Screening And Identification Of MicroRNAs Related To The Occurrence And Development Of Oral Mucosal Carcinoma In Chinese Hamsters And Functional Analysis In Vitro

Objective:An animal model of oral mucosal cancer in Chinese hamsters was established,and the development of oral mucosal cancer in animal models was divided into four stages according to the WHO classification criteria: Normal,Simple hyperplasia,Abnormal hyperplasia,and Squamous cell carcinoma;and high-throughput sequencing technology and Quantitative real-time PCR(qRT-PCR)was used to perform in-depth sequencing and verification of various stages in the development of oral mucosal cancer in Chinese hamsters,constructs its microRNA(miRNA)expression profile,predicts its target genes,and through further bioinformatics functional analysis,Screen out key GO entries,signaling pathways,and possible biomarkers that may play an important role in this process.In addition,the effects of candidate genes miRNA-181a-5p on human oral squamous cell carcinoma cell lines Tca-8113 and CAL-27 were further clarified by in vitro cell experiments,and their target genes and downstream related interaction genes were verified.From the perspective of non-coding RNA level and gene expression,to explore the possible regulatory mechanisms in the development of oral squamous cell carcinoma,and to reveal the signaling pathways and candidate miRNAs involved in tumor apoptosis,cycle,proliferation,migration and invasion,and the clinical guidance significance.Methods:In this study,experimental animal models were established using the chemical drug induction method.First of all,DMBA(9,10-Dimethyl-1,2-Benzanthracene)was smeared on the oral cheek pouch mucosa of Chinese hamsters at a frequency of three times a week to establish Chinese hamster cheek pouch mucosa Human Disease Animal Model of Squamous Cell Carcinoma.The miRNA-seq technology was used to construct miRNA expression profiles of Chinese hamster oral mucosa cancer at various stages.Bioinformatics methods were used to analyze the differentially expressed miRNAs in each group,and the corresponding GO,KEGG and other functional analyses were performed,meanwhile,the qRT-PCR technology was used to verify the differentially expressed genes.In in vitro experiments,transient transfection technology was used to construct a miRNA-181a-5p over-expression and knock-down expression cell model.Meanwhile,the two selected oral cancer cells were divided into five groups: control(blank control group),mimics(mimic group),mimic NC(mimic negative control group),inhabitor(inhibitor group),and inhabitor NC(inhibitor negative).CCK8 test,Transwell test,and Matrigel invasion test were used to detect the proliferation,migration,and invasion of cells in each group after transfection.Flow cytometry was used to detect cell cycle and apoptosis changes.Subsequently,the miRNA target gene prediction tool(miRWalk)was used to predict possible target genes of miRNA-181a-5p and detect them using qRT-PCR.Finally,qRT-PCR was used to study the mechanism of miR-181a-5p / TIMP 1 axis inhibiting the biological behavior of oral cancer cell lines.Results:An animal model of Chinese hamster oral cancer was successfully established.The pathological results showed that during the occurrence and development of oral hamster squamous cell carcinoma in Chinese hamsters,they experienced the four stages: normal epithelium,simple hyperplasia of epithelium,abnormal proliferation of epithelium,and squamous cell carcinoma.Differentially expressed miRNA profiles were constructed at each stage,and differentially expressed miRNAs in each group were analyzed in depth using high-throughput sequencing technology combined with bioinformatics methods.There were 77,76,and 76 significantly different miRNAs in the stages of simple epithelial hyperplasia,abnormal epithelial hyperplasia,and squamous cell carcinoma;interaction analysis showed that in the simple hyperplasia stage,abnormal hyperplasia stage and squamous cell carcinoma stage were unique with 3,2 and 10 significantly different miRNAs.In the functional analysis of differential miRNAs at each stage: in the simple epithelial hyperplasia stage,abnormal epithelial hyperplasia stage,and squamous cell carcinoma stage,there were 11,10,42 biological processes,24,36,18 cellular components,42,43,48 molecular components,and 107、107 、 110 significantly enriched KEGG signal pathways,respectively.Interaction analysis shows that,there were uniquely 3 cellular components,2 molecular functions,and 3 KEGG signaling pathways in the simple epithelial hyperplasia stage,and there is no separate biological process at this stage;in the abnormal hyperplasia stage,there were uniquely 4 cellular components,2 molecular functions,and 2 KEGG signaling pathways,there was also no separate biological process at this stage;in the squamous cell carcinoma stage,there are 29 uniquely enriched biological processes,6 molecular functions,and 9 KEGG signaling pathways and there is no separate cellular component.Finally we found that miR-181a-5p had high expression abundance in each stage,and at the same time its expression level decreased in each stage of oral cancer development,thus miR-181a-5p was selected as the focus for subsequent functional verification.The vitro cell transfection experiments showed that high expression of miR-181a-5p can significantly inhibit the proliferation,colony formation,invasion,migration and cycle of oral cancer cell lines Tca-8113 and CAL-27,but significantly promote apoptosis.However,when miR-181a-5p expression was inhibited,it showed a completely opposite result to the biological behavior of the two oral cancer cell lines.Target gene prediction software shows that TIMP 1 is one of the potential targets of the miR-181a-5p.In subsequent verification experiments,compared with the blank control group and the negative control group,it was found that,In the miR-181a-5p mimic group,E-cadherin and Bax are overexpressed,while the target genes TIMP 1 and MMP2,MMP9,Vimentin,Ki67,BCL2,cyclin D1,CDK6 And E2F1 were downregulated.However,in the inhibitor group of miR-181a-5p,all genes showed completely opposite expression trends.It is speculated that miR-181a-5p / TIMP 1 axis may regulate the biological behavior of oral cancer cells by affecting the expression of invasion and migration-related genes,cycle and proliferation-related genes,and even apoptosis-related genes.Conclusion:In this study,a Chinese hamster oral buccal pouch mucosa cancer of human disease animal model with four stages of normal epithelium,simple epithelial hyperplasia,epithelial hyperplasia,and squamous cell carcinoma was successfully constructed using the smear method.And every stage animal models were sequenced and comprehensive bioinformatics analyzed.Key miRNAs,key GO entries,and key KEGG signaling pathways in the development of oral mucosal cancer in Chinese hamsters were discovered.In vitro cell experiments found that miR-181a-5p / TIMP 1 axis may be an important signaling pathway for miR-181a-5p to inhibit the development of oral cancer,and miR-181a-5p may through the miR-181a-5p / BCL2 / Bax signal Transduction axis to regulate oral cancer cell apoptosis.