CAJ | 학술논문

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.
목적 관찰미소RNA-338-3p(miR-338-3p)대인결직장암세포증식급조망적영향.방법 구건만병독개도적miR-338-3p급기억제제적표체재체병전염결직장암세포주SW-620,류식세포의사선건립은정과표체miR-338-3p급기억제제적SW-620아세포주,이용실시정량역전록취합매련반응(RT-qPCR)검측miR-338-3p표체,Western blot검측기파기인Smoothened(SMO)단백표체수평,통과새서람비색법급류식세포의관찰세포적증식화조망상태.결과 경쌍매절감정화측서증실,성공구건료포함miR-338-3p급기억제제적만병독표체재체,형광현미경하관찰가견SW-620세포표체록색형광.은정과표체miR-338-3p적SW-620아세포주중,miR-338-3p표체수평현저고우음성대조조(상대표체량3.91 ±0.51비2.36±0.44,P＜0.01),SMO단백표체수평명현하강,차종류세포증식능력감약[세포증식억제솔(CPIR):(61.9±5.2)％비(41.6±4.8)％,P＜0.01],세포조망증가.은정과표체miR-338-3p억제제적SW-620아세포주중,miR-338-3p표체수평현저저우음성대조조(상대표체량0.92±0.29비2.36±0.44,P＜0.01),SMO단백표체수평명현승고,차종류세포증식능력증강[CPIR:(19.2±3.8)％비(41.6±4.8)％,P＜O.01],세포조망감소；이차충촉증식작용가피anti-SMO-siRNA부분역전,설명차충생물학효응학유SMO소개도.결론 miR-338-3p가통과억제결직장암세포중SMO단백표체이억제종류세포생장.
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.