摘要
恶性黑素瘤是一种高度恶性的黑素细胞肿瘤,皮肤是其主要累及部位之一。皮肤恶性黑素瘤的发病率逐年增加,其死亡例数占所有皮肤肿瘤的80%。BAG-1是一种多功能蛋白,在多种肿瘤的发生、发展及放、化疗治疗反应中有重要的生物学作用。但BAG-1蛋白在皮肤黑素瘤组织中的表达情况及其临床意义尚未见报道,BAG-1在黑素瘤发生、发展中的具体作用及其机制更不清楚。
目的
探讨BAG-1在皮肤黑素瘤的表达及其与临床病理特征的相关性;RNAi抑制BAG-1基因表达,研究其对小鼠黑素瘤B16F10细胞株的细胞周期、增殖、凋亡及多西紫杉醇治疗反应的影响。
材料与方法
第一部分:免疫组化检测BAG-1、p53、Bcl-2在皮肤恶性黑素瘤、色素痣、正常人皮肤的表达,结合患者临床资料(性别、年龄、Clark分级等)进行分析;检测BAG-1在黑素瘤细胞株M14和B16F10细胞的表达。
第二部分:以pRNAT-U6.1/Neo为载体,构建重组shRNA质粒pRNAT-U6.1/Neo-BAG-1-1、-2、-3,酶切及PCR测序鉴定;利用lipofectamin~(TM) 2000将重组质粒转染小鼠黑素瘤B16F10细胞,设阴性对照组(转染阴性对照质粒Neg)和空白对照组(未转染组),转染48 h后,荧光显微镜下检测转染效率,G418筛选阳性克隆并扩增;用RT-PCR、western blot法分别检测BAG-1基因mRNA、蛋白的表达,筛选干扰效率最高的shRNA质粒pRNAT-U6.1/Neo-BAG-1-3。
第三部分:分别培养空白对照组、阴性对照组细胞及转染pRNAT-U6.1/Neo-BAG-1-3的细胞,流式细胞仪分析细胞周期,MTT法和平皿克隆形成实验检测细胞增殖能力,TUNEL法检测细胞凋亡,并在显微镜下计数每500个细胞中凋亡细胞的数值;MTT法计算多西紫杉醇对B16F10细胞的增殖抑制率,进一步以多西紫杉醇10 umol/L的浓度分别处理空白对照组、阴性对照组及转染pRNAT-U6.1/Neo-BAG-1-3组细胞,于加药后24 h、48 h,流式细胞仪检测细胞周期,TUNEL法检测细胞凋亡。
结果
第一部分:BAG-1在正常皮肤不表达,在皮肤恶性黑素瘤的阳性表达率为78%,显著高于色素痣组15%(P＜0.001),BAG-1蛋白在皮肤恶性黑素瘤中的表达与Clark分级呈正相关(r=0.47,P＜0.05)。
第二部分:酶切及测序结果证实重组质粒pRNAT-U6.1/Neo-BAG-1-1、-2、-3构建成功。质粒在B16F10细胞的转染效率为20～30%。阴性对照组的BAG-1基因表达水平与空白对照组间差异无统计学意义(P＞0.05);与阴性对照及空白对照组相比,shRNA质粒转染组的BAG-1基因表达显著下降(P＜0.05),其中以pRNAT-U6.1/Neo-BAG-1-3抑制效率最高,抑制率在mRNA和蛋白水平分别为(77±4)%和(62±2)%。
第三部分:与阴性对照组及空白对照组细胞相比,转染pRNAT-U6.1/Neo-BAG-1-3组细胞出现以下变化:细胞周期S期细胞增多;生长速度减慢,形成的细胞克隆数值减少;凋亡细胞数值增多,差异有统计学意义(P＜0.05)。
多西紫杉醇10 umol/L处理后24 h,各组细胞均表现为G2期细胞百分比升高;处理后48 h,转染pRNAT-U6.1/Neo-BAG-1-3组的G2期细胞百分比明显高于阴性对照及空白对照组(P＜0.05)。多西紫杉醇10 umol/L处理后24 h及48 h,转染pRNAT-U6.1/Neo-BAG-1-3组凋亡细胞数值均明显高于阴性对照及空白对照组(P＜0.05)。
结论
BAG-1在皮肤恶性黑素瘤组织中的表达显著升高,其表达水平与Clark分级呈正相关;针对小鼠BAG-1基因的shRNA真核表达质粒pRNAT-U6.1/Neo-BAG-1-3能高效、特异地抑制小鼠黑素瘤B16F10细胞BAG-1基因的表达:干扰BAG-1基因表达能影响小鼠黑素瘤B16F10细胞的细胞周期、细胞增殖、细胞凋亡及其对多西紫杉醇的治疗反应,表现为明显的S期阻滞、细胞生长速度和克隆形成能力明显降低、凋亡细胞数目显著增加、对多西紫杉醇处理所诱导的细胞周期G2期阻滞、细胞凋亡的敏感性明显增加。
Malignant melanoma is one of the most malignant tumors. Cutaneous malignant melanoma accounts for 80% mortality of all cutaneous tumours, and the incidence of malignant melanoma is rising rapidly. As a multifunctional protein, BAG-1 has many important biological activities in tumor genesis, development and therapy response. Interestingly, there's rare report about the expression of BAG-1 in cutaneous malignant melanoma tissues and its relationship with clinicopathologic characteristics, and the exact effects of BAG-1 on malignant melanoma is still unclear.
Objective
Detect the expression of BAG-1 protein in cutaneous malignant melanoma and explore its correlation with clinicopathological characteristics. Further investigate the effect of BAG-1 gene inhibition on cell proliferation, cell cycle, cell apoptosis and cellular response to docetaxel in mouse melanoma cell line B16F10.
Materials and methods
Part 1: Immunohistochemistry was done to examine the expression of BAG-1, p53, and Bcl-2 in paraffin-embedded tissue blocks containing 32 cutaneous malignant melanoma, 20 pigemented nevi and 10 nomal skin tissues. The correlations between BAG-1 and clinicopathological characteristics were analyzed. The expression of BAG-1 in melanoma cell lines M14 and B16F10 was also analyzed.
Part 2: The recombinant plasmids of BAG-1, pRNAT-U6.1/Neo-BAG-1-1, -2, -3, were constructed and transfected into melanoma cell line B16F10, the positive clone was selected by G418 method. RT-PCR and western blot were performed to detect the expression of BAG-1 mRNA and protein in transfected, negative control and untransfected cells. The most effective shRNA plasmid was selected to next step.
Part 3: Flow cytometry was used to analyze cell cycle, MTT assay and clone formation assay to confirm cell proliferation, TUNEL assay to determine cell apoptosis. After using MTT assay to determine proliferation inhibition rate, docetaxel was added to cells including untransfected, transfected negative control and most effective shRNA plasmid in 10 umol/l. Flow cytometry was done to analyze cell cycle, TUNEL assay to determine cell apoptosis.
Results
Part 1: The positive rate of BAG-1 expression was significantly higher in malignant melanoma than pigmented nevi and normal skin tissues (78% vs. 15%, 0%, P<0.001) . Statistically significant association was observed between BAG-1 expression and Clark level (r=0.47, P<0.05). Expression of BAG-1 was not correlated with p53 and Bcl-2 (r=0.05, 0.11, P>0.05) . The positive expression of BAG-1 was confirmed in melanoma cell lines M14 and B16F10.
Part 2: The eukaryotic expression plasmids-pRNAT-U6.1/Neo-BAG-1-1, -2, -3 were constructed and verified by restriction enzyme digestion and DNA sequencing. The transfection rates in B16F10 cells was 20～30%. Comparing with the untransfected and negative control cells, the expressions of BAG-1 mRNA and protein in shRNA plasmid transfected cells were significantly inhibited by BAG-1 shRNA (P<0.05) . The most effective shRNA plasmid was pRNAT-U6.1 /Neo-BAG-1 -3, its inhibition rates were (77±4) % and (62±2) %, respectively.
Part 3: Statistically significant differences were found in cell cycle, cell proliferation and cell apoptosis among transfected, negative control and untranfected groups (P<0.05) . Cells transfected with pRNAT-U6.1/Neo-BAG-1-3 exhibited higher percentge of S-phase cell, lower proliferation rapid, less cell clones and more apoptotic cells.
24 h after docetaxel added, cells exhibited higher G2-phase percentage. Statistically significant difference was not found among groups; 48 h later, G2-phase percentage in cells transfected with pRNAT-U6.1/Neo-BAG-1-3 was higher than negative control and untranfected cells (P<0.05). About cell apoptosis, the number of apoptotic cells was higher in cells transfected with pRNAT-U6.1/Neo-BAG-1-3 than negative control and untranfected cells both 24 h later and 48 h later (P<0.05) .
Conclusions
BAG-1 protein is highly expressed in cutaneous malignant melanoma, and its expression is positively corrected with Clark level. The shRNA plasmid pRNAT-U6.1/Neo-BAG-1-3 can predomiantly and specifically inhibit the expression of BAG-1 gene in mouse melanoma cell line B16F10. BAG-1 can affect the cell cycle, cell proliferation, cell apoptosis and cellular response to docetaxel of mouse melanoma cell line B16F10. After the expression of BAG-1 gene was inhibited, B16F10 cell exhibited S-phase stasis, lower proliferation ability, more apoptotic cells, and more sensitive to docetaxel induced G2-phase stasis and apoptosis.
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