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乳腺癌是全球女性中最常见的恶性癌症之一,也是导致女性癌症相关死亡的第二大原因[1]。尽管开发了早期诊断,根治性手术和辅助治疗等治疗方法,但乳腺癌病人的预后仍然较差[2]。了解乳腺癌的分子机制有助于寻找乳腺癌治疗的新靶点,因此,迫切需要了解乳腺癌的调控网络,发现新的分子靶点以改善乳腺癌病人的不良预后。微小核糖核酸(micro ribonucleic acid,miRNA)是一类内源性、非蛋白质编码的小RNA分子,可通过直接切割靶信使核糖核酸(messenger RNA,mRNA)或通过抑制其翻译来负调节转录后基因的表达。研究[3-4]表明,异常表达的miRNA参与多种疾病的发生和进展,包括癌症。有研究[5-6]显示,miR-99b-5p在各种癌症的发病机制中发挥重要作用。近期一项基于miRNA和组织特异性预测乳腺癌的潜在药物的研究[7]指出,miR-99b-5p在乳腺癌中呈低表达,提示miR-99b-5p可能与乳腺癌的发生和进展有关。但miR-99b-5p对下游靶基因的调控作用及机制尚不清楚。Tribbles同源蛋白1(Tribbles pseudokinase 1,TRIB1)属于Tribbles家族成员,参与多种肿瘤的发展进程[8]。前期预实验通过生物信息学软件预测发现,TRIB1可能是miR-99b-5p的直接靶基因。miR-99b-5p是否通过靶向TRIB1调控乳腺癌细胞的增殖和凋亡仍不明确。本研究探讨miR-99b-5p靶向TRIB1对乳腺癌MCF-7细胞增殖和凋亡的影响,以期为乳腺癌的靶向治疗提供新的分子靶点。
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与人乳腺上皮细胞HBL-100比较,乳腺癌MCF-7细胞中miR-99b-5p的表达量明显降低(P < 0.01),而TRIB1蛋白的表达明显升高(P < 0.01)(见图 1、表 1)。
细胞系 miR-99b-5p TRIB1 HBL-100 1.00±0.09 0.17±0.02 MCF-7 0.20±0.02 0.52±0.05 t 26.03 19.50 P < 0.01 < 0.01 表 1 HBL-100细胞和MCF-7细胞中miR-99b-5p表达量及TRIB1蛋白表达水平比较(ni=9;x±s)
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转染48 h后,miR-99b-5p组MCF-7细胞中miR-99b-5p的表达量高于Control组和miR-NC组(P < 0.05),TRIB1蛋白表达水平低于Control组和miR-NC组(P < 0.05);miR-NC组与和Control组中miR-99b-5p和TRIB1蛋白表达差异均无统计学意义(P>0.05)(见图 2、表 2)。
分组 miR-99b-5p TRIB1 Control 1.00±0.10 0.51±0.05 miR-NC 0.96±0.09 0.52±0.05 miR-99b-5p 2.05±0.02*# 0.23±0.02*# F 557.56 135.50 P < 0.01 < 0.01 MS组内 0.006 0.002 q检验:与Control比较*P < 0.05;与miR-NC比较#P < 0.05 表 2 各组MCF-7细胞中miR-99b-5p表达量及TRIB1蛋白表达水平比较(ni=9;x±s)
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MTT实验结果显示,miR-99b-5p组MCF-7细胞在转染48 h和72 h时OD值均低于miR-NC组和Control组(P < 0.05);Control组和miR-NC组MCF-7细胞在转染24、48、72 h时OD值差异均无统计学意义(P>0.05)(见表 3)。
分组 OD值 24 h 48 h 72 h Control 0.22±0.02 0.50±0.05 0.74±0.08 miR-NC 0.21±0.02 0.48±0.05 0.70±0.07 miR-99b-5p 0.21±0.02 0.31±0.03*# 0.42±0.04*# F 0.75 49.88 63.63 P > 0.05 < 0.01 < 0.01 MS组内 0.001 0.002 0.004 q检验:与Control比较*P < 0.05;与miR-NC比较#P < 0.05 表 3 各组MCF-7细胞在转染24、48、72 h时OD值比较(ni=9;x±s)
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AnnexinV-FITC/PI双染色法结果显示,miR-99b-5p组MCF-7细胞凋亡率高于miR-NC组和Control组(P < 0.05);Control组和miR-NC组MCF-7细胞凋亡率差异无统计学意义(P>0.05)(见图 3、表 4)。
分组 凋亡率/% F P MS组内 Control 3.08±1.13 191.60 < 0.01 8.618 miR-NC 3.22±1.20 miR-99b-5p 26.61±4.81*# q检验:与Control比较*P < 0.05;与miR-NC比较#P < 0.05 表 4 各组MCF-7细胞凋亡率比较(ni=9;x±s)
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TargetScan等生物信息学在线软件预测发现,miR-99b-5p与TRIB1 3′UTR存在互补配对碱基(见图 4)。双荧光素酶报告基因实验结果显示,共转染miR-99b-5p mimics和TRIB1-Wt后,与miR-NC组比,miR-99b-5p组MCF-7细胞相对荧光素酶活性明显降低(P < 0.01);共转染miR-99b-5p mimics和TRIB1-Mut后,miR-NC组和miR-99b-5p组MCF-7细胞相对荧光素酶活性差异无统计学意义(P>0.05)(见表 5)。
分组 TRIB1-Wt TRIB1-Mut miR-NC 1.00±0.09 1.00±0.10 miR-99b-5p 0.20±0.02 0.98±0.09 t 26.03 0.45 P < 0.01 > 0.05 表 5 各组MCF-7细胞相对荧光素酶活性比较(ni=9;x±s)
miR-99b-5p通过靶向TRIB1基因调控乳腺癌MCF-7细胞增殖和凋亡的研究
miR-99b-5p regulates the proliferation and apoptosis of breast cancer MCF-7 cells by targeting TRIB1 gene
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摘要:
目的探讨miR-99b-5p靶向Tribbles同源蛋白1(Tribbles pseudokinase 1,TRIB1)基因调控人乳腺癌MCF-7细胞增殖和凋亡。 方法采用RT-PCR检测人乳腺上皮细胞HBL-100和乳腺癌MCF-7细胞中miR-99b-5p的表达水平,采用Western blotting检测人乳腺上皮细胞HBL-100和乳腺癌MCF-7细胞中TRIB1蛋白表达量。在MCF-7细胞中转染miR-99b-5p mimics或mimics对照,分别记为miR-99b-5p组和miR-NC组,RT-PCR检测细胞中miR-99b-5p表达变化,Western blotting检测TRIB1蛋白表达量,MTT法检测细胞增殖能力,AnnexinV-FITC/PI双染色法检测细胞凋亡水平,双荧光素酶报告基因实验检测miR-99b-5p和TRIB1的靶向关系。 结果与人乳腺上皮细胞HBL-100比较,乳腺癌MCF-7细胞中miR-99b-5p的表达量明显降低(P < 0.01),而TRIB1蛋白的表达明显升高(P < 0.01)。与miR-NC组比较,miR-99b-5p组MCF-7细胞中miR-99b-5p的表达量升高(P < 0.05),TRIB1蛋白表达水平降低(P < 0.05),细胞增殖能力减弱(P < 0.05),细胞凋亡率升高(P < 0.05)。双荧光素酶报告基因检测实验结果显示,miR-99b-5p能够明显降低TRIB1-Wt MCF-7细胞相对荧光素酶活性(P < 0.01),而对TRIB1-Mut MCF-7细胞相对荧光素酶活性无明显影响(P>0.05)。 结论miR-99b-5p在乳腺癌MCF-7细胞中呈低表达,TRIB1蛋白呈高表达,过表达miR-99b-5p能够靶向抑制TRIB1阻碍MCF-7细胞增殖并促进细胞凋亡。 -
关键词:
- 乳腺肿瘤 /
- miR-99b-5p /
- Tribbles同源蛋白1 /
- 增殖 /
- 凋亡
Abstract:ObjectiveTo investigate the regulation of proliferation and apoptosis of human breast cancer MCF-7 cells by miR-99b-5p targeting Tribbles pseudokinase 1(TRIB1) gene. MethodsThe expression of miR-99b-5p in human breast epithelial cells HBL-100 and breast cancer MCF-7 cells was detected by RT-PCR, and the expression of TRIB1 protein in human breast epithelial cells HBL-100 and breast cancer MCF-7 cells was detected by Western blotting.After transfection with miR-99b-5p mimics or mimics control in MCF-7 cells, which was named as miR-99b-5p group and miR-NC group, respectively, the expression of miR-99b-5p in the cells was detected by RT-PCR, the expression of TRIB1 protein was detected by Western blotting, the cell proliferation was detected by MTT assay, the apoptosis level was detected by AnnexinV-FITC/PI double staining, and the targeting relationship between miR-99b-5p and TRIB1 was detected by dual-luciferase reporter gene assay. ResultsCompared with human breast epithelial cells HBL-100, the expression of miR-99b-5p in breast cancer MCF-7 cells was significantly decreased(P < 0.01), while the expression of TRIB1 protein was significantly increased(P < 0.01).Compared with miR-NC group, the expression of miR-99b-5p in MCF-7 cells in miR-99b-5p group increased(P < 0.05), the expression level of TRIB1 protein decreased(P < 0.05), the cell proliferation ability decreased(P < 0.05), and the apoptosis rate increased(P < 0.05).The results of dual-luciferase reporter gene assay showed that miR-99b-5p significantly reduced the relative luciferase activity in TRIB1-Wt MCF-7 cells(P < 0.01), but had no significant effect on the relative luciferase activity in TRIB1-Mut MCF-7 cells(P>0.05). ConclusionsMiR-99b-5p is down-regulated in breast cancer MCF-7 cells, and TRIB1 protein is up-regulated.Overexpression of miR-99b-5p can inhibit the proliferation and promote apoptosis in MCF-7 cells by targeted inhibiting TRIB1. -
Key words:
- breast neoplasms /
- miR-99b-5p /
- Tribbles pseudokinase 1 /
- proliferation /
- apoptosis
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表 1 HBL-100细胞和MCF-7细胞中miR-99b-5p表达量及TRIB1蛋白表达水平比较(ni=9;x±s)
细胞系 miR-99b-5p TRIB1 HBL-100 1.00±0.09 0.17±0.02 MCF-7 0.20±0.02 0.52±0.05 t 26.03 19.50 P < 0.01 < 0.01 表 2 各组MCF-7细胞中miR-99b-5p表达量及TRIB1蛋白表达水平比较(ni=9;x±s)
分组 miR-99b-5p TRIB1 Control 1.00±0.10 0.51±0.05 miR-NC 0.96±0.09 0.52±0.05 miR-99b-5p 2.05±0.02*# 0.23±0.02*# F 557.56 135.50 P < 0.01 < 0.01 MS组内 0.006 0.002 q检验:与Control比较*P < 0.05;与miR-NC比较#P < 0.05 表 3 各组MCF-7细胞在转染24、48、72 h时OD值比较(ni=9;x±s)
分组 OD值 24 h 48 h 72 h Control 0.22±0.02 0.50±0.05 0.74±0.08 miR-NC 0.21±0.02 0.48±0.05 0.70±0.07 miR-99b-5p 0.21±0.02 0.31±0.03*# 0.42±0.04*# F 0.75 49.88 63.63 P > 0.05 < 0.01 < 0.01 MS组内 0.001 0.002 0.004 q检验:与Control比较*P < 0.05;与miR-NC比较#P < 0.05 表 4 各组MCF-7细胞凋亡率比较(ni=9;x±s)
分组 凋亡率/% F P MS组内 Control 3.08±1.13 191.60 < 0.01 8.618 miR-NC 3.22±1.20 miR-99b-5p 26.61±4.81*# q检验:与Control比较*P < 0.05;与miR-NC比较#P < 0.05 表 5 各组MCF-7细胞相对荧光素酶活性比较(ni=9;x±s)
分组 TRIB1-Wt TRIB1-Mut miR-NC 1.00±0.09 1.00±0.10 miR-99b-5p 0.20±0.02 0.98±0.09 t 26.03 0.45 P < 0.01 > 0.05 -
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