RAD54L 在口腔鳞状细胞癌中的表达及功能研究

Study on the expression and function of RAD54L in oral squamous cell carcinoma

目的探讨DNA修复重组RAD54样蛋白(RAD54-like protein,RAD54L)在口腔鳞状细胞癌(oral squa-mous cell carcinoma,OSCC)中的表达及功能作用。方法利用癌症基因组图谱(The Cancer Genome Atlas,TC-GA)数据库中OSCC相关数据,通过Mann-Whitney U检验分析RAD54L在OSCC与对照样本中的表达差异,并采用受试者工作特征曲线评估RAD54L在OSCC诊断中的潜在价值。通过卡方检验分析RAD54L mRNA表达水平与OSCC患者临床病理数据之间的关系。将OSCC样本根据RAD54L mRNA表达中位值分为高低表达两组后,采用Cox回归分析比较两组的预后差异;采用DESeq2软件包筛选组间的差异表达基因,并利用cluster-Profiler包进行KEGG通路富集分析。采用Spearman相关性分析展示RAD54L与同源重组修复途径中基因表达的相关性。在明确RAD54L的生物信息学意义后,在人OSCC细胞株HSC-3中进行RAD54L基因敲低实验,并通过qRT-PCR验证敲低效率。转染后,通过CCK-8、5-乙炔基-2'-脱氧尿苷(5-ethynyl-2'-deoxyuridine,EdU)染色、细胞划痕、细胞凋亡及细胞周期实验,评估HSC-3细胞增殖、迁移、凋亡及周期的变化。结果生物信息学分析结果显示,RAD54L mRNA在OSCC中的表达高于正常对照组(P<0.001),且对预后不良的预测具有较高价值(AUC=0.927)。RAD54L高表达组男性患者比例增加(P=0.032),具有更高的T分期(P=0.040)、临床分期(P=0.027)及病理学分级(P=0.013)。以RAD54L mRNA表达中位值将OSCC样本分为高低表达两组后,RAD54L高表达组的预后较低表达组差(P=0.049);RAD54L高低表达两组间的差异表达基因主要富集于神经活性配体与受体的相互作用、细胞因子-细胞因子受体相互作用、钙信号通路、细胞周期、胃癌、细胞外基质受体相互作用、化学致癌-DNA加合物、DNA复制、同源重组和错配修复途径(P<0.05),在同源重组修复途径中RAD54L的表达与BRCA1、BLM、EME1、XRCC2、POLD1、TOPBP1、RAD51、BRIP1、RAD54B、BRCA2和SYCP3的表达呈正相关(P<0.05),其中与BRCA1、BLM和EME1的表达呈强正相关(R>0.8,P<0.05)。体外实验结果表明,RAD54L在HSC-3细胞中的表达被敲低至约25%(P<0.001),与对照组相比,RAD54L敲低组的增殖率降低(P<0.05),EdU阳性细胞比例下降(P<0.001),划痕闭合比例降低(P<0.001),G1期细胞比例增加(P<0.001),S期细胞比例降低(P<0.001),细胞凋亡比例增加(P<0.001)。结论RAD54L在OSCC中高表达且与不良预后相关。下调RAD54L表达可抑制HSC-3细胞的增殖和迁移,促进细胞凋亡,并阻碍细胞周期进程。

Objective To investigate the expression and role of the DNA repair and recombination protein RAD54-like(RAD54L)in oral squamous cell carcinoma(OSCC).Methods Using OSCC-related data from The Cancer Genome Atlas(TCGA)database,the difference in RAD54L expression between OSCC and control samples was analysed using the Mann-Whitney rank sum test,and the potential value of RAD54L mRNA in OSCC diagnosis was assessed using the receiver operator characteristic curve.The correlation between RAD54L expression levels and clinicopathological data of OSCC patients was analysed using the chi-square test.Once OSCC samples were divided into two groups of high and low expression based on the median value of RAD54L mRNA expression,Cox regression analysis was used to compare the prognostic differences between the two groups.The differentially expressed genes between the groups were subse-quently screened using the DESeq2 package,and KEGG pathway enrichment analysis was performed using the cluster-Profiler package.The correlation between RAD54L mRNA and gene expression in the homologous recombination repair pathway was demonstrated by Spearman correlation analysis.After clarifying the bioinformatics significance of RAD54L,RAD54L knockdown experiments were performed in human oral squamous carcinoma cell line HSC-3,and the knock-down efficiency was verified through real-time quantitative polymerase chain reaction.After transfection,the changes in proliferation,migration,apoptosis,and cycle of HSC-3 cells were assessed by CCK-8,5-ethynyl-2'-deoxyuridine(EdU)staining,wound healing,apoptosis,and cell cycle assays.Results Bioinformatics analysis showed that the expression of RAD54L mRNA was higher in OSCC than in normal controls(P<0.001)and had a high value in predicting poor prognosis(AUC=0.927).The high RAD54L expression group was associated with an increased proportion of male pa-tients(P=0.032),having a higher T-stage(P=0.040),clinical stage(P=0.027),and pathological grading(P=0.013).Once OSCC samples were divided into two groups of high and low expression using the median value of RAD54L mRNA expression,the prognosis of the group with high expression of RAD54L was poorer than that of the group with low expression(P=0.049).The differentially expressed genes between the high and low RAD54L expression groups two groups were mainly enriched in neuroactive ligand-receptor interactions,cytokine-cytokine receptor interactions,calci-um signaling pathway,cell cycle,gastric cancer,extracellular matrix receptor interactions,chemical carcinogenesis-DNA adducts,DNA replication,homologous recombination,and mismatch repair pathways(P<0.05).In the homolo-gous recombination repair pathway,the expression of RAD54L was positively correlated with the expression of BRCA1,BLM,EME1,XRCC2,POLD1,TOPBP1,RAD51,BRIP1,RAD54B,BRCA2,and SYCP3(P<0.05),and was strongly positively correlated with the expression of BRCA1,BLM,and EME1(R>0.8,P<0.05).The results of in vitro experi-ments showed that RAD54L expression was knocked down to approximately 25%in HSC-3 cells(P<0.001).Compared with the control group,the RAD54L knockdown group showed a lower proliferation rate(P<0.05),a lower proportion of EdU-positive cells(P<0.001),a lower proportion of wound closure(P<0.001),a higher proportion of G1-phase cells(P<0.001),a lower proportion of S-phase cells(P<0.001),and a higher proportion of apoptotic cells(P<0.001).Conclusion RAD54L is highly expressed in OSCC and correlates with poor prognosis.Down-regulation of RAD54L expression inhibits the proliferation and migration of HSC-3 cells,promotes apoptosis,and impedes cell cycle progres-sion.