G蛋白偶联受体81基因沉默联合顺铂诱导与喉癌能量代谢相关因子的研究

Study on G-protein-coupled receptor 81 gene silencing combined with the factors of cisplatin induction related to energy metabolism in laryngeal carcinoma cells

摘要:

目的 探讨G蛋白偶联受体81（GPR81）基因沉默联合顺铂诱导对喉癌细胞中能量代谢相关因子及细胞凋亡抑制蛋白表达的影响。方法 Hep2细胞在5 μg/mL顺铂浓度下诱导0、12、24、48 h。构建shRNA-GPR81干扰质粒，转染shRNA-scramble质粒作对照，转染至Hep2细胞后经不同浓度的顺铂诱导Hep2细胞24 h，经蛋白印迹分析（Western Blot）检测单羧酸转运蛋白4（MCT4）、细胞凋亡抑制蛋白（survivin）及Na⁺-葡萄糖共转运蛋白（SGLT-1）在蛋白质水平的变化。结果 GPR81基因沉默的Hep2经不同浓度顺铂诱导后，MCT4整体表达下调，在1 μg/mL顺铂诱导的shRNA-GPR81组中MCT4的表达显著低于shRNA-scramble组中MCT4的表达，约为后者的0.65倍（P<0.05）。SGLT-1经不同浓度顺铂诱导后先上调，在2 μg/mL达到最大值，约为0 μg/mL的3.38倍，其后开始下降，其中2 μg/mL顺铂诱导SGLT-1在shRNA-GPR81组中表达显著高于shRNA-scramble对照组，约为后者的4.5倍（P<0.05）。Survivin经不同浓度顺铂诱导后，GPR81基因沉默的Hep2细胞中，表达呈现下调趋势，其中5 μg/mL的浓度刺激使得其表达量最低，约为0 μg/mL的0.36倍，差异具有统计学意义（P<0.05），survivin在shRNA-scramble组表达的抑制程度比shRNA-GPR81组弱，在2 μg/mL和5 μg/mL顺铂诱导后，后者约为前者的0.62倍和0.64倍。特定浓度顺铂诱导Hep2细胞中MCT4、survivin、SGLT-1的表达随时间呈现递减趋势，其蛋白水平48 h表达量与0 h相比，差异具有统计学意义（P<0.05）。结论 GPR81基因沉默联合顺铂诱导可抑制MCT4表达，促进SGLT-1表达，并抑制survivin的表达。

Abstract:

Objective To investigate the effects of G-protein-coupled receptor81 (GPR81) gene silencing combined with cisplatin induction on expression of energy metabolism-related factors and apoptotic inhibitory proteins in laryngeal carcinoma cells. Methods Hep2 cells were induced at 5μg/mL cisplatin concentration for 0, 12, 24, 48 h. shRNA-GPR 81 vector and shRNA-scramble vector were constructed, and transfected into Hep2 cells. After 48h of transfection, Hep2 cells were incubated with cisplatin of different concentrations for 24 h. The protein levels of monocarboxylate transporter 4 (MCT4), apoptotic inhibitory protein(survivin), and Na⁺/glucose co-transporter (SGLT-1) were detected by Western Blot. Results The expression of MCT4 was significantly down-regulated in Hep2 silencing-GPR81 combined with the treatment of cisplatin at different concentrations. The expression of MCT4 in shRNA-GPR81 group induced by 1μg/mL cisplatin was significantly lower(0.65 times) than that of the shRNA-scramble group (P<0.05). The expression of SGLT-1 was initially up-regulated, and then down-regulated by different concentrations of cisplatin. The expression of SGLT-1 was about 3.38 times after treatment of 2 μg/mL than that of 0 μg/mL, which was the maximum value at 2 μg/mL cisplatin. The expression of SGLT-1 at 2 μg/mL induced cisplatin in shRNA-GPR81 group was significantly 4.5 times higher than that in shRNA-scramble control group (P<0.05). The expression of survivin in Hep2 cells transfected with shRNA-GPR81 was exhibited a downtrend after stimulated with cisplatin, which was the lowest at 5 μg/mL(0.36 times compared to 0 μg/mL), and there was statistical significance between two groups(P<0.05). The expression of survivin in the shRNA-scramble group was less inhibited than that in the shRNAGPR81 group. And the expression of survivin at 2 and 5 μg/mL of cisplatin induction in shRNA-GPR81 group compared to shRNA-scramble group were about 0.62-fold and 0.64 fold(P<0.05). The expressions of MCT4, survivin and SGLT-1 in Hep2 cells induced by cisplatin at a specific concentration showed a decreasing trend in time. The expression levels of MCT4, Survivin and SGLT-1 were statistically difference in Hep2 cells at 48h compared to at 0h (P<0.05). Conclusion GPR81 silencing combined with cisplatin incubation could inhibit the expression of MCT4 and survivin, but enhance the expression of SGLT-1 in laryngeal cancer.