臭椿酮抑制ENST00000441270/miR-149轴影响结直肠癌细胞增殖和凋亡的实验研究

目的探讨臭椿酮对结直肠癌细胞增殖和凋亡的影响。方法根据检测结果实施分组,采用低(0.2μmol/L)、中(0.4μmol/L)、高剂量(0.6μmol/L)的臭椿酮干预结直肠癌SW1116细胞24 h,细胞计数试剂盒(CCK-8)检测细胞活力,集落形成实验检测克隆形成数,流式细胞术检测细胞凋亡,实时荧光定量聚合酶链反应(RT-qPCR)检测ENST00000441270和微小RNA-149(miR-149)的表达水平。转染ENST00000441270小干扰RNA(si-ENST00000441270)至SW1116细胞,采用上述方法检测干扰ENST00000441270表达对SW1116细胞活力、克隆形成以及凋亡的影响。双荧光素酶报告基因实验验证ENST00000441270与miR-149之间靶向关系。两组间比较采用t检验,多组间比较采用单因素方差分析和LSD-t检验。结果臭椿酮低剂量组SW1116细胞的活力(0.84±0.05)比(0.84±0.05),t=0.529,P>0.05]、克隆形成数(110.00±3.56)个比(111.33±3.86)个,t=0.499,P>0.05]、凋亡率(7.56±0.80)%比(7.35±0.63)%,t=0.307,P>0.05]、ENST00000441270(0.98±0.06)比(0.99±0.06),t=0.238,P>0.05]和miR-149(1.01±0.07)比(0.99±0.07),t=0.303,P>0.05]表达与对照组比较,差异均无统计学意义。臭椿酮中、高剂量组SW1116细胞的活力(0.63±0.04、0.40±0.03比0.84±0.05,t=7.667、12.667,P<0.05]、克隆形成数(85.67±2.87)个、(58.33±2.62)个比(111.33±3.86)个,t=9.487、24.666,P<0.05]、ENST00000441270表达(0.69±0.05)、(0.41±0.03)比(0.99±0.06),t=7.138、13.799,P<0.05]低于对照组,差异均有统计学意义,细胞凋亡率(12.61±0.91)%、(21.66±0.97)%比(7.35±0.63)%,t=7.715、12.036,P<0.05]、miR-149表达(1.48±0.08)、(1.92±0.10)比(0.99±0.07),t=7.415、14.074,P<0.05]高于对照组,差异均有统计学意义。si-ENST00000441270组SW1116细胞活力(0.35±0.02)比(0.88±0.05),t=17.047,P<0.05]、克隆形成数(49.67±2.87)个比(112.67±3.40)个,t=24.525,P<0.05]低于阴性对照(si-NC)组,细胞凋亡率(22.62±0.82)%比(7.58±0.65)%,t=24.896,P<0.05]高于si-NC组,差异均有统计学意义。miR-149是ENST00000441270的靶基因。结论0.4、0.6μmol/L的臭椿酮能够抑制结直肠癌细胞SW1116增殖,诱导细胞凋亡,其机制可能与抑制ENST00000441270/miR-149分子轴有关。

Ailanthone affects colorectal cancer cell proliferation and apoptosis by inhibiting ENST00000441270/microRNA-149 axis

Objective To explore the effect of Ailanthone on proliferation and apoptosis of colorectal cancer(CRC)cells.Methods The low(0.2μmol/L),medium(0.4μmol/L)and high(0.6μmol/L)doses of Ailanthone were used to treat CRC SW1116 cells for 24 h.The cell counting kit(CCK-8)test was used to detect the cell viability,colony formation experiment to colony formation,flow cytometry to apoptosis,and real-time fluorescence polymerase chain reaction(RT-qPCR)to the expression levels of ENST00000441270 and micro RNA-149.ENST00000441270 small interfering RNA(si-ENST00000441270)was transfected into SW1116 cells,and the above methods were used to detect the effect of si-ENST00000441270 on the viability,clone formation and apoptosis of SW1116 cells.The dual luciferase reporter gene experiment verified the targeting relationship between ENST00000441270 and miR-149.The comparison between the two groups was performed by t test,and the comparison between multiple groups was performed by one-way ANOVA and LSD-t test.Results The viability of SW1116 cells(0.84±0.05)vs.(0.84±0.05),t=0.529,P>0.05],the number of clone formation(110.00±3.56)vs.(111.33±3.86),t=0.499,P>0.05],apoptosis rate(7.56±0.80)%vs.(7.35±0.63)%,t=0.307,P>0.05],ENST00000441270(0.98±0.06)vs.(0.99±0.06),t=0.238,P>0.05]and miR-149(1.01±0.07)vs.(0.99±0.07),t=0.303,P>0.05]expression in low-dose Ailanthone group showed no statistically significant difference from those in the control group.The viability of SW1116 cells(0.63±0.04,0.40±0.03 vs.0.84±0.05,t=7.667,12.667,P<0.05),the number of colonies(85.67±2.87),(58.33±2.62)vs.(111.33±3.86),t=9.487,24.666,P<0.05]and ENST00000441270 expression(0.69±0.05),(0.41±0.03)vs.(0.99±0.06),t=7.138,13.799,P<0.05]in Ailanthone medium and high dose groups were lower,the apoptosis rate(12.61±0.91)%,(21.66±0.97)%vs.(7.35±0.63)%,t=7.715,12.036,P<0.05]and miR-149 expression(1.48±0.08,1.92±0.10 vs.0.99±0.07,t=7.415,14.074,P<0.05)were significantly higher than those in the control group.The cell viability of SW1116(0.35±0.02)vs.(0.88±0.05),t=17.047,P<0.05]and the number of colonies(49.67±2.87 vs.112.67±3.40,t=24.525,P<0.05)in si-ENST00000441270 group were significantly reduced,and cell apoptosis rate(22.62±0.82)%vs.(7.58±0.65)%,t=24.896,P<0.05]was significantly higher than those in si-NC group.miR-149 was the target gene of ENST00000441270.Conclusion A total of 0.4 and 0.6μmol/L of Ailanthone could inhibit the proliferation of CRC SW1116 cells and induce apoptosis.The mechanism may be related to the inhibition of the molecular axis of ENST00000441270/miR-149.