MiR-30a Inhibits The Proliferation Of Oral Squamous Cell Carcinoma Cells By Directly Targeting LRP6

Objective:Our previous study found that miR-30 a is a potential miRNA targeting LRP6.In order to further study the effect and mechanism of miR-30 a targeting LRP6 on the proliferation of oral squamous cell carcinoma cells,this study explored the correlation between miR-30 a expression and LRP6 expression in oral squamous cell carcinoma cell lines,and taken oral squamous cell carcinoma cell line HSC-3,Cal-27 cells as the research object to investigate the effect of miR-30 a on the cell proliferation by directly targeting LRP6.This study may find miR-30a/LRP6 signaling as a new pathogenic mechanism of oral squamous cell carcinoma and might provide novel ideas and targets for the diagnosis and treatment of oral squamous cell carcinoma,as well as might provide a new reference for treatment of other malignant tumors.Methods:(1)The expression of LRP6 and miR-30 a in six oral squamous cell carcinoma cell lines(HSC-4,HSC-3,Cal-27,Um1,Um2 and SCC9)and normal oral squamous cell HOK were detected by Western blot and QPCR,the relationship between miR-30 a and LRP6 expression were detected by using Pearson analysis;(2)The online bioinformatics database,TragetScan,was used to predict the regulation between miR-30 a and LRP6;dual-luciferase reporter assay was used to verify the regulation between miR-30 a and LRP6;the LRP6 protein and mRNA level in HSC-3 and Cal-27 cells were detected by Western blot and QPCR,respectively,after overexpressing miR-30 a.(3)Constructing pcDNA3.1-LRP6 plasmid to overexpress LRP6,and transfected into HSC-3 and Cal-27 cells to establish the overexpression LRP6 cells;MTT and cell clonoy formation assay were used to detect the cell proliferation and colony formation ability of HSC-3 and Cal-27 cells overexpressing miR-30 a or miR-30 a combinated with exogenously overexpression of LRP6;(4)Transfected with siLRP6 to construct HSC-3 and Cal-27 cells silencing LRP6,MTT and cell colony formation assay were used to detect the effect of silencing LRP6 on proliferatory ability of HSC-3 and Cal-27 cells,respectively.(5)Western blot was used to detect the expression of LRP6 and Wnt/β-catenin signaling pathway key proteins,such as β-catenin,Cyclin D1 and FGF8,after silencing LRP6,overexpression of miR-30 a and overexpression of miR-30 a in combination with exogenous LRP6.Results:(1)Western blot showed that the expression of LRP6 in normal oral squamous cell HOK cell was lower than that in six oral squamous cell carcinoma cell lines(HSC-4,HSC-3,Cal-27,Um1,Um2 and SCC9);QPCR showed that miR-30 a was overexpressed in normal oral squamous cell HOK cell while general low expressed in six oral squamous cell carcinoma cell lines;(2)The online database TargetScan found that LRP6 is a potential target of miR-30 a.The 3’UTR of LRP6 contains nucleotide sites complementary to miR-30 a.Dual luciferase reporter assays showed that the luciferase activity in the pmirGLO-LRP6-3’ UTR-WT group was significantly decreased after transfection with miR-30 a,while the miR-30 a binding site in 3’UTR of LRP6 was mutated,its luciferase activity showed no significant change.The expression of LRP6 mRNA and protein in HSC-3 and Cal-27 cells was significantly down-regulated after transfection with miR-30 a,confirming that LRP6 is the target of miR-30 a in oral squamous cell carcinoma;(3)Overexpression of miR-30 a in HSC-3 and Cal-27 cells significantly inhibited the ability of cell proliferation and colony formation;and the cell proliferatory and clonogenic capacity was partially restored after overexpressing miR-30 a combined with exogenous expression of LRP6;(4)the cell proliferatory and clonogenic capacity of HSC-3 and Cal-27 cells were significantly inhibited after silencing LRP6,which was consistent with that in miR-30 a overexpressing cells.(5)MiR-30 a could inhibit LRP6 and its downstream Wnt/β-catenin signaling pathway protein β-catenin,Cyclin D1 and FGF8 expression in HSC-3 cells.Conclusions: MiR-30 a inhibits oral squamous cell carcinoma by directly targeting LRP6 to block its downstream Wnt/β-catenin signal pathway,which revealing a potential new molecular mechanism leading to the progress of oral squamous cell carcinoma,and providing new ideas and new targets for its clinical diagnosis and treatment.