| Research background:Lung cancer is among the most prevalent types of cancer worldwide and represents a serious threat to human health.The most common subtype of lung cancer is NSCLC,which comprises ~85% of all lun g cancer cases.Due to a lack of early screening and diagnosis based on speci fic biological markers,and the nonobvious clinical symptoms of early lung ca ncer,the majority of lung cancer cases are diagnosed at a late stage.The 5-y ear survival rate for lung cancer is <10%.Therefore,it is important to identi fy novel biomarkers to provide more accurate diagnosis and individualized trea tment regimens for patients with lung cancer.micro RNAs(mi RNA/mi Rs)are non-coding single-stranded RNAs of appro ximately 19-22 nucleotides in length.Numerous studies have demonstrated that mi RNAs are involved in the processes of tumorigenesis and tumor developm ent,and serve similar oncogene roles in cell colonization,apoptosis,migration and other biological processes.Recent studies have indicated that a number o f mi RNAs,including mi R-126 and mi R-16-1,are involved in the initiation an d progression of NSCLC through the regulation of target genes.The aim of the present study was to observe the function of mi R-939 in NSCLC and its mechanism of action,in order to provide data to potentially aid in the early diagnosis and clinical treatment of NSCLC.Additionally,it aimed to provide an effective intervention target and a theoretical basis for the development of novel drugs against genes associated with NSCLC.Methods: 1.Build the mi RNA-939 slow virus expression vector and the correspondi ng blank against the carrier,and then,each in the two kinds of carrier to inf ection of cells,NSCLC cell lines(H1299 SPCA1,A549,H358)and normal l ung epithelial cell line(16HBE)in mi R-939 expression level in q RT-PCR test ing and validation of micrornas-939 expression level.2.The effects of mi R-939 on the invasion ability of NSCLC cells were detected by transwell invasion.3.Western blot was used to test the expression level of EMT related ma rkers e-cadherin and Vimentin.4.The potential target genes of mi R-939 in lung cancer cells were select ed by combining preliminary experimental results and biological online predicti on software.5.The expression of TIMP2 in the mi R-939 inhabitor and inhabitor NC was studied,and the techniques used were qrt-pcr and western blot.6.The luciferase reporter gene of mi R-939 and the 3 ’UTR region of TI MP2 was reported,and the activity of the reporter gene was detected after 24 h.Results: 1.The expression levels of mi R-939 in NSCLC cell lines(H1299,SPCA1,A549,H358)and normal pulmonary epithelial cell lines(16HBE)were dete cted by qrt-pcr.The results showed that the expression level of mi R-939 in N SCLC cell lines(H1299,SPCA1,A549,H358)was significantly higher than that of 16 HBE,and the results supported the clinical analysis results.2.The difference of mi R-939 expression is associated with lymph node metastasis,to explore the mi R-939 in vitro regulation mechanisms of invasi on and metastasis of NSCLC cells,this study will mi R-939 inhibitor(inhibitor)and inhibitors in control group(inhibitor NC)transfection cell line respectivel y.q RT-PCR method was used to verify the transfection efficiency,and the re sults showed that mi R-939 expression was significantly reduced in the cells tr ansfected with mi R-939 inhibitor(* P <0.05)compared with the control inhibi tor NC.3.Western blot experiment showed that the expression level of E-cadheri n decreased after mi R-939 was down-regulated,and the expression level of Vi mentin decreased.The results show that down-regulation of mi R-939 can inhib it EMT process.4.With the help of online software such as Target Scan and mi Rtarbase,t he bioinformatics analysis of mir-939 was conducted to predict the possible ta rget genes downstream of mir-29 c.The potential target genes of mir-939 in lu ng cancer cells were selected as TIMP2.mi R-939 plays a regulatory role by combining the 3 ’-UTR of TIMP2.5.Transwell invasion results showed that the number of cells in the cells with filtration membranes decreased significantly,and the difference was stati stically significant(p < 0.05).The SPCA1 has similar results.The above resul ts suggest that the expression of TIMP2 can inhibit the invasion ability of H1299 and SPCA1 cells.It is speculated that the possible mechanism of mi R-939 inhibiting invasion and metastasis of lung cancer cells is related to the targ eted inhibition of TIMP2.Conclusions: The present study demonstrated that mi R-939,a novel oncogene,could re gulate NSCLC cell proliferation and invasion.It was also identified that mi R-939 negatively regulated TIMP2 by binding to its 3’UTR.Therefore,mi R-939 may be a potential target in the treatment of NSCLC. |
