| [Background and Objective]Primary pulmonary carcinoma is the cancer with high morbidity and mortality both in China and the world.Although there have been major advances in early diagnosis and treatment in recent years,poor survival for lung cancer patients remains with 5-year survival rate of approximately 10%.The majority of lung malignant tumor patients are non-small cell lung cancer(NSCLC),accounting for almost 90%of total primary pulmonary carcinoma patients.Over 90%cancer-related deaths from solid malignancies,including NSCLC,are reported to be caused by metastasis.Therefore,it is crucial and urgent to reveal the metastatic mechanism of lung cancer.EMT,known as epithelial-mesenchymal transformation,which is an early event in tumor metastasis,is essential for the transformation of early tumors into aggressive malignancies.Transforming growth factor β(TGF-β)is identified as an primary inducer of EMT in NSCLC cells,it plays a pivotal role in cancer cells invasion and metastasis.MicroRNAs(miRNAs)are a kind of key molecules to regulate the expression of various genes in cancers,including non-small cell lung cancer(NSCLC).Radiotherapy is an effective therapeutic measures for advanced NSCLC,and radioresistance is a key factor leading to radiotherapy failure,increasing the mortality of patients.Radiation treatment can increases neuroendocrine differentiation(NED)in NSCLC,which is the key reason for NSCLC to develop radioresistance.All tumors with NED function can secret neuropeptides,which are widely involved in the biological process of tumors.Snail is not only a key factor in TGF-β-induced EMT,but also plays an important role in the formation of radioresistance development.The purpose of this study is to to explore the regulation mechanism of miRNA in Snail mediated TGF-β induced EMT,as well as the regulation mechanism of neuropeptide in lung cancer radioresistance development,so as to provide new ideas and strategies for the treatment of NSCLC.[Methods](1)Ninety-one paired NSCLC tissues and adjacent noncancerous lung tissues were collected.Real-time qRT-PCR was used to detect the levels of miR-940 and Snail mRNA in tissue samples from patients,and statistical software analyzed their correlation and their relationship with clinical parameters.(2)Real-time qRT-PCR was used to detect the expression of miRNA and mRNA in cells,the protein expression of Snail and other genes in cells was detected by western blot.(3)Transient transfection technique transfects miRNA,siRNA or plasmid into cell lines.(4)The direct targeting relationship between miR-940 and Snail 3’-UTR was detected by dual-luciferase report gene plasmid technology.(5)Small RNA interference was used to knock down mRNA expression in lung cancer cell lines.(6)Transwell migration and invasion assays used to detect cell invasion ability.(7)Gene expression in cell lysate and serum was detected by ELISA assay.(8)MTT method was used to detect the cell growth after radiation treatment.(9)The single-cell electrophoresis assay(comet assay)was used to detect the changes of DNA repair ability of cells after radiotherapy.(10)Gene expression in xenografts in vivo was detected by immunohistochemical assay.[Results]Part1 MiR-940 inhibits TGF-β-induced epithelial-mesenchymal transition in nonsmall cell lung cancer(1)The level of miR-940 was decreased,while the level of Snail was increased in pulmonary malignancy tissue samples from patients.And the ratio of miR-940 levels were reversely correlated with that of Snail mRNA levels in NSCLC tissues.The comparison results showed that miR-940 was down-regulated in NSCLC tissues with lymph node metastasis,advanced TNM stages and poor cell differentiation,in which,on the contrary,the expression of Snail was up-regulated.(2)In NSCLC cells,miR-940 significantly inhibited the relative intensity of Renilla fluorescence in lung cancer cells transfected with Snail 3’-UTR wild-type plasmid,but could not suppress the relative intensity of Renilla fluorescence in cells transfected with Snail 3’-UTR mutant plasmid.In addition,overexpression of miR-940 markedly inhibited the expression levels of Snail mRNA and protein in NSCLC cells.In conclusion,the results proved that miR-940 can negatively regulates Snail expression through the 3’-UTR-binding to Snail transcript.(3)After the overexpression of miR-940 in NSCLC cells,the decrease of E-cadherin protein induced by TGF-β and the increase of Snail,N-cadherin and Vimentin were inhibited.In addition,overexpression of miR-940 prevented the cells from migrating and invading in the presence of TGF-β1.(4)After inhibiting Snail expression in NSCLC cells,the decrease of Ecadherin protein induced by TGF-β and the rise of Snail,N-cadherin and Vimentin were inhibited.Moreover,phenotype experiments indicated that the decreasing trend of invasion ability of lung cancer cells was the same as the result of miR-940 overexpression.Part2 BK and VIP promote NSCLC radioresistance development by upregulating Snail(1)Radioresistant cell lines A549R26 and H157R24 were produced by the method of cumulative radiation,and the expression of neuroendocrine markers in radiation-resistant cells was significantly higher than A549 and H157 cells.MTT and comet assay indicated that the radioresistance of A549R26 and H157R24 cells was significantly stronger than A549 and H157 cells.(2)Among the 8 common neuropeptides,bradykinin(BK)and vasoactive intestinal peptide(VIP)increased most significantly in radiation-resistant cells compared with parental cells,and the expression of both receptors and the concentration of cell lysate showed the same trend.(3)BK and VIP and their receptors were significantly increased in parental cells 4h after radiation,and BK and VIP promoted NED in NSCLC.(4)After overexpression of BK and VIP in parental cells,the survival rate and DNA repair ability of cells after radiation were increased;In addition,the radioresistance of radiation-resistant cells decreased after inhibiting the expression of BK and VIP.(5)The expression of Snail in radioresistant cells was higher than parental cells.(6)BK and VIP can raise the expression level of Snail in NSCLC.(7)After inhibiting Snail expression in radioresistant lung cancer cells,the radioresistance properties of the cells were weakened.(8)The concentrations of BK and VIP in serum of mice bearing A549R26-xenografts were higher than those in A549 group,and the concentrations of BK and VIP in serum of mice bearing A549-xenografts were increased after radiation.The expressions of BK,VIP,BKB2,VPAC1 and Snail in tumor tissues of A549R26-xenografts were higher than those in A549-xenografts,and the expressions of BK,VIP,BKB2,VPAC1 and Snail in tumor tissues of A549-xenografts after radiation were higher than unirradiated group,and were positively correlated with the concentrations of BK and VIP in serum.[Conclusions]In the first part of this study,we found that miR-940 can directly target Snail and inhibit TGF-β-induced EMT and invasion in NSCLC cells.Our results reveal a new mechanism of NSCLC cell invasion and metastasis,and provide a new approach for the treatment of advanced NSCLC.In the second part,we revealed that neuropeptides BK and VIP can promote NSCLC radioresistance development through activation of Snail pathway,providing a new method for improving NSCLC radiotherapy effect. |
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