Study On The Mechanism Of MiR-181a-5p Regulating Autophagy Of Oral Cancer By Targeting BCL2

Objective:Oral cancer is the most common malignant tumor in oral and maxillofacial region.Autophagy is a conserved catabolic mode involving lysosomal degradation of cells,which maintains cell activity by removing damaged macromolecules and organelle and regenerating metabolic precursor substances.Studies have reported that microRNA and autophagy play important regulatory roles in the occurrence and development of cancer.In our previous studies,we found that miR-181a-5p plays an important role in the pathogenesis of oral cancer.At present,there are no reports on the mechanism of miR-181a-5p and autophagy in the pathogenesis of oral cancer.Therefore,this study explored the effect of miR-181a-5p on autophagy of oral cancer and its potential mechanism at the cellular and animal levels.Methods:1.The overexpressed miR-181a-5p vector was synthesized and stable overexpressed miR181a-5p cell line(LV-miR-181a-5p)was constructed by lentivirus transfection.The empty vector was transfected as negative Control group(LV-NC)and CAL27 cell line was blank control group(Control).The stable transfected LV-miR-181a-5p and LV-NC cell lines were screened using purinomycin,and the transfection efficiency was detected by laser confocal microscopy and qRT-PCR,respectively.The effect of miR-181a-5p on the morphology and structure of CAL27 cells was detected by HE staining after cell slides.The effects of miR-181a-5p on the proliferation of CAL27 cells were detected by CCK-8 assay and clonal formation assay.The effects of miR-181a-5p on the migration and invasion of CAL27 were detected by cell scratch assay and Transwell invasion and migration assay.2.Bioinformatics databases such as miRTar Base,Target Scan,ENCORI and miRDB were used to screen the target genes of miR-181a-5p.Pathway enrichment analysis of key target genes was performed by Metascape software,and the result bubble map was drawn.Dual luciferase reporter gene system was used to verify the targeting relationship between miR-181a-5p and BCL2.qRT-PCR and Western Blot were used to detect the gene and protein expression of BCL2 in cells.The interaction between BCL2 and Beclin1 was predicted by the String online platform and KEGG database.3.The expression of LC3Ⅱ/Ⅰ,p62,Beclin1,ATG5 and ATG7 genes and proteins in different groups of cells were detected by qRT-PCR and Western Blot.The formation of autophagosomes in cells was observed by transmission electron microscopy.4.The LV-NC group was taken as the negative control and the LV-miR-181a-5p group as the experimental group.The tumor formation model of nude mice was established,and the effect of miR-181a-5p on autophagy of oral cancer was detected at the animal level.The expression of miR-181a-5p in tumor tissues was detected by qRT-PCR.Pathological changes of tumor were observed by HE staining.The mRNA and protein expression levels of autophagy related LC3Ⅱ,p62,BCL2,Beclin1,ATG5 and ATG7 in tumor tissues were detected by qRT-PCR,Western Blot and immunohistochemical staining(IHC).Results:1.qRT-PCR showed that the expression level of miR-181a-5p in LV-miR-181a-5p cells was about 2.8 times than that of LV-NC and CAL27 cells,indicating that the overexpression miR-181a-5p stable cell line was successfully constructed.Compared with Control cells and LV-NC cells,the proliferative power,colony formation,migration and invasion of LV-miR-181a-5p cells were generally decreased.2.Four kinds of bioinformatics databases were used to predict the target genes of miR-181a-5p,and it was found that BCL2 was the intersection gene of all the prediction software.In addition,targeted prediction revealed multiple binding sites to miR-181a-5p seed sequences in its 3’UTR region,suggesting that BCL2 may be the direct target gene of miR-181a-5p.Double luciferase reporter gene assay further confirmed the direct targeting relationship between miR-181a-5p and BCL2.qRT-PCR and Western Blot showed that miR-181a-5p down-regulated the expression of BCL2 mRNA and protein.The Protein-Protein Interaction(PPI)network showed the interaction between BCL2 and Beclin1.3.The mRNA and protein expressions of LC3Ⅱ/Ⅰ,ATG5,ATG7 and Beclin1 in LV-miR-181a-5p cells were significantly higher than those in LV-NC cells by qRT-PCR and Western Blot.The mRNA and protein expressions of p62 were significantly lower than those of LV-NC cells.The number of autophagosomes and autophagic vesicles in LV-miR-181a-5p cells was significantly higher than that in LV-NC cells under transmission electron microscopy.4.Tumor formation in nude mice showed that the subcutaneous tumor formation ability of nude mice in LV-miR-181a-5p group was significantly lower than that in LV-NC group,and compared with LV-NC group,The mRNA and protein expression levels of LC3Ⅱ,Beclin1,ATG5 and ATG7 in LV-miR-181a-5p group were significantly increased(P<0.05),while the expression of p62 was significantly decreased(P<0.05),which was consistent with the results at the cellular level.The results showed that the autophagy ability of LV-miR-181a-5p group was enhanced.Conclusion:In this study,CAL27 cell line with stable overexpression of miR-181a-5p was successfully constructed by lectin virus transfection,and it was found that miR-181a-5p could promote the autophagy of oral cancer cell,confirming the targeting relationship between miR-181a-5p and BCL2.In vitro and in vivo experiments showed that overexpression of miR-181a-5p had a regulatory effect on autophagy related genes.These results suggest that miR-181a-5p may regulate the occurrence of autophagy in oral cancer cells by targeting BCL2,providing new targets and ideas for the treatment of oral cancer.