RKIP在人乳腺癌组织及细胞中的表达及其通过NF-κB信号通路对乳腺癌细胞迁移和侵袭作用的研究
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摘要
目的探讨Raf激酶抑制蛋白(RKIP)在人乳腺癌组织及细胞中的表达及其通过核因子-κB(NF-κB)信号通路对乳腺癌细胞迁移和侵袭的作用。方法收集良性乳腺肿瘤组织28例和恶性乳腺肿瘤组织35例,采用免疫组织化学法检测两种组织中RKIP的表达水平。将含有RKIP基因的cDNA插入GV141载体中构建RKIP质粒,作为RKIP过表达组的转染质粒;以空白GV141载体构建空白质粒,作为对照组的转染质粒。将上述两组质粒转染乳腺癌MDA-MB-435细胞,应用Transwell小室实验检测RKIP过表达组和对照组细胞的迁移和侵袭能力,蛋白质印迹法(Western blot)检测RKIP蛋白的表达水平,逆转录聚合酶链反应(RT-PCR)检测NF-κB mRNA的表达水平。结果良性乳腺肿瘤组织中RKIP的表达水平较高,积分光密度(IOD)值为(0.527±0.057),高于恶性乳腺肿瘤组织的(0.217±0.048),差异有统计学意义(P<0.05)。RKIP过表达组迁移细胞数目和侵袭细胞数目均明显少于对照组,差异均有统计学意义(P<0.01)。Western blot检测结果显示,RKIP过表达组的RKIP表达水平为(0.059±0.005),明显高于对照组的(0.009±0.002),差异有统计学意义(P<0.01)。RT-PCR检测结果显示,RKIP过表达组中NF-κB mRNA的相对表达量为(0.120±0.016),明显低于对照组的(0.380±0.025),差异有统计学意义(P<0.01)。结论 RKIP与乳腺癌的恶化程度有关,RKIP能够抑制乳腺癌细胞的迁移和侵袭,且其作用机制可能是通过对NF-κB的负调控而实现。
Objective To investigate the expression of Raf kinase inhibitor protein(RKIP) in human breast cancer tissues and cells, and to explore its role in the migration and invasion of breast cancer via NF-κB signal pathway. Method Benign(n=28) and malignant(n=35) breast cancer tissue specimens were collected, the expression of RKIP was detected by immunehistochemical method and compared between the two groups. cDNA containing RKIP gene was inserted into GV141 vector to construct RKIP plasmid, which was used as the transfected plasmid of RKIP overexpression group;blank plasmid was constructed with blank GV141 vector as the transfected plasmid of control group; then the two kinds of plasmids were transfected into MDA-MB-435 cells. The migration and invasion ability of MDA-MB-435 cells in the RKIP overexpression group and the control group were tested by Transwell chambers experiment, and the expression of RKIP protein and mRNA expression of NF-κB gene in both groups were detected by Western blot and RT-PCR. Result The expression of RKIP in benign breast cancer tissues(IOD=0.527±0.057) was significantly higher than that in malignant breast cancer tissues(IOD=0.217±0.048)(P<0.05). The migration and invasion cells of RKIP overexpression group were significantly less than those of the control group(P<0.01). The result of Western blot showed that the RKIP protein expression in RKIP overexpression group(0.059±0.005) was significantly higher than that(0.009±0.002) in control group(P<0.01), while the mRNA expression of NF-κB gene in RKIP overexpression group(0.120±0.016) was significantly lower than that(0.380±0.025) in control group(P<0.01). Conclusion RKIP is associated with the progression of breast cancer, and can inhibit the migration and invasion of breast cancer, which may be performed via its negative regulation on NF-κB.
Objective To investigate the expression of Raf kinase inhibitor protein(RKIP) in human breast cancer tissues and cells, and to explore its role in the migration and invasion of breast cancer via NF-κB signal pathway. Method Benign(n=28) and malignant(n=35) breast cancer tissue specimens were collected, the expression of RKIP was detected by immunehistochemical method and compared between the two groups. cDNA containing RKIP gene was inserted into GV141 vector to construct RKIP plasmid, which was used as the transfected plasmid of RKIP overexpression group;blank plasmid was constructed with blank GV141 vector as the transfected plasmid of control group; then the two kinds of plasmids were transfected into MDA-MB-435 cells. The migration and invasion ability of MDA-MB-435 cells in the RKIP overexpression group and the control group were tested by Transwell chambers experiment, and the expression of RKIP protein and mRNA expression of NF-κB gene in both groups were detected by Western blot and RT-PCR. Result The expression of RKIP in benign breast cancer tissues(IOD=0.527±0.057) was significantly higher than that in malignant breast cancer tissues(IOD=0.217±0.048)(P<0.05). The migration and invasion cells of RKIP overexpression group were significantly less than those of the control group(P<0.01). The result of Western blot showed that the RKIP protein expression in RKIP overexpression group(0.059±0.005) was significantly higher than that(0.009±0.002) in control group(P<0.01), while the mRNA expression of NF-κB gene in RKIP overexpression group(0.120±0.016) was significantly lower than that(0.380±0.025) in control group(P<0.01). Conclusion RKIP is associated with the progression of breast cancer, and can inhibit the migration and invasion of breast cancer, which may be performed via its negative regulation on NF-κB.
引文
[1]钱飚,鲁洪丰,励超,等.终末期乳腺癌患者高危型HPV感染与Stat3活性及IL-17表达意义研究[J].中华医院感染学杂志, 2017, 27(23):5373-5376.
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[8]陶善东,王春玲,陈月,等. Btk和NFκB在急性淋巴细胞白血病中的表达与意义[J].中国实验血液学杂志, 2016, 24(4):969-974.
[9]宋思幽,吴英杰,李冬华. Raf激酶抑制蛋白、NF-κBp65及VEGF-C在卵巢上皮性肿瘤中的表达及相关性[J].中国临床研究, 2013, 26(6):525-527; 530.
[10]王发亮,侯金超. miRNA在乳腺癌分型及耐药判断中的研究进展[J].癌症进展, 2017, 15(4):354-357; 362.
[11] Duffy MJ, Harbeck N, Nap M, et al. Clinical use of biomarkers in breast cancer:updated guidelines from the European Group on Tumor Markers(EGTM)[J]. Eur J Cancer, 2017, 75:284-298.
[12] Kim HS, Park SJ, Lee KY, et al. Reduced Raf-1 kinase inhibitor protein expression predicts less favorable outcomes in patients with hepatic colorectal metastasis[J].Oncol Rep, 2012, 28(1):161-171.
[13] Hagan S, Al-Mulla F, Mallon E, et al. Reduction of Raf-1kinase inhibitor protein expression correlates with breast cancer metastasis[J]. Clin Cancer Res, 2005, 11(20):7392-7397.
[14]姚志强,周鸿飞,崔明. RKIP、NF-κBp65蛋白在乳腺癌组织中的表达及其临床意义[J].江西医药, 2010, 45(12):1185-1188.
[2] Drossaert CH, Boer H, Seydel ER. Monitoring women’s experience during three rounds of breast cancer screening:results form a longitudinal study[J]. J Med Screen, 2002, 9(4):168-175.
[3]陈玮,吴库生.乳腺癌术后患者心理弹性及其影响因素分析[J].中国医药科学, 2017, 7(20):24-29.
[4]王歆光,刘毅强,何英剑,等.同侧多原发灶乳腺癌病灶间异质性的研究[J].中国癌症杂志, 2017, 27(10):809-814.
[5] Wang A, Duan G, Zhao C, et al. Reduced RKIP expression levels are associated with frequent non-small cell lung cancer metastasis and STAT3 phosphorylation and activation[J]. Oncol Lett, 2017, 13(5):3039-3045.
[6]赫莉,李芳芳,曹山虎,等.喉癌组织RKIP基因甲基化表达调控研究[J].中国耳鼻咽喉颅底外科杂志, 2016, 22(3):235-238.
[7]张磊,江浩. Raf激酶抑制蛋白在鼻咽癌中的研究进展[J].中华全科医学, 2016, 14(4):659-661.
[8]陶善东,王春玲,陈月,等. Btk和NFκB在急性淋巴细胞白血病中的表达与意义[J].中国实验血液学杂志, 2016, 24(4):969-974.
[9]宋思幽,吴英杰,李冬华. Raf激酶抑制蛋白、NF-κBp65及VEGF-C在卵巢上皮性肿瘤中的表达及相关性[J].中国临床研究, 2013, 26(6):525-527; 530.
[10]王发亮,侯金超. miRNA在乳腺癌分型及耐药判断中的研究进展[J].癌症进展, 2017, 15(4):354-357; 362.
[11] Duffy MJ, Harbeck N, Nap M, et al. Clinical use of biomarkers in breast cancer:updated guidelines from the European Group on Tumor Markers(EGTM)[J]. Eur J Cancer, 2017, 75:284-298.
[12] Kim HS, Park SJ, Lee KY, et al. Reduced Raf-1 kinase inhibitor protein expression predicts less favorable outcomes in patients with hepatic colorectal metastasis[J].Oncol Rep, 2012, 28(1):161-171.
[13] Hagan S, Al-Mulla F, Mallon E, et al. Reduction of Raf-1kinase inhibitor protein expression correlates with breast cancer metastasis[J]. Clin Cancer Res, 2005, 11(20):7392-7397.
[14]姚志强,周鸿飞,崔明. RKIP、NF-κBp65蛋白在乳腺癌组织中的表达及其临床意义[J].江西医药, 2010, 45(12):1185-1188.