RSK4在结直肠癌中作用的初步探讨

发布时间：2018-07-29 16:18

【摘要】：P90核糖体S6激酶(ribosomal S6Kinase, RSK)是信号通路Raf-MEK-ERK下游重要的作用靶点,属于苏氨酸丝氨酸激酶家族,迄今已发现此家族有4个成员RSK1-4,他们在调节细胞增殖、运动、生长、生存及侵袭等各种生命活动发挥着重要的作用。有关RSK1-3与肿瘤的关系的研究报道较多,在分子和细胞水平上的作用已比较清晰,而RSK4在结直肠癌研究较少,其功能和作用尚不明确。我们用免疫组织化学系统地分析了103例结直肠癌及相匹配的46例癌旁组织和正常组织中RSK4蛋白的表达水平。结果发现,RSK4蛋白在结直肠癌和癌旁组织和正常结直肠中表达相差较大,在结直肠癌组织中表达水平最低,RSK4蛋白的表达情况与结直肠癌肿瘤的恶性程度、淋巴结的转移数目和临床分期早晚紧密相关。单因素,多因素Cox风险模型发现RSK4低表达是结直肠癌众多预后因素中一个相对独立的预后较差的因子。在结直肠癌细胞SW480和HCT116中,我们用qRT-PCR和Western Blot检测到RSK4呈低表达状态,利用Lipofectamin2000技术,我们观察到转染RSK4基因过表达后,用MTT法检测到RSK4可以明显抑制结直肠癌细胞的体外生长,随着时间的延长,抑制效果越明显,48至60小时达高峰,抑制率为60-70%。流式细胞仪检测到转染RSK4基因后,SW480和HCT116细胞的细胞周期发生了显著的变化,G0/1期细胞比例上升较为明显,DNA合成期(S期)细胞比例显著下降。为了进一步观察肿瘤的侵袭能力,Transwell实验结果显示,转染RSK4基因的SW480和HCT116细胞侵袭能力明显低于对照组。RSK4对肿瘤的侵袭转移有重要的调节作用,而临床和基础实验均证实肿瘤的侵袭转移和肿瘤的间质上皮转化密不可分。本文选择了EMT相关分子Claudin-2, E-cadherin, P53, Snail,TGF-β,Twist及Vimentin,观察转染RSK4基因后各EMT相关分子的mRNA及蛋白的表达情况进行了分析,结果发现E-cadherin在转染RSK4组呈高表达,Snail在SW480, HCT116两株细胞呈低表达,而Claudin-2, P53, TGF-β,Twist, Vimentin的mRNA表达情况变化不大。其蛋白表达也有类似的情况,说明RSK4对肿瘤的侵袭转移减弱可能是通过调节E-cadherin和Snail来实现的。本研究证实了RSK4可能为结直肠癌的抑癌基因,抑制结直肠癌的侵袭转移,靶向RSK4有望成为结直肠癌的另一有效治疗靶点。
[Abstract]:Ribosomal S6 kinase (RSK) is an important target in the downstream of Raf-MEK-ERK signaling pathway and belongs to the threonine serine kinase family. Up to now, four members of the family RSK1-4 have been found to regulate cell proliferation, movement and growth. Survival and invasion and other life activities play an important role. There are many reports about the relationship between RSK1-3 and tumor, the role of RSK4 in the molecular and cellular level has been more clear, but RSK4 in colorectal cancer research is less, its function and role is not clear. We systematically analyzed the expression of RSK4 protein in 103 cases of colorectal cancer and 46 matched adjacent tissues and normal tissues by immunohistochemistry. The results showed that the expression of RSK4 protein was significantly different in colorectal cancer and adjacent tissues and normal colorectal tissues. The expression level of RSK4 protein was the lowest in colorectal cancer tissues and the malignant degree of colorectal cancer. The number of lymph node metastases is closely related to the clinical stage. Univariate and multivariate Cox risk models showed that low expression of RSK4 was a relatively independent and poor prognostic factor among many prognostic factors in colorectal cancer. In SW480 and HCT116 cells of colorectal cancer, we detected the low expression of RSK4 by qRT-PCR and Western Blot. Using Lipofectamin2000 technique, we observed that after transfection of RSK4 gene, RSK4 could significantly inhibit the growth of colorectal cancer cells in vitro by MTT assay. With the prolongation of time, the inhibitory effect reached the peak at 48 ~ 60 hours, and the inhibition rate was 60-70. Flow cytometry showed that the cell cycle of SW480 and HCT116 cells changed significantly after transfection of RSK4 gene. The proportion of G0 / 1 phase cells increased significantly and the proportion of HCT116 cells decreased significantly in S phase. In order to further observe the invasiveness of tumor and the results of Transwell experiment, it was found that the invasion ability of SW480 and HCT116 cells transfected with RSK4 gene was significantly lower than that of control group. RSK4 had an important regulatory effect on tumor invasion and metastasis. Both clinical and basic experiments have confirmed that invasion and metastasis of tumor are closely related to the transformation of interstitial epithelium. The EMT related molecules Claudin-2, E-cadherin, p53, Snail-TGF- 尾 Twist and Vimentin were selected to analyze the expression of mRNA and proteins of EMT related molecules after transfection of RSK4 gene. The results showed that E-cadherin was highly expressed in RSK4 group and low in HCT116 cells. However, the expression of mRNA in Claudin-2, p53, TGF- 尾 Twistand Vimentin was not changed. The expression of RSK4 protein is similar, suggesting that the decrease of invasion and metastasis of RSK4 may be achieved by regulating E-cadherin and Snail. This study confirmed that RSK4 may be a tumor suppressor gene for colorectal cancer and inhibit invasion and metastasis of colorectal cancer. Targeted RSK4 may become another effective therapeutic target for colorectal cancer.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34

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