The Role Of MiR-9-5P In Non-small Cell Lung Cancer And Vasculogenic Mimicry

[Background and purpose]In 1999,Maniotis[1]first identified vasculogenic mimicry(VM),a new channel-like structure,in a highly aggressive melanomas case.The tubular walls that make up the VM,such as laminin,heparin sulfate proteoglycans,? collagen,etc.,are extracellular matrix enriched with PAS stain positive.Its vascular endothelial cells have negative stains to specific immunohistochemical markers such as FVlllRag,Ules,cD31,CD34,KDR,etc.VM's channels are connected to the tumor vasculature and provide blood to the tumor tissue.The most significant difference between VM and endothelium-dependent vessels is that endothelial cells enclose vessels which are positive to CD31 and negative to PAS,whereas VM is reverse with negative to CD31 and positiveto PAS.miRNAs and VM play an important role in the metastasis of malignant tumors.Related researches have discovered that miRNAs may have the ability to regulate the formation of VM and sequentially changes in the aggressiveness and metastatic ability of malignant tumors.However,the specific relationship between VM and miRNAs,as well as the regulatory signaling pathways between the two,are not clear.Our team's previous researches have shown in lung cancers:comparing with the patients with benign lung tumors,miR-9-5P is significantly up-regulated in the blood of lung cancer patients;and miR-9-5P is significantly up-regulated in tumor tissues compared to distal normal lung tissues.Such relationship is not well researched in lung cancer especially.In this study,we tried to research the regulation of miR-9-5p and VM's formation mechanism in non-small cell lung cancer'tissue.To investigate the effects of different regulation of miR-9-5P on proliferation,migration,invasion and VM formation in NSCLC by vivo and vitro experiments.This research can serve as a preliminary exploration of miR-9-5P target genes and related pathways,exploring and discuss the role of miR-9-5P and its mechanism of action in NSCLC,so that we can provide an experimental basis for lung cancer diagnosis and targeted drug design.[Methods]1.Expression of miR-9-5P in human 's NSCLC tissues,VM formation in NSCLC tissues and clinical significance.Sixty cases of NSCLC tissues and those distal normal lung tissues were surgically collected.qPCR was applied to detect the expression of miR-9-5P in human NSCLC frozen tissues and paracancerous normal lung frozen tissues.Then we analyzed the relationship between miR-9-5P and clinicopathological features.In addition,immunohistochemistry and CD31/PAS double staining were applied to detect the formation of VM in human NSCLC frozen tissues.Then we analyzed the relationship between VM and clinicopathological features.2.Effect of miR-9-5P on the biological function of NSCLC cellsUsing A549 and NCI-H1299 as experimental cell lines,miR-9-5P was up-regulated and down-regulated by plasmid transfection.We screened the corresponding stably transfected cell lines.qPCR is used to verify the transfection efficiency of miR-9-5P.The effects of miR-9-5P on proliferation,cloning,migration,invasion,apoptosis and pipeline formation in non-small cell lung cancer were examined in CCK-8 assay,cell clone formation assay,scratch assay,Transwell invasion assay,apoptosis detection assay and three-dimensional cell culture experiments,respectively.3.Molecular mechanism of miR-9-5P in NSCLC cell characterization and VM formationTargetScan,PicTar and miRanda gene prediction software were applied to screen the target genes of miR-9-5P,and then validated with dual luciferase reporter gene assay;Western Blot was used to detect the expression of E-cadherin in NSCLC cell lines with stable up-and down-regulation of miR-9-5P;Western Blot was performed to detect the expression of E-cadherin in lung cancer tissues with different miR-9-5P levels and to verify the correlation between miR-9-5P and E-cadherin.Western Blot was performed to detect the expression of VM-related proteins such as VE-cadherin,MMP2.Western Blot examined the expression of VE-cadherin,MMP2 and MMP9 in lung cancer tissues with different miR-9-5P levels to verify the effect of miR-9-5P on angiogenic mimetic-related proteins in human NSCLC cancer tissues.The effect of miR-9-5P on VM-related proteins in human NSCLC tissues.4.Effect of miR-9-5P on VM formation in transplanted tumors and that in nude miceA549 and NCI-H1299,which stably up-and down-regulated miR-9-5P,were used as experimental cell lines to construct a subcutaneous transplantation tumor model in nude mice,to compare the effects of up-and down-regulation of miR-9-5P on the tumorigenic ability of NSCLC cells in nude mice.Immunohistochemistry and CD31/PAS double staining were applied to detect the formation of VM in transplanted tumors at different miR-9-5P levels,and to compare the effects of up-and down-regulation of miR-9-5P on the formation of VM in transplanted tumors of nude mice.The expression of E-cadherin,a key protein for EMT,was measured by immunohistochemistry and Western Blot in nude mice transplanted tumors.The expression of VE-cadherin,MMP2 and MMP9 in transplanted tumors of nude mice was measured by immunohistochemistry and Western Blot,to compare the effects of up-and down-regulation of miR-9-5P on VM-related proteins in nude mouse transplanted tumors,for further validation of the effect of miR-9-5P on NSCLC cell characteristics and VM formation.[Results]1.Expression of miR-9-5P in human's NSCLC tissues,VM formation in NSCLC tissues and clinical significance.miR-9-5P was significantly upregulated in cancer tissues of NSCLC patients compared with normal lung tissues which adjacent to cancer.The comparison between the group which miR-9-5P was significantly upregulated and the group without miR-9-5P upregulation:higher rate of squamous carcinoma and higher rate of stage ?,but the difference was not statistically significant(P>0.05);much higher rate of hypofractionated carcinoma(P<0.05).VM formation can be found in cancer tissue of NSCLC patients.VM was more likely to be formed in poorly differentiated tumors than in moderately or highly differentiated tumors(P<0.01);stage ? tumors were more likely to form VM than stage ?+? tumors(P<0.01).Compared to the VM-negative group,the miR-9-5P was dramatically upregulated in cancer tissues in the VM-positive group.So miR-9-5P and VM can be regarded as an indicator of poor prognosis in NSCLC,Both are positively correlated.2.Effect of miR-9-5P on the biological function of NSCLC cellsCompared to the control group,there was upregulation of miR-9-5P promoted the proliferative capacity of A549 and H1299 cells,and the difference became more pronounced with longer culture time.Up-regulation of miR-9-5P promoted the clonogenic ability,migratory ability,invasive ability and pipeline formation ability of A549 and H1299 cells;down-regulation of miR-9-5P resulted in the opposite of the above results.However,no significant apoptosis was observed in A549 and H1299 cells after up-regulation and down-regulation of miR-9-5P.3.Molecular mechanism of miR-9-5P in NSCLC cell characterization and VM formationBiological information predicted that E-cadherin is one of the potential target genes of miR-9-5P,and a dual luciferase reporter gene assay verified that miR-9-5P could directly target E-cadherin.Western Blot detection of VM-associated proteins in NSCLC tissues revealed that the expression of E-cadherin in cancer tissues was higher in the miR-9-5P upregulated group than in the non-upregulated group,the expression of VE-cadherin,MMP2 and MMP9 was lower than that of the non-significantly up-regulated group,Western blot detection of A549 and H1299 cell lines shows that Cell lines with upregulated miR-9-5P showed a significant decrease in E-cadherin protein expression,while MMP2,MMP9,and VE-cadherin expression showed an increase,what's more down-regulation of miR-9-5P cell lines illustrates a significant increase in E-cadherin protein expression,while MMP2,MMP9,and VE-cadherin expression showed a decrease.4.Effect of miR-9-5P on VM formation in transplanted tumors and that in nude miceNude mice transplantation tumor experiments revealed compared to the control group that there was no difference in body weight growth and tumorigenic ability between the up-regulated and down-regulated miR-9-5P groups;the down-regulated miR-9-5P group showed slower growth rate,smaller tumor size and lighter mass;however,no difference was observed between the up-regulated miR-9-5P group and the control group.Immunohistochemistry and CD31/PAS double staining of transplanted tumor tissues from nude mice revealed that there was VM formation in transplanted tumor tumors.But,there was no difference between the groups,respectively,due to the small number of cases(6 cases per group),Immunohistochemistry and Western Blot detection of E-cadherin,a key protein for EMT development,and VM-related protein findings in transplanted tumour tissue from nude mice:reduced expression of E-cadherin,a key protein for EMT development,in the up-regulated miR-9-5P group;Elevated expression of VM-related proteins MMP2,MMP9 and VE-cadherin,the results of the down-regulated miR-9-5P group were opposite to those described above.[Conclusion]1.miR-9-5P and VM formation were both negatively correlated with the degree of NSCLC tumor differentiation and pathological stage.miR-9-5P and VM formation are positively correlated with each other,which suggests that miR-9-5P and VM formation may be an indicator of poor clinical prognosis.2.Inhibition of miR-9-5P could inhibit the proliferation,cloning,migration,invasion and duct formation ability of non-small cell lung cancer,and slowed down the growth of transplanted tumors in nude mice,suggesting that miR-9-5P may be a potential target for lung cancer treatment.3.miR-9-5P decreased the expression of E-cadherin,a key protein for EMT development in NSCLC,and increased the expression of VM-related proteins:VE-cadherin,MMP2,and MMP9.The EMT pathway may be one of the signaling pathways through which miR-9-5P regulates NSCLC growth and VM formation.