NONO基因沉默对乳腺癌细胞生物学特性影响研究

Effects of NONO silencing on the biological characteristics of breast cancer cells

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 known as muhifunction- al proteins involved in many nuclear processes. It was shown that p54nrb/NONO was closely related to the occurrence of some tumors. This study explored the effects of NONO silencing on the biological characteristics of breast cancer cells （SKBR3） including the proliferation, migration and invasion in vitro. METHODS SKBR3 cells were infected with lenti- viruses containing NONO-specific shRNA, then the cells were especially cultured in media containing puromyein and es- tablished stable NONO-silencing SKBR3 cell line. The efficiency of NONO silencing was determined by Western blotting. The proliferation ability of NONO-silencing SKBR3 cells was assessed by the Cell Counting Kit-8 （CCK-8） assay and plate clone formation assay. The migration ability of NONO silencing SKBR3 cells was measured by Transwell motility assay, and the invasion ability of NONO-silencing SKBR3 cells was measured by Transwell invasion assay. RESULTS Western blotting showed that the relative gray value of NONO protein in SKBR3, NONO-silencing SKBR3（SKBR3-NO- NO-si） and negative control SKBR3 （SKBR3-NC） cells was 0.716 7-0. 225, 0. 103-0. 045, 0. 727±0. 127 （F= 16. 699, P=0. 004）, which demonstrated that stable NONO-silencing SKBR3 cell line was established. The CCK-8 assay showed that CCK-8 absorbance in SKBR3, SKBR3-NONO-si and SKBRa-NC cells after the cells were cultured for 7 days was 1. 717±0. 233, 0.566±0.158, 1.110±0.187 （F=78.469,P〈0.001）. Plate clone formation assay showed that the number of clone in SKBR3, SKBR3-NONO-si and SKBR3-NC ceils after the cells were cultured for 14 days was 64. 667±14.84, 21. 167±7. 414, 53. 667t 11. 587 （F=78.469,P〈0.001）. All above results demonstrated that knockdown of NONO inhibited the proliferation of SKBR3 cells. In vitro Transwell motility assay displayed that the number of migrated cells in SKBR3, SKBR3-NONO-si and SKBR3 NC cells was 62. 333±7. 767, 44. 667±7. 637, 85. 333±11. 930 respec- tively （F=14. 338,P = 0. 005）, which demonstrated that knockdown of NONO significantly inhibited the migration of SKBR3 cells. In vitro Transwell invasion assay showed that the number of invaded cells in SKBR3, SKBR3-NONO-si and SKBR3-NC cells was 27. 667±7. 024, 24. 666±3. 512 and 81. 000±5. 568 respectively （F=97. 558, P〈0. 001）, which also demonstrated that knockdown of NONO significantly inhibited the invasion of SKBR3 cells. CONCLUSION Knock down of NONO significantly inhibited the proliferation, migration and invasion of SKBR3 cells, which indicated that NO- NO may be a key regulation protein promoting the proliferation, migration and invasion of breast cancer.