鞘氨醇-1-磷酸转运体在胃癌组织的表达及其对胃癌细胞增殖和迁移的影响

Expression of Spns2 in gastric cancer tissue and its effect on the proliferation and migration of gastric cancer cells

目的鞘氨醇-1-磷酸转运体(Spns2)与肿瘤相关研究报道较少。文中旨在比较Spns2在胃癌和癌旁组织中的表达,并探讨Spns2对人胃癌细胞SGC-7901增殖、克隆形成和迁移能力的影响。方法收集2016年2月至8月江苏省肿瘤医院消化内科胃癌术后病理标本20对(癌组织和癌旁组织)。通过免疫组化法比较Spns2蛋白在胃癌和癌旁组织中的表达差异。构建过表达Spns2的p EX-1真核载体和小干扰RNA(siRNA),转染人胃癌SGC-7901细胞,根据转染的质粒载体不同分2组:p EX-1-Spns2组(转染p EX-1-Spns2真核表达载体)、p EX-1组(转染空载体p EX-1)。进行siRNA制备与转染,根据转染的siRNA不同分2组:siRNA-Spns2组(加入针对Spns2的脂质体介导的siRNA)、siRNA-NC组(加入不与人任何基因序列同源的siRNA)。采用RT-PCR和Western blot分别检测转染后各组Spns2 mRNA和蛋白的表达水平;采用CCK-8、克隆形成实验、Transwell小室和划痕实验检测Spns2过表达和下调对SGC-7901细胞的增殖、克隆形成和迁移能力的影响。结果胃癌组织Spns2蛋白表达(0.39±0.04)明显低于癌旁组织(0.45±0.02),差异有统计学意义(P<0.05)。p EX-1-Spns2组Spns2 mRNA(21.96±2.08)、蛋白表达(10.71±2.78)较p EX-1组[(0.88±0.12)、(0.83±0.16)]明显升高(P<0.05)。siRNA-Spns2组Spns2mRNA (0.35±0.07)、蛋白表达(0.53±0.07)较siRNA-NC组[(0.91±0.09)、(0.92±0.08)]明显降低(P<0.05)。转染后第48、72、96小时,p EX-1-Spns2组SGC-7901细胞增殖能力较p EX-1组明显降低(P<0.05)。siRNA-Spns2组SGC-7901细胞增殖能力较siRNA-NC组明显增强(P<0.05)。p EX-1-Spns2组克隆形成率较p EX-1组明显降低[(33.30±3.81)%vs (48.00±4.60)%],差异有统计学意义(P<0.05)。siRNA-Spns2组克隆形成率较siRNA-NC组明显升高[(48.38±4.22)%vs (26.25±4.88)%],差异有统计学意义(P<0.05)。p EX-1-Spns2组穿膜细胞数明显低于p EX-1组; siRNA-Spns2组穿膜细胞数则明显高于siRNA-NC组,差异均有统计学意义(P<0.05)。p EX-1-Spns2组细胞迁移率较p EX-1组降低[(1.60±0.19)%vs (3.25±0.37)%],siRNASpns2组较siRNA-NC组升高[(3.16±0.40)%vs (1.41±0.15)%],差异有统计学意义(P<0.05)。结论 Spns2在胃癌组织中低表达,过表达Spns2可降低胃癌SGC-7901细胞的增殖、克隆形成和迁移能力;下调Spns2则可促进胃癌SGC-7901细胞的增殖、克隆形成和迁移,提示Spns2可能起着抑制胃癌发生发展的作用。

Objective Few studies are reported on the correlation of Spinster homolog2(Spns2)with cancer.This study aims to investigate the expression of Spns2 in gastric cancer and the adjacent tissue and explore its effects on the proliferation,clone formation and migration of human gastric cancer cell line SGC-7901. Methods Samples of gastric cancer and the adjacent tissue were collected from 20 patients(12 males and 8 females)after surgery between February 2016 and August 2016.The expression of Spns2 in the gastric cancer and adjacent tissues was determined by immunohistochemistry.Eukaryotic expression vector pEX-1(pGCMV/MCS/EGFP/Neo)-Spns2 and siRNA were constructed and transfected into the human gastric cancer cell line SGC-7901.The cells were divided into a pEX-1-Spns2(siRNA-Spns2)group and a negative control pEX-1(siRNA-NC)group.The mRNA and protein expressions of Spns2 were detected by RT-PCR and Western blot,respectively.And the effects of Spns2 on the migration,clone formation and proliferation of the SGC-7901 cells after Spns2 over-expressed and down-regulated were evaluated by cell counting kit-8(CCK-8)assay,clone formation assay,transwell experiment and wound healing scratch assay. Results The expression of Spns2 was significantly lower in the gastric cancer than in the adjacent tissue(0.39±0.04 vs 0.45±0.02,P<0.05).After transfection,both Spns2 mRNA and protein levels were remarkably higher in the pEX-1-Spns2(21.96±2.08 and 10.71±2.78)than in the pEX-1 group(0.88±0.12 and 0.83±0.16)(P<0.05),but markedly lower in the siRNA-Spns2(0.35±0.07 and 0.53±0.07)than in the siRNA-NC group(0.91±0.09 and 0.92±0.08)(P<0.05).At 48,72 and 96 hours,the proliferation of the SGC-7901 cells was significantly decreased in the pEX-1-Spns2 group as compared with the pEX-1 group(P<0.05)but increased in the siRNA-Spns2 group in comparison with the siRNA-NC group(P<0.05);the clone formation rate was reduced in the pEX-1-Spns2 group as compared with the pEX-1 group([33.30±3.81]%vs[48.00±4.60]%,P<0.05)but elevated in the siRNA-Spns2 group in comparison with the siRNA-NC group([48.38±4.22]%vs[26.25±4.88]%,P<0.05). Conclusion Spns2 is lowly expressed in gastric cancer,and its over-expression in SGC-7901 cells can reduce its proliferation,clone formation and migration.Spns2 may play a role in inhibiting the development and progression of gastric cancer.