| [Background and Objective]:Although there are many treatments for lung cancer at present,lung cancer is still a malignant tumor with the highest morbidity and mortality in the world.Its 5-year overall survival rate is only 10-20%,and it is prone to recurrence,drug resistance and metastasis after treatment.MicroRNA is an important regulatory gene commonly found in organisms,and it is closely related to malignant tumor.The study found that microRNA-338-3p is an important tumor suppressor gene.NRP1 is a multifunctional transmembrane glycoprotein,as a co-receptor of multiple growth factors or other ligands,which plays an important role in promoting angiogenesis and invasion and metastasis of tumors.At present it has not been reported in the study of lung cancer targeted relations between microRNA-338-3p and NRP1,so the research goal of this article is mainly clear the relationship of microRNA-338-3p and NRP1,and study the role and mechanism of microRNA-338-3p-NRP1 signaling axis in the development and progression of lung cancer.[Methods]:(1)The expression levels of microRNA-338-3p in lung cancer(n=55)and adjacent normal mucosa tissues(n=55)were determined using quantitative PCR,and the expression of miRNA in lung cancer cell lines(A549,H226 and HCC827)and normal lung mucosa cell line(BEAS-2B)were also detected by quantitative PCR.(2)A549 and H226 cells were transfected with microRNA-338-3p mimics and their corresponding negative controls to detect NSCLC proliferation and invasion and metastasis using CCK-8 assay,colony formation,transwell Cell migration and invasion experiment and wound healing test.(3)The target gene of microRNA-338-3p was predicted to be NRP1 by using biological prediction software.The biluciferase activity test was used to verify whether microRNA-338-3p is directly bound to the seed sequence of NRP1 3’UTR region to clarify the targeted regulation effect of microRNA-338-3p on NRP1.(4)The expression levels of NRP1 mRNA in lung cancer(n=55)and adjacent normal mucosa tissues(n=55)were determined using quantitative PCR,and the expression of NRP1 in lung cancer cell lines(A549,H226 and HCC827)and normal lung mucosa cell line(BEAS-2B)were also detected by quantitative PCR.Meanwhile,we performed regression analysis of the microRNA-338-3p and NRP1 expression to explore their correlation.(5)The expression of NRP1 mRNA and protein expression before and after transfection was determined by building sh-NRP1 virus transfection A549 and H226 cells.The growth and proliferation of cells were observed by CCK-8,flow cytometry and plate cloning experiments,and the invasion and migration of cells were observed by transwell cell migration and invasion experiment and wound healing test.(6)Primary tumor growth was examined after the orthotopic injection of 1×106 A549 cells with the forced expression of microRNA-338-3p or cont-microRNA and the growth of the tumor was observed and the mRNA levels of microRNA-338-3p and NRP1 in the tumor were detected.(7)The expression levels of the phosphorylated EGFR,Akt and FAK were detected by Western blot,and the changes of EMT related proteins were detected in A549 and H226 cells with microRNA-338-3p.(8)A549 and H226 cells with sh-NRP1 or NRP1 overexpression were detected expression of relevant signaling pathways and EMT related markers by Western blot method.(9)A549 cells,infected by sh-NRP1 lentivirus,injected into the tail vein and A549 cells were infected by blank vector sh-NC.A lung metastatic tumor model was established to observe the effect of NRP1 on the metastasis ability of lung cancer in vivo.(10)To observe proliferation changes in lung cancer cell using sh-NRPl joint EGFR-TKI or FAK inhibitors respectively.[Results]:(1)The results of qRT-PCR showed that the expression of microRNA-338-3p in 3 lung cancer cell lines was significantly lower than that of normal lung epithelial cells(p<0.05),and the expression of microRNA-338-3p in 55 lung cancer tissues was also significantly lower than that in 55 adjacent tissues(p<0.05).The results were consistent with the results of previous pre-experiment tissue microRNA chips.(2)The expression level of mature microRNA-338-3p in the lung cancer tissues was significantly lower than in the adjacent normal mucosa tissues(p<0.05).MicroRNA-338-3p was decreased in the lung cancer cell lines(A549,H226 and HCC827)compared with the normal lung mucosa cell line(BEAS-2B)(p<0.05).The results were consistent with the results of pre-experimental tissue microRNA chip.(3)The co-transfection of microRNA-338-3p and the wild-type NRP1 3’UTR caused a significant decrease in luciferase units compared with the controls(p<0.05).However,the co-transfection of microRNA-338-3p and the mutant NRP13’UTR did not cause a decrease in luciferase units compared with the controls.(4)Compared with BEAS-2B,the expression of NRP1 in NSCLC cell lines A549,H226 and HCC827 was up-regulated.The expression of NRP1 was also up-regulated in 55 cases of NSCLC tissue samples.In NSCLC,microRNA-338-3p is low expression.(5)The CCK 8 cells,plate cloning experiments,the testing and transwell Chambers attack experiment and scratches the experimental results show that the overexpression of microRNA-338-3p in lung cancer cell line A549 and H226 growth,proliferation and invasion and metastasis ability significantly lower than the control group(p<0.05).(6)In vivo experiment,compared with the control group the tumor size of the nude mice,treated with microrna-338-3p,was significantly smaller(p<0.05).The RNA level of NRP1 in the tumor tissues after microRNA-338-3p was significantly reduced compared with the control group,indicating that microRNA-338-3p inhibits the growth of nude mouse transplanted tumor by inhibiting the expression of NRP1.(7)Western blot test results showed that compared with the control group,the expressions of phosphorylated EGFR,FAK and Akt in the overexpressed microRNA-338-3p group were significantly reduced(p<0.05).The remediation experiment proved that overexpression of NRP1 could partially restore the expression levels of phosphorylated EGFR,FAK and Akt in the overexpressed microRNA-338-3p group,indicating that microRNA-338-3p could regulate the relevant signal pathway by inhibiting the expression of NRP1.(8)Western blot test results showed that the phosphorylation levels of MMP2,MMP9,Smad3,Vimentin,N-cadherin and Snail protein in the NRP1 interference group were significantly reduced compared with the control group(p<0.05),while overexpression of NRP1 promoted up-regulation of MMP2,MMP9,Smad3,Vimentin,N-cadherin and Snail.It showed that NRP1 could regulate the metastasis of lung cancer and the process of epithelial-mesenchymal transforamtion.In the overexpressed microRNA-338-3p group,Smad3 phosphorylation level,Vimentin,N-cadherin and Snail protein were significantly reduced compared with the control group(p<0.05),suggesting that microRNA-338-3p,like sh-NRP1,can inhibit lung cancer metastasis and the process of epithelial-mesenchymal transformation.(9)After interfering with NRP1,the experimental results of lung metastatic tumor in vivo showed that pulmonary metastatic nodules were significantly reduced compared with the control group(p<0.05).(10)Interference with NRP1 can increase the sensitivity of lung cancer cells to EGFR-TKI or FAK inhibitors.[Conclusion]:microRNA-338-3p as a novel tumor suppressor gene was decreased in lung cancer,and NRP1 is an oncogene that is highly expressed in lung cancer and can inhibit metastasis and migration of lung cancer after interfering with NRP1.Overexpression NRP1 can promote proliferation and metastasis.Meanwhile,microRNA-338-3p can decrease migratory,invasive and proliferative behaviors,as well as lung cancer EMT,by attenuating the expression of NRP1 through EGFR,FAK and Akt pathways.Interference with NRP1 can increase the sensitivity of lung cancer cells to EGFR-TKI or FAK inhibitors. |
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