The Mechanism Of MiR-10b Regulating Autophagy In Oral Squamous Cell Carcinoma

Objective: Oral squamous cell carcinoma(OSCC)is one of the most common malignant tumors in oral cancer.Although the diagnosis and treatment technology has improved,it still cannot improve the overall survival rate of OSCC.Autophagy maintains cell homeostasis and participates in different stages of tumor progression.At present,a variety of micro RNAs(miRNAs)are involved in the regulation of autophagy.miR-10 b plays a carcinogenic effect in a variety of tumors,but the mechanism of miR-10 b on OSCC cell autophagy is still unclear.This study will explore the potential mechanism of miR-10 b in OSCC cell autophagy.Methods: 1.We used RT-qPCR to verify the expression of miR-10 b in 20 pairs of OSCC tissues and OSCC cell lines.2.The cell model was constructed by transfecting micro RNA inhibitor or micro RNA mimics to knock down or overexpress the expression of miR-10 b,respectively.Then we used wound healing and transwell assay to detect the effect of miR-10 b on the migration and invasion of OSCC cells.3.Western Blot,transmission electron microscopy(TEM)and sens GFP-stub RFP-LC3 were used to detect the effect of miR-10 b on autophagy in OSCC cells.4.We knock down the expression of the ATG5 in OSCC cells by si RNA-ATG5,and then wound healing and transwell assay was used to observe the effect of miR-10 b on the migration and invasion ability of OSCC cells after inhibiting autophagy.5.Bioinformatics analysis was used to predict potential target genes of miR-10 b,and further detected by RT-qPCR.Then,the candidate gene was verified by luciferase reporter assay,Western Blot and immunohistochemistry.6.The overexpression plasmid of the target gene and wound healing and transwell assay was used to verify whether the target gene is involved in the migration and invasion of OSCC cells by miR-10 b.7.We used western blot,TEM and sens GFP-stub RFP-LC3 to detect whether miR-10 b mediated autophagy in OSCC cells through the target gene.Results: 1.Compared to adjacent tissues,the expression of miR-10 b was elevated in OSCC,and the expression of miR-10 b was higher in OSCC cell lines SCC-15,TCA-8113,CAL-27,and SCC-25 than in HOK cell lines.The OSCC cell lines SCC-25 which was highest expression of miR-10 b was used for subsequent cytological experiments.2.miR-10 b inhibitor and mimics could effectively downregulate or upregulate the expression of miR-10 b in SCC-25 cells.Compared with the control group,low expression of miR-10 b would significantly inhibit the migration and invasion ability of SCC-25 cells.The upregulation of miR-10 b could promote the migration and invasion ability of SCC-25 cells.3.Inhibition of miR-10 b will inhibit the autophagy of SCC-25 cells,and overexpression of miR-10 b will enhance the autophagy of SCC-25 cells.4.Silencing ATG5 could abolished the effects on SCC-25 invasion and migration caused by miR-10 b overexpression.5.We initially screened out 11 candidate genes(RORA,CREB1,FIGN,SOBP,BDNF,TFAP2 C,GATA6,BCL2L11,EBF2,HOXA3,CADM2)by analyzing the four databases of Target Scan,miRDB,micro T_CDS,and EIMMo,and then we further verified candidate genes by RTqPCR.We found BCL2L11(Bim)was the direct target of miR-10 b in OSCC.6.When the expression of Bim was upregulated by Bim plasmid in SCC-25 cells,we found that the migration and invasion ability of SCC-25 was inhibited.When miR-10 b mimics were cotransfected,the inhibition of Bim on SCC-25 cells could be lifted.7.Overexpression of Bim could significantly inhibit autophagy in SCC-25 cells,and co-transfection of miR-10 b mimics can reverse the inhibitory effect of Bim on autophagy.Conclusion: The high expression of miR-10 b could regulate autophagy and promote migration and invasion of OSCC cells.Moreover,Bim is a direct target gene of miR-10 b in OSCC cells.Overexpression of Bim can inhibit the autophagy and migration and invasion of OSCC cells.Therefore,miR-10 b can be used as a potential therapeutic target for OSCC.