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目的无POU域八聚体结合蛋白(non-POU domain containing octamer-binding protein,NONO)是一种参与多种核内事件的多功能核蛋白,NONO与某些肿瘤的发生密切相关。本研究旨在探讨NONO基因沉默对乳腺癌细胞(SKBR3)增殖、迁移和侵袭生物学特性的影响。方法用含有靶向人NONO基因shRNA的慢病毒感染SKBR3细胞,并用嘌呤霉素进行筛选,通过蛋白质印迹法检测SKBR3细胞中NONO蛋白表达,建立稳定的NONO基因沉默的SKBR3细胞系;采用CCK-8法和平板克隆实验检测NONO基因沉默对SKBR3细胞增殖的影响;通过体外Transwell迁移和侵袭实验检测NONO基因沉默对SKBR3细胞迁移和侵袭能力的影响。结果蛋白质印迹法实验结果显示,SKBR3、NONO基因沉默的SKBR3(SKBR3-NONO-si)和阴性对照SKBR3(SKBR3-NC)组细胞NONO蛋白表达相对灰度值分别为0.716 7±0.225、0.103±0.045和0.727±0.127,差异有统计学意义,F=16.699,P=0.004,表明成功地建立了稳定的NONO基因沉默的SKBR3细胞系。CCK-8实验结果显示,细胞接种后7 d,SKBR3、SKBR3-NONO-si和SKBR3-NC细胞CCK-8吸光度值分别为1.717±0.233、0.566±0.158和1.110±0.187,差异有统计学意义,F=78.469,P<0.001;平板克隆形成实验结果显示,细胞培养14d后,SKBR3、SKBR3-NONO-si和SKBR3-NC组形成的克隆数分别为64.667±14.84、21.167±7.414和53.667±11.587,差异有统计学意义,F=16.333,P<0.001;以上结果均说明,NONO基因沉默抑制SKBR3细胞增殖能力。Transwell迁移实验结果显示,细胞接种后20h,SKBR3、SKBR3-NONO-si和SKBR3-NC组迁移细胞数分别为62.333±7.767、44.667±7.637和85.333±11.930,差异有统计学意义,F=14.338,P=0.005;侵袭实验结果显示,细胞接种后40h,SKBR3、SKBR3-NONO-si和SKBR3-NC组侵袭细胞数分别为27.667±7.024、24.666±3.512和81.000±5.568,差异有统计学意义,F=97.558,P<0.001;与SKBR3-NC细胞相比,NONO基因沉默的SKBR3-NONO-si细胞迁移和侵袭能力均明显降低。结论 NONO基因沉默抑制SKBR3细胞增殖、迁移和侵袭,提示NONO可能是促进乳腺癌细胞增殖、转移和侵袭一个新的重要调控蛋白。
Objective Non-POU domain containing octamer-binding protein (NONO) is a multifunctional nuclear protein involved in a variety of nuclear events. NONO is closely related to the occurrence of some tumors. This study aimed to investigate the effects of NONO gene silencing on the biological characteristics of breast cancer cells (SKBR3) in proliferation, migration and invasion. Methods SKBR3 cells were infected with lentivirus containing shRNA targeting human NONO gene. The cells were screened with puromycin. The expression of NONO protein in SKBR3 cells was detected by Western blotting to establish a stable SKBR3 cell line with silenced NONO gene. CCK-8 The effect of NONO gene silencing on the proliferation of SKBR3 cells was assayed by plate-cloning assay and the effect of NONO gene silencing on the migration and invasion of SKBR3 cells was detected by Transwell migration and invasion assay in vitro. Results The results of Western blotting showed that the relative gray value of NONO protein in SKBR3 and SKBR3-NC cells with SKON3 and NONO gene silencing was 0.716 7 ± 0.225 and 0.103 ± 0.045 And 0.727 ± 0.127, respectively. The difference was statistically significant, F = 16.699, P = 0.004, indicating that a stable SKBR3 cell line with silenced NONO gene was successfully established. The results of CCK-8 assay showed that the CCK-8 absorbance of SKBR3, SKBR3-NONO-si and SKBR3-NC cells were 1.717 ± 0.233, 0.566 ± 0.158 and 1.110 ± 0.187 respectively at 7 days after inoculation, the difference was statistically significant F = 78.469, P <0.001. The results of plate clone formation assay showed that the number of colonies formed by SKBR3, SKBR3-NONO-si and SKBR3-NC group after culture for 14 days were 64.667 ± 14.84, 21.167 ± 7.414 and 53.667 ± 11.587, The difference was statistically significant, F = 16.333, P <0.001; The above results indicate that NONO gene silencing inhibits SKBR3 cell proliferation. The results of Transwell migration assay showed that the numbers of migrated cells in SKBR3, SKBR3-NONO-si and SKBR3-NC groups were 62.333 ± 7.767, 44.667 ± 7.637 and 85.333 ± 11.930, respectively, with a statistically significant difference at F = 14.338, P = 0.005. The results of invasion assay showed that the number of invasive cells in SKBR3, SKBR3-NONO-si and SKBR3-NC groups was 27.667 ± 7.024,24.666 ± 3.512 and 81.000 ± 5.568 at 40 h after inoculation, respectively, with significant difference = 97.558, P <0.001. Compared with SKBR3-NC cells, the migration and invasion ability of SKBR3-NONO-si cells with NONO gene silencing were significantly decreased. Conclusion NONO gene silencing inhibits the proliferation, migration and invasion of SKBR3 cells, suggesting that NONO may be a new important regulatory protein that promotes the proliferation, metastasis and invasion of breast cancer cells.