MiR-675-5p Enhances Cell Proliferation And Migration Through Targeting FBXL16 In Nasopharyngeal Carcinoma

Objective:To investigate the effect of miR-675-5p on proliferation and migration of nasopharyngeal carcinoma cells by targeting FBXL16.Methods:1.Effect of miR-675-5p on cell biological functionRT-PCR was used to verify the transfection efficiency of miR-675-5p mimic,miR-675-5p inhibitor and corresponding controls in nasopharyngeal carcinoma CNE-2 cells.The effects of miR-675-5p over-expression or silencing on proliferation and migration of nasopharyngeal carcinoma CNE-2cells were detected by CCK-8 proliferation assay and scratch assay,respectively.2.Expression and prognosis of FBXL16 in head and neck squamous cell carcinomaThe differential expression of FBXL16 in head and neck squamous cell carcinoma(HNSCC)and adjacent normal tissues was analyzed by TCGA database,and the protein expression level of FBXL16 in HNSCC was analyzed by the Human Protein Atlas.Using Kaplan-meier Plotter to explore the correlation between FBXL16 expression level and prognosis of patients with head and neck squamous cell carcinoma.3.Effect of FBXL16 on cell biological functionAfter FBXL16 over-expressed plasmid or FBXL16 sh RNA was transfected into nasopharyngeal carcinoma CNE-2 cells for 48 h,the transfection efficiency was detected at both transcriptional and translational levels by RT-PCR and Western Blotting.The effects of FBXL16 up-regulation or down-regulation on proliferation and migration of nasopharyngeal carcinoma CNE-2 cells were detected by CCK-8 and scratch assay respectively.4.Prediction and validation of the targeting relationship between miR-675-5p and FBXL16Target Scan predicted the presence of targeted binding sites in the 3 ’ UTR of miR-675-5p and FBXL16,RT-PCR and Western Blotting verified the inhibitory effect of miR-675-5p on FBXL16,Dual luciferase reporter gene assay confirmed the direct targeting relationship between miR-675-5p and FBXL16,and re-expression of FBXL16 partially reversed the function of miR-675-5p.Results:1.RT-PCR and WB showed that transfection with miR-675-5p mimic or miR-675-5p inhibitor up-regulated or silenced miR-675-5p expression in nasopharyngeal carcinoma CNE-2 cells.miR-675-5p increased the proliferation of CNE-2 cells,while silencing of miR-675-5p cells reduced the proliferation ability.In addition,scratch experiments showed that over-expression of miR-675-5p increased the mobility of CNE-2 cells while silencing of miR-675-5p inhibited the mobility of CNE-2 cells.2.FBXL16 is under-expressed in head and neck squamous cell carcinoma compared with normal adjacent tissues,and high expression of FBXL16 is associated with a good prognosis in patients with head and neck squamous cell carcinoma.3.Similarly,transfection of FBXL16 over-expressed plasmid or FBXL16 sh RNA can also up-regulated or down-regulated the expression of FBXL16.Up-regulation of FBXL16 inhibited the proliferation of CNE-2 cells,while silencing FBXL16 promoted the proliferation.Scratch test showed that the cell migration rate was slowed down when FBXL16 was over-expressed,while the cell migration rate was increased when FBXL16 was silenced.4.Target Scan indicated the possible presence of miR-675-5p binding sites in the 3 ’ UTR of FBXL16.RT-PCR and WB demonstrated the inhibitory effect of miR-675-5p on FBXL16 from both RNA and protein levels.Dual luciferase reporting assay showed that the wild-type luciferase activity of FBXL16 was significantly decreased after miR-675-5p expression was enhanced,while the luciferase activity of FBXL16 mutant was not different,suggesting a direct targeting relationship between miR-675-5p and FBXL16.Moreover,re-expression of FBXL16 partially reversed the effect of miR-675-5p on proliferation and migration of nasopharyngeal carcinoma CNE-2 cells.Conclusion:miR-675-5p promotes proliferation and migration of nasopharyngeal carcinoma CNE-2 cells by targeting FBXL16.