Effect Of MiRNA-9-5p Targeting Notch2 On Proliferation Invasion And Migration Of Esophageal Squamous Cancer Cells

BackgroundMicroRNAs(miRNAs)are composed of 17 to 23 nucleotides(nt)and regulate transcription of target genes.The miRNA targets the mRNA 3’-uTR(non-coding base domain)to achieve direct degradation of mRNA,or indirect inhibition.The miRNA member miR-9-5p is involved in the biological process of tumors.However,the mechanism of operation of miR-9-5p in ESCC is not completely clear.By observing the targeted regulation of ESCC by miR-9-5p and Notch2,we provide new ideas for ESCC diagnosis and treatment.ObjectiveTo investigate the effects of microRNA9-5p on proliferation,invasion and migration of ESCC by targeting Notch 2.Method1.RT-PCR was used to detect the expression of miRNA-9-5p in 50 cases of esophageal squamous cell carcinoma,serum and esophageal squamous cell lines TE-1,ECA109,KYSE410 and human normal esophageal epithelial cell strains HEEC.2.The KYSE410 cell lines expressed by miRNA-9-5p were constructed,and the effects of miRNA-9-5p expression on the proliferation,invasion and migration ability of KYSE410 cells were detected by MTT method and Transwell chamber experiment.3.Detection of the expression of Notch2 mRNA in esophageal carcinoma by RT-PCR and detection of Notch2 mRNA and protein in esophageal carcinoma cells by RT-PCR and Western blot.4.Bioinformatics prediction and dual fluorescein enzyme reporting gene experiment to detect the targeted relationship between miRNA-9-5p and Notch2,the regulatoryrelationship between RT-PCR and Western blot detection miR-9-5p and Notch2.5.The effects of increased Notch2 on the proliferation,invasion and migration of mirna-9-5p cells expressed by KYSE410 were observed after the miRNA-9-5p of KYSE410 cells were transmitted and pcDNA3.0-Notch2 expressed vector plasmid.Result1.The expression level of miRNA-9-5p in esophageal squamous cell carcinoma tissue and serum was significantly lower than that in normal control group(P<0.05),while the expression of Notch2 mRNA in esophageal carcinoma tissue was significantly higher than that in normal control group(P<0.05).2.Correlation analysis showed that the expression of miRNA-9-5p and Notch2 in esophageal squamous cell carcinoma was negatively correlated.3.Compared with HEEC cells,the expression of miRNA-9-5p in TE-1,ECA109 and KYSE410 cells decreased significantly,while the expression of Notch2 mRNA and protein increased significantly(P<0.05),and the change of KYSE410 cells was the most significant.4.miRNA-9-5p expression can obviously inhibit the proliferation,migration and invasion ability of KYSE410 cells.5.The results of double fluorescein gene experiment confirmed that Notch2 was the target gene of miRNA-9-5p,and the results of RT-PCR and Western blot showed that miRNA-9-5p could be negatively regulated Notch2 expressed.6.Increased Notch2 expression can significantly promote the proliferation,invasion and migration of KYSE410 cells with high expression of miRNA-9-5p.Conclusion1.There is a targeted regulatory relationship between microRNA-9-5p and Notch2.2.MiR-9-5p inhibits ESCC cell proliferation,migration and invasion by targeting Notch2...