SPHK1作为鼻咽癌预后因子及潜在治疗靶点的研究

发布时间：2018-01-31 00:07

本文关键词： SPHK1 鼻咽癌预后 FTY720 增强放疗敏感性　出处：《第三军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的鞘脂类的代谢产物如神经酰胺(Cer)、鞘氨醇(Sph)、1磷酸鞘氨醇(S1P)等,在肿瘤的发生发展过程中发挥重要作用,而鞘氨醇激酶-1(sphingosine kinase 1,SPHK1)是调节鞘脂类平衡的关键酶。研究表明SPHK1在多种实体瘤中高表达,通过SPHK1/S1P途径促进肿瘤进展、影响预后。FTY720(芬戈莫德)是鞘氨醇的类似物,具有很强的免疫抑制活性。最近研究发现,FTY720可以抑制某些肿瘤细胞增殖,促进凋亡。本实验探讨SPHK1在鼻咽癌中的表达及其与患者分期分级及预后的关系,以及SPHK1作为鼻咽癌潜在治疗靶点的可能性。研究方法运用q RT-PCR,Western blot技术检测SPHK1 m RNA和蛋白在新鲜鼻咽癌组织与鼻咽部炎症组织中的表达情况;石蜡切片免疫组化检测142鼻咽癌组织和10例鼻咽部炎症组织中SPHK1的表达,并进行评分,运用SPSS统计学分析SPHK1表达与患者临床病理参数和预后的关系。在细胞实验中,鼻咽癌细胞株CNE1和CNE2中转染siRNA,运用q RT-PCR,Western blot检测siRNA转染后SPHK1的表达;运用CCK-8法检测干扰SPHK1对细胞增殖的影响及FTY720对细胞活性和放疗敏感性的影响;流式细胞术检测干扰SPHK1和FTY720对细胞周期及凋亡的影响;运用SPHK1活性检测ELISA试剂盒检测FTY720对SPHK1酶活性的影响。研究结果qRT-PCR和Western blot检测表明SPHK1在新鲜鼻咽癌组织中的表达高于炎症组织。免疫组化检测发现142例鼻咽癌中93例(65.5%)SPHK1高表达,10例非肿瘤性组织中,SPHK1低表达或不表达。卡方检验发现SPHK1高表达与临床分期(P=0.001)、T分期(P=0.029),N分期(P=0.002)、局部复发(P=0.018)和远处转移(P=0.001)相关。Kaplan-Meier生存分析和Log-rank检验分析发现SPHK1高表达鼻咽癌患者较低表达者生存时间短(P0.001)。多因素方差分析表明SPHK1是鼻咽癌患者独立的预后因子(P0.05)。鼻咽癌细胞中转染si RNA后,q RT-PCR和WB检测转染组细胞中SPHK1 mRNA和蛋白表达均降低。鼻咽癌细胞转染si-SPHK1后抑制细胞增殖,阻滞细胞周期,促进细胞凋亡(均P0.05)。FTY720可以抑制SPHK1酶的活性,抑制鼻咽癌细胞的增殖,促进凋亡,增强细胞对放疗的敏感性。结论我们的研究结果表明SPHK1在鼻咽癌中高表达,且SPHK1表达与患者临床分期、局部复发和预后相关。干扰SPHK1抑制鼻咽癌细胞增殖,阻滞细胞周期,促进凋亡;FTY720可以抑制SPHK1酶的活性,抑制鼻咽癌细胞增殖,促进凋亡,增强鼻咽癌细胞的放疗敏感性。SPHK1有望成为一个鼻咽癌的预后分子标志物和潜在治疗靶点。
[Abstract]:Background and objective the metabolites of sphingolipids such as ceramide Ceran and sphingosine 1 sphingosine phosphate S1P play an important role in the carcinogenesis and development of tumors. Sphingosine kinase-1 sphingosine kinase 1 (SPHK1) is the key enzyme to regulate the lipid balance of sphingosine. Studies have shown that SPHK1 is highly expressed in many solid tumors. SPHK1/S1P pathway promotes tumor progression and affects prognosis. FTY720 is a sphingosine analogue with strong immunosuppressive activity. FTY720 can inhibit the proliferation of some tumor cells and promote apoptosis. This study was designed to investigate the expression of SPHK1 in nasopharyngeal carcinoma and its relationship with stage, grade and prognosis. And the possibility of SPHK1 as a potential therapeutic target for nasopharyngeal carcinoma. Q RT-PCR was used in the study. Western blot technique was used to detect the expression of SPHK1 m RNA and protein in nasopharyngeal carcinoma and nasopharyngeal inflammation. The expression of SPHK1 in 142NPC tissues and 10 cases of nasopharyngeal inflammation was detected by immunohistochemical staining in paraffin sections. The relationship between the expression of SPHK1 and clinicopathological parameters and prognosis was analyzed by SPSS. In the cell experiment, siRNA was transfected into CNE1 and CNE2. The expression of SPHK1 after siRNA transfection was detected by Q RT-PCR blot. The effect of interfering SPHK1 on cell proliferation and the effect of FTY720 on cell activity and radiosensitivity were detected by CCK-8 assay. The effects of interfering SPHK1 and FTY720 on cell cycle and apoptosis were detected by flow cytometry. The effect of FTY720 on the activity of SPHK1 was detected by ELISA kit of SPHK1 activity. Results qRT-PCR and Western. Blot showed that the expression of SPHK1 in fresh nasopharyngeal carcinoma was higher than that in inflammatory tissue. The expression of SPHK1 was high. In 10 cases of non-tumor tissues, the expression of SPHK1 was low or not. Chi-square test showed that the high expression of SPHK1 and the clinical stage P0. 001T stage were P0. 029). N stage P0. 002). Local recurrence (P0. 018) and distant metastasis (P0. 001). Kaplan-Meier survival analysis and Log-rank test showed that the survival time of patients with high expression of SPHK1 was shorter than that of patients with low expression of SPHK1 (P0.001). Multivariate analysis of variance showed that SPHK1 was an independent prognostic factor in patients with nasopharyngeal carcinoma (NPC). After transfection of si RNA into nasopharyngeal carcinoma cells. The expression of SPHK1 mRNA and protein in transfected cells was decreased by Q RT-PCR and WB. The proliferation and cell cycle of nasopharyngeal carcinoma cells were inhibited by si-SPHK1 transfection. Promote apoptosis (P0.05. FTY720) can inhibit the activity of SPHK1 enzyme, inhibit the proliferation of nasopharyngeal carcinoma cells, promote apoptosis. Conclusion our results indicate that SPHK1 is highly expressed in nasopharyngeal carcinoma, and the expression of SPHK1 is related to the clinical stage of patients. Local recurrence is related to prognosis. Interfering with SPHK1 inhibits the proliferation of nasopharyngeal carcinoma cells, blocks cell cycle and promotes apoptosis. FTY720 can inhibit the activity of SPHK1 enzyme, inhibit the proliferation of nasopharyngeal carcinoma cells and promote apoptosis. Enhancing the radiosensitivity of nasopharyngeal carcinoma cells. SPHK1 is expected to be a prognostic molecular marker and a potential therapeutic target for nasopharyngeal carcinoma.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.63

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