reporting vectors,the results showed that miR-9-5p transfection could significantly decrease the luciferase activity of WT containing 3’UTR reporter vector.In contrast,the luciferase activity of 3’UTR containing mutant carriers was not decreased.The Western blot test suggested that miR-9-5pRNA could bind directly to the 3’UTR of wild type TGFBR2 to bind and inhibit its expression,and further reduce the phosphorylation and activation of Smad2 and Smad3 proteins.Conclusions:1.The expression of miR-9-5p in human lung cancer tissue was significantly decreased compared with that in normal tissues.Compared with lymph node metastasis,lung cancer specimens showed a significant decrease in mir-9-5p levels in lung cancer specimens without lymph node metastasis.MiR-9-5pmRNA levels were negatively correlated with lung cancer metastasis ability.2.MiR-9-5p gene can regulate the proliferation and migration of lung cancer cells in vitro and in vivo,by liposome encapsulated miR-9-5p 2000 vector construction,transfer to the lung A549 cells and SK-MES-1 cells,can inhibit the migration and proliferation of human lung cancer cell line A549 and SK-MES-1.3.MiR-9-5p inhibits migration and proliferation of lung cancer cells in vivo and in vitro,possibly through inhibition of the TGFBR2 pathway.