Effects Of MiR-664b-5p On The Proliferation,Apoptosis,Invasion And Migration Of Esophageal Cancer Cells

Object: We have found that miR-664b-5p expression was significantly increased in Kazakh esophageal squamous cell carcinoma through gene chip technology in the previous study,suggesting that miR-664b-5p may play an important role in the development of Kazakh esophageal squamous cell carcinoma.In this study,we verified miR-664b-5p expression firstly in Kazakh esophageal squamous cell carcinoma tissues,and further explored the effects of miR-664b-5p on the biological behavior of esophageal cancer cells EC9706 and TE-1 in vitro.Methods:(1)The expression of miR-664b-5p in formalin-fixed paraffin-embedded tissues was determined by Quantitative real-time PCR(qRT-PCR),including 23 cases of Xinjiang Kazakh esophageal squamous cell carcinoma and 30 cases of Kazakh esophagitis.And the relationship between clinicopathological characteristics with the expression of miR-664b-5p was analyzed in Kazakh patients with squamous cell carcinoma.(2)After transfected with miR-664b-5p mimic or inhibitor in esophageal cancer cells EC9706 and TE-1,cell proliferation was assessed by CCK-8 and colony formation;Migration and invasion of esophageal cancer cells were observed by the transwell chamber assay;The expression of apoptosis-related proteins was detected by Western blot,including Bax and Bcl-2.And the cell cycle distribution was measured using flow cytometry assay after transfected with the miR-664b-5p mimic in EC9706 and TE-1.(3)The target gene of miR-664b-5p was predicted through Targetscan and miRDB database,the GO function and KEGG pathway of the target gene were analyzed via DAVID online website,and the protein-protein interaction network of the target proteins was constructed in STRING website and Metascape website,and the core molecule of the network was analyzed by Cytoscape’s plug-in of Cyto Hubba.(4)The correlation between the predicted target m RNA and miR-664b-5p expression was analyzed in the result of gene chip about Kazakh esophageal cancer,and the target gene expression was detected by qRT-PCR after over-expressed miR-664b-5p in EC9706 and TE-1.Results:(1)Compared with Kazakh esophagitis tissue,miR-664b-5p expression was increased in Kazakh esophageal squamous cell carcinoma tissues(P <0.05),and the expression of miR-664b-5p was related to the tumor grade(P <0.05).(2)Overexpression of miR-664b-5p inhibited the proliferation,colony formation,migration and invasion,promotes cell apoptosis,induced G1 phase arrest in cells,while the silencing of miR-664b-5p promoted the proliferation,migration and invasion and inhibited cell apoptosis in ESCC cells EC9706 and TE-1(P <0.05).(3)The results of GO annotations showed that the molecular function was mainly enriched in protein binding,the biological process was mainly enriched in regulation of gene expression,and the results of KEGG pathway showed that the pathway of target genes was mainly enriched in pathways in cancer.The results of Cyto Hubba and Metascape’s website showed that PIK3R1,KDR,RAC1,FLT1,and CRK were located at the core nodes of the protein-protein interaction network(P<0.05).(4)The results of gene chip showed that the expression of miR-664b-5p was positively correlated with CCNE2 and CDK expression in Kazakh esophageal squamous cell carcinoma(P <0.05).The results of qRT-PCR showed that the m RNA expression of CCNE2 was significantly reduced after transfected with the miR-664b-5p mimic in EC9706 and TE-1.(P <0.05).Conclusions:(1)The expression of miR-664b-5p is higher in Kazakh esophageal squamous cell carcinoma tissues compared with the Kazakh esophagitis tissue,which is significantly related to tumor grade.(2)miR-664b-5p inhibits the proliferation,migration and invasion,and induces apoptosis in esophageal squamous cell carcinoma cells EC9706 and TE-1.CCNE2 may be a target gene of miR-664b-5p.