MicroRNA-99a-5p Regulates The Proliferation,invasion And Metastasis Of Oral Squamous Cell Carcinoma By Targeting FGFR3

Objective: OSCC is the most common epithelial malignancy in oral and maxillofacial region,incidence rate is about 90% of oral cancer,and its mortality rate accounts for sixth of the world’s cancer mortality.OSCC often occurs in 40-60 years old adults,the incidence rate of male is more than that of female,the most frequent sites are tongue,gum,cheek and palate.The main clinical manifestations are lymph node metastasis,poor prognosis,strong local invasion,etc.because OSCC has the characteristics of occult disease,rapid disease progress,high degree of malignancy,easy recurrence,and most of the patients have been diagnosed in the middle and late stage.Most of the diseases have invaded important areas such as blood vessels,nerves,bone or skull base,causing discomfort symptoms such as pain,numbness,bleeding,and serious dysfunction in swallowing,speech,breathing,etc.These symptoms make the living difficult to OSCC patients,sometimes cause the death.In terms of treatment,the comprehensive treatment based on surgery is still the most effective treatment at present.With the continuous progress of related research,the cure rate of OSCC patients has improved,but the prognosis of OSCC patients is still poor,which brings difficulties to clinical treatment.Therefore,how to achieve early prevention,early diagnosis and explore the sensitive diagnostic markers of OSCC and effective treatment of individualized treatment scheme to improve the clinical cure rate and survival rate is one of the issues concerned by the modern medical community.MiRNA is a kind of small noncoding RNA.By acting on the 3 ′ untranslated region of the target gene,miRNA silences the expression of the target gene,participates in the regulation of gene expression at the post transcriptional level,thus affecting the stability of mRNA or interfering with protein translation,thus playing the role of oncogene or tumor suppressor gene.A large number of studies have shown that miRNA plays an important role in the evolution of tumor,and as a tumor suppressor gene or proto oncogene participates in the occurrence and development of tumor.In recent years,many studies have reported that mirna-99a-5p can be used as a tumor suppressor gene to inhibit the proliferation and invasion of a variety of tumor cells.As a new kind of miRNA,its regulatory mechanism is not completely clear.Therefore,this study will transfer the research of mirna-99a-5p to the pathogenesis of OSCC,explore its function in OSCC and further explore the mechanism of action,which is of great significance.In this study,we first used the Cancer Genome Atlas(TCGA)database,combined with survival analysis and hierarchical analysis of clinicopathological data,to screen and analyze prognosis related miRNA(miRNA-99a-5p).To study the role of miRNA-99a-5p)in oral squamous cell carcinoma cells by cell function experiment.According to the prediction of bioinformatics,the potential target(FGFR3)of FGFR3 was verified,and the significance of target regulation was discussed by knocking out the target molecules.Methods: 1.Data collection: download the clinical case data of HNSCC patients through the database website(https://portal.gdc.cancer.gov/).Using programming software R language to carry out systematic arrangement and statistical analysis of downloaded data.The miRNA expression profile was analyzed by active Perl,R and R studio software,and the differentially expressed miRNA was screened by single factor and multi factor Cox regression analysis.2.Kaplan Meier and ROC curve analysis were used for survival analysis and risk model for prognosis was established;meanwhile,bioinformatics function annotation and pathway analysis and prediction were carried out for selected differentially expressed mi RNAs by David online tool;target gene target analysis and molecular pathway prediction were carried out by Miranda,Targetscan 7.1 and Pictar network database software.3.The expression of mi R-99a-5p gene was detected by real-time quantitative PCR.The effects of miR-99a-5p on the proliferation,migration and invasion of OSCC cells were observed by CCK-8,scratch and transwell experiments.4.It was predicted that there might be a targeted binding site between mi R-99a-5p and FGFR3 by combining database and website,and further confirmed the targeting relationship between FGFR3 and miR-99a-5p by double Luciferase Report.Real time PCR and Western blot were used to detect the expression of FGFR3 in normal oral keratinocytes and different oral squamous cell carcinoma cell lines.CCK-8,scratch and transwell experiments were used to detect the effects of FGFR3 knockdown on the proliferation,migration and invasion of OSCC cells,further proving that mi R-99a-5p can mediate the effects of FGFR3 on the biological properties of OSCC.5.Statistical analysis: experimental results and data analysis were analyzed by SPSS 22.0 Inc.,Chicago,IL,USA software,P < 0.05,indicating that there was statistical difference between groups.Results: 1.Based on the analysis of TCGA database,we found three miRNAs(hsa-mir-99a(P = 0.0078),hsa-mir-499a(P=0.0023),hsa-mir-1911(P=0.0304)),which are independent risk factors significantly related to the survival of HNSCC patients.2.The survival analysis of miRNA signature risk score showed that hsa-mir-499 a and hsamir-1911 coefficients were positive,indicating that their high expression was related to short survival and poor prognosis,while hsa-mir-99a-5p coefficients were negative,indicating that their high expression was related to long survival and good prognosis.Hsamir-99a-5p,as a protective factor,was highly expressed in the low-risk group,while hsamir-499 a and hsa-mir-1911,as risk factors,were highly expressed in the high-risk group.3.KEGG and go analysis suggested that HNSCC might be related to multiple molecular signaling pathways.The changes of JAK-STAT signaling pathway,tyrosine metabolism and hormone related protein gene may be closely related to HNSCC.4.The expression of mir-99a-5p in OSCC was low,and the relative expression of mir-99a-5p in OSCC was 0.46 times lower than that in normal tissues.5.The expression level of mir-99a-5p in four OSCC cell lines Cal-27,Tca8113,HSC-3 and SCC-4 was significantly lower than that of Hok cell line.The relative expression of HOK was 0.48 times,0.30 times,0.16 times and 0.19 times higher than that of Hok(P < 0.01).6.Overload of miR-99a-5p mimics could significantly reduce the invasion and migration function of OSCC(*P<0.05),but had no significant effect on cell proliferation(P>0.05).7.The predicted results of bioinformatics indicate that there is a targeted binding site between the 3′UTR region of FGFR3 gene and mir-99a-5p,and FGFR3 may be an important target molecule of mir-99a-5p.8.The expression of FGFR mRNA and FGFR3 protein in OSCC cell lines Cal-27 and Tca8113 was significantly higher than that in normal human oral keratinocytes.9.Pearson correlation analysis showed that the expression level of FGFR3 was negatively correlated with the expression level of mir-99a-5p,and the overload of mir-99a-5p could significantly reduce FGFR3.10.Double luciferase assay also confirmed that there was a target gene binding site between mi R-99a-5p and downstream FGFR3.11.micro-RNA-99a-5p can negatively regulate FGFR3,and the proliferation,metastasis and invasion of OSCC decreased after FGFR3 targeted knockdown.The results suggested that microrna-99a-5p could regulate the proliferation,invasion and metastasis of OSCC by targeting FGFR3.Conclusions: 1.Based on the bioinformatics analysis and experimental results of TCGA database,we found that mir-99a-5p,as one of the specific expression miRNAs of OSCC,is low expression in tumor,which is an independent risk factor related to survival;mir-99a-5p has the function of reducing the invasion and migration of OSCC cells,suggesting that it has the potential ability to inhibit invasion and metastasis.2.The double luciferase experiment confirmed that there was a target gene binding site between miR-99a-5p and downstream FGFR3;miR-99a-5p mimics could significantly down regulate FGFR3 after overload;mi R-99a-5p could negatively regulate FGFR3;after targeted knockdown of FGFR3,the proliferation,transfer and invasion function of OSCC decreased.These results suggest that microrna-99a-5p regulates the proliferation,invasion and metastasis of OSCC by targeting FGFR3.