口腔鳞状细胞癌中SALL4调控相关miRNAs的鉴定及其抑制肿瘤转移的机制

Identification of SALL4-regulating miRNAs in oral squamous cell carcinoma and its mechanism inhibiting tumor metastasis

目的 探讨口腔鳞状细胞癌(OSCC)中人婆罗双树样基因4(SALL4)调控相关mi RNAs的鉴定及其抑制肿瘤转移的机制。方法 取对数期生长期的人OSCC Tca8113细胞构建负载mi RNAs前体序列的慢病毒重组体转染Tca8113细胞,将上、下调Tca8113细胞内mi RNA表达丰度的细胞设为上调组与下调组、单纯Tca8113细胞作为对照组;采用半定量逆转录多聚酶链式反应(RT-PCR)和蛋白质免疫印迹(Western blot)方法检测各组细胞转染前后SALL4 m RNA和蛋白表达,采用双荧光素酶报告系统验证mi RNA对SALL4的靶向调控,并鉴定mi RNA-S,检测SALL4调控相关的mi RNA对Tca8113细胞侵袭、转移能力的影响。结果 上调组Tca8113细胞中SALL4 m RNA表达分别高于下调组和对照组,下调组低于对照组,差异均有统计学意义(P<0.05);上调组和下调组Tca8113细胞SALL4蛋白水平差异无统计学意义(P>0.05),但均高于对照组,且差异均有统计学意义(P<0.05);双荧光素酶报告系统结果表明,经过鉴定的mi RNA为mi RNA-S;上调组和下调组Tca8113细胞干预后迁移及侵袭细胞数差异均无统计学意义(P>0.05),但均少于对照组,且差异均有统计学意义(P<0.05)。结论SALL4调控相关mi RNAs在人OSCC中呈低表达,能抑制肿瘤细胞的侵袭、转移,有望成为口腔鳞状细胞癌治疗新靶点。

Objective To investigate the identification of miRNAs which regulate human sal-like gene gene 4(SALL4) and the mechanism by which idenitified miRNAs inhibit metastasis in oral squamous cell carcinoma(OSCC).Methods Human OSCC Tca8113 cells in logarithmic growth phase were transfected with lentiviral recombinant carrying miRNA precursor sequences Tca8113 cells.Three groups were set in this study: Tca8113 cells that up-regulated miRNA expression as upregulation group,in contrast,Tca8113 cells that down-regulated miRNA expression as downregulation group;Tca8113 cells as control group.Real-time fluorescent PCR(RT-PCR) and Western blot were used to detect the expression of SALL4 mRNA and protein before and after transfection in each group.The dual luciferase reporter system was used to verify that SALL4 was a target of identified miRNA.The effect of identified miRNA-S on the invasion and migration of Tca8113 cells were examined.Results SALL4 mRNA levels were ranked in the order of upregulation group > control group > downregulation group(P<0.05).SALL4 protein level were similar in both upregulation and downregulation groups(P >0.05),but higher than that in the control group(P<0.05).The dual luciferase reporter assay showed that the identified miRNA was miRNA-S.Although cell numbers in migration and invasion were not significantly different between upregulation and downregulation groups(P>0.05),they were less than those in the control group(P<0.05).Conclusion miRNAs that regulates SALL4 lowly express in human OSCC and inhibit tumor cell invasion and migration.Identified miRNA is expected to become a new target for the treatment of OSCC.