钠氢交换子调节因子-1及β-联蛋白在皮肤黑素瘤组织及细胞株中的表达和意义
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摘要
皮肤黑素瘤是一种高度恶性的黑素细胞肿瘤,不易受化疗药物的干扰且易发生转移,预后不良。对其发病机制、治疗方法的研究是国内外皮肤肿瘤研究的热点。钠氢交换子调节因子-1(NHERF1)是一种支架蛋白,其在调节离子转运、运输中发挥着重要作用,同时它还与膜-细胞骨架连接蛋白相互作用,可以稳定跨膜蛋白,并参与构成上皮顶端微绒毛结构。它还是一种调节蛋白,能与30余种蛋白质作用并调节其功能。随着近年来研究的深入,有学者发现其在多种肿瘤中发挥着重要作用：其基因的突变或杂合性丢失可导致肿瘤细胞的恶性程度增高。其在正常细胞中通常为胞膜表达,发挥抑癌作用；胞膜不表达或在胞浆及胞核中过表达则提示肿瘤恶性程度增加。然而,尽管越来越多的研究表明其在癌症中发挥着重要的作用,但目前对其在肿瘤、尤其在皮肤恶性黑素瘤中的作用及作用机制仍知之甚少。在进一步的研究中除了不断探索其抗肿瘤的机制,其对肿瘤生物学行为的影响及临床意义将成为研究重点。
(1) NHERF1及β-联蛋白在皮肤黑素瘤及痣细胞痣组织中的表达及意义
为了探讨NHERF1是否参与了皮肤黑素瘤的发生发展过程,我们收集47例皮肤黑素瘤组织及37例痣细胞痣组织,应用免疫组化方法对NHERF1和β-联蛋白在组织中的表达模式(膜表达、浆表达及核表达)进行检测。结果显示：原位皮肤黑素瘤组织中NHERF1胞浆中等/强阳性表达率为38%；而侵袭性皮肤黑素瘤组织中NHERF1胞浆中等/强阳性表达率为71%,两组相比具有统计学显著性差异。同时,全部皮肤黑素瘤组织中NHERF1胞浆中等/强阳性表达率为60%,而痣细胞痣组织中NHERF1胞浆中等/强阳性表达率为0,有明显统计学差异。原位皮肤黑素瘤组织中可见β-联蛋白胞浆中等/强阳性表达率为50%；而侵袭性皮肤黑素瘤组织中可见β-联蛋白胞浆中等/强阳性表达率为84%。全部皮肤黑素瘤组织中β-联蛋白胞浆中等/强阳性表达率为72%,而在痣细胞痣组织中β-联蛋白胞浆中等/强阳性表达率为30%,两组相比具有统计学显著性差异。NHERF1及β-联蛋白在痣细胞痣、原位黑素瘤及侵袭性黑素瘤组织中的胞浆阳性表达率均随着病变组织的恶性程度增加而升高。为了进一步探讨NHERF1对β-联蛋白及黑素瘤的影响,我们将在实验的第三部分,利用shRNA干扰技术进行深入的了解。
(2) NHERF1及β-联蛋白在黑素瘤细胞株A375,M14,MV3中的表达及意义
从第一部分的研究中观察到NHERF1及β-联蛋白在痣细胞痣、原位黑素瘤及侵袭性黑素瘤组织中的异常胞浆阳性表达率随着病变组织的恶性程度增加而升高。为了研究NHERF1及β-联蛋白在黑素瘤细胞株A375,M14,MV3中的表达及意义。培养黑素瘤细胞株A375、M14、MV3及黑素细胞；使用免疫细胞化学法、实时荧光定量-PCR法、免疫印迹法对黑素瘤细胞株A375、M14、MV3及黑素细胞中NHERF1及β-联蛋白的表达情况进行观察；并使用MTT法对黑素瘤细胞株A375、M14、MV3及黑素细胞的增殖情况进行检测。结果发现：NHERF1及β-联蛋白在黑素细胞中同时表现为细胞膜阳性表达,而在黑素瘤细胞株A375, M14, MV3中均出现膜表达的缺失,同时呈现不同程度的细胞浆和/或细胞核阳性表达。在三株黑素瘤细胞株中,MV3细胞中的NHERF1免疫细胞化学染色仅呈少部分细胞胞质弱阳性表达,实时荧光定量-PCR检测NHERF1的mRNA表达水平最低,免疫印迹法中未见NHERF1蛋白表达条带,而其在MTT法中检测到的的增殖速度最快,明显高于另外两株细胞；A375细胞中的NHERF1免疫细胞化学染色呈细胞浆及细胞核的强阳性表达,实时荧光定量-PCR检测NHERF1的mRNA表达水平最高,免疫印迹法中可见NHERF1蛋白最强阳性表达,而其增殖速度却明显慢于MV3细胞。同时,p-联蛋白在MV3细胞中免疫细胞化学染色呈细胞浆及细胞核的强阳性表达,实时荧光定量-PCR检测的mRNA表达水平最高,免疫印迹法检测到蛋白最强阳性表达；而A375细胞及M14细胞中,p-联蛋白在免疫细胞化学染色呈相对弱的细胞浆阳性表达,实时荧光定量-PCR检测的mRNA表达水平明显较低；免疫印迹法中的蛋白表达较MV3中的表达低。与正常黑素细胞相比,黑素瘤细胞A375, M14, MV3中的NHERF1的mRNA表达水平都呈不同程度升高,而β-联蛋白表达水平(mRNA表达水平及蛋白水平)亦存在不同程度升高,同时,黑素瘤细胞中的NHERF1与p-联蛋白存在着差异性表达,黑素瘤细胞胞浆中的NHERF1表达量可能与细胞的增殖速度相关,NHERF1在黑素瘤细胞中表达程度越低则细胞的增殖可能越快。为了深入探讨NHERF1在黑素瘤细胞中对β-联蛋白及黑素瘤细胞增殖的影响,我们将在实验的第三部分,利用慢病毒载体shRNA干扰技术进行深入的了解。
(3) A375细胞中NHERF1的基因沉默对β-联蛋白的表达及A375细胞增殖、凋亡的影响作用
从第二部分的研究中观察到,黑素瘤细胞中的NHERF1与β-联蛋白存在着差异性表达。黑素瘤细胞胞浆中的NHERF1表达量可能与细胞的增殖速度相关,NHERF1在黑素瘤细胞中表达程度越低则细胞的增殖可能越快。因此在本部分实验中,我们设计并合成NHERF1的shRNA干扰片段,并载入pLVX-shRNA-puro载体中,载体转化到Ecoli感受态中。采用PCR及基因测序等方法对重组质粒进行鉴定。慢病毒包装,慢病毒感染靶细胞,多克隆细胞的验证,单克隆筛选稳定干扰NHERF1的A375细胞株,并采用酶切鉴定、基因测序、qPCR和Western blotting对转染效果进行鉴定。使用Western blotting检测稳定干扰NHERF1的A375细胞株中NHERF1及β-联蛋白的蛋白表达量,以对照序列干扰细胞为对照,并使用MTT法检测两种细胞的增殖情况,Annexin-V/PI双染,流式细胞仪检测细胞凋亡。结果发现设计的shRNA-NHERF1干扰片段载入pLVX-shRNA-puro载体中,并稳定转染A375细胞后,经qPCR和Western blotting检测NHERF1表达明显下降,NHERF1沉默的A375细胞中β-联蛋白较对照细胞中的表达增高,同时A375细胞的增殖速度较对照细胞明显增快。所设计的shRNA-NHERF1干扰片段对A375细胞中NHERF1的表达起到了明显的抑制作用,NHERF1基因沉默导致β-联蛋白的表达增高,细胞增殖加快,细胞凋亡减少。
结论
在皮肤黑素瘤细胞中NHERF1与β-联蛋白呈差异性表达,并可能通过与β-联蛋白的相互作用而调节β-联蛋白的表达水平,从而影响细胞增殖和凋亡。NHERF1在皮肤黑素瘤组织及细胞中的表达较良性痣细胞痣组织及正常黑素细胞均呈不同程度的异常细胞浆阳性表达模式。
Cutaneous malignant melanoma is a highly lethal tumor, originating from melanocyte and resistant to medical intervention of any kind in advanced disease. Initiatives have concentrated on the mechanism and the therapy of melanoma for decades. Na+/H+exchanger regulatory factor-1(NHERF1) is a scaffolding protein, playing an important role in regulating of ion transport, and stabilizing the transmenbrane protein involved in epithelial microvilli structure. It is also a regulatory protein in signaling and trafficking of more than30kinds of proteins. In recent years, it has been found that NHERF1has an emerging role in various tumors. NHERF1gene mutations or loss of heterozygosity can lead to increased degree of malignancy of the tumor cells. In normal tissue, NHERF1is usually cyto-membrane expressed demonstrated by immunohistochemistry and plays a tumor suppressor role, on the other hand, negative expression in cyto-membrane or overexpression in the cytoplasm or nucleus correlate with increased degree of malignancy. Although a growing number of studies have shown that NHERF1plays an important role in oncogensis, its role in anti-tumorigenesis is still poorly understood. Further studies, in addition to the continuate exploring of its tumor suppressor mechanism, willbe focused on its role in the tumor biological behavior and the clinical correlation.
Expression of NHERF1and β-catenin in cutaneous malignant melanoma and melanocytic nevus tissues
To detect the expression of NHERF1and β-catenin in cutaneous malignant melanoma and melanocytic nevus. The expression of NHERF1and β-catenin in47cutaneous malignant melanomas and37melanocytic nevi were examined with immunohistochemistry. The cytoplasm moderate/strong positive expression rate of both NHERF1and β-catenin were gradually increased from melanocytic nevus group, melanoma in situ group to invasive melanoma group. Correlation analysis with Spearman's rho test for NHERF1and β-catenin expression showed no significant correlation between the expression in melanoma tissue samples and melanocytic nevus tissue samples. The cytoplasm moderate/strong positive expression rate of both NHERF1and β-catenin were gradually increased with the increase of the degree of malignancy of lesions from melanocytic nevus group, melanoma in situ group to invasive melanoma group.
Expresssion and significance of NHERF1and β-catenin in malignant melanoma cell lines A375, M14and MV3
To investigate the expression of NHERF1and3-catenin in human melanoma cell lines A375, M14and MV3, as well as their significance. The human melanoma cell lines A375, M14, MV3and human melanocytes were cultured. Immunocellulochemistry, Real-time PCR and Western blotting were performed to detect the expression of NHERF1and β-catenin in these cells respectively; the proliferation rates of these cell lines were measured by the MTT method. In the immunocytochemistry, NHERF1and β-catenin were showed both membranous staining in the melanocytes; while they were absent in the membranous staining in the melanoma cell lines A375, M14and MV3, but presented cytoplasmic and/or nuclear staining with different intensity. Among the3melanoma cell lines, the MV3cells showed partly weak cytoplasmic staining of NHERF1, with the lowest level of the relative quantity of mRNA and absence of immunoblotting NHERF1protein expression. But, its proliferation rate was the highest by MTT assay, much higher than that of the other two cells; while the A375cells showed intensive cytoplasmic and nuclear staining of NHERF1, with the highest expression of the relative quantity of mRNA and intensive immunoblotting NHERF1protein expression. But, their proliferation rate were much lower than that of the MV3cells. Meanwhile, the MV3cells showed intensive cytoplasmic and nuclear staining of β-catenin, with the highest level of the relative quantity of mRNA and intensive immunoblotting NHERF1protein expression; while the A375cells and the M14cells showed less intensive cytoplasmic staining of NHERF1, with the much lower level of the relative quantity of mRNA and less immunoblotting NHERF1protein expression. The level of NHERF1cytoplasm expression in melanoma cell lines may be associated with the proliferation rate of these cells. NHERF1and β-catenin were differentially expressed in the melanoma cell lines.
The influence in the expression of β-catenin as well as the cell proliferation and apoptosis of melanoma cell line A375by the shRNA knockdown of NHERF1
To investigate the influence of NHERF1knockdown in melanoma cell line A375in the expression of β-catenin and the cell proliferation and apoptosis. The shRNA interference fragment of NHERF1was designed and synthesized, then loaded in the pLVX-shRNA-puro vector. The vector was transfected into E. coli competent. The recombinant plasmids were identified by using Real-time PCR and gene sequencing. Lentiviral packaging, lentivirus infection of target cells, polyclonal cells verification and monoclonal screening of stable NHERF1knockdown A375cell were performed. By using gene sequencing, restriction enzyme digestion, qPCR and Western blotting, the effect of transfection was identified. Then, the expression of NHERF1and β-Catenin in NHERF1-knockdown A375cells were detected with Western blotting, in comparing with the shRNA-control A375cells and the untreated A375cells. MTT assay and flow cytometry were performed to detect cell proliferation and apoptosis. After the designed interference fragment of shRNA-NHERF1was loaded in the pLVX-shRNA-puro vector and transfected A375cells, the expression of NHERF1in the transfected cells was decreased significantly as detected by qPCR and Western blotting, while the expression of β-catenin was increased in the transfected A375cells. The cell proliferation of transfected A375cells was faster than that of the controls. The designed shRNA-NHERF1interference fragment inhibits the expression of NHERF1in A375cells, leading to increased β-Catenin expression, acceleration of cell proliferation, and decrease of cell apoptosis.
Conclusions
In the melanoma cell lines, NHERF1and β-catenin were differentially expressed, and NHERF1may regulate the expression of β-catenin through their interaction, thereby modulating cell proliferation and apoptosis. The expression of NHERF1in cutaneous melanoma tissues and melanoma cell lines showed aberrant cytoplasm positive expression in comparing with benign melanocyte nevi and normal melanocytes, respectively.
(1) NHERF1及β-联蛋白在皮肤黑素瘤及痣细胞痣组织中的表达及意义
为了探讨NHERF1是否参与了皮肤黑素瘤的发生发展过程,我们收集47例皮肤黑素瘤组织及37例痣细胞痣组织,应用免疫组化方法对NHERF1和β-联蛋白在组织中的表达模式(膜表达、浆表达及核表达)进行检测。结果显示：原位皮肤黑素瘤组织中NHERF1胞浆中等/强阳性表达率为38%；而侵袭性皮肤黑素瘤组织中NHERF1胞浆中等/强阳性表达率为71%,两组相比具有统计学显著性差异。同时,全部皮肤黑素瘤组织中NHERF1胞浆中等/强阳性表达率为60%,而痣细胞痣组织中NHERF1胞浆中等/强阳性表达率为0,有明显统计学差异。原位皮肤黑素瘤组织中可见β-联蛋白胞浆中等/强阳性表达率为50%；而侵袭性皮肤黑素瘤组织中可见β-联蛋白胞浆中等/强阳性表达率为84%。全部皮肤黑素瘤组织中β-联蛋白胞浆中等/强阳性表达率为72%,而在痣细胞痣组织中β-联蛋白胞浆中等/强阳性表达率为30%,两组相比具有统计学显著性差异。NHERF1及β-联蛋白在痣细胞痣、原位黑素瘤及侵袭性黑素瘤组织中的胞浆阳性表达率均随着病变组织的恶性程度增加而升高。为了进一步探讨NHERF1对β-联蛋白及黑素瘤的影响,我们将在实验的第三部分,利用shRNA干扰技术进行深入的了解。
(2) NHERF1及β-联蛋白在黑素瘤细胞株A375,M14,MV3中的表达及意义
从第一部分的研究中观察到NHERF1及β-联蛋白在痣细胞痣、原位黑素瘤及侵袭性黑素瘤组织中的异常胞浆阳性表达率随着病变组织的恶性程度增加而升高。为了研究NHERF1及β-联蛋白在黑素瘤细胞株A375,M14,MV3中的表达及意义。培养黑素瘤细胞株A375、M14、MV3及黑素细胞；使用免疫细胞化学法、实时荧光定量-PCR法、免疫印迹法对黑素瘤细胞株A375、M14、MV3及黑素细胞中NHERF1及β-联蛋白的表达情况进行观察；并使用MTT法对黑素瘤细胞株A375、M14、MV3及黑素细胞的增殖情况进行检测。结果发现：NHERF1及β-联蛋白在黑素细胞中同时表现为细胞膜阳性表达,而在黑素瘤细胞株A375, M14, MV3中均出现膜表达的缺失,同时呈现不同程度的细胞浆和/或细胞核阳性表达。在三株黑素瘤细胞株中,MV3细胞中的NHERF1免疫细胞化学染色仅呈少部分细胞胞质弱阳性表达,实时荧光定量-PCR检测NHERF1的mRNA表达水平最低,免疫印迹法中未见NHERF1蛋白表达条带,而其在MTT法中检测到的的增殖速度最快,明显高于另外两株细胞；A375细胞中的NHERF1免疫细胞化学染色呈细胞浆及细胞核的强阳性表达,实时荧光定量-PCR检测NHERF1的mRNA表达水平最高,免疫印迹法中可见NHERF1蛋白最强阳性表达,而其增殖速度却明显慢于MV3细胞。同时,p-联蛋白在MV3细胞中免疫细胞化学染色呈细胞浆及细胞核的强阳性表达,实时荧光定量-PCR检测的mRNA表达水平最高,免疫印迹法检测到蛋白最强阳性表达；而A375细胞及M14细胞中,p-联蛋白在免疫细胞化学染色呈相对弱的细胞浆阳性表达,实时荧光定量-PCR检测的mRNA表达水平明显较低；免疫印迹法中的蛋白表达较MV3中的表达低。与正常黑素细胞相比,黑素瘤细胞A375, M14, MV3中的NHERF1的mRNA表达水平都呈不同程度升高,而β-联蛋白表达水平(mRNA表达水平及蛋白水平)亦存在不同程度升高,同时,黑素瘤细胞中的NHERF1与p-联蛋白存在着差异性表达,黑素瘤细胞胞浆中的NHERF1表达量可能与细胞的增殖速度相关,NHERF1在黑素瘤细胞中表达程度越低则细胞的增殖可能越快。为了深入探讨NHERF1在黑素瘤细胞中对β-联蛋白及黑素瘤细胞增殖的影响,我们将在实验的第三部分,利用慢病毒载体shRNA干扰技术进行深入的了解。
(3) A375细胞中NHERF1的基因沉默对β-联蛋白的表达及A375细胞增殖、凋亡的影响作用
从第二部分的研究中观察到,黑素瘤细胞中的NHERF1与β-联蛋白存在着差异性表达。黑素瘤细胞胞浆中的NHERF1表达量可能与细胞的增殖速度相关,NHERF1在黑素瘤细胞中表达程度越低则细胞的增殖可能越快。因此在本部分实验中,我们设计并合成NHERF1的shRNA干扰片段,并载入pLVX-shRNA-puro载体中,载体转化到Ecoli感受态中。采用PCR及基因测序等方法对重组质粒进行鉴定。慢病毒包装,慢病毒感染靶细胞,多克隆细胞的验证,单克隆筛选稳定干扰NHERF1的A375细胞株,并采用酶切鉴定、基因测序、qPCR和Western blotting对转染效果进行鉴定。使用Western blotting检测稳定干扰NHERF1的A375细胞株中NHERF1及β-联蛋白的蛋白表达量,以对照序列干扰细胞为对照,并使用MTT法检测两种细胞的增殖情况,Annexin-V/PI双染,流式细胞仪检测细胞凋亡。结果发现设计的shRNA-NHERF1干扰片段载入pLVX-shRNA-puro载体中,并稳定转染A375细胞后,经qPCR和Western blotting检测NHERF1表达明显下降,NHERF1沉默的A375细胞中β-联蛋白较对照细胞中的表达增高,同时A375细胞的增殖速度较对照细胞明显增快。所设计的shRNA-NHERF1干扰片段对A375细胞中NHERF1的表达起到了明显的抑制作用,NHERF1基因沉默导致β-联蛋白的表达增高,细胞增殖加快,细胞凋亡减少。
结论
在皮肤黑素瘤细胞中NHERF1与β-联蛋白呈差异性表达,并可能通过与β-联蛋白的相互作用而调节β-联蛋白的表达水平,从而影响细胞增殖和凋亡。NHERF1在皮肤黑素瘤组织及细胞中的表达较良性痣细胞痣组织及正常黑素细胞均呈不同程度的异常细胞浆阳性表达模式。
Cutaneous malignant melanoma is a highly lethal tumor, originating from melanocyte and resistant to medical intervention of any kind in advanced disease. Initiatives have concentrated on the mechanism and the therapy of melanoma for decades. Na+/H+exchanger regulatory factor-1(NHERF1) is a scaffolding protein, playing an important role in regulating of ion transport, and stabilizing the transmenbrane protein involved in epithelial microvilli structure. It is also a regulatory protein in signaling and trafficking of more than30kinds of proteins. In recent years, it has been found that NHERF1has an emerging role in various tumors. NHERF1gene mutations or loss of heterozygosity can lead to increased degree of malignancy of the tumor cells. In normal tissue, NHERF1is usually cyto-membrane expressed demonstrated by immunohistochemistry and plays a tumor suppressor role, on the other hand, negative expression in cyto-membrane or overexpression in the cytoplasm or nucleus correlate with increased degree of malignancy. Although a growing number of studies have shown that NHERF1plays an important role in oncogensis, its role in anti-tumorigenesis is still poorly understood. Further studies, in addition to the continuate exploring of its tumor suppressor mechanism, willbe focused on its role in the tumor biological behavior and the clinical correlation.
Expression of NHERF1and β-catenin in cutaneous malignant melanoma and melanocytic nevus tissues
To detect the expression of NHERF1and β-catenin in cutaneous malignant melanoma and melanocytic nevus. The expression of NHERF1and β-catenin in47cutaneous malignant melanomas and37melanocytic nevi were examined with immunohistochemistry. The cytoplasm moderate/strong positive expression rate of both NHERF1and β-catenin were gradually increased from melanocytic nevus group, melanoma in situ group to invasive melanoma group. Correlation analysis with Spearman's rho test for NHERF1and β-catenin expression showed no significant correlation between the expression in melanoma tissue samples and melanocytic nevus tissue samples. The cytoplasm moderate/strong positive expression rate of both NHERF1and β-catenin were gradually increased with the increase of the degree of malignancy of lesions from melanocytic nevus group, melanoma in situ group to invasive melanoma group.
Expresssion and significance of NHERF1and β-catenin in malignant melanoma cell lines A375, M14and MV3
To investigate the expression of NHERF1and3-catenin in human melanoma cell lines A375, M14and MV3, as well as their significance. The human melanoma cell lines A375, M14, MV3and human melanocytes were cultured. Immunocellulochemistry, Real-time PCR and Western blotting were performed to detect the expression of NHERF1and β-catenin in these cells respectively; the proliferation rates of these cell lines were measured by the MTT method. In the immunocytochemistry, NHERF1and β-catenin were showed both membranous staining in the melanocytes; while they were absent in the membranous staining in the melanoma cell lines A375, M14and MV3, but presented cytoplasmic and/or nuclear staining with different intensity. Among the3melanoma cell lines, the MV3cells showed partly weak cytoplasmic staining of NHERF1, with the lowest level of the relative quantity of mRNA and absence of immunoblotting NHERF1protein expression. But, its proliferation rate was the highest by MTT assay, much higher than that of the other two cells; while the A375cells showed intensive cytoplasmic and nuclear staining of NHERF1, with the highest expression of the relative quantity of mRNA and intensive immunoblotting NHERF1protein expression. But, their proliferation rate were much lower than that of the MV3cells. Meanwhile, the MV3cells showed intensive cytoplasmic and nuclear staining of β-catenin, with the highest level of the relative quantity of mRNA and intensive immunoblotting NHERF1protein expression; while the A375cells and the M14cells showed less intensive cytoplasmic staining of NHERF1, with the much lower level of the relative quantity of mRNA and less immunoblotting NHERF1protein expression. The level of NHERF1cytoplasm expression in melanoma cell lines may be associated with the proliferation rate of these cells. NHERF1and β-catenin were differentially expressed in the melanoma cell lines.
The influence in the expression of β-catenin as well as the cell proliferation and apoptosis of melanoma cell line A375by the shRNA knockdown of NHERF1
To investigate the influence of NHERF1knockdown in melanoma cell line A375in the expression of β-catenin and the cell proliferation and apoptosis. The shRNA interference fragment of NHERF1was designed and synthesized, then loaded in the pLVX-shRNA-puro vector. The vector was transfected into E. coli competent. The recombinant plasmids were identified by using Real-time PCR and gene sequencing. Lentiviral packaging, lentivirus infection of target cells, polyclonal cells verification and monoclonal screening of stable NHERF1knockdown A375cell were performed. By using gene sequencing, restriction enzyme digestion, qPCR and Western blotting, the effect of transfection was identified. Then, the expression of NHERF1and β-Catenin in NHERF1-knockdown A375cells were detected with Western blotting, in comparing with the shRNA-control A375cells and the untreated A375cells. MTT assay and flow cytometry were performed to detect cell proliferation and apoptosis. After the designed interference fragment of shRNA-NHERF1was loaded in the pLVX-shRNA-puro vector and transfected A375cells, the expression of NHERF1in the transfected cells was decreased significantly as detected by qPCR and Western blotting, while the expression of β-catenin was increased in the transfected A375cells. The cell proliferation of transfected A375cells was faster than that of the controls. The designed shRNA-NHERF1interference fragment inhibits the expression of NHERF1in A375cells, leading to increased β-Catenin expression, acceleration of cell proliferation, and decrease of cell apoptosis.
Conclusions
In the melanoma cell lines, NHERF1and β-catenin were differentially expressed, and NHERF1may regulate the expression of β-catenin through their interaction, thereby modulating cell proliferation and apoptosis. The expression of NHERF1in cutaneous melanoma tissues and melanoma cell lines showed aberrant cytoplasm positive expression in comparing with benign melanocyte nevi and normal melanocytes, respectively.
引文
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[36]Hayashi Y, Molina J R, Hamilton S R, et al. NHERF1/EBP50 is a new marker in colorectal cancer[J]. Neoplasia,2010,12(12):1013-1022.
[1]Taipale J, Beachy P A. The Hedgehog and Wnt signalling pathways in cancer[J]. Nature,2001,411(6835):349-354.
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[1]Taipale J, Beachy P A. The Hedgehog and Wnt signalling pathways in cancer[J]. Nature,2001,411(6835):349-354.
[2]刘启方,周晓燕,徐玉荣,等.甲磺酸伊马替尼对黑素瘤细胞Hs294T生物学活性的影响[J].中华皮肤科杂志,2012,45(6)：404-407.
[3]刘启方,廖文俊.皮肤肿瘤中Wnt/β-联蛋白信号通路的研究进展[J].国际皮肤性病学杂志,2011,37(1)：41-44.
[4]殷河慧,廖文俊,高滢,等.β联蛋白和淋巴样增强因子1在恶性黑素瘤中的表达[J].中华皮肤科杂志,2005,38(11)：665-667.
[5]Bienz M. beta-Catenin:a pivot between cell adhesion and Wnt signalling[J]. Curr Biol,2005,15(2):R64-R67.
[6]杨松,李慎秋.β连环蛋白与恶性黑素瘤的研究现状[J].国外医学(皮肤性病学分册),2005,31(1)：59-60.
[7]Dai J L, Wang L, Sahin A A, et al. NHERF (Na+/H+exchanger regulatory factor) gene mutations in human breast cancer[J]. Oncogene,2004,23(53):8681-8687.
[8]Pan Y, Wang L, Dai J L. Suppression of breast cancer cell growth by Na+/H+ exchanger regulatory factor 1 (NHERF1)[J]. Breast Cancer Res,2006,8(6):R63.
[9]Pan Y, Weinman E J, Dai J L. Na+/H+ exchanger regulatory factor 1 inhibits platelet-derived growth factor signaling in breast cancer cells [J]. Breast Cancer Res,2008,10(1):R5.
[10]Bellizzi A, Mangia A, Malfettone A, et al. Na+/H+ exchanger regulatory factor 1 expression levels in blood and tissue predict breast tumour clinical behaviour[J]. Histopathology,2011,58(7):1086-1095.
[11]Song J, Bai J, Yang W, et al. Expression and clinicopathological significance of oestrogen-responsive ezrin-radixin-moesin-binding phosphoprotein 50 in breast cancer[J]. Histopathology,2007,51(1):40-53.
[12]Shibata T, Chuma M, Kokubu A, et al. EBP50, a beta-catenin-associating protein, enhances Wnt signaling and is over-expressed in hepatocellular carcinoma[J]. Hepatology,2003,38(1):178-186.
[13]Hayashi Y, Molina J R, Hamilton S R, et al. NHERF1/EBP50 is a new marker in colorectal cancer[J]. Neoplasia,2010,12(12):1013-1022.
[14]Molina J R, Morales F C, Hayashi Y, et al. Loss of PTEN binding adapter protein NHERF1 from plasma membrane in glioblastoma contributes to PTEN inactivation[J]. Cancer Res,2010,70(17):6697-6703.
[15]Kreimann E L, Morales F C, de Orbeta-Cruz J, et al. Cortical stabilization of beta-catenin contributes to NHERF1/EBP50 tumor suppressor function[J]. Oncogene,2007,26(36):5290-5299.
[16]Wheeler D S, Barrick S R, Grubisha M J, et al. Direct interaction between NHERF1 and Frizzled regulates beta-catenin signaling[J]. Oncogene,2011,30(1):32-42.
[1]Georgescu M M, Morales F C, Molina J R, et al. Roles of NHERF1/EBP50 in cancer[J]. Curr Mol Med,2008,8(6):459-468.
[2]Mamonova T, Kurnikova M, Friedman P A. Structural basis for NHERF1 PDZ domain binding[J]. Biochemistry,2012,51(14):3110-3120.
[3]Rendina D, De Filippo G, Strazzullo P. NHERF1 mutations and responsiveness of renal parathyroid hormone[J]. N Engl J Med,2008,359(24):2616,2616-2617.
[4]Wheeler D S, Barrick S R, Grubisha M J, et al. Direct interaction between NHERF1 and Frizzled regulates beta-catenin signaling[J]. Oncogene,2011,30(1):32-42.
[5]Mangia A, Chiriatti A, Bellizzi A, et al. Biological role of NHERF1 protein expression in breast cancer [J]. Histopathology,2009,55(5):600-608.
[6]Giallourakis C, Cao Z, Green T, et al. A molecular-properties-based approach to understanding PDZ domain proteins and PDZ ligands[J]. Genome Res,2006,16 (8):1056-1072.
[7]Neisch A L, Fehon R G. Ezrin, Radixin and Moesin:key regulators of membrane-cortex interactions and signaling[J]. Curr Opin Cell Biol,2011,23 (4):377-382.
[8]Dai J L, Wang L, Sahin A A, et al. NHERF (Na+/H+exchanger regulatory factor) gene mutations in human breast cancer[J]. Oncogene,2004,23(53):8681-8687.
[9]Song J, Bai J, Yang W, et al. Expression and clinicopathological significance of oestrogen-responsive ezrin-radixin-moesin-binding phosphoprotein 50 in breast cancer[J]. Histopathology,2007,51(1):40-53.
[10]Pan Y, Wang L, Dai J L. Suppression of breast cancer cell growth by Na+/H+ exchanger regulatory factor 1 (NHERF1)[J]. Breast Cancer Res,2006,8(6):R63.
[11]Pan Y, Weinman E J, Dai J L. Na+/H+ exchanger regulatory factor 1 inhibits platelet-derived growth factor signaling in breast cancer cells [J]. Breast Cancer Res,2008,10(1):R5.
[12]Bellizzi A, Mangia A, Malfettone A, et al. Na+/H+ exchanger regulatory factor 1 expression levels in blood and tissue predict breast tumour clinical behaviour[J]. Histopathology,2011,58(7):1086-1095.
[13]Shibata T, Chuma M, Kokubu A, et al. EBP50, a beta-catenin-associating protein, enhances Wnt signaling and is over-expressed in hepatocellular carcinoma[J]. Hepatology,2003,38(1):178-186.
[14]Hayashi Y, Molina J R, Hamilton S R, et al. NHERF1/EBP50 is a new marker in colorectal cancer[J]. Neoplasia,2010,12(12):1013-1022.
[15]Molina J R, Morales F C, Hayashi Y, et al. Loss of PTEN binding adapter protein NHERF1 from plasma membrane in glioblastoma contributes to PTEN inactivation[J]. Cancer Res,2010,70(17):6697-6703.
[16]Kreimann E L, Morales F C, de Orbeta-Cruz J, et al. Cortical stabilization of beta-catenin contributes to NHERF1/EBP50 tumor suppressor function[J]. Oncogene,2007,26(36):5290-5299.
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[5]http://seer.cancer.gov/statfacts/html/melan.html。[Z].2012.
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[8]Dai J L, Wang L, Sahin A A, et al. NHERF (Na+/H+exchanger regulatory factor) gene mutations in human breast cancer[J]. Oncogene,2004,23(53):8681-8687.
[9]Song J, Bai J, Yang W, et al. Expression and clinicopathological significance of oestrogen-responsive ezrin-radixin-moesin-binding phosphoprotein 50 in breast cancer[J]. Histopathology,2007,51(1):40-53.
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[11]Pan Y, Weinman E J, Dai J L. Na+/H+ exchanger regulatory factor 1 inhibits platelet-derived growth factor signaling in breast cancer cells [J]. Breast Cancer Res,2008,10(1):R5.
[12]Bellizzi A, Mangia A, Malfettone A, et al. Na+/H+ exchanger regulatory factor 1 expression levels in blood and tissue predict breast tumour clinical behaviour[J]. Histopathology,2011,58(7):1086-1095.
[13]Shibata T, Chuma M, Kokubu A, et al. EBP50, a beta-catenin-associating protein, enhances Wnt signaling and is over-expressed in hepatocellular carcinoma[J]. Hepatology,2003,38(1):178-186.
[14]Hayashi Y, Molina J R, Hamilton S R, et al. NHERF1/EBP50 is a new marker in colorectal cancer[J]. Neoplasia,2010,12(12):1013-1022.
[15]Molina J R, Morales F C, Hayashi Y, et al. Loss of PTEN binding adapter protein NHERF1 from plasma membrane in glioblastoma contributes to PTEN inactivation[J]. Cancer Res,2010,70(17):6697-6703.
[16]Kreimann E L, Morales F C, de Orbeta-Cruz J, et al. Cortical stabilization of beta-catenin contributes to NHERF1/EBP50 tumor suppressor function[J]. Oncogene,2007,26(36):5290-5299.
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