miR-199b-5p调控C1GALT1对胃癌细胞增殖、迁移及侵袭的影响

Effects of mir-199b-5p regulation of C1GALT1 on proliferation, migration and invasion of gastric cancer BGC-823 cells

目的 C1GALT1已被报道在多种癌症中呈现高表达,miR-199b-5p对肿瘤的发生有抑制作用,但在胃癌中的作用机制尚不明确。文章旨在探讨miR-199b-5p通过调控C1GALT1对胃癌BGC-823细胞增殖、迁移和侵袭影响。方法 利用生物信息学分析C1GALT1在胃癌中的表达情况。细胞进行分组(1)SiRNA转染:空白对照组1、Si-NC组、Si组(3、5、8μM);(2)双荧光素酶实验分组:MI-NC+h-C1GALT1-3UTR-wt组、MI+h-C1GALT1-3UTR-wt组、MI-NC+h-C1GALT1-3UTR-mu组、MI+h-C1GALT1-3UTR-mu组;(3)miRNA转染:MI组、 MI-NC组、空白对照组2;IN组、IN-NC组,空白对照组3;(4)共转染:空载体组、Si-C1GALT1组、Si-C1GALT1+miR-199b-5p inhibitor。Western blot检测SiRNA抑制C1GALT1的表达,CCK8法、细胞划痕和Transwell实验分别检测SiRNA干扰C1GALT1对胃癌BGC-823细胞的增殖、迁移和侵袭能力。预测能与其存在负向调控的miRNAs,双荧光素酶报告基因实验验证C1GALT1与miR-199b-5p之间的靶向关系。转染miR-199b-5pmimics/inhibitor后用Western blot检测转染后C1GALT1的表达情况,CCK8法、细胞划痕和Transwell实验分别检测miR-199b-5p对胃癌BGC823细胞的增殖、迁移和侵袭能力。通过Si-C1GALT1和miR-199b-5p inhibitor共转染检测其对胃癌BGC-823细胞的增殖、迁移和侵袭能力影响。结果 生物信息学提示C1GALT1在胃癌中存在高表达,通过SiRNA抑制C1GALT1表达后,发现C1GALT1低表达会使胃癌BGC-823细胞增殖、迁移和侵袭能力[(0.270±0.005)%、(534.0±24.1)个、(156.0±42.5)个]低于空白对照组1[(0.519±0.012)%、(783.0±12.8)个、(460.0±19.4)个]与Si-NC组[(0.523±0.001)%、(744.0±8.6)个、(477.0±26.9)个],差异具有统计学意义(P<0.05)。双荧光素报告显示,miR-199b-5p能靶向结合C1GALT1。miR-199b-5p mimics降低BGC-823细胞中C1GALT1表达水平(P<0.01);miR-199b-5p inhibitor提高BGC-823细胞中C1GALT1表达水平(P<0.05)。miR-199b-5p mimics组BGC-823细胞的增殖,迁移和侵袭能力[(0.256±0.002)%、(145±5.7)个、(368±57.6)个]与空白对照组2[(0.529±0.003)%、(651.0±35.7)个、(668.0±21.5)个]和miR-199b-5p mimic NC组[(0.514±0.004)%、(641.0±17.3)个、(662.0±36.1)个]相比均降低(P<0.05);miR-199b-5p inhibitor组BGC-823细胞的增殖,迁移和侵袭能力[(0.984±0.001)%、(1 089.0±114.4)个、(894.0±15.4)个]与空白对照组3[(0.529±0.003)%、(651.0±35.7)个、(668.0±21.5)个]和miR-199b-5p inhibitor NC[(0.532±0.011)%、(630.0±17.7)个、(672.0±48.5)个]相比均提高(P<0.05)。Si-C1GALT1和miR-199b-5p inhibitor共转染可部分逆转Si-C1GALT1对胃癌BGC-823细胞增殖、迁移和侵袭抑制作用。结论 miR-199b-5p可能通过调控C1GALT1抑制胃癌BGC-823细胞的增殖,迁移和侵袭。

Objective C1GALT1 has been reported to be highly expressed in a variety of cancers, and mir-199b-5p has an inhibitory effect on the occurrence of tumors, but the mechanism in gastric cancer is still unclear. This study aimed to investigate the effects of mir-199b-5p on the proliferation, migration and invasion of GASTRIC cancer BGC-823 cells by regulating C1GALT1. Methods The expression of C1GALT1 in gastric cancer was analyzed by bioinformatics.(1)SiRNA transfection: control group 1, Si-NC group, Si group(3, 5, 8μM);(2) The double lucifase experiment was divided into: MI-NC+h-C1GALT1-3 UTR-wt group, MI +h-C1GALT1-3 UTR-wt group, MI-NC+h-C1GALT1-3 UTR-mu group and MI +h-C1GALT1-3 UTR-mu group.(3)miRNA transfection: MI group, MI-NC group and control group 2;IN group, IN-NC group, control group 3;(4) Co-transfection: empty vector group, Si-C1GALT1 group, Si-C1GALT1+mir-199b-5p inhibitor. Western blot was used to detect the inhibition of C1GALT1 expression by SiRNA. CCK8 assay, cell scratch assay and Transwell assay were used to detect the proliferation, migration and invasion of C1GALT1 by SiRNA on GC-823 cells. The negative regulatory miRNAs were predicted, and the targeting relationship between C1GALT1 and mir-199b-5p was verified by dual luciferase reporter assay. Western blot was used to detect the expression of C1GALT1 after transfection with mir-199b-5pmimics/inhibitor. CCK8 assay, cell scratch assay and Transwell assay were used to detect the proliferation, migration and invasion of mir-199b-5p on gastric cancer BGC-823 cells, respectively. The effects of Si-C1GALT1 and mir-199b-5p inhibitor co-transfection on the proliferation, migration and invasion of GASTRIC cancer BGC-823 cells were detected. Results Bioinformatics suggested that C1GALT1 was highly expressed in gastric cancer. After inhibiting the expression of C1GALT1 by SiRNA, it was found that low expression of C1GALT1 could induce the proliferation, migration and invasion of gastric cancer BGC-823 cells [(0.270±0.005)%,(534.0±24.1),(156.0±42.5)] lower than that of the control group 1 [(0.519±0.012)%,(783.0±12.8),(460.0±19.4)] and Si-NC group[(0.523±0.001)%,(744.0±8.6),(477.0±26.9)] with statistically significant difference(P<0.05). The dual fluorescein report showed that mir-199b-5p could bind C1GALT1 in a targeted manner. mir-199b-5p mimics reduced the expression level of C1GALT1 in BGC-823 cells(P<0.01);mir-199b-5p inhibitor increased the expression level of C1GALT1 in BGC-823 cells(P<0.05). Proliferation, migration and invasion of BGC-823 cells in mir-199b-5p mimics group [(0.256±0.002)%,(145±5.7),(368±57.6)] were lower(P<0.05), compared with control group 2 [(0.529±0.003)%,(651.0±35.7),(668.0±21.5)] and mir-199b-5p mimic NC group [(0.514 + 0.004) %,(641.0±17.3),(662.0±36.1)];Proliferation, migration and invasion of BGC-823 cells in mir-199b-5p inhibitor group [(0.984±0.001)%,(1089.0±114.4),(894.0±15.4)] were increased, compared with control group 3 [(0.529±0.003)%,(651.0±35.7),(668.0±) 21.5)] and mir-199b-5p inhibitor NC group[(0.532±0.011)%,(630.0±17.7),(672.0±48.5)](P<0.05). Inhibition of proliferation, migration and invasion of GASTRIC cancer BGC-823 cells by Si-C1GALT1 and mir-199b-5p inhibitor co-transfection was partially reversed. Conclusion mir-199b-5p may inhibit the proliferation, migration and invasion of GASTRIC cancer BGC-823 cells by regulating C1GALT1.