Objective: To explore the effect of growth arrest-specific5 (GAS5) inhibition on the proliferation, colony formation, invasion, migration andepithelial-mesenchymal transition(EMT), cancer cell stem of HCT-116 and its mechanism. Methods: The colorectal carcinoma (CRC) cell HCT116 was divided into blank control, negative control (NC), si-GAS5 and si-GAS5+ miR-21 inhibitor groups. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expressions of miR-21 and GAS5 at 48 h after transfection. The binding site of GAS5 and miR-21 was determined by luciferase reporter array. Cell proliferation ability was detected by CCK-8 assay. Cell colony ability was detected by colony formation assay. Cell invasion and migration abilities were detected by Transwell assay. Cell cycle and apoptosis were examined by flow cytometer (FCM). The protein levels of EMT associated factors including Snail, N-cadherin, vimentin, E-cadherin, stem cell related factors including CD44, SOX2, Oct2, and PTEN/Akt signal pathway associated factors were examined by western blotting. Results: The expression levels of miR-21 in blank, NC, si-GAS5 group were 1.00±0.10, 1.00±0.10, 1.80±0.20, the absorbance values were 0.51±0.02, 0.50±0.01 and 0.65±0.01, the cell clones were 90±4, 91±5, 200±8, the invaded cells were 118±3, 119±3, 150±4, the migrated cells were 110±2, 108±2, 127±2, the cell ratios in G(1) phase were (49.3±2.1)%, (50.1±2.0)% and (42.2±1.1)%, the cell ratios in S phase were (19.2±1.2)%, (20.2±1.1)% and (28.3±2.2)%, the cell apoptotic ratios were (14.4±2.2)%, (14.5±2.1)% and (7.2±1.3)%. These results indicated that inhibition of GAS5 up regulated the expression level of miR-21, promoted cell proliferation, invasion and migration, decreased G(1)-phase cells and increased S-phase cells, and suppressed cell apoptosis (P<0.05). Moreover, inhibition of GAS5 up regulated the expressions of Snail, N-cadherin, vimentin, Sox2, CD44, Oct2 and p-Akt in HCT-116 cells (P<0.05), while down regulated the expressions of E-cadherin and PTEN (P<0.05). Inhibition of miR-21 reversed the impact of GAS5 knockdown on PTEN/Akt signaling pathway (P<0.05). Conclusion: GAS5 can act as a competing endogenous RNA for miR-21, and down regulation of GAS5 can promote the development of CRC by activating the miR-21/PTEN/Akt signaling pathway and promoting the acquisition of EMT and tumor cell stemness.
目的： 探讨抑制长链非编码RNA生长停滞敏感基因5(GAS5)对结肠癌细胞生物学特性的影响及其机制。 方法： 取结肠癌HCT116细胞，分为空白对照组(加入磷酸盐缓冲液)、阴性对照组(转染si-GAS5NC)、si-GAS5组(转染si-GAS5)和si-GAS5+miR-21抑制剂组(转染si-GAS5联合miR-21抑制剂反义寡核苷酸has-miR-21-5p)。采用实时荧光定量聚合酶链反应检测转染48 h后各组细胞中GAS5和miR-21的表达水平，荧光素酶基因报告实验验证GAS5与miRNA-21的结合位点，细胞计数试剂盒8法检测各组细胞的增殖情况，克隆形成实验观察细胞的克隆形成能力，Transwell实验检测细胞的侵袭和迁移能力，流式细胞术检测细胞周期和凋亡，Western blot检测上皮间质转化(EMT)相关蛋白(Snail、N-cadherin、vimentin、E-cadherin)、干细胞相关因子(Sox2、CD44、Oct2)和磷酸酶与张力蛋白同源物(PTEN)/Akt信号通路相关蛋白的表达情况。 结果： 转染后48 h，空白对照组、阴性对照组和si-GAS5组HCT-116细胞miR-21表达水平分别为1.00±0.10、1.00±0.10和1.80±0.20，吸光度分别为0.51±0.02、0.50±0.01和0.65±0.01，细胞克隆形成数分别为(90±4)个、(91±5)个和(200±8)个，侵袭细胞数分别为(118±3)个、(119±3)个和(150±4)个，迁移细胞数分别为(110±2)个、(108±2)个和(127±2)个，G(1)期细胞比例分别为(49.3±2.1)%、(50.1±2.0)%和(42.2±1.1)%，S期细胞比例分别为(19.2±1.2)%、(20.2±1.1)%和(28.3±2.2)%，细胞凋亡率分别为(14.4±2.2)%、(14.5±2.1)%和(7.2±1.3)%，表明GAS5下调后HCT-116细胞miR-21表达水平升高，增殖、侵袭和迁移能力增强，G(1)期细胞减少，S期细胞增加，细胞凋亡率降低(均P<0.05)。GAS5下调后，HCT-116细胞中Snail、N-cadherin、vimentin、Sox2、CD44、Oct2和p-Akt的表达水平升高(均P<0.05)，E-cadherin和PTEN的表达水平降低(均P<0.05)。抑制miR-21后，可以逆转GAS5下调引起的PTEN/Akt信号通路相关蛋白表达水平的变化(均P<0.05)。 结论： GAS5可作为miR-21的竞争内源性RNA，下调GAS5可通过激活miR-21/PTEN/Akt信号通路，促进EMT和肿瘤细胞干性的获得，从而促进结肠癌的发生发展。.
Keywords: Colonic neoplasms; Epithelial-mesenchymal transition; Growth arrest-specific5; Neoplastic Stem Cells; miR-21.