GPRC5A对肝癌细胞增殖、凋亡和氧化应激的影响及作用机制探讨

目的　通过检测G蛋白偶联受体家族C群5成员A（G protein coupled receptor C type group 5 member A, GPRC5A）在肝癌组织及细胞中的表达，探讨GPRC5A对肝癌细胞增殖、凋亡、氧化应激的影响及其相关作用机制。方法　收集2018年12月～2019年11月在解放军联勤保障部队第九六九医院行手术治疗的38例肝癌患者癌组织及配对癌旁正常组织样本；另选取人正常肝上皮细胞株HL-7702和人肝癌细胞株（HepG2，HCCLM3，HuH-7和MHCC-97H）进行研究。采用实时荧光定量聚合酶链反应（qRT-PCR）检测肝癌组织、癌旁正常组织及肝癌细胞、正常肝上皮细胞中GPRC5A表达量。通过转染pcDNA3.1-GPRC5A质粒构建过表达GPRC5A肝癌细胞株，采用MTT实验和V-FITC/PI凋亡试剂盒分别检测过表达GPRC5A对肝癌细胞增殖、凋亡的影响。采用活性氧指示剂DCFH-DA检测细胞中氧化应激相关因子活性氧（ROS），NAD+/NADH和三磷酸腺苷（ATP）水平。采用Western blot实验检测凋亡相关蛋白caspase-3及上皮生长因子（VEGF）蛋白表达水平。结果　肝癌组织中GPRC5A表达较癌旁正常组织显著降低（0.34±0.09 vs 0.73±0.10），差异有统计学意义（t=17.869，P<0.01）。肝癌细胞HepG2（0.35±0.06），HCCLM3（0.38±0.10），HuH-7（0.53±0.07），MHCC-97H（0.67±0.06）中GPRC5A表达量显著低于人正常肝上皮细胞HL-7702中的GPRC5A表达量（1.00±0.08），差异有统计学意义（t=5.716~11.258, 均P<0.05）。pcDNA3.1-GPRC5A组转染36 h细胞增殖吸光度（A）值（0.94±0.16）显著低于对照组（1.49±0.05）和pcDNA3.1组（1.45±0.07）（F=25.640，P<0.01）。GPRC5A过表达组VEGF（0.98±0.04）表达量较对照组（2.94±0.15）和pcDNA3.1组（2.89±0.11）显著降低（F=310.450，P<0.001）。pcDNA3.1-GPRC5A组细胞中ROS（182.12±13.42）水平显著高于对照组（101.23±11.20）和pcDNA3.1组（98.30±10.26）（F=49.577，P<0.001）；NAD+/NADH（35.24±6.43）及ATP（55.34±6.51）水平显著低于对照组（100.25±12.41，100.17±14.36）和pcDNA3.1组（97.86±9.67，102.23±11.02）（F=42.338，17.077，P<0.05）。GPRC5A过表达组细胞凋亡率高于对照组和pcDNA3.1组；细胞凋亡蛋白caspase-3（2.61±0.16）表达量也高于对照组（1.00±0.11）和pcDNA3.1组（1.01±0.02）（F=202.843，P<0.001）。pcDNA3.1-GPRC5A组细胞中p-STAT3表达量（0.43±0.06）显著低于对照组（1.00±0.13）和pcDNA3.1组（1.03±0.12）（F=29.476，P<0.001）；pcDNA3.1-GPRC5A组下游基因Socs3（0.47±0.05），c-MYC（0.54±0.06）表达量也显著低于对照组（1.01±0.05，1.00±0.04）和pcDNA3.1组（0.98±0.09，1.02±0.06）（F=63.275，75.409，P<0.001）。肝癌组织中STAT3与GPRC5A表达量呈显著负相关（r=-0.746，P<0.01）。过转染pcDNA3.1-STAT3或pcDNA3.1-NLRP3后显著逆转了pcDNA3.1-GPRC5A对肝癌细胞的抑制作用（P<0.05）。结论　GPRC5A低表达可能通过调节STAT3/Socs3/c-MYC信号和炎症反应抑制了肝癌细胞的增殖，诱导肝癌细胞的氧化应激和凋亡。

Effects of GPRC5A on Proliferation,Apoptosis and Oxidative Stress of Hepatocellular Carcinoma Cells and Its Mechanism

Objective　To explore the effects of GPRC5A on the proliferation, apoptosis and oxidative stress of liver cancer cells and its related mechanism by detecting the expression of GPRC5A in liver cancer tissues and cells. Methods　A total of 38 patients with liver cancer who underwent surgical treatment in the 969th Hospital of the Joint Logistics Support Force of the People’s Liberation Army from December 2018 to November 2019 were collected for their cancer tissue samples and matched adjacent normal tissue samples. Human normal liver epithelial cell lines HL-7702 and human hepatocellular carcinoma cell lines (HepG2, HCCLM3, Huh-7 and MHCC-97H) were selected for the study. QRT-PCR was used to detect the expression of GPRC5A in liver cancer tissues, adjacent normal tissues, liver cancer cells and normal liver epithelial cells. The overexpressed GPRC5A hepatoma cell line was constructed by transfection of pcDNA3.1-GPRC5A plasmid. The effects of overexpression of GPRC5A on the proliferation and apoptosis of hepatoma cells were detected by MTT assay and V-FITC/PI apoptosis kit, respectively. The levels of reactive oxygen species (ROS), NAD+/NADH and adenosine triphosphate (ATP) were detected by DCFH-DA, a reactive oxygen indicator. Western blot assay was used to detect the expression levels of apoptosis-related protein caspase-3 and epithelial growth factor (VEGF). Results　The expression of GPRC5A in HCC tissues was significantly lower than that in adjacent normal tissues (0.34±0.09 vs 0.73±0.10), the difference was statistically significant(t=17.869, P<0.01). The expression level of GPRC5A in HepG2 cells (0.35±0.06), HCClM3 cells (0.38±0.10), Huh-7 cells (0.53±0.07), and MHCC-97H (0.67±0.06) was significantly lower than that in HL-7702 (1.00±0.08), the differences were statistically significant(t=5.716~11.258, P<0.05). The A value of pcDNA3.1-GPRC5A group (0.94±0.16) at 36 h after transfection was significantly lower than that of Control group (1.49±0.05) and pcDNA3.1 group (1.45±0.07) (F=25.640, P<0.01). The expression of VEGF in GPRC5A overexpression group was significantly decreased (0.98±0.04) compared with that in control group (2.94±0.15) and PCDNA3.1 group (2.89±0.11) (F=310.450, P<0.001). The level of ROS (182.12±13.42) in PCDNA3.1-GPRC5A group was significantly higher than that in control group (101.23±11.20) and PCDNA3.1 group (98.30±10.26) (F=49.577, P<0.001). NAD+/NADH (35.24±6.43) and ATP (55.34±6.51) levels were significantly lower than those in control group (100.25±12.41, 100.17±14.36) and PCDNA3.1 group (97.86±9.67, 102.23±11.02) (F=42.338, 17.077, P<0.05). The apoptosis rate of GPRC5A overexpression group was higher than that of control group and pcDNA3.1 group. The expression level of apoptosis protein caspase-3 (2.61±0.16) was also higher than that of control group (1.00±0.11) and PCDNA3.1 group (1.01±0.02) (F=202.843, P<0.001). The expression level of p-STAT3 in PCDNA3.1-GPRC5A group (0.43±0.06) was significantly lower than that in control group (1.00±0.13) and PCDNA3.1 group (1.03±0.12) (F=29.476, P<0.001). The expression levels of downstream gene SOCS3 and c-myc in pcDNA3.1-GPRC5A group were also significantly lower than those in control group(1.01±0.05, 1.00±0.04) and pcDNA3.1 group (0.98±0.09, 1.02±0.06) (F=63.275 and 75.409, all P<0.001). There was a significant negative correlation between STAT3 and GPRC5A expression in liver cancer (r=-0.746, P<0.01). The overtransfection of pcDNA3.1-STAT3 or pcDNA3.1-NLRP3 significantly reversed the inhibitory effect of pcDNA3.1-GPRC5A on HCC cells (P<0.05). Conclusion　GPRC5A may inhibit the proliferation of HCC cells by regulating STAT3/ SOCS3 / c-MYC signaling and inflammatory response, and induce oxidative stress and apoptosis of HCC cells. The low expression of GPRC5A may inhibit the proliferation of HCC cells by regulating STAT3/ SOCS3 / c-MYC signaling and inflammatory response, and induce oxidative stress and apoptosis of HCC cells.