Downregulation Of MiR-126-3p Inhibits Proliferation,migration And Invasion In Nasopharyngenal Carcinoma

Research Background:MicroRNAs(miRNAs)are a class of highly conserved small non-coding RNAs that are involved in regulating multiple biological behaviors of cells by binding to the 3'UTR of target genes.More and more studies have reported that miRNAs play an important role in the development of nasopharyngeal carcinoma and participate in the proliferation,cycle regulation,apoptosis and metastasis of nasopharyngeal carcinoma cells.Although miR-126-3p has been reported in many tumors,the function and molecular mechanism of miR-126-3p has not been reported in nasopharyngeal carcinoma.Nasopharyngeal carcinoma(NPC)is a malignant tumor derived from nasopharyngeal mucosa.It occurs in Southeast Asia and southern China.At present,it is believed that the occurrence of nasopharyngeal carcinoma may be related to EB virus infection,environmental factors,genetic susceptibility and other factors.More and more research shows that abnormal expression of miRNA plays an important role in the development of nasopharyngeal carcinoma.For example,EB virus-encoded microRNA BART7-3p promotes nasopharyngeal carcinoma cell metastasis and cell mesenchymal transition by modulating PTEN-mediated signaling pathways.miR-3188 inhibits the proliferation of nasopharyngeal carcinoma cells through the mTOR-pPI3K/AKT-c-JUN loop.Research purposes:To explore the abnormal expression of miR-126-3p in nasopharyngeal carcinoma and its biological function.Research content and methods:1.Quantitative detection of miR-126-3p expression in nasopharyngeal carcinoma tissues and cells by fluorescence quantitative PCR2.Effect of miR-126-3p on biological function of nasopharyngeal carcinoma cells1)The effect of miR-126-3p on proliferation of nasopharyngeal carcinoma cells was detected by CCK8 cell proliferation assay,plate colony formation assay,and subcutaneous tumorigenesis in nude mice;miR-126-was verified by scratch wound callback experiments and transwell chamber experiments.Effect of 3p on metastasis and invasion of nasopharyngeal carcinoma cells.Western blot was used to determine whether miR-126-3p could reverse nasopharyngeal carcinoma cells by down-regulating the effect of miR-126-3p on E-cadherin,N-cadherin and Vimentin protein expression.Epithelial-mesenchymal transition,in order to determine the function of miR-126-3p in nasopharyngeal carcinoma cells;2)In order to clearly down-regulate the mechanism of miR-126-3p inhibiting the proliferation,invasion and metastasis of nasopharyngeal carcinoma cells,Western blot was used to detect the effect of miR-126-3p on the expression of p-AKT and AKT protein.3.miR-126-3p target gene validation1)Bioinformatics software predicts that the 3'UTR of the PTPN9 gene is highly complementary to the seed region of miR-126-3p;2)After transfection of miR-126-3p Inhibitor,RT-PCR was used to detect the mRNA level of PTPN9 in nasopharyngeal carcinoma cells.3)After transfection of miR-126-3p Inhibitor,the protein level of PTPN9 in nasopharyngeal carcinoma cells was detected by Western blot.4)Double luciferase reporter assays verify that miR-126-3p directly targets PTPN9 gene.Research result:1.Fluorescent quantitative PCR results showed that the expression of miR-126-3p in nasopharyngeal carcinoma tissues and cells was higher than that of nasopharyngitis and nasopharyngeal carcinoma cells,respectively.2.Down-regulation of miR-126-3p inhibits proliferation,invasion,metastasis and epithelial-mesenchymal transition in nasopharyngeal carcinoma cells.1)In vitro functional assay CCK8,plate colony formation assay results showed that miR-126-3p down-regulation in 5-8F and HONE1 cell lines significantly inhibited the proliferation of nasopharyngeal carcinoma cells.2)Subcutaneous tumorigenicity experiments in nude mice showed that the ability of transfected miR-126-3p Inhibitor nasopharyngeal carcinoma cells in vivo significantly reduced the ability to tumorigenesis,and the expression of ki67 and PCNA was also significantly reduced compared to the empty control,indicating that Down-regulation of miR-126-3p inhibits tumor formation in vivo;3)Scratch callus experiments and Transwell chamber experiments demonstrated that miR-126-3p down-regulation of nasopharyngeal carcinoma cells in vitro migration and invasion ability was significantly reduced;4)Western blotting showed that the expression of E-cadherin was up-regulated in nasopharyngeal carcinoma cells that down-regulated miR-126-3p,while the expression of N-cadherin and Vinmintin was down-regulated.This indicates that down-regulating the expression of miR-126-3p can reverse nasopharyngeal carcinoma.Epithelial-mesenchymal transition of cells.Mechanism studies showed that the down-regulation of miR-126-3p expression in nasopharyngeal carcinoma cells 5-8F and HONE1 down-regulated the change of p-AKT,but there was no significant difference in total AKT,indicating that down-regulation of miR-126-3p may pass through PI3K/AKT signaling pathway to inhibit proliferation,metastasis,and cell-mesenchymal transition in nasopharyngeal carcinoma cells.4.miR-126-3p can target PTPN9 gene1)Using TargetScan,a bioinformatics software,it is predicted that PTPN9 is a target gene of miR-126-3p;2)Fluorescent quantitative PCR results showed that the expression of PTPN9 was up-regulated in 5-8F and HONE 1 cells that down-regulated miR-126-3p;3)Western blot results showed that the expression of PTPN9 was up-regulated in 5-8F and HONE1 cells that down-regulated miR-126-3p;4)The pmirGLO/PTPN9 wt 3'UTR plasmid and the pmirGLO/PTPN9 mt 3'UTR plasmid were successfully constructed,and 293T cells were co-transfected with miR-126-3p mimics.The pmirGLO empty plasmid was used as a control and the luciferase activity was detected.24h after dyeing.The results showed that the activity of luciferase was significantly decreased after co-transfection of the wild-type plasmid pmirGLO/PTPN9 wt 3'UTR with miR-126-3p mimics;the mutant plasmid pmirGLO/PTPN9 mt 3'UTR was co-transformed with miR-126-3p mimics.After staining,there was no significant change in luciferase activity.The above results indicate that miR-126-3p can specifically bind PTPN9 3' UTR.Conclusion:1.miR-126-3p is highly expressed in nasopharyngeal carcinoma tissues and cells;2.Down-regulation of miR-126-3p inhibits proliferation,metastasis and cell-mesenchymal transition of nasopharyngeal carcinoma cells through inactivation of PI3K/AKT signaling pathway;3.miR-126-3p directly targets the PTPN9 gene.