LINC01285促进结直肠癌细胞的增殖和转移

LINC01285 promotes proliferation and metastasis of colorectal cancer cells by regulating epithelial-mesenchymal transition

目的探究长链非编码RNA LINC01285在结直肠癌(CRC)中的表达特点及临床意义,阐明潜在的生物学功能及调控机制。方法基于Starbase生物数据库检索LINC01285在CRC及癌旁组织中的表达量;收集本单位结直肠癌患者的CRC样本及周围正常样本各70例,分组为肿瘤组与非肿瘤组;利用荧光定量PCR实验(RT-qPCR)验证本中心CRC队列、肿瘤细胞中LINC01285的表达量,并分析其与患者临床病理参数及无瘤生存时间的关系。采用脂质体转染技术构建LINC01285低表达细胞株并分为si-Control(对照组)、si-LINC01285-1(敲低组1)、si-LINC01285-2(敲低组2)3个组,采用CCK-8实验、流式细胞学、Transwell法及蛋白印迹法检测LINC01285对结直肠癌细胞的增殖、凋亡及转移能力的影响及潜在的调控机制。结果Starbasev3.0公共数据库获取TCGA-COAD转录组测序数据发现LINC01285在癌组织中的表达量明显高于正常组织(P=0.00016);RT-qPCR结果显示:与非肿瘤组相比,LINC01285在肿瘤组表达水平显著上调(P=0.0002),其表达量与肿瘤组织学分化程度(P=0.036)、T分期(P=0.000)、淋巴结转移(P=0.001)、TNM分期(P=0.000)、Duke分期(P=0.009)和无瘤生存时间(P=0.0102)密切相关。相比于正常结直肠粘膜细胞,CRC细胞(尤其在SW620和HT-29)内LINC01285的表达水显著高表达(P<0.001)。相比于si-Control组,脂质体转染的细胞(si-LINC01285-1、si-LINC01285-2)内LINC01285表达量显著下降(P<0.001)。同时相比于si-Control组,LINC01285敲低CRC细胞组的增殖能力明显降低(P<0.001)、早期凋亡、晚期凋亡及总凋亡率明显升高(P<0.05)。相比于si-Control组,LINC01285敲低组的CRC细胞迁移和侵袭能力显著下降(P<0.001),且上皮间质转化相关通路的E-钙粘蛋白表达量显著升高(P<0.001)、N-钙粘蛋白显著下调(P<0.001)。结论LINC01285的表达与CRC的预后高度相关,可调节上皮-间质转化通路影响CRC细胞的增殖、凋亡与转移能力。

Objective To clarify the mechanism by which LINC01285 regulates proliferation and migration of colorectal cancer(CRC)cells and the clinical implications.Methods We analyzed the expression of LINC01285 in CRC tissues and normal tissues using data from Starbase public database.We also examined the expression levels of LINC01285 in 70 pairs of CRC and adjacent tissue samples collected from our center and in different CRC cell lines using RT-qPCR,and analyzed the correlation of LINC01285 expression with the clinicopathological parameters and tumor-free survival time of the patients.In CRC cell lines(SW620 and HT-29),the changes in cell proliferation,apoptosis,metastasis and epithelial-mesenchymal transition(EMT)phenotype following LINC01285 knockdown were analyzed using CCK-8 assay,flow cytometry,Transwell assay and Western blotting.Results The TCGA-COAD transcriptome sequencing data obtained from the Starbasev3.0 public database revealed a significantly higher expression level of LINC01285 in CRC tissues than in adjacent tissues(P=0.00016),which was verified by RT-qPCR results of the clinical samples(P=0.0002).In CRC patients,the expression level of LINC01285 was closely correlated with histological differentiation of the tumor(P=0.036),T classification(P=0.000),lymph node metastasis(P=0.001),TNM stage(P=0.000),Duke stage(P=0.009)and relapse-free survival(P=0.0102).In SW620 and HT-29 cells,which expressed significantly higher levels of LINC01285 than normal colorectal mucosal cells(P<0.001),LINC01285 knockdown significantly inhibited cell proliferation(P<0.001),increased early apoptosis,late apoptosis and total apoptosis rates(P<0.05),suppressed cell migration and invasion(P<0.001),upregulated the expression of E-cadherin(P<0.001),and downregulated the expression of N-cadherin(P<0.001).Conclusion The expression level of LINC01285,which modulates the EMT pathway to regulate the proliferation,apoptosis and metastasis of CRC cells,is closely correlated with the prognosis of CRC patients.