靶向于瘦素受体的siRNA对SK-BR-3细胞增殖和凋亡的影响及机制探讨
摘要
背景和目的：
一直以来，肥胖被认为是乳腺癌发病的危险因素并与预后不良及耐药相关[1,2]。瘦素(leptin，OB)是肥胖基因（obese gene ob）编码的蛋白质产物，瘦素作为一种多肽激素，在生物体中具有广泛的生物学功能，除了是体质量调节中枢，还在促进细胞增殖与分化，增加细胞侵袭能力以及促进血管生成等方面发挥重要作用，因此瘦素与肿瘤的发生发展有着密切的关系。瘦素通过与其受体（leptinreceptor ObR）结合发挥一系列生物学效应，瘦素与其受体结合后激活了一系列信号转导途径，包括JAK（janus kinase）/STAT（signal transducer and activator oftranscription），MAPK（mitogen activated protein kinase），PI3K(phosphoinositede 3kinase )等。有研究发现在乳腺癌SK-BR-3细胞株中，OB可以通过表皮生长因子HER1或JAK2信号途径反式激活HER2。本文旨在探讨（1）OB/ObR及HER2在乳腺癌组织中的表达是否相关；（2）OB/ObR表达与乳腺癌临床病理特征及预后的关系；（3）靶向于ObR的siR N A（small interfering RNA）对乳腺癌细胞株SK-BR-3增殖及凋亡的影响及其机制探讨。
方法：
1．免疫组织化学法检测乳腺癌组织中OB、ObR的表达并分析其与临床病理特征及预后的关系；
2．构建靶向于ObR的小片段RNA（siRNA）干扰质粒，将siRNA干扰质粒瞬时转染HER2及ObR共同表达的乳腺癌细胞株SK-BR-3；
3．用qRT-PCR方法检测转染后ObR mRNA表达水平的改变；
4．用Western-blot方法检测转染后ObR蛋白表达水平的改变；
5．用四甲基偶氮唑蓝法(MTT)检测siRNA干扰后对细胞增殖率的影响；
6．分别用Annexin V/PI流式细胞术及TUNEL染色法检测siRNA干扰后对细胞凋亡率的影响；
7．用Western-blot方法检测siRNA干扰前后HER2蛋白表达水平的改变。
结果：
1．OB和ObR在大多数乳腺癌组织中均有表达，阳性率分别为79.8%和85.1%；OB和ObR的表达与患者的年龄、绝经状态、肿瘤大小、肿瘤病理学分型、淋巴结转移及远处转移均无关( P>0.05)；与ER、PR、P53的表达状态也无关（P>0．05）；而ObR阳性组与阴性组HER2表达的差异有统计学意义，P=0.04；
2．OB阳性者总生存时间（OS）短，P=0.008；在分亚组讨论中，三苯氧胺治疗组、绝经后组、三阴乳腺癌组和淋巴结转移组中OB阳性组OS均短于阴性组，P值分别为0.05、0.036、0.005和0.005；
3．qRT-PCR及Western-blot法分别证实了靶向ObR的siRNA干扰SK-BR-3细胞后，ObR的mRNA表达水平及蛋白表达水平均下调；
4．MTT法证实了靶向ObR的siRNA干扰SK-BR-3细胞后，细胞的增殖率明显下降；
5．Annexin V/PI流式细胞术及TUNEL染色法均证实靶向ObR的siRNA干扰SKBR-3后，细胞的凋亡率明显上升；
6．Western-blot法证实了靶向ObR的siRNA干扰SK-BR-3后，HER2的蛋白表达水平显著下调。
结论：
ObR与HER2在乳腺癌组织中的表达具有一致性，下调ObR的表达能明显抑制SK-BR-3细胞的增殖，诱导其凋亡，其机制可能与抑制HER2的活性相关。
Background and Objective：Obesity is a risk factor for breast cancer and isassociated with poor prognosis and drug resistance.Leptin,the protein product of theobese gene, which controlling food intake and energy balance.In cellular models,leptin has been shown to activate proliferation, angiogenesis,and invasion.Leptinaction is mediated through the transmembrane leptin receptor ObR. The signalingpathways known to be activated by ObR include JAK/STAT,MAPK andPI3K,et,al.Recent studie suggested that in SK-BR-3 breast cancer cell line,leptin cantransactivate HER2 through both the epidermal growth factor receptor HER1 andJAK2 pathways.Here,we first tested whether the ObR and HER2 can be coexpressedin breast cancer biopsies and the relationship of OB/ObR expression with theclinicopathlogical features and prognosis;then we studied the effect of siRNA againstObR on the proliferation and apoptosis of SK-BR-3 cell line and explored themechanisms.
Methods：(1) The expression of OB and ObR in breast cancer tissues wasevaluated with immunohistochemistry;(2) A specific ObR siRNA oligonucleotidewere constructed. The ObR siRNA was transient transfected into SK-BR-3 cell line that have coexpression of ObR and HER2;(3) The results of transfections wereconfirmed by qreal-time PCR and Western blotting;(4) The effect of siRNA on theproliferation of SK-BR-3 cell lines was evaluated using MTT assay;(5) The effect ofsiRNA on the apoptosis of SK-BR-3 cell line was evaluated by Annexin V andTUNEL;(6) Change of HER2 protein expression was detected by western blot assay.
Results：(1) Positive expressions of OB and ObR were noted in 79.8% and 85.1%of the samples. The expression of OB and ObR in breast cancer tissue was notsignificantly related to the clinicopathological characteristics,including ages,menopause, tumor size,pathological type,lymph node metastasis and distantmetastasis,( P>0.05). The expression of OB and ObR was not significantly related tothe ER,PR and P53 status,（P>0.05）.The expression of ObR showed a significantcorrelation with the HER2 status ,(P=0.04);(2) The expression of OB wassignificantly related to the overall survival(OS),(p=0.008). For the tamoxifen-treatedpatients, postmenopause patients, triple negative patients and lymph node metastasispatents ,the OS of the OB-positive group was significantly shorter than that of thenegative group, (p=0.05, p=0.036, p=0.005, p=0.005);(3)ObR-targeted siRNA transfectionremarkably decreased the ObR expression at the mRNA and proteinlevels;(4)ObR-targeted siRNA transfection remarkably decreased the growth of SKBR-3 cells;(5)ObR-targeted siRNA transfection remarkably increased the apoptosis ofSK-BR-3 cells;(6) ObR-targeted siRNA transfection remarkably decreased HER2expression at the protein level.
Conclusion：ObR and HER2 coespression in breast cancer tissues,；downregulationof ObR expression with ObR-targeted siRNA remarkably decreased thegrowth and increased the apoptosis of SK-BR-3 cells;and the inhibition of HER2expression might be related to the mechanisms.
一直以来，肥胖被认为是乳腺癌发病的危险因素并与预后不良及耐药相关[1,2]。瘦素(leptin，OB)是肥胖基因（obese gene ob）编码的蛋白质产物，瘦素作为一种多肽激素，在生物体中具有广泛的生物学功能，除了是体质量调节中枢，还在促进细胞增殖与分化，增加细胞侵袭能力以及促进血管生成等方面发挥重要作用，因此瘦素与肿瘤的发生发展有着密切的关系。瘦素通过与其受体（leptinreceptor ObR）结合发挥一系列生物学效应，瘦素与其受体结合后激活了一系列信号转导途径，包括JAK（janus kinase）/STAT（signal transducer and activator oftranscription），MAPK（mitogen activated protein kinase），PI3K(phosphoinositede 3kinase )等。有研究发现在乳腺癌SK-BR-3细胞株中，OB可以通过表皮生长因子HER1或JAK2信号途径反式激活HER2。本文旨在探讨（1）OB/ObR及HER2在乳腺癌组织中的表达是否相关；（2）OB/ObR表达与乳腺癌临床病理特征及预后的关系；（3）靶向于ObR的siR N A（small interfering RNA）对乳腺癌细胞株SK-BR-3增殖及凋亡的影响及其机制探讨。
方法：
1．免疫组织化学法检测乳腺癌组织中OB、ObR的表达并分析其与临床病理特征及预后的关系；
2．构建靶向于ObR的小片段RNA（siRNA）干扰质粒，将siRNA干扰质粒瞬时转染HER2及ObR共同表达的乳腺癌细胞株SK-BR-3；
3．用qRT-PCR方法检测转染后ObR mRNA表达水平的改变；
4．用Western-blot方法检测转染后ObR蛋白表达水平的改变；
5．用四甲基偶氮唑蓝法(MTT)检测siRNA干扰后对细胞增殖率的影响；
6．分别用Annexin V/PI流式细胞术及TUNEL染色法检测siRNA干扰后对细胞凋亡率的影响；
7．用Western-blot方法检测siRNA干扰前后HER2蛋白表达水平的改变。
结果：
1．OB和ObR在大多数乳腺癌组织中均有表达，阳性率分别为79.8%和85.1%；OB和ObR的表达与患者的年龄、绝经状态、肿瘤大小、肿瘤病理学分型、淋巴结转移及远处转移均无关( P>0.05)；与ER、PR、P53的表达状态也无关（P>0．05）；而ObR阳性组与阴性组HER2表达的差异有统计学意义，P=0.04；
2．OB阳性者总生存时间（OS）短，P=0.008；在分亚组讨论中，三苯氧胺治疗组、绝经后组、三阴乳腺癌组和淋巴结转移组中OB阳性组OS均短于阴性组，P值分别为0.05、0.036、0.005和0.005；
3．qRT-PCR及Western-blot法分别证实了靶向ObR的siRNA干扰SK-BR-3细胞后，ObR的mRNA表达水平及蛋白表达水平均下调；
4．MTT法证实了靶向ObR的siRNA干扰SK-BR-3细胞后，细胞的增殖率明显下降；
5．Annexin V/PI流式细胞术及TUNEL染色法均证实靶向ObR的siRNA干扰SKBR-3后，细胞的凋亡率明显上升；
6．Western-blot法证实了靶向ObR的siRNA干扰SK-BR-3后，HER2的蛋白表达水平显著下调。
结论：
ObR与HER2在乳腺癌组织中的表达具有一致性，下调ObR的表达能明显抑制SK-BR-3细胞的增殖，诱导其凋亡，其机制可能与抑制HER2的活性相关。
Background and Objective：Obesity is a risk factor for breast cancer and isassociated with poor prognosis and drug resistance.Leptin,the protein product of theobese gene, which controlling food intake and energy balance.In cellular models,leptin has been shown to activate proliferation, angiogenesis,and invasion.Leptinaction is mediated through the transmembrane leptin receptor ObR. The signalingpathways known to be activated by ObR include JAK/STAT,MAPK andPI3K,et,al.Recent studie suggested that in SK-BR-3 breast cancer cell line,leptin cantransactivate HER2 through both the epidermal growth factor receptor HER1 andJAK2 pathways.Here,we first tested whether the ObR and HER2 can be coexpressedin breast cancer biopsies and the relationship of OB/ObR expression with theclinicopathlogical features and prognosis;then we studied the effect of siRNA againstObR on the proliferation and apoptosis of SK-BR-3 cell line and explored themechanisms.
Methods：(1) The expression of OB and ObR in breast cancer tissues wasevaluated with immunohistochemistry;(2) A specific ObR siRNA oligonucleotidewere constructed. The ObR siRNA was transient transfected into SK-BR-3 cell line that have coexpression of ObR and HER2;(3) The results of transfections wereconfirmed by qreal-time PCR and Western blotting;(4) The effect of siRNA on theproliferation of SK-BR-3 cell lines was evaluated using MTT assay;(5) The effect ofsiRNA on the apoptosis of SK-BR-3 cell line was evaluated by Annexin V andTUNEL;(6) Change of HER2 protein expression was detected by western blot assay.
Results：(1) Positive expressions of OB and ObR were noted in 79.8% and 85.1%of the samples. The expression of OB and ObR in breast cancer tissue was notsignificantly related to the clinicopathological characteristics,including ages,menopause, tumor size,pathological type,lymph node metastasis and distantmetastasis,( P>0.05). The expression of OB and ObR was not significantly related tothe ER,PR and P53 status,（P>0.05）.The expression of ObR showed a significantcorrelation with the HER2 status ,(P=0.04);(2) The expression of OB wassignificantly related to the overall survival(OS),(p=0.008). For the tamoxifen-treatedpatients, postmenopause patients, triple negative patients and lymph node metastasispatents ,the OS of the OB-positive group was significantly shorter than that of thenegative group, (p=0.05, p=0.036, p=0.005, p=0.005);(3)ObR-targeted siRNA transfectionremarkably decreased the ObR expression at the mRNA and proteinlevels;(4)ObR-targeted siRNA transfection remarkably decreased the growth of SKBR-3 cells;(5)ObR-targeted siRNA transfection remarkably increased the apoptosis ofSK-BR-3 cells;(6) ObR-targeted siRNA transfection remarkably decreased HER2expression at the protein level.
Conclusion：ObR and HER2 coespression in breast cancer tissues,；downregulationof ObR expression with ObR-targeted siRNA remarkably decreased thegrowth and increased the apoptosis of SK-BR-3 cells;and the inhibition of HER2expression might be related to the mechanisms.
引文
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