上调基因-4对结肠癌细胞增殖的影响

Effects of up-regulated gene-4 on the proliferation of colonic cancer cells

目的探讨上调基因-4（URG-4）对结肠癌细胞增殖的影响。方法筛选高表达URG-4的结肠癌细胞。构建URG-4基因siRNA的逆转录病毒载体及阴性对照，经PT67细胞包装后，获得可表达人URG-4基因siRNA逆转录病毒及阴性对照。RT—PCR和Westernblot法检测MKN45、SW480、LoVo、HCT116、HT29细胞中URG-4mRNA和蛋白表达水平，筛选稳定转染细胞株。分别使用重组病毒（干扰组）、原始病毒（阴性对照组）和等量PBS（空白对照组）转染结肠癌LoVo细胞。MTT法检测各组结肠癌LoVo细胞生长情况。计量资料比较采用单因素方差分析和t检验。结果测序结果证实表达siRNA的逆转录病毒成功构建。URG-4mRNA在MKN45、SW480、LoVo、HCT116和HT29细胞中的相对表达量分别为0．58±0．02、0．63±0．03、0．81±0．01、1．01±0．02和0．91±0．04；URG-4蛋白相对表达量分别为0．73±0．02、0．85±0．03、1．42±0．01、0．80±0．03和0．80±0．04。结肠癌LoVo细胞高表达URG-4。干扰组中URG-4mRNA表达为0．55±0．03，显著低于阴性对照组的1．15±0．02和空白对照组的1．15±0．01（t=-5．179，-9．285，P〈0．05）。干扰组URG—dmRNA的抑制率为52．6％。干扰组中URG-4蛋白表达为0．82±0．05，显著低于阴性对照组的1．46±0．07和空白对照组的1．54±0．04（t=-4．239，-3．704，P〈0．05）。干扰组URG-4蛋白的抑制率为43．6％。各组结肠癌LoVo细胞呈指数生长。与阴性对照组比较，第3～6天干扰组细胞增殖受到明显抑制，差异有统计学意义（t=-6．436，-6．045，-6．434，-4．285，P〈0．05）。结论干扰URG-4表达能够抑制LoVo细胞的生长。

Objective To study the effects of up-regulated gene-4 （URG-4） on colon cancer cell proliferation. Methods Colon cancer cell line with high expression of URG-4 was selected. The recombinant URG-4 siRNA retroviral vector was constructed and packaged by PT67 cell, then retroviral particles which can express URG-4 siRNA in mammal cell and its negative control were obtained. Expressions of URG-4 in MKN45, SW480, LoVo, HCT116, HT29 were detected by RT-PCR and Western blot, respectively. Recombinant virus （interference group） , original virus （negative control group） and the same amount of PBS （blank group ） were used to transfect LoVo cells respectively. Stably transfected cell lines were screened. The growth condition of cell lines in each group was assayed by MTT. All data were analyzed by the one-way analysis of variance and the t test. Results Sequencing results confirmed the successful construction of retroviral which expressed siRNA, the relative expression levels of URG-4 mRNA in MKN45, SW480, LoVo, HCTll6, HT29 were 0.58 ＋0.02, 0.63 _＋ 0.03, 0.81 ±0.01, 1.01 ±0.02, 0.91 ＋-0.04 and the expression levels of URG-4 protein in the 5 cell lines were 0.73 - 0.02, 0.85 ,＋ 0.03, 1.42 ± 0.01, 0.80 ,＋ 0.30, 0.80 ,＋ 0.04, respectively. High expression of URG-4 was observed in the LoVo cells. The expression of URG-4 mRNA in the LoVo cells in the interference group was 0.55 ±0. 03, which was significantly lower than 1.15 ± 0.02 of the negative control group and 1.15±0.01 of the blank group （t =-5. 179, -9.285, P〈0.05） . The inhibition rate of URG-4 mRNA in the interference group was 52.6%. The expression of URG-4 protein in the interference group was 0.82 ± 0.05, which was significantly lower than 1.46 ± 0.07 of the negative control group and 1.54± 0.04 of the blank group （ t =- 4. 239, - 3. 704, P 〈 0.05）. The inhibition rate of URG-4 protein in the interference group was 43.6%. The LoVo cells in each group grew exponentially. Compared with the negative control group, the cell growth of the interference group was inhibited during day 3 to day 6, - 6. 045, - 6. 434, - 4. 285, P 〈0. 05 ）. Conclusion growth of LoVo cells. which had statistical significant difference （ t = - 6. 436, Interference of the expression of URG-4 can inhibit the growth of LoVo cells.