The Study Of MiR-497 Modulating Laryngeal Squamous Cell Carcinoma Growth Through CDK6,PlexinA4 In Vitro And In Vivo

Objective: To investigate miR-497 from expression profiles of microRNAs in laryngeal squamous cell carcinoma(LSCC).To study the expression and correlation of miR-497 and target gene CDK6?Plexin A4 in fresh specimens and adjacent normal mucosa and cell lines of laryngeal squamous cell carcinoma,and to evaluate the association of miR-497 and its target gene CDK6 ? Plexin A4 expression with clinicopathologic factors and their prognostic value in LSCC.To predict target gene of miR-497 by bioinformatics methods and to verify the relationship between them.To probe the effects of miR-497 on cell proliferation,migration,invasion,apoptosis and cell cycle of laryngeal squamous cell carcinoma,and to expound the effects of CDK6 ? Plexin A4 on the proliferation,migration and invasion of laryngeal squamous cell carcinoma,and to clarify their biological functions.Methods: Quantitative RT-PCR and immunohistochemistry were used to examine m RNA and protein levels in LSCC specimens and adjacent normal mucosa.To evaluate the relationship between miR-497 and CDK6?Plexin A4 expression with clinical pathologic parameters and postoperative follow-up data,and to evaluate their clinical significance and prognosis.The bioinformatics was used in prediction of regulation between miR-497 and CDK6 ? Plexin A4 which was validated by dual luciferase reporter gene assay.The gene transfection technology was used to explore miR-497 mimics on the biological function of laryngeal squamous cell carcinoma in vitro by MTT assay,colony formation assay,transwell assay and flow cytometry.The tumor inhibition effect of miR-497 was observed in nude mice transplanted tumor model.Si RNA CDK6 and Si RNA plexin A4 was transfected into laryngeal squamous cell carcinoma cells by RNA interference technique,and the effect of Si RNA CDK6 and Si RNA plexin A4 on the proliferation of laryngeal squamous cell carcinoma cells was observed by MTS method.The effect of pc DNA3.1(+)CDK6 and pc DNA3.1(+)Plexin A4 on the growth of laryngeal squamous cell carcinoma cells was observed by MTS method.Results: The low expression of miR-497 was significantly higher in lower degree of differentiation and distant metastasis(P<0.05).On the contrary,the low expression of miR-497 were significantly lower in higher degree of differentiation(P<0.05).There was no significant difference in the expression of miR-497 with age,sex,primary sites,tumor size and preoperative smoking(P>0.05).Kaplan-Meier survival analysis revealed that the prognosis of patients with high expression of miR-497 was better than those with low expression of miR-497(?2=10.342,P=0.001).Cox regression analysis show the relative risk for low expression of miR-497 was 2.262(95%CI ? 0.151~0.732,P=0.033),which was the independent prognostic factors of LSCC.There were significant differences in the changes of the fluorescence activity of the wild type((CDK6 Wt 3'-UTR group and plexin A4 Wt3'-UTR group)compared with that of the control group(P<0.01).There was no significant differencein the fluorescence activity of the mutant(CDK6 Mut 3'-UTR group and plexin A4 Mut 3'-UTR group)group compared with the control group(P>0.05).In vitro,after transfection into Hep-2 and TU-212 cells by miR-497 mimics,the proliferation,clone,invasion of miR-497 group was significantly inhibited(P<0.05).The percentage of G0/G1 cells and early apoptotic cells of miR-497 group was increased(P<0.05).In vivo,the tumor growth rate of miR-497 agomir group was slower than NC agomir group and Blank group(P<0.01).After transfection into Hep-2 and TU-212 cells by CDK6 si RNA2 and plexin A4 si RNA2,the cell growth was significantly inhibited(P<0.05).After transfection into Hep-2 and TU-212 cells by pc DNA3.1(+)/CDK6,pc DNA3.1(+)/plexin A4 The number of cells of pc DNA3.1(+)/CDK6,pc DNA3.1(+)/plexin A4 group was significantly higher than that of control group(P<0.01).Conclusions: miR-497 was significantly down-regulated,play a role in the role of tumor suppressor genes,and its target gene CDK6 and plexin A4 was significantly up-regulated,play the role of oncogene in laryngeal squamous cell carcinoma.Low expression of miR-195 in individuals has poor prognosis.It can be used as an independent risk factor for the prognosis of patients with laryngeal squamous cell carcinoma.CDK6 and plexin A4 are target genes of miR-497.In vitro,miR-497 has inhibitory effect on cell proliferation,cloning,invasion of laryngeal squamous cell carcinoma.It can promote cell apoptosis and arrest in G0/G1 phase.In vivo,miR-497 has inhibitory effect on growth of hep-2 human laryngeal carcinoma xe nograft tumor in nude mice.Si RNA CDK6 and Si RNA plexinA4 has inhibitory effect on cell proliferation,invasion of laryngeal squamous cell carcinoma.pc DNA3.1(+)/CDK6,pc DNA3.1(+)/plexin A4 restore they.miR-497 is involved in the development and invasion of laryngeal squamous cell carcinoma by regulating its target gene CDK6 and plexin A4,which provides a new molecular marker for the early diagnosis,early treatment and prognosis evaluation of LSCC.