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胆囊癌是一种发生在胆囊组织的恶性肿瘤,其发病率在我国消化道肿瘤高居第五[1]。胆囊癌早期临床症状不明显,诊断困难,且由于其恶性程度高、病情发展迅速、预后差等特点,使胆囊癌病人的死亡率较高,五年生存率不足5%[2-3]。近年黏蛋白(mucin,MUC)对于肿瘤的影响逐渐得到学术界的重视[4]。MUC是一种通过上皮细胞分泌,具有单次跨膜结构的高度糖基化蛋白,在信号转导通路、免疫应答等生理病理过程中均具有一定调控作用[5]。MUC16基因位于染色体19p13.2位点,是卵巢癌的特异性诊断标志物之一。但当前关于胆囊癌细胞与MUC16间相互作用的研究仍不深入。本文采用RNA干扰技术观察MUC16对人胆囊癌细胞GBC-SD增殖和迁移的影响,探讨其可能的作用机制,以期为后续研究提供参考。现作报道。
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GBC-SD+MUC16-siRNA组MUC16 mRNA表达水平均明显低于GBC-SD组和GBC-SD+NC组(P<0.01),GBC-SD+NC组和GBC-SD组MUC16 mRNA表达差异无统计学意义(P>0.05)(见表 1)。
分组 MUC16 mRNA GBC-SD组 0.62±0.09 GBC-SD+NC组 0.59±0.09 GBC-SD+MUC16-siRNA组 0.40±0.07**## F 11.14 P <0.01 MS组内 0.007 q检验:与GBC-SD组比较**P<0.01;与GBC-SD+NC组比较##P<0.01 表 1 3组细胞中MUC16 mRNA相对表达水平比较(n=6;x±s)
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GBC-SD+MUC16-siRNA组细胞增殖率低于GBC-SD组(P<0.05),而GBC-SD+MUC16-siRNA组、GBC-SD组和GBC-SD+NC组细胞增殖率差异均无统计学意义(P>0.05)(见表 2)。
分组 增值率/% GBC-SD组 76.37±15.01 GBC-SD+NC组 74.78±14.86 GBC-SD+MUC16-siRNA组 55.32±10.73* F 4.41 P <0.05 MS组内 187.084 q检验:与GBC-SD+NC组比较*P<0.05 表 2 3组GBC-SD细胞增殖率比较(n=6;x±s)
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培养48 h后,GBC-SD+MUC16-siRNA组GBC-SD细胞迁移数均低于GBC-SD组和GBC-SD+NC组(P<0.05),GBC-SD+NC组与GBC-SD组差异无统计学意义(P>0.05)(见图 1、表 3)。
分组 迁移数目/(×104) GBC-SD组 9.12±1.56 GBC-SD+NC组 8.92±1.38 GBC-SD+MUC16-siRNA 3.68±0.70*# F 35.48 P <0.01 MS组内 1.609 q检验:与GBC-SD组比较*P<0.05;与GBC-SD+NC组比较#P<0.05 表 3 3组GBC-SD细胞迁移数目比较(n=6;x±s)
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GBC-SD+MUC16-siRNA组MUC16、CD44蛋白表达均低于GBC-SD组、GBC-SD+NC组(P<0.05~P<0.01),PCNA蛋白表达亦低于GBC-SD+NC组(P<0.05),GBC-SD组和GBC-SD+NC组MUC16、PCNA、CD44蛋白表达差异均无统计学意义(P>0.05)(见表 4、图 2)。
分组 MUC16 PCNA CD44 GBC-SD组 0.87±0.22 0.81±0.25 0.78±0.22 GBC-SD+NC组 0.84±0.18 0.79±0.21 0.74±0.21 GBC-SD+MUC16-siRNA组 0.46±0.17*## 0.48±0.19# 0.41±0.16**## F 8.57 4.31 6.28 P <0.01 <0.05 <0.01 MS组内 0.037 0.048 0.039 q检验:与GBC-SD组比较*P<0.05;与GBC-SD+NC组比较#P<0.05,##P<0.01 表 4 3组MUC16、PCNA、CD44蛋白表达比较(n=6;x±s)
siRNA沉默MUC16对胆囊癌细胞增殖和迁移的影响
Effect of siRNA silencing MUC16 on the proliferation and migration of gallbladder carcinoma cells
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摘要:
目的探讨siRNA沉默黏蛋白(mucin,MUC)16基因对人胆囊癌细胞(GBC-SD)增殖和迁移的影响及其分子机制。 方法采用慢病毒感染和siRNA干扰技术沉默GBC-SD中MUC16基因(GBC-SD+MUC16-siRNA组),同时设立阴性对照组(GBC-SD+NC组)和空白对照组(GBC-SD组)。RT-PCR检测各组GBC-SD细胞中MUC16表达情况,噻唑蓝实验检测GBC-SD细胞增殖能力,划痕实验检测GBC-SD细胞迁移能力,蛋白免疫印迹实验检测GBC-SD细胞中PCNA和CD44蛋白表达情况。 结果GBC-SD+MUC16-siRNA组细胞增殖率和细胞迁移数目均低于GBC-SD组(P < 0.05);GBC-SD+MUC16-siRNA组MUC16、CD44蛋白表达均低于GBC-SD组、GBC-SD+NC组(P < 0.05~P < 0.01),PCNA蛋白表达亦低于GBC-SD+NC组(P < 0.05)。 结论siRNA沉默MUC16具有一定的抑制胆囊癌细胞增殖和迁移作用,可为胆囊癌的治疗提供新的实验证据。 Abstract:ObjectiveTo explore the effects of siRNA silencing mucin 16(MUC16) on the proliferation and migration of gallbladder carcinoma GBC-SD cells, and its molecular mechanisms. MethodsThe GBC-SD+MUC16-siRNA group was constructed using the lentivirus infection and siRNA interference technique to silence MUC16 gene in GBC-SD cells, and the negative control group(GBC-SD+NC group) and blank control group(GBC-SD group) were set up simultaneously.The RT-PCR was used to detect the expression of MUC16 in GBC-SD cells, the MTT and scratch assays were used to detect the proliferation and migration ability of GBC-SD cells, and the Western blotting was used to detect the protein expressions of CD44 and PCNA in GBC-SD cells. ResultsThe cell proliferation rate and cell migration number in GBC-SD+MUC16-SiRNA group were lower than those in GBC-SD group(P < 0.05).The expression levels of MUC16 and CD44 protein in GBC-SD+MUC16-SIRNA group were lower than those in GBC-SD group and GBC-SD+NC group(P < 0.05 to P < 0.01), and the expression level of PCNA protein in GBC-SD+MUC16-SIRNA group was also lower than that in GBC-SD+NC group(P < 0.05). ConclusionsSiRNA silencing MUC16 can inhibit the proliferation and migration of gallbladder cancer cells, which may provide the new experimental evidence for the treatment of gallbladder cancer. -
Key words:
- gallbladder neoplasms /
- mucin /
- cell proliferation /
- cell migration
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表 1 3组细胞中MUC16 mRNA相对表达水平比较(n=6;x±s)
分组 MUC16 mRNA GBC-SD组 0.62±0.09 GBC-SD+NC组 0.59±0.09 GBC-SD+MUC16-siRNA组 0.40±0.07**## F 11.14 P <0.01 MS组内 0.007 q检验:与GBC-SD组比较**P<0.01;与GBC-SD+NC组比较##P<0.01 表 2 3组GBC-SD细胞增殖率比较(n=6;x±s)
分组 增值率/% GBC-SD组 76.37±15.01 GBC-SD+NC组 74.78±14.86 GBC-SD+MUC16-siRNA组 55.32±10.73* F 4.41 P <0.05 MS组内 187.084 q检验:与GBC-SD+NC组比较*P<0.05 表 3 3组GBC-SD细胞迁移数目比较(n=6;x±s)
分组 迁移数目/(×104) GBC-SD组 9.12±1.56 GBC-SD+NC组 8.92±1.38 GBC-SD+MUC16-siRNA 3.68±0.70*# F 35.48 P <0.01 MS组内 1.609 q检验:与GBC-SD组比较*P<0.05;与GBC-SD+NC组比较#P<0.05 表 4 3组MUC16、PCNA、CD44蛋白表达比较(n=6;x±s)
分组 MUC16 PCNA CD44 GBC-SD组 0.87±0.22 0.81±0.25 0.78±0.22 GBC-SD+NC组 0.84±0.18 0.79±0.21 0.74±0.21 GBC-SD+MUC16-siRNA组 0.46±0.17*## 0.48±0.19# 0.41±0.16**## F 8.57 4.31 6.28 P <0.01 <0.05 <0.01 MS组内 0.037 0.048 0.039 q检验:与GBC-SD组比较*P<0.05;与GBC-SD+NC组比较#P<0.05,##P<0.01 -
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