实用肿瘤学杂志 ›› 2020, Vol. 34 ›› Issue (5): 404-411.doi: 10.11904/j.issn.1002-3070.2020.05.004

• 基础研究 • 上一篇    下一篇

SKA1对肾细胞癌细胞增殖凋亡的影响及其分子机制研究

韩静, 蒲艳, 柳惠斌   

  1. 新疆医科大学附属肿瘤医院肿瘤防治研究所(乌鲁木齐 830011)
  • 收稿日期:2020-03-22 出版日期:2020-10-28 发布日期:2020-11-02
  • 通讯作者: 柳惠斌,E-mail:1047893820@qq.com
  • 作者简介:韩静,女,(1987-),硕士,实验员,从事肿瘤分子生物学的研究。
  • 基金资助:
    国家自然科学基金(编号:81760453)

Effects of SKA1 on cell proliferation and apoptosis of renal cell carcinoma and its molecular mechanism

HAN Jing, PU Yan, LIU Huibin   

  1. Institute of Cancer Research,the Affiliated Tumor Hospital of Xinjiang Medical University,Urumqi 830011,China
  • Received:2020-03-22 Online:2020-10-28 Published:2020-11-02

摘要: 目的 研究SKA1在肾细胞癌中的表达情况,探讨其表达对肾细胞癌细胞增殖凋亡的影响及其分子机制。方法 从癌症基因组图谱(TCGA)数据库收集肾细胞癌数据集,分析SKA1在肾细胞癌组织和正常肾组织中的表达差异及其与临床病理特征的关系和对预后的影响。收集2016年1月—2018年9月在新疆医科大学附属肿瘤医院就诊的72例肾透明细胞癌患者切除的肿瘤组织及癌旁组织,利用实时荧光定量PCR法检测SKA1的表达水平。体外培养的人肾细胞癌细胞系786-O和ACHN分别用shSKA1干扰慢病毒和对照病毒载体感染。CCK-8法检测细胞增殖活性;平板克隆实验检测细胞克隆形成能力;Annexin V-FITC检测细胞凋亡率。运用IPA@在线生物信息学软件,对基因表达谱芯片检测的差异基因进行信号传导通路及生物学功能分析。Western blot法检测各组细胞TNF-α、cleaved-caspase 3、cleaved-caspase 9、p-Akt、p-mTOR蛋白水平变化。结果 在各病理亚型肾细胞癌组织中SKA1的表达水平明显高于正常肾组织(P<0.01),与肾透明细胞癌和肾乳头状细胞癌患者的T、N、M(TNM)及AJCC分期有关(P<0.01)。SKA1高表达患者总生存期明显短于低表达患者(P<0.001)。组织样本研究结果证实,SKA1在肾透明细胞癌组织中表达水平高于癌旁组织,与TCGA数据库分析结果一致。与对照组相比,感染shSKA1慢病毒的肾细胞癌细胞增殖能力明显下降,凋亡细胞比例增加(P<0.01)。IPA@分析发现,生物功能富集排名第一的是细胞生长与增殖,TNF被强烈激活,PI3K/Akt/mTOR通路信号分子被显著抑制。Western blot实验结果证实,SKA1干扰组中TNF-α、Caspase 3和Caspase 9表达水平显著上升,而与细胞增殖相关的通路信号分子Akt和mTOR的磷酸化表达水平显著下调,与富集分析结果相一致。结论 SKA1是肾细胞癌预后不良因素。降低肾细胞癌中SKA1表达能减弱肾细胞癌细胞增殖,同时促进凋亡,其机制可能与TNF-α激活和PI3K/Akt/mTOR通路分子磷酸化水平抑制有关。

关键词: 肾细胞癌, SKA1, 细胞增殖与凋亡, 分子机制

Abstract: Objective The objectives of this study were to investigate the expression of SKA1 in renal cell carcinoma(RCC)and its effect on cell proliferation and apoptosis of RCC as well as its molecular mechanism.Methods The data set of RCC was collected from the Cancer Genome Atlas(TCGA).The expression of SKA1 in renal cell carcinoma tissues and normal renal tissues,and its relationship with clinicopathological characteristics and prognosis were analyzed.The samples of 72 patients with RCC were collected from the Affiliated Tumor Hospital of Xinjiang Medical University from January 2016 to September 2018.The expression of SKA1 was detected by quantitative real-time PCR.786-O and ACHN cells were transfected with lentivirus vector(shSKA1)and control virus vector(shNC),respectively.A CCK-8 assay was used to detect cell proliferation;a colony formation assay was used to detect the ability of cell clone formation;annexin V-FITC was used to detect the apoptotic rate.IPA@ online bioinformatics software was used to analyze the signal transduction pathway and biological function of differential genes detected by gene expression profile chip.The levels of TNF-α,cleaved-caspase-3,cleaved-caspase-9,p-Akt,and p-mTOR proteins were detected by Western blot.Results The expression of SKA1 in RCC was significantly higher than that in normal renal tissues(P<0.01),which was related to T,N,M(TNM)and AJCC stages of renal clear cell carcinoma and renal papillary cell carcinoma(P<0.01).The overall survival time of patients with high expression of SKA1 was significantly shorter than that of patients with low expression of SKA1(P<0.001).The results of tissue sample study confirmed that the expression of SKA1 in renal clear cell carcinoma was higher than that in adjacent tissues,which was consistent with the results of TCGA database analysis.Compared with the control group,the proliferation of RCC cells transfected with shSKA1 lentivirus decreased significantly,and the proportion of apoptotic cells increased(P<0.01).IPA@ analysis showed that cell growth and proliferation ranked the first in terms of biological function enrichment,TNF was strongly activated,and PI3K/Akt/mTOR signaling molecules were significantly inhibited.The results of Western blot showed that the expression of TNF-α,cleaved-caspase-3 and cleaved-caspase-9 in the SKA1 interference group was significantly increased,while the phosphorylation levels of Akt and mTOR,which were related to cell proliferation,were significantly down-regulated,which was consistent with enrichment analysis result.Conclusion SKA1 is a poor prognostic factor for RCC.The mechanism may be related to the activation of TNF-α and the inhibitory phosphorylation of PI3K/Akt/mTOR pathway.

Key words: Renal cell carcinoma, SKA1, Cell proliferation and apoptosis, Molecular mechanism

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