【摘要】 目的 探讨circ-SOX4表达与非小细胞肺癌(NSCLC)起始细胞(TICs)干性的关系以及其促进NSCLC进展的机制。方法 利用数据库获得NSCLC原发肿瘤的环状RNA(circRNA)表达信息并验证。采用分子生物学技术检测circ-SOX4对肺癌TICs干性、增殖及凋亡的影响,并检测不同通路抑制剂对上述作用的影响。结果 数据库结果显示：在ALDH1~+肿瘤细胞中,hsa＿circ＿0000497、hsa＿circ＿0018082、hsa＿circ＿0024105、hsa＿circ＿0044360及hsa＿circ＿0131457表达显著上调。实时荧光定量-聚合酶链式反应(qRT-PCR)检测结果显示：相较于CD133~-肺癌细胞,hsa＿circ＿0131457(circ-SOX4)在CD133~+细胞中的表达显著上调,差异均具有统计学意义(P<0.05)。与阴性对照相比,过表达circSOX4使CD133~-H-1944/Calu-1细胞中CD133表达显著上调,而敲减circ-SOX则显著下调CD133~+细胞中CD133表达,差异均具有统计学意义(P<0.01)。与阴性对照相比,敲减circ-SOX4能显著抑制体外培养48、72、96 h时CD133~+H-1944/Calu-1细胞的增殖,减少CD133~+H-1944/Calu-1细胞的克隆形成,增加CD133~+H-1944/Calu-1细胞的凋亡,下调干性基因OCT4、Nanog、SOX2和SOX4的表达,差异均具有统计学意义(P<0.05)。与阴性对照相比,过表达circ-SOX4能显著促进体外培养48、72、96 h时CD133~-Calu-1细胞的增殖,增加CD133~-Calu-1细胞克隆形成,抑制CD133~-Calu-1细胞凋亡,差异均具有统计学意义(P<0.05)。Wnt通路抑制剂KYA1797 k可逆转过表达circ-SOX4对上述细胞的影响。相较于单纯过表达circ-SOX4,过表达circ-SOX4联合KYA1797 k能显著抑制体外培养48、72和96 h时CD133~-Calu-1细胞的增殖,减少CD133~-Calu-1细胞克隆形成,并增加CD133~-Calu-1细胞凋亡,差异均具有统计学意义(P<0.05)。而RO4929097(Notch通路抑制剂)、LY294002(PI3K/AKT通路抑制剂)和CHS828(NF-kB通路抑制剂)对上述转染细胞的增殖和凋亡无显著影响,差异均无统计学意义(P>0.05)。结论 circ-SOX4在NSCLC起始细胞中表达显著上调并正向调控TICs干性,circ-SOX4通过Wnt通路促进NSCLC增殖并抑制其凋亡。更多还原

【Abstract】 Objective To discuss the correlation between circ-SOX4 expression and the stemness of non-small cell lung cancer (NSCLC) tumor-initiating cells (TICs) and the mechanism of circ-SOX4 in promoting the progression of NSCLC.Methods The expression of circular RNA (circRNA) in primary tumors of NSCLC was obtained from the database and verified.Molecular biological techniques were used to detect the effects of circ-SOX4 on the stemness,proliferation and apoptosis of lung cancer TICs,and the effects of different pathway inhibitors on the above effects were detected.Results The database results showed that hsa＿circ＿0000497,hsa＿circ＿0018082,hsa＿circ＿0024105,hsa＿circ＿0044360 and hsa＿circ＿0131457 expressions were significantly upregulated in ALDH1~+tumor cells.Quantitative real-time polymerase chain reaction (qRT-PCR) assay showed that the expression of hsa＿circ＿0131457 (circ-SOX4) was significantly upregulated in CD133~+cells compared to CD133~-lung cancer cells,and the differences were all statistically significant (P<0.05).Overexpression of circ-SOX4 significantly upregulated CD133 expression in CD133~-H-1944/Calu-1 cells compared to negative controls,whereas knockdown of circ-SOX significantly downregulated CD133 expression in CD133~+cells.All differences were statistically significant (P<0.01).Compared with the negative controls,knockdown of circ-SOX4significantly inhibited the proliferation of CD133~+H-1944/Calu-1 cells in vitro at 48,72 and 96 h of culture,decreased the clone formation of CD133~+H-1944/Calu-1 cells,increased the apoptosis of CD133~+H-1944/Calu-1 cells,and down-regulated the stemness genes OCT4,Nanog,SOX2 and SOX4 expression.The differences were all statistically significant (P<0.05).Compared with the negative controls,overexpression of circ-SOX4significantly promoted the proliferation of CD133~-Calu-1 cells,increased CD133~-Calu-1 cell clone formation and inhibited CD133~-Calu-1 cell apoptosis at 48,72 and 96 h in vitro culture.The differences were all statistically significant (P<0.05).The effect of Wnt pathway inhibitor KYA1797 k reversible overexpression of circ-SOX4 on the above cells.Compared with circ-SOX4 overexpression alone,overexpression of circ-SOX4 combined with KYA1797 k significantly inhibited the proliferation of CD133~-Calu-1 cells,decreased CD133~-Calu-1 cell clone formation,and increased CD133~-Calu-1 cell apoptosis at 48,72,and 96 h in vitro cultures.The differences were all statistically significant (P<0.05).In contrast,RO4929097 (Notch pathway inhibitor),LY294002 (PI3K/AKT pathway inhibitor) and CHS828 (NF-kB pathway inhibitor) had no significant effect on the proliferation and apoptosis of the above transfected cells,and none of the differences were statistically significant (P>0.05).Conclusion circ-SOX4 expression is significantly up-regulated in NSCLC TICs and positively regulates TICs stemness.circ-SOX4 promotes proliferation and inhibits apoptosis of NSCLC through Wnt pathway.更多还原