RNA结合蛋白NONO通过影响SKP2、E2F8表达调控乳腺癌细胞增殖的研究

RNA-binding protein NONO regulates breast cancer cell proliferation by affecting the expression of SKP2 and E2F8

目的探讨不含POU结构域的八聚体核苷酸结合蛋白(NONO)是否通过影响S期激酶相关蛋白2(SKP2)和E2F转录因子8(E2F8)表达从而调控乳腺癌细胞增殖。方法收集2019年6月至2020年6月于该院接受乳腺癌根治术治疗的43例乳腺癌患者的乳腺癌组织及癌旁组织石蜡块。采用免疫组织化学SP染色检测NONO、SKP2和E2F8在组织中的表达。将MCF-7细胞分为MCF-7组(无特殊处理)、sh-NC-MCF-7组(加入sh-NC序列正义链的慢病毒LV3液)和sh-NONO-MCF-7组(加入含sh-NONO序列正义链的慢病毒LV3液)。检测sh-NONO的沉默效率,筛选稳定株。分析沉默NONO对乳腺癌细胞增殖能力、细胞周期以及SKP2和E2F8表达的影响。结果乳腺癌组织中NONO、SKP2和E2F8阳性表达率显著高于癌旁组织(P<0.05);与MCF-7和sh-NC-MCF-7组比较,sh-NONO-MCF-7组NONO蛋白表达水平降低(P<0.05),提示成功构建NONO沉默MCF-7细胞株;与MCF-7和sh-NC-MCF-7组比较,sh-NONO-MCF-7组增殖能力降低(P<0.05);与MCF-7和sh-NC-MCF-7组比较,sh-NONO-MCF-7组G_(0)/G_(1)期细胞比例显著增加,S期和G_(2)/M期细胞比例显著降低(P<0.05);与MCF-7和sh-NC-MCF-7组比较,sh-NONO-MCF-7组SKP2和E2F8蛋白表达水平以及SKP2和E2F8 mRNA相对表达水平均显著降低(P<0.05)。结论沉默NONO表达可通过下调SKP2和E2F8表达,诱导细胞周期阻滞,从而抑制乳腺癌细胞增殖。

Objective To investigate whether the non-POU domain containing octamer-binding protein(NONO)regulates breast cancer cell proliferation by affecting the expression of S-phase kinase-related protein 2(SKP2)and E2F transcription factor 8(E2F8).Methods The paraffin blocks of breast cancer tissue and paracancerous tissue of 43 breast cancer patients who received radical mastectomy in the hospital from June 2019 to June 2020 were collected.The expressions of NONO,SKP2 and E2F8 in tissues were detected by immunohistochemical SP staining.MCF-7 cells were divided into MCF-7 cell group(no special treatment),sh-NC-MCF-7 cell group(treated with lentivirus LV3 carring the sense chain of sh-NC sequence)and sh-NONO-MCF-7 cells group(treated with lentivirus LV3 carring the sense sequence of sh-NONO).The silencing efficiency of sh-NONO was detected,and stable strains were screened.The effects of silencing NONO on proliferation,cell cycle and expression of SKP2 and E2F8 in breast cancer cells were analyzed.Results The positive expression rates of NONO,SKP2 and E2F8 in breast cancer tissues were significantly higher than those in adjacent normal tissues(P<0.05).Compared with MCF-7 and sh-NC-MCF-7 group,the expression of NONO protein in sh-NONO-MCF-7 group was significantly reduced(P<0.05),indicating a success in constructing NONO-silencing MCF-7 cell line.Compared with MCF-7 and sh-NC-MCF-7 group,the proliferation ability significantly decreased in sh-NONO-MCF-7 group(P<0.05),meanwhile the sh-NONO-MCF-7 group had a significantly higher proportion of cells arrested at G_(0)/G_(1) phase,and a significantly lower proportion of cells arrested at S and G_(2)/M phases than those of MCF-7 and sh-NC-MCF-7 group(P<0.05).Both protein and mRNA expression levels of SKP2 and E2F8 were significantly lower in MCF-7 and sh-NC-MCF-7 group than in sh-NONO-MCF-7 group(P<0.05).Conclusion Silencing NONO expression can induce cell cycle arrest by down-regulating the expression of SKP2 and E2F8,thereby inhibiting breast cancer cell proliferation.