The Role And Mechanism Of HNRNPA2B1 In Oral Squamous Cell Carcinoma

Objective:To clarify the role of N6-adenylate methylation(m~6A)modification in oral squamous cell carcinoma(OSCC),and to investigate the role and mechanism of HNRNPA2B1,the key regulatory protein of m~6A modification associated with OSCC prognosis.To study its role and mechanism in the progression of OSCC,to provide a theoretical basis for exploring new methods and approaches for the treatment of OSCC.Methods:1)Based on the m RNA expression data and clinical data of OSCC patients in TCGA database,bioinformatics analysis was conducted to explore the role of m~6A modification in the progression of OSCC.m~6A RNA Methylation Assay was used to determine the level of m~6A in OSCC tumor tissue samples and paired normal oral mucosa samples.2)Machine learning and regression analysis were used to screen out HNRNPA2B1,a key m~6A modified regulatory protein associated with OSCC prognosis,and the relationship between its expression level and patients’age,gender,TNM grade,pathological grade and prognosis was investigated in TCGA database and clinical tissue samples.3)The OSCC cell lines(SCC4 and CAL27)with stable knockdown and high expression of HNRNPA2B1 were constructed.The effects of HNRNPA2B1 expression on the proliferation,migration and invasion ability of OSCC cells were explored by in vitro functional experiments.The SCC4 xenograft mice model was constructed to verify the effect of HNRNPA2B1 knockdown on tumor growth4)The biological mechanism of HNRNPA2B1 promoting OSCC proliferation and metastasis was explored by gene set enrichment analysis and EMT scoring,and its downstream target gene LINE-1 was screened based on previous research results.The expression of LINE-1 and markers related to EMT process were detected by Western blot in OSCC cell lines with stable knockdown/overexpression of HNRNPA2B1 and xenograft mice tumors.Immunohistochemical(IHC)was used to detect the expression of LINE-1 and markers related to EMT process in xenograft tumor samples.5)The specific molecular mechanism of HNRNPA2B1 targeting regulation of LINE-1 was investigated by RNA-binding protein immunoprecipitation(RIP)and m~6A modification site prediction.6)In OSCC cell lines with stable knockdown and high expression of HNRNPA2B1,the expression of m~6A demethylase FTO and methyltransferase METTL3 were modified by specific si RNA interference respectively.The modification level of m~6A on LINE-1m RNA was detected by Me RIP-q PCR.The expression levels of LINE-1and EMT process related markers were detected by WB,and the proliferation,migration and invasion abilities of cells were detected by in vitro functional assay.The SCC4 xenograft mice model was constructed to observe the effect of continuous feeding of Meclofenamic acid(MA),a functional inhibitor of FTO,on tumor growth rate,and the expression levels of LINE-1 and EMT process related markers were detected by WB and IHC.Results:1)m~6A modification level was abnormally high in OSCC tumor tissues.And the level of m~6A modification was significantly correlated with the prognosis and metastasis of OSCC patients,and there were differences among different clinical grades and T grades.2)HNRNPA2B1 was a key regulatory gene of m~6A modification significantly associated with OSCC prognosis of.Its expression level increased successively in normal oral mucosa,OSCC tumor tissues and metastatic lymph nodes,and there were differences in different clinical grades,T grades and pathological grades.Its high expression was significantly correlated with OSCC metastasis and poor prognosis.3)In vitro,down-regulated HNRNPA2B1 expression reduced OSCC cell proliferation,migration and invasion,while up-regulated HNRNPA2B1 expression had the opposite effect.In mice,down-regulation of HNRNPA2B1 inhibits OSCC tumor growth.4)Compared with the low expression group of HNRNPA2B1,the genes in the high expression group were mainly enriched in the pathway related to EMT process with higher EMT score.In OSCC cell lines and nude mice,reduction of HNRNPA2B1 expression could inhibit the expression of LINE-1 and EMT process related markers,while up-regulation of HNRNPA2B1 expression resulted in opposite results.5)HNRNPA2B1 could directly bind to LINE-1 m RNA,and there were multiple m~6A motifs on line-1 m RNA.6)In OSCC cell lines with stable knockdown of HNRNPA2B1,interference with FTO expression increased the m~6A modified level of LINE-1 m RNA.Knockdown HNRNPA2B1 expression partially rescued the inhibition of LINE-1 expression,EMT process and OSCC cell proliferation,migration and invasion.However,in OSCC cell lines with stably overexpression of HNRNPA2B1,interference with METTL3expression could reduce the enrichment of m~6A on LINE-1 m RNA,and partially rescue the promoting role of LINE-1 expression,EMT process and proliferation,migration and invasion of OSCC cells after enhanced HNRNPA2B1 expression.In nude mice,continuous feeding of FTO inhibitor MA partially rescued the inhibitory effect of HNRNPA2B1knockdown on LINE-1 expression,EMT process and tumor growth.Conclusion:(1)Compared with normal oral mucosa,overall m~6A modification levels were significantly increased in OSCC tumor tissue and were significantly associated with poor prognosis and metastasis.(2)HNRNPA2B1 is a key m~6A modification regulatory gene related to the prognosis of OSCC.Its high expression is significantly correlated with OSCC metastasis and poor prognosis.(3)HNRNPA2B1 promotes OSCC proliferation and metastasis by targeting LINE-1/TGFβ1/Snail mediated EMT process in a m~6A dependent manner.19 figures,3 tables,124 references...