摘要
[RASSF1A基因在黑色素瘤细胞和组织中表达下调]
RASSFIA是定位于肿瘤高频杂合性丢失区域3p21.3位点的抑瘤基因,编码一个微管相关蛋白。该基因在多种实体瘤组织中因启动子高甲基化而表达沉默,是迄今为止在肿瘤组织中甲基化程度最高的基因之一。前期多项研究指出,除了在上皮性肿瘤中发生甲基化之外,RASSF1A基因在恶性黑色素瘤细胞中也存在高频甲基化,提示该基因在黑色素瘤的发生过程中也可能存在表达异常。在本研究中,我们首次采用免疫组织化学及western blot的方法研究了RASSFIA基因在正常皮肤组织、皮肤色素痣以及恶性黑色素瘤组织和细胞系中的表达,结果发现,RASSF1A蛋白在正常皮肤色素细胞和色素痣细胞中高表达,而在恶性黑色素瘤组织中表达下调甚至缺失,并与肿瘤的转移呈负相关。RASSFIA蛋白在非转移性黑色素瘤细胞系中有微弱表达,而在转移性黑色素瘤细胞系中表达缺失。该结果证实了RASSFIA在黑色素瘤组织和细胞中表达下调,强烈提示RASSFIA参与了黑色素瘤的发生与演进过程。
[RASSFIA基因具有抑制黑色素瘤细胞生长、增殖的作用]
以往关于RASSFIA基因功能的研究绝大部分都是在上皮性肿瘤中开展的,为了进一步探讨RASSF1A基因是否参与了恶黑的发生发展,我们在RASSF1A基因表达缺失的恶黑细胞系中转染RASSF1A基因,恢复其表达,建立稳定转染的细胞系,研究该基因对恶黑细胞生物学行为的影响。细胞生长曲线测定结果表明,稳定转染RASSF1A基因抑制了A375细胞的生长与增殖能力；平板集落形成实验表明该基因同时抑制了A375细胞的克隆集落形成能力；进一步采用裸鼠移植瘤的方法证实,稳定表达RASSF1A基因不仅抑制了A375细胞的体外增殖能力,而且抑制了该细胞在裸鼠体内形成移植瘤的能力。以上结果表明：RASSF1A基因具有抑制恶黑细胞生长增殖的能力,是一个黑色素瘤抑瘤基因。
[RASSF1A基因通过促进细胞凋亡,引起细胞周期G1-S期阻滞抑制黑色素瘤细胞的生长与增殖]
肿瘤细胞的生长与增殖受抑制,通常是通过影响细胞周期进程或者凋亡过程而实现。为了研究RASSF1A抑制恶黑的机制,我们对细胞周期及细胞凋亡进行了分析。Hochest 33258染色发现稳定表达RASSF1A基因的A375细胞存在细胞核固缩、浓染,是典型的细胞凋亡的形态；通过流式细胞仪检测进一步证明,稳定表达RASSF1A的A375细胞凋亡率增加；细胞周期进程分析结果表明,稳定表达RASSF1A基因的A375细胞停留于G1期的细胞比例增加,而S期细胞比例降低,两者相比具有统计学差异,说明RASSF1A稳定表达引起A375细胞周期G1-S期阻滞。Western blot检测发现RASSF1A抑制了cyclin D1和磷酸化Rb蛋白的表达；RASSF1A抑制cyclin D1蛋白表达是通过降低其蛋白稳定性。以上结果说明,RASSF1A是通过同时影响细胞凋亡与细胞周期而抑制恶黑细胞的增殖能力与致瘤性。
[RASSF1A基因对黑色素瘤细胞A375基因表达谱的影响]
为了研究RASSF1A基因稳定表达对A375细胞基因表达谱的影响,采用Affimatrix公司全基因组表达谱芯片筛查受RASSF1A表达所调控的差异表达基因。筛选出受RASSF1A基因调控黑色素瘤A375细胞中差异表达的基因共210个,其中上调184个,下调26个基因。为了验证基因芯片的数据的可靠性,采用realtime RT-PCR方法检测部分差异表达的基因,结果表明realtime RT-PCR检测结果与芯片检测结果一致,说明芯片数据可靠。通过生物信息学分析差异表达基因的功能归类,发现RASSF1A调控的差异基因功能涉及转录与信号通路调控、细胞生长与增殖调控、细胞周期、细胞凋亡、细胞间粘附、细胞免疫应答等过程,提示RASSF1A可能通过影响这些基因的表达从而影响A375细胞相应的生物学过程。
[RASSF1A基因通过上调ASK1表达活化P38 MAPK,激活线粒体凋亡途径]
基因芯片筛查发现RASSF1A基因稳定表达引起细胞凋亡相关分子ASK1表达上调,结合前期发现RASSF1A稳定表达促进A375细胞凋亡,我们认为RASSF1A可能是通过影响ASK1的表达从而促进恶黑细胞凋亡。通过realtime RT-PCR和western blot检测证实RASSF1A转染后ASK1的表达水平上调。由于ASK1是JNK、P38 MAPK的上游激酶,我们采用western blot分析了JNK、P38 MAPK的表达,结果表明磷酸化P38 MAPK表达增加,total P38 MAPK表达水平没有改变；在RASSF1A转染细胞和空载体对照细胞中,我们没有检测到磷酸化的JNK表达。说明RASSF1A引起ASK1表达上调促进了P38 MAPK的活化；western blot分析发现RASSF1A下调Bcl2表达,并引起cytochromec从线粒体漏出至胞浆,促进了caspase 3的表达与活化,说明RASSF1A通过活化P38 MAPK激活线粒体凋亡途径,是其促进细胞凋亡的分子机制。在RASSF1A稳定表达的细胞中采用RNAi的方法抑制ASK1表达,结果细胞凋亡率下降,说明RASSF1A促进恶黑细胞凋亡的作用部分依赖于ASK1。
[RASSF1A负调控AKT/mTOR信号通路]
RASSF1A抑制恶黑细胞增殖、促进凋亡的作用有可能是通过影响细胞信号传导。采用western blot分析与增殖、凋亡密切相关的细胞信号通路的表达与活化,结果发现RASSF1A抑制了AKT的磷酸化,并引起其下游mTOR通路的p70S6K与eIF4E磷酸化水平降低,对其总蛋白表达没有影响,表明RASSF1A抑制了A375细胞AKT/mTOR通路的活化。在RASSF1A稳定表达的细胞中瞬时转染myc-AKT1质粒,结果发现过表达AKT1可以逆转RASSF1A对凋亡、细胞周期相关分子的调节,具体表现为：瞬时转染分子myc-AKT1引起RASSF1A/A375细胞中磷酸化P38 MAPK表达下调,而磷酸化eIF4E、Bcl2和cycling D1表达水平上调,说明过表达AKT1可以逆转RASSF1A对这些分子的调控作用,也就是说,RASSF1A抑制eIF4E磷酸化、Bcl2和cycling D1表达的作用是通过抑制AKT的活性而实现,因此RASSFIA抑制AKT/mTOR信号通路是其调控细胞凋亡、细胞周期进程的上游分子事件。
[Background of RASSF1A]
Malignant melanoma (MM) accounts for 80 percent of deaths from skin cancer, only 14 percent of patients with metastatic melanoma survive for five years. Numerous molecular events, many of them revealed by genomic and proteomic methods, have been associated with the development of MM. Several group reported that high frequency of Ras association domain family 1, isoform A (RASSF1A) gene promoter hypermethylation was detected in MM cell lines, tissues and serum of MM patients, which suggested that aberrant alteration of RASSFIA gene is also involved in melanoma progression. RASSFIA is a member of a new group of RAS effectors thought to regulate cell proliferation and apoptosis. It has been shown that introduction of RASSF1A into lung cancer cell lines lacking RASSF1A gene expression can reduce colony formation, growth in soft agar, and tumorigenesis in nude mice. At present, understanding the physiological role of RASSFIA is still in its early stages. The effect of RASSFIA expression on melanoma tumorigenic potential in vitro and in vivo has not been reported.
[Downexpression of RASSF1A protein in melanoma cell lines and tissues]
In this study, we analyzed the expression level of RASSFIA protein in MM tissues and cell lines. Firstly, we have screened the expression levels of RASSFIA in MM biopsies, navus pigmentosus and normal skins. By immunohistochemical analysis,10 of 10 (100%) paraffin-embedded archival normal skins showed positive to strong cytoplasmic staining of RASSFIA in most of the melanocyte cells.8 of 9 (88.9%) of paraffin-embedded navus pigmentosus showed strong cytoplasmic staining of RASSFIA in pigmentosus nest. Only 8 of 23 (34.8%) showed weak positive to moderate staining of RASSFIA in MM samples. Thus the percentage of MM tissues with a positive to strong staining intensity was significantly lower than that observed in normal or benign melanocytes(P<0.01). The relative importance of RASSF1A expression levels and clinical parameters of the patients were analyzed. The positive expression rate of RASSFIA was 57.1%(8/14) in the group of 14 MM patients without metastasis, while it was 0%(0/9) in the group of 9 MM patients with metastasis (P=0.007). Thus the positive ratio of RASSF1A expression was higher in the non-metastasis group than that in the metastasis group.
[Exogenous expression of RASSFIA suppresses melanoma tumorigenecity in vitro and in vivo]
We have screened the expression levels of RASSFIA in several melanoma cell lines, it shows a absent pattern in metastatic melanoma cell lines(1205Lu, MeWo, A375SM, M14 and A375 cell lines), while a weak expression pattern in non-metastatic melanoma cells(WM1552C, WM1341D, WM793, WM164). To identify the effect of RASSFIA expression on melanoma tumorigenic potential in vitro and in vivo, the wild-type RASSF1A was stablely introducted into melanoma A375 cells. By quantitative RT-PCR, variable levels of RASSFIA expression were observed in RASSF1A-expressing clones. In contrast, undetectable level of RASSFIA expression was found in pIRESneo3 transfected cells. RASSFIA protein expression of the pooled clones was further confirmed by Western blotting. To investigate the effect of RASSFIA in cell growth, we then examined the proliferation rate of the RASSF1A-expressing cells. Expression of wild-type RASSFIA in the stable transfectants significantly reduced the number of colonies formed. The number of colonies formed in the RASSF1A-expressing clones was decreased markedly (-43%) (P=0.005). The proliferation rate of the RASSF1A-transfected clones were retarded when we compared to the vector control. The in vivo tumorigenicity potential of transfectants was then tested in athymic nude mice. Monitoring the injected mice showed that RASSF1A-expressing clones resulted in a drastic reduction in tumor sizes when compared to vector control. The wet weight of tumors in RASSF1A-expressing cells was also decreased significantly by the time of sacrifice (P-0.005).
[Exogenous expression of RASSFIA induces apoptosis and cell cycle G1-S phase arrest in melanoma cells]
RASSFIA may function as a mediator of apoptosis. To characterize the putative antioncogenic properties of RASSFIA on melanoma cells, we then examined whether inhibition of tumorigenicity was through RASSF1A-induced apoptosis. We used Hoechst33258 staining to measure the apoptosis of RASSF1A expressing A375 cell. RASSF1A-expressing cells showed a increase in apoptosis relative to control cells. Samilar result was obtained from FCAs analysis. We also used propidium iodide incorporation to investigate the effect of RASSF1A on cell cycle progression in the melanoma cell line A375. RASSF1A expression resulted in an increase of cells in the G1 phase while a decrease of cells in the S phase of the cell cycle compared with the mocked-transfected cells. This was correlated to decreased levels of cyclin Dl and phosphorylated Rb protein level in RASSF1A-expressing cells.
[Analysis of RASSF1A target genes in melanoma cell lines]
In order to identify genes modulated by RASSF1A in melanoma, the expression profile of RASSF1A expressing melanoma cells and controls were investigated. High-density oligonucleotide microarray was performed on well-characterized RASSF1A-expressing pooled clones and the vector control clones. Change of gene expression between transfected clones and control cells with a minimum of two-fold was scored as significant. Using these selection criteria, we identified 210 differentially expressed genes in the microarray analysis. Totally,26 genes were downregulated whereas 184 were upregulated. Microarray analysis of melanoma cells stably expressing RASSF1A identified expression changes consistent with the observed phenotypic effects such as increased apoptosis modifiers (ASK1, MST1 and RASSF2), reduced cell cycle effectors (cyclins D2 and GAS1), and alterations in the levels of a number of cell adhesion molecules.
[RASSF1A mediates mitochondrion apoptosis pathway through upregulation of ASK1 in melanoma cells]
Quantitative RT-PCR analysis confirmed changes in the mRNA levels of apoptosis signal-regulating kinase 1(ASK1) and RASSF2 in accordance with the microarray data. RASSF1A mediated up-regulation of ASK1 mRNA levels was also reflected by increases in the level of ASK1 protein. ASK1 activated stress-activated protein kinase (SAPK, also known as JNK; c-Jun amino-terminal kinase) and P38 subgroups of MAP kinases, respectively. Hence we questioned whether up-regulation ASK1 by overexpression of RASSF1A would promote activation of JNK and P38 MAP kinase. Indeed, although basal expression of P38 was unaffected in RASSF1A overexpressing cells, phosphorylation of P38 was found to be increased in cells overexpressing RASSF1A. However, activation of JNK was undetectable both in cultured RASSF1A overexpressing A375 cells and vector control cells. RASSF1A also induced a decrease of Bcl2 protein. Cleaved caspase 3, a marker of active effector caspase activity, appeared at elevated levels in RASSF1A expressing cells. RASSF1A expression also promoted precocious cytochrome c release, as determined by subcellular fraction and western blot. To assess the relevance of ASK1 for the RASSF1A-mediated apoptotic cell death, we tested the effect of reducing ASK1 expression on RASSF1A-induced apoptosis. To that end, A375 cells expressing RASSF1A were transfected with either ASK 1-specific siRNA or irrelevant siRNA. Two distinct ASK1-specific siRNAs were pooled and used, and expression of ASK1-specific siRNAs significantly impaired ASK1 expression. As expected, silencing ASK1 with siRNAs partially rescued cells from RASSF1A induced apoptosis, as revealed by FCAs analysis. These data indicate that up-regulation of ASK1 contributes to RASSF1A-induced apoptosis in MM cells.
[Exogenous expression of RASSF1A blocks the AKT-p70S6K-eIF4E pathway]
To identify the pathway upstream of RASSF1A-induced apoptosis and cell cycle arrest, we did Western blot analysis. It is interesting that phosphorylated AKT was downregulated in the RASSF1A expressing A375 cells. This result is consistent with a previous report, which showed that the exogenous expression of RASSF1A down-regulated the expression of phosphorylated AKT. The downstream mediators of AKT were also examined by Western blot analysis. The exogenous expression of RASSF1A inhibited the phosphorylation of p70S6K and eIF4E. To analyze whether overexpression of AKT in RASSF1A transfected cells could restore PI3K/AKT/eIF4E signaling, we exogenously expressed a myc-tagged AKT in RASSF1A stable transfected A375 cells. Exogenous expression of myc-AKT1 increased the phosphorylation of eIF4E and expression level of Bcl2 and cyclin D1, while the expression level of phosphorylated P38 MAPK decreased. Thus these result indicates that RASSF1A inhibits AKT-mTOR (p70S6K and eIF4E) signal pathway in melanoma A375 cells, which at least partially contribute to mediate the expression level of apoptosis regulator Bcl2 and cell cycle molecules such as cyclin D1, which in turn induced apoptosis and cell cycle G1-S arrest.
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