细胞周期蛋白K促进食管鳞癌细胞增殖的机制研究

Mechanism of cyclin K promoting proliferation of esophageal squamous cell carcinoma cells

目的:探讨细胞周期蛋白K(CCNK)促进食管鳞状细胞癌(ESCC)发生发展的机制。方法:对中国ESCC高发区155例患者的转录组数据及GSE53625数据库进行CCNK基因差异表达分析;通过转染慢病毒分别在KYSE150、KYSE30及KYSE180细胞中构建CCNK基因稳定敲减(CCNK-SH)和阴性对照(NC)细胞株;利用集落形成实验和CCK-8法检测敲减CCNK基因对ESCC细胞增殖的影响;应用流式细胞术检测敲减CCNK基因对ESCC细胞周期的影响;分析155例ESCC患者转录组数据,对CCNK差异表达基因进行信号通路富集;应用免疫荧光染色检测DNA损伤的指标γ-H2AX;分析155例ESCC患者转录组数据和RT-qPCR实验验证CCNK基因与DNA损伤修复相关基因表达的情况。结果:CCNK基因在ESCC组织中高表达(P<0.01);与NC组相比,CCNK-SH组细胞增殖和集落形成能力显著降低(P<0.01);敲减CCNK基因能够将ESCC细胞阻滞于G2/M期(P<0.01);免疫荧光染色结果显示,与NC组相比,CCNK-SH组γ-H2AX信号显著增强(P<0.01);CCNK基因表达与DNA损伤相关基因存在显著相关性。结论:CCNK可能通过DNA损伤修复机制参与细胞周期调控,从而影响ESCC细胞的增殖。

AIM:To explore the role and mechanism of cyclin K(CCNK)in the development of esophageal squamous cell carcinoma(ESCC).METHODS:Transcriptome data of 155 patients with ESCC in China and GSE53625 database were analyzed for the differential expression of CCNK gene.Cell lines with stable knockdown of CCNK gene(CCNK-SH)and negative control(NC)cell lines were constructed in KYSE150,KYSE30 and KYSE180 cells via transfecting the lentivirus.The proliferation ability was detected by clonal formation assay and CCK-8 assay.The cell cycle was detected by flow cytometry.Pathway enrichment associated with CCNK was analyzed using the transcriptome data of 155 ESCC cases.The DNA damage marker was detected by immunofluorescence staining.The correlations between CCNK and DNA damage-related genes were verified through the transcriptome data of 155 cases and RT-qPCR assay.RESULTS:CCNK was highly expressed in ESCC tumor tissues compared with normal tissues(P<0.01).Its knockdown significantly inhibited cell proliferation and colony formation and induced cell cycle arrest in G2/M phase compared with NC group(P<0.01).Bioinformatics analysis suggested that CCNK was associated with DNA damage repair.Immunofluorescence staining results showed thatγ-H2AX signal was significantly enhanced in CCNK-SH group compared with NC group(P<0.01).The expression of CCNK was significantly correlated with DNA damage-related genes.CONCLUSION:CCNK might be involved in cell cycle regulation of ESCC cell lines via DNA damage repair pathway.