Tang Wei-wei, Wan Gui-ping, Wan Yi-cong, Zhang Lin, Cheng Wen-jun
Department of Gynecology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Zhonghua Fu Chan Ke Za Zhi. 2013 May;48(5):364-9.
To investigate the effects of miR-135a on HOXA10 expression, proliferation and apoptosis of SKOV3 cells.
(1) Through computer-aided algorithms,the predicted target gene of miR-135a (HOXA10)were determined. (2) miR-135a mimics, miR-135a inhibitor and negative control were transfected into SKOV3 cells, respectively.Reverse transcription (RT)-PCR, western blot analysis were used to examine the expression levels of HOXA10 at different times (24, 48 and 72 hours). (3) A luciferase reporter assay was used to confirm the direct regulation between miR-135a and HOXA10. (4) SKOV3 cells proliferation at different times (24,48 and 72 hours) was detected by methyl thiazolyl tetrazolium (MTT) assay [quantified by absorbance(A)]. Western blot was used to examine the expression of apoptosis-associated protein bcl-2, bax and caspase-3 in SKOV3 cells after 48 hours transfection.
(1) HOXA10 was predicted to be the target gene of miR-135a by computer-aided algorithms. (2) RT-PCR shown that HOXA10 mRNA levels were decreased over time (24, 48 and 72 hours) after miR-135a mimics transfection in SKOV3 cells (0.94 ± 0.04 vs 0.78 ± 0.03 vs 0.70 ± 0.03, P < 0.05). While, the expression of HOXA10 mRNA was increased over time after miR-135a inhibitor transfection (1.14 ± 0.05 vs 1.16 ± 0.03 vs 2.60 ± 0.08,P < 0.05). After transfected with miR-135a mimics or miR-135a inhibitor over 48 and 72 hours, the HOXA10 expression levels in SKOV3 cells were significantly lower or higher than each control group, respectively (all P < 0.01). Western blot analysis of HOXA10 expression in SKOV3 cells confirmed the results of RT-PCR detected. (3) After cotransfection of miR-135a plasmid and pMIR-REPORT luciferase plasmid containing HOXA10, luciferase reporter assays showed that the luciferase activity reduced by 67.8% (P < 0.01). (4) MTT showed that SKOV3 cells growth after miR-135a mimics transfection for 48 and 72 hours were significantly lower than those in control group (0.38 ± 0.03 vs 0.52 ± 0.05, 0.67 ± 0.05 vs 0.75 ± 0.06;respectively,all P < 0.05).While, SKOV3 cells transfected with miR-135a inhibitor for 72 hours grew significantly faster than that in control group (0.95 ± 0.05 vs 0.75 ± 0.06, P < 0.01). After miR-135a mimics transfection, the level of bcl-2 protein was significantly lower than that in control group (0.28 ± 0.06 vs 0.76 ± 0.09,P < 0.01). The activity of caspase-3 was significantly higher than that in control group (115.0 ± 2.4 vs 95.4 ± 2.1, P < 0.01). While, there was no statistical difference of bax expression (P = 0.142). However, after miR-135a inhibitor transfection, the expression level of bcl-2 protein was significantly higher than that in control group (0.92 ± 0.03 vs 0.76 ± 0.09, P = 0.037) and the activity of caspase-3 was significantly lower than that in control group (59.5 ± 4.1 vs 95.4 ± 2.1, P < 0.01). There was also no statistical difference of bax expression (P = 0.066).
miR-135a may play an important role in cell proliferation and apoptosis of ovarian cancer cells by regulating HOXA10 and its downstream pathways.
探讨miR-135a对SKOV3细胞中HOXA10表达、增殖及凋亡的影响。
（1）通过计算机辅助算法确定miR-135a的预测靶基因（HOXA10）。（2）将miR-135a模拟物、miR-13�a抑制剂及阴性对照分别转染至SKOV3细胞。采用逆转录（RT）-PCR、蛋白质免疫印迹分析检测不同时间点（24、48及72小时）HOXA10的表达水平。（3）采用荧光素酶报告基因检测法证实miR-135a与HOXA10之间的直接调控关系。（4）采用甲基噻唑基四氮唑（MTT）法检测不同时间点（24、48及72小时）SKOV3细胞的增殖情况[以吸光度（A）进行定量分析]。转染48小时后，采用蛋白质免疫印迹法检测SKOV3细胞中凋亡相关蛋白bcl-2、bax及caspase-3的表达。
（1）通过计算机辅助算法预测HOXA10为miR-135a的靶基因。（2）RT-PCR结果显示，转染miR-135a模拟物后，SKOV3细胞中HOXA10 mRNA水平随时间（24、48及72小时）逐渐降低（0.94±0.04 vs 0.78±0.03 vs 0.70±0.03，P<0.05）。而转染miR-135a抑制剂后，HOXA10 mRNA表达随时间增加（1.14±0.05 vs 1.16±0.03 vs 2.60±0.08，P<0.05）。转染miR-135a模拟物或miR-135a抑制剂48及72小时后，SKOV3细胞中HOXA10表达水平分别显著低于或高于各对照组（均P<0.01）。蛋白质免疫印迹法分析SKOV3细胞中HOXA10的表达，证实了RT-PCR检测结果。（3）共转染miR-135a质粒与含HOXA10的pMIR-REPORT荧光素酶质粒后，荧光素酶报告基因检测显示荧光素酶活性降低了67.8%（P<0.01）。（4）MTT结果显示，转染miR-135a模拟物48及72小时后，SKOV3细胞的生长显著低于对照组（0.38±0.03 vs 0.52±0.05，0.67±0.05 vs 0.75±0.06；均P<0.05）。而转染miR-135a抑制剂72小时后，SKOV3细胞的生长显著快于对照组（0.95±0.05 vs 0.75±0.06，P<0.01）。转染miR-135a模拟物后，bcl-2蛋白水平显著低于对照组（0.28±0.06 vs 0.76±0.09，P<0.01）。caspase-3活性显著高于对照组（115.0±2.4 vs 95.4±2.1，P<0.01）。而bax表达无统计学差异（P=0.142）。然而，转染miR-135a抑制剂后，bcl-2蛋白表达水平显著高于对照组（0.9^2±0.03 vs 0.76±0.09，P=0.037），caspase-3活性显著低于对照组（59.5±4.1 vs 95.4±2.1，P<0.01）。bax表达也无统计学差异（P=0.066）。
miR-135a可能通过调控HOXA10及其下游通路在卵巢癌细胞的增殖和凋亡中发挥重要作用。
