Department of Geriatric Gastroenterology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China.
World J Gastroenterol. 2013 Feb 28;19(8):1239-46. doi: 10.3748/wjg.v19.i8.1239.
To investigate the effect of high mobility group A2 (HMGA2) gene silencing on gastric cancer MKN-45 cells in vitro.
HMGA2 short hairpin RNA (shRNA) expression plasmids were constructed, including a pair of random scrambled sequences. Human gastric cancer cell line MKN-45 cells were divided into three groups: blank control group (non-transfected cells), transfected group (cells transfected with HMGA2 shRNA recombinant plasmid) and scrambled sequence group (transfected with random scrambled plasmid). Cells were transfected with HMGA2 shRNA recombinant plasmids and scrambled plasmid in vitro, and the cells transfection efficiency was assayed by fluorescence microscopy. The HMGA2 messenger RNA (mRNA) expression was detected by reverse transcription polymerase chain reaction, gastric cancer cells apoptosis was detected by flow cytometry, cell proliferation was detected by methyl thiazol tetrazolium, and the protein expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), P27, caspase-9 and B-cell leukemia/lymphoma-2 (Bcl-2) were analyzed by Western blotting.
Compared with the blank control group and the scrambled sequence group, the levels of HMGA2 mRNA and protein expression in the transfected group were significantly reduced (P < 0.05). The relative HMGA2 mRNA expression levels of the blank control group, transfected group and scrambled sequence group were 0.674 ± 0.129, 0.374 ± 0.048 and 0.689 ± 0.124, respectively. The relative HMGA2 protein expression levels of the blank control group, transfected group and scrambled sequence group were 0.554 ± 0.082, 0.113 ± 0.032 and 0.484 ± 0.123, respectively. Moreover, transfection with the scrambled sequence had no effect on the expression of HMGA2. After being transfected with shRNA for 24, 48 and 72 h, the cell apoptotic rates of the transfected group were 21.65% ± 0.28%, 39.98% ± 1.82% and 24.51% ± 0.93%, respectively, which significantly higher than those of blank control group (4.72% ± 1.34%, 5.83% ± 0.13% and 5.22% ± 1.07%) and scrambled sequence group (4.28% ± 1.33%, 7.87% ± 1.43% and 6.71% ± 0.92%). After 24, 48 and 72 h, the cell proliferation inhibition rates in the transfected group were 31.57% ± 1.17%, 39.45% ± 2.07% and 37.56% ± 2.32%, respectively; the most obvious cell proliferation inhibition appeared at 48 h after transfection. Compared with the blank control group and scrambled sequence group, after transfection of shRNA for 72 h, the protein expression levels of PI3K (0.042 ± 0.005 vs 0.069 ± 0.003, 0.067 ± 0.05), Akt (0.248 ± 0.004 vs 0.489 ± 0.006, 0.496 ± 0.104) and Bcl-2 (0.295 ± 0.084 vs 0.592 ± 0.072, 0.594 ± 0.109) were significantly reduced. The protein expression levels of P27 (0.151 ± 0.010 vs 0.068 ± 0.014, 0.060 ± 0.013) and caspase-9 (0.136 ± 0.042 vs 0.075 ± 0.010, 0.073 ± 0.072) were significantly upregulated.
HMGA2 shRNA gene silencing induces apoptosis and suppresses proliferation of MKN-45 cells.
研究高迁移率族蛋白 A2（HMGA2）基因沉默对体外胃癌 MKN-45 细胞的影响。
构建 HMGA2 短发夹 RNA（shRNA）表达质粒，包括一对随机的乱序序列。将人胃癌细胞系 MKN-45 细胞分为三组：空白对照组（未转染细胞）、转染组（转染 HMGA2 shRNA 重组质粒的细胞）和乱序序列组（转染随机乱序质粒的细胞）。体外转染 HMGA2 shRNA 重组质粒和乱序质粒，荧光显微镜检测细胞转染效率。逆转录聚合酶链反应检测 HMGA2 信使 RNA（mRNA）表达，流式细胞术检测胃癌细胞凋亡，噻唑蓝比色法检测细胞增殖，Western blot 分析磷脂酰肌醇 3-激酶（PI3K）、蛋白激酶 B（Akt）、P27、半胱天冬酶-9（caspase-9）和 B 细胞淋巴瘤/白血病-2（Bcl-2）的蛋白表达。
与空白对照组和乱序序列组相比，转染组的 HMGA2 mRNA 和蛋白表达水平明显降低（P<0.05）。空白对照组、转染组和乱序序列组的相对 HMGA2 mRNA 表达水平分别为 0.674±0.129、0.374±0.048 和 0.689±0.124，相对 HMGA2 蛋白表达水平分别为 0.554±0.082、0.113±0.032 和 0.484±0.123。此外，转染乱序序列对 HMGA2 的表达没有影响。转染 shRNA 24、48 和 72 h 后，转染组的细胞凋亡率分别为 21.65%±0.28%、39.98%±1.82%和 24.51%±0.93%，明显高于空白对照组的 4.72%±1.34%、5.83%±0.13%和 5.22%±1.07%和乱序序列组的 4.28%±1.33%、7.87%±1.43%和 6.71%±0.92%。转染 shRNA 24、48 和 72 h 后，转染组的细胞增殖抑制率分别为 31.57%±1.17%、39.45%±2.07%和 37.56%±2.32%，转染后 48 h 时细胞增殖抑制最明显。与空白对照组和乱序序列组相比，转染 shRNA 72 h 后，PI3K（0.042±0.005 比 0.069±0.003、0.067±0.05）、Akt（0.248±0.004 比 0.489±0.006、0.496±0.104）和 Bcl-2（0.295±0.084 比 0.592±0.072、0.594±0.109）的蛋白表达水平明显降低，P27（0.151±0.010 比 0.068±0.014、0.060±0.013）和半胱天冬酶-9（0.136±0.042 比 0.075±0.010、0.073±0.072）的蛋白表达水平明显上调。
HMGA2 shRNA 基因沉默诱导 MKN-45 细胞凋亡并抑制增殖。
