摘要
目的 观察微小RNA-338-3p（miR-338-3p）对人结直肠癌细胞增殖及凋亡的影响.方法 构建慢病毒介导的miR-338-3p及其抑制剂的表达载体并转染结直肠癌细胞株SW-620,流式细胞仪筛选建立稳定过表达miR-338-3p及其抑制剂的SW-620亚细胞株,利用实时定量逆转录聚合酶链反应（RT-qPCR）检测miR-338-3p表达,Western blot检测其靶基因Smoothened（SMO）蛋白表达水平,通过噻唑蓝比色法及流式细胞仪观察细胞的增殖和凋亡状态.结果 经双酶切鉴定和测序证实,成功构建了包含miR-338-3p及其抑制剂的慢病毒表达载体,荧光显微镜下观察可见SW-620细胞表达绿色荧光.稳定过表达miR-338-3p的SW-620亚细胞株中,miR-338-3p表达水平显著高于阴性对照组（相对表达量3.91 ±0.51比2.36±0.44,P＜0.01）,SMO蛋白表达水平明显下降,且肿瘤细胞增殖能力减弱[细胞增殖抑制率（CPIR）：（61.9±5.2）％比（41.6±4.8）％,P＜0.01],细胞凋亡增加.稳定过表达miR-338-3p抑制剂的SW-620亚细胞株中,miR-338-3p表达水平显著低于阴性对照组（相对表达量0.92±0.29比2.36±0.44,P＜0.01）,SMO蛋白表达水平明显升高,且肿瘤细胞增殖能力增强[CPIR：（19.2±3.8）％比（41.6±4.8）％,P＜O.01],细胞凋亡减少；而此种促增殖作用可被anti-SMO-siRNA部分逆转,说明此种生物学效应确由SMO所介导.结论 miR-338-3p可通过抑制结直肠癌细胞中SMO蛋白表达而抑制肿瘤细胞生长.
Objective To investigate the regulative effect of microRNA-338-3p （miR-338-3p） on proliferation and apoptosis of colorectal carcinoma （CRC） cells.Methods The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed following the protocols recommended by commercial corporation.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRC-derived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein （GFP ＋） cells.The expression of miR-338-3p was detected by using real-time reverse transcriptase-polymerase chain reaction （RT-qPCR）,and Western blotting was used to detect the expression of the smoothened （SMO） protein in SW-620 cells.The proliferation and apoptosis of CRC cells were detected by methyl thiazol tetrazolium （MTT） assay and flow cytometry,respectively.Results Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.The expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased （relative expression：3.91 ± 0.51 vs.2.36 ± 0.44,P 〈 0.01）.Furthermore,over-expression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [the inhibitory rate of cell proliferation （CPIR）：（61.9 ± 5.2） % vs.（41.6 ±4.8） %,P 〈0.01].The expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased （relative expression：0.92 ± 0.29 vs.2.36 ± 0.44,P 〈 0.01）.Moreover,the down-regulated expression of miR-338-3p caused the up-regulated expression of the SMO protein in SW-620 cells,which showed significantly enhanced proliferation ability [CPIR：（19.2 ± 3.8） % vs.（41.6 ± 4.8） %,P 〈 0.01].However,anti-SMO-siRNA largely,but not completely,reversed the effects induced by blockage of miR-338-3p,suggesting that the regulative effect of miR-338-3p was indeed mediated by SMO.Conclusion MiR-338-3p could suppress the growth of CRC cells by inhibiting SMO protein expression.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第11期2311-2314,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金青年科学基金资助项目（81101896）
教育部高等学校博士学科点专项科研基金项目（20124433110010）
