Rho GTP酶家族在黑素瘤细胞株中的表达及RhoD过表达对A375细胞骨架和迁移侵袭的影响及机制初探
发布时间：2018-08-07 18:45
【摘要】：皮肤恶性黑素瘤(cutaneous malignant melanoma, CMM)是恶性程度最高的皮肤肿瘤,近年来CMM发病率在世界范围内以3%-7%比例逐年上升,具有进展速度快、易于转移、放化疗不敏感、治疗后易复发和预后差等特点。侵袭和转移是影响该疾病患者生存的首要因素,是治疗的最大障碍,也是造成该疾病临床预后极差的关键因素,目前CMM的侵袭和转移机制尚未完全阐明。细胞骨架重组是细胞迁移和侵袭所必需的,Rho GTPases家族是细胞骨架的关键调控因子,在肿瘤发生发展多个步骤和过程均可发挥重要作用。因此,我们以M14、A375和MV3三株人黑素瘤细胞和人黑色素细胞(Melanocyte, MC)为研究对象,探讨Rho GTPases家族不同成员的表达及与细胞骨架、细胞迁移、侵袭的关系,然后选取该家族中在肿瘤细胞系及MC中表达差异最明显的RhoD为进一步研究的目标,构建慢病毒介导的过表达RhoD的A375细胞株,探讨RhoD过表达对A375细胞株细胞骨架、迁移、侵袭等生物学功能的影响及可能的机制。第一部分Rho GTPases家族在黑素瘤细胞骨架和迁移运动中的作用及分子机制目的：黑素瘤迁移和侵袭转移是影响患者生存的关键因素之一,细胞骨架重组是细胞迁移和侵袭所必需的,Rho GTPases家族是细胞骨架关键调控因子。本研究拟探讨Rho GTPases家族不同成员在黑素瘤细胞中的表达,观察细胞骨架的异同,揭示Rho GTPases家族通过调节细胞骨架在黑素瘤迁移和侵袭中发挥的作用和分子机制。方法：1、霍夫曼显微镜下观察M14、A375和MV3三株人黑素瘤细胞与人黑色素细胞(Melanocyte, MC)形态学上的差异；罗丹明标记的鬼笔环肽染色,在超高分辨率显微镜下观察以肌动蛋白丝为主要组成成分的丝状伪足、板状伪足、应力纤维和粘着斑在四种细胞中的差异。2、Transwell迁移实验检测M14、A375和MV3和MC四种细胞的迁移能力。3、实时荧光定量聚合酶链反应(Quantitative Real-time Polymerase Chain Reaction, QPCR)检测Rho GTPases家族各成员的mRNA转录水平；免疫印迹法(western blotting, WB)检测RhoD、Diaph2(diaphanous related formin2)、丝切蛋白Cofilin和磷酸化丝切蛋白(P-Cofilin)蛋白的表达。结果：1、M14、A375和MV3和MC四种细胞丝状伪足、板状伪足、应力纤维和粘着斑具有明显差异。MC无应力纤维和粘着斑,M14、A375、MV3三种黑素瘤细胞虽然形态学不同,但均含有应力纤维和粘着斑；MV3应力纤维的数量少于M14和A375,但较后两者粗；四种细胞都具有极细且短的丝状伪足。2、Transwell迁移实验结果显示M14和A375细胞跨膜迁移能力类似,迁移率高；MV3细胞迁移率低于M14和A375,MC无跨膜迁移能力。3、Rho GTPases家族的不同成员在四种细胞中的QPCR结果各不相同,并且呈现与细胞骨架免疫荧光相对应的变化；WB结果显示Rho GTPases家族成员之一的RhoD在M14.A375.MV3中基本不表达,与QPCR水平变化一致,其下游效应因子Diaph2亦出现了相对应的变化；肌动蛋白丝重要调控因子cofilin蛋白虽没有显著变化,但是其磷酸化水平P-Cofilin在黑素瘤细胞中明显高于黑色素细胞。结论：Rho GTPases通过调节以肌动蛋白丝为主要组成成分的丝状伪足、板状伪足、应力纤维、粘着斑进而影响黑素瘤细胞迁移和侵袭；黑色素和黑素瘤细胞骨架和细胞迁移运动是不同Rho GTPases家族成员精准调节的结果。第二部分慢病毒介导的过表达RhoD人黑素瘤A375细胞株的构建及鉴定目的：构建人RhoD基因的慢病毒载体并进行慢病毒包装和鉴定,体外转染人黑素瘤细胞A375,使其过表达RhoD蛋白,为后续研究RhoD在黑素瘤中的作用奠定基础。方法：1、利用Gateway技术构建携带增强型绿色荧光蛋白EGFP的RhoD慢病毒载体,经PCR及基因测序鉴定后,与辅助质粒pLV/helper-SL3、pLV/helper-SL4及pLV/helper-SL5混合采用脂质体法制备DNA-Lipofectamine2000复合物,并共同转染293FT细胞进行慢病毒包装,产生相应慢病毒颗粒,通过定量PCR方法测定病毒滴度。2、利用包装好的慢病毒转染人黑素瘤A375细胞,建立RhoD过表达的人黑素瘤A375细胞株,荧光显微镜下观察荧光表达情况,流式细胞仪检测转染效率；实验分为A375(未处理对照组)、A375-EGFP(不含目的基因的空病毒对照组)和A375-RhoD(含RhoD基因的病毒组)三组,采用实时荧光定量PCR (QPCR)及免疫印记法(Western blot, WB)验证RhoD在A375-RhoD组细胞中的过表达。结果： 1、 通过PCR、 基因测序证实, 慢病毒表达载体pLV[Exp]-EGFP/Neo-CMVhRhoD构建成功。与辅助质粒共转染293FT细胞包装出具高效感染力的慢病毒,经测定病毒滴度为(5.13±2)×108TU/ml。2、转染A375细胞后,在细胞内及细胞膜表面都有明显的绿色荧光表达,流式检测转染效率大于80%；A375-RhoD组RhoD mRNA及蛋白水平均较A375-EGFP组和A375组细胞中明显增高(P0.05)。结论：成功构建RhoD慢病毒表达载体,包装出具高效感染力的慢病毒颗粒并成功转染人黑素瘤A375细胞,为进一步研究RhoD在黑素瘤中的作用提供实验基础。第三部分过表达RhoD对黑素瘤细胞A375细胞骨架和迁移侵袭的影响及作用机制的探讨目的：探讨RhoD对人黑素瘤细胞株A375细胞骨架、迁移和侵袭能力等生物学行为的影响及其作用机制。方法：采用罗丹明-鬼笔环肽染色观察细胞骨架、Transwell小室实验检测细胞迁移及侵袭能力,流式细胞技术检测细胞周期,观察空白对照组A375、阴性对照慢病毒载体转染组A375-EGFP及过表达RhoD慢病毒载体转染组A375-RhoD三组细胞的体外生物学行为改变；采用Western blot免疫印迹法比较三组细胞中Diaph2、 cofilin和P-cofilin的表达情况,分析RhoD影响黑素瘤细胞骨架和运动、侵袭功能的可能机制。结果：1、细胞骨架染色显示,与A375和A375-EGFP组细胞对比,A375-RhoD组细胞体积增大,应力纤维变细,软弱无力,粘着斑增多；丝状伪足形成增加；皱褶缘和板状伪足无明显变化。2、Transwell小室迁移实验显示：A375、A375-EGFP和A375-RhoD组平均每视野穿膜细胞数分别为(149.67±11.93)、(152.67±11.23)和(72.67.25±5.03),RhoD过表达可以显著抑制黑素瘤细胞A375的跨膜运动和迁移能力(P0.05); Transwell小室人工基底膜侵袭试验显示：A375、A375-EGFP和A375-RhoD组平均每视野穿膜细胞数分别为(83±7)、(78.33±12.34)和(9±1), RhoD过表达可以显著抑制黑素瘤细胞A375的侵袭能力(P0.05)。3、流式细胞技术分析细胞周期结果表明：A375、A375-EGFP和A375-RhoD三组细胞间G1期、S期和G2期细胞所占百分比无统计学差异(P=0.685、0.138和P=0.410),过表达RhoD和阴性对照慢病毒载体对细胞周期均无影响。5、WB免疫印迹结果显示,A375-RhoD细胞可在RhoD的刺激下激活下游信号效应分子Diaph2,促进其表达,而对照组未能激活。RhoD过表达和阴性对照慢病毒载体对cofilin和P-cofilin均无影响。结论：RhoD在黑素瘤A375细胞的细胞骨架以及运动和迁移侵袭中发挥一定作用,有可能成为黑素瘤治疗中的靶向目标。
[Abstract]:Cutaneous malignant melanoma (CMM) is the most malignant skin tumor. In recent years, the incidence of CMM in the world is increasing year by year in the proportion of 3%-7%. It has the characteristics of rapid progress, easy transfer, insensitivity to radiotherapy and chemotherapy, easy to relapse after treatment and poor prognosis after treatment. Invasion and metastasis are the factors affecting the survival of the disease patients. The most important factor, the biggest obstacle to treatment, is the key factor in the poor clinical prognosis of the disease. At present, the mechanism of invasion and metastasis of CMM has not been fully elucidated. The cytoskeleton recombination is essential for cell migration and invasion. The Rho GTPases family is the key regulating factor of the cytoskeleton, and many steps and processes in the development of the tumor. Therefore, we take M14, A375 and MV3 three human melanoma cells and human melanocytes (Melanocyte, MC) as research objects to investigate the expression of different members of the Rho GTPases family and the relationship with cytoskeleton, cell migration and invasion, and then select the most distinct R expression in the family of the tumor cell lines and MC. HoD is the goal of further research to construct a A375 cell line of RhoD mediated by lentivirus, and to explore the effects of RhoD overexpression on the biological functions of cytoskeleton, migration and invasion of A375 cell lines and the possible mechanisms. The first part of the Rho GTPases family in the cytoskeleton and migration of melanoma and the molecular mechanism of the molecular mechanism: Black The migration and invasion of vegetarian tumor are one of the key factors that affect the survival of the patients. The cytoskeleton recombination is necessary for cell migration and invasion. The Rho GTPases family is a key regulatory factor in the cytoskeleton. This study intends to explore the expression of the different members of the Rho GTPases family in the melanoma cells, observe the similarities and differences of cytoskeleton, and reveal the Rho GTPases The role and molecular mechanism of the family by regulating the cytoskeleton in the migration and invasion of melanoma. Methods: 1, the morphological differences between M14, A375 and MV3 three melanoma cells and human melanocytes (Melanocyte, MC) were observed under the Hoffman microscope; the Luo Danming labeled phallus cytosine staining was observed under the ultrahigh resolution microscope. The difference between the actin filaments as the main components of the filamentous pseudo foot, the plate-like pseudo foot, the stress fiber and the plaque in the four cells was detected by.2. The migration tests of the four cells of M14, A375 and MV3 and MC were detected by the Transwell migration test, and the real-time fluorescence quantitative polymerase chain reaction (Quantitative Real-time Polymerase Chain Reaction) was detected. The mRNA transcriptional level of the members of the Rho GTPases family; Western blotting (WB) for the detection of RhoD, Diaph2 (diaphanous related formin2), the expression of filamentin Cofilin and phosphorylated silk cut proteins. Results: 1. The three melanoma cells of.MC, M14, A375, and MV3 were different in morphology, but they all contained stress fibers and adhesion spots, and the number of MV3 stress fibers was less than M14 and A375, but the latter two were coarsely. The four cells all had very thin and short filamentous pseudfoot.2, Transwell migration experimental results showed M14 and A375 cells. The transmembrane migration ability was similar and the migration rate was high; the migration rate of MV3 cells was lower than that of M14 and A375, and the migration ability of MC without transmembrane was.3. The QPCR results of the different members of the Rho GTPases family were different in the four cells, and the corresponding changes in the cytoskeleton immunofluorescence were presented; WB results showed that RhoD in the Rho GTPases family members was MV3 was basically not expressed, consistent with the change of QPCR level, and its downstream effect factor Diaph2 also changed. Although the important regulatory factor of actin filament cofilin protein did not change significantly, the phosphorylation level P-Cofilin was obviously higher in melanoma cells than black vegetarian cells. Conclusion: Rho GTPases is regulated by muscle movement. The main components of the filaments are filamentous pseudo foot, plate-like pseudo foot, stress fiber, and adhesion spots that affect the migration and invasion of melanoma cells; the cytoskeleton and cell migration of melanoma and melanoma are the result of the precise regulation of Rho GTPases family members. The second part of lentivirus mediated overexpression of RhoD human melanoma A375 cells Construction and identification of the plant: construct the lentivirus vector of human RhoD gene and carry out lentivirus packaging and identification, transfect human melanoma cell A375 in vitro, make it overexpress RhoD protein, and lay the foundation for the follow-up study of the role of RhoD in melanoma. Method: 1, using Gateway technique to construct RhoD with enhanced green fluorescent protein EGFP. The lentivirus vector was identified by PCR and gene sequencing, and DNA-Lipofectamine2000 complex was prepared by liposomes mixed with auxiliary plasmid pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5, and 293FT cells were transfected together to package lentivirus, produce corresponding lentivirus particles, determine the virus titer.2 by quantitative PCR, and use packaging. A good lentivirus was transfected into human melanoma A375 cells to establish a human melanoma A375 cell line overexpressed by RhoD, the fluorescence expression was observed under the fluorescence microscope, and the transfection efficiency was detected by flow cytometry. The experiment was divided into A375 (untreated control group), A375-EGFP (free virus control group without target gene) and A375-RhoD (containing RhoD gene virus group). Three groups, using real time fluorescence quantitative PCR (QPCR) and immune imprint (Western blot, WB) to verify the overexpression of RhoD in the A375-RhoD group cells. Results: 1, through PCR, gene sequencing confirmed that the Lentivirus Expression Vector pLV[Exp]-EGFP/Neo-CMVhRhoD construction was successful. The slow disease of the high effective infection force was issued with the adjuvant plasmids co transfected 293FT cells. The virus titer was (5.13 + 2) x 108TU/ml.2. After transfection of A375 cells, there was a clear green fluorescence expression on both cell and cell membrane surface, and the transfection efficiency of flow detection was more than 80%. The level of RhoD mRNA and protein in group A375-RhoD was significantly higher than that in A375-EGFP and A375 groups (P0.05). Conclusion: RhoD lentivirus was successfully constructed. The expression vector, packed with highly effective lentivirus particles and successfully transfected human melanoma A375 cells, provided an experimental basis for further study of the role of RhoD in melanoma. The third part overexpressed the effect of RhoD on the A375 cytoskeleton and migration and invasion of melanoma cells and the mechanism to explore the effect of RhoD on human melanin. The effect and mechanism of biological behavior such as cytoskeleton, migration and invasion ability of tumor cell line A375. Methods: the cytoskeleton was observed by Luo Danming phallus cyclin staining, cell migration and invasion ability was detected by Transwell chamber test, cell cycle was detected by flow cytometry, A375 in blank control group and negative control lentivirus were observed. The biological behavior changes in the transfected group of A375-EGFP and RhoD lentivirus vector transfected group A375-RhoD three were compared, and the expression of Diaph2, cofilin and P-cofilin in the three groups was compared by Western blot immunoblotting, and the possible mechanism of RhoD affecting the bone frame and movement of melanoma cells and the possible mechanism of invasion was analyzed. 1, cytoskeleton staining showed that, compared with A375 and A375-EGFP groups, the cell volume of the A375-RhoD group increased, the stress fiber became thinner, the soft weak, the adhesion plaque increased, the formation of filamentous pseudo foot increased, the folds margin and the plate like pseudo foot had no obvious changes of.2, and the Transwell chamber migration experiment showed that A375, A375-EGFP, and A375-RhoD groups averaged every film in each field of vision. The number of cells was (149.67 + 11.93), (152.67 + 11.23) and (72.67.25 + 5.03). The overexpression of RhoD could significantly inhibit the transmembrane movement and migration of A375 in melanoma cells (P0.05). The invasion test of artificial basement membrane in the Transwell compartment showed that the average number of membrane cells in each field of A375-EGFP and A375-RhoD was (83 + 7), respectively (78.33 + 12.34). And (9 + 1), RhoD overexpression could significantly inhibit the invasiveness of the melanoma cell A375 (P0.05).3. Flow cytometry analysis of cell cycle results showed that A375, A375-EGFP and A375-RhoD three groups had G1 phase, and there was no statistical difference in the percentage of S and G2 cells (P =0.685,0.138 and negative), over expression and negative control lentivirus The carrier had no effect on the cell cycle of.5, and the WB immunoblotting showed that A375-RhoD cells could activate the downstream signal effector Diaph2 and promote its expression under the stimulation of RhoD, while the control group failed to activate.RhoD overexpression and negative control lentivirus vector with no influence on cofilin and P-cofilin. Conclusion: RhoD is in the thinner A375 cell of melanoma. Cytoskeleton may play a role in motility, migration and invasion, and may become a target in melanoma therapy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.5
[Abstract]:Cutaneous malignant melanoma (CMM) is the most malignant skin tumor. In recent years, the incidence of CMM in the world is increasing year by year in the proportion of 3%-7%. It has the characteristics of rapid progress, easy transfer, insensitivity to radiotherapy and chemotherapy, easy to relapse after treatment and poor prognosis after treatment. Invasion and metastasis are the factors affecting the survival of the disease patients. The most important factor, the biggest obstacle to treatment, is the key factor in the poor clinical prognosis of the disease. At present, the mechanism of invasion and metastasis of CMM has not been fully elucidated. The cytoskeleton recombination is essential for cell migration and invasion. The Rho GTPases family is the key regulating factor of the cytoskeleton, and many steps and processes in the development of the tumor. Therefore, we take M14, A375 and MV3 three human melanoma cells and human melanocytes (Melanocyte, MC) as research objects to investigate the expression of different members of the Rho GTPases family and the relationship with cytoskeleton, cell migration and invasion, and then select the most distinct R expression in the family of the tumor cell lines and MC. HoD is the goal of further research to construct a A375 cell line of RhoD mediated by lentivirus, and to explore the effects of RhoD overexpression on the biological functions of cytoskeleton, migration and invasion of A375 cell lines and the possible mechanisms. The first part of the Rho GTPases family in the cytoskeleton and migration of melanoma and the molecular mechanism of the molecular mechanism: Black The migration and invasion of vegetarian tumor are one of the key factors that affect the survival of the patients. The cytoskeleton recombination is necessary for cell migration and invasion. The Rho GTPases family is a key regulatory factor in the cytoskeleton. This study intends to explore the expression of the different members of the Rho GTPases family in the melanoma cells, observe the similarities and differences of cytoskeleton, and reveal the Rho GTPases The role and molecular mechanism of the family by regulating the cytoskeleton in the migration and invasion of melanoma. Methods: 1, the morphological differences between M14, A375 and MV3 three melanoma cells and human melanocytes (Melanocyte, MC) were observed under the Hoffman microscope; the Luo Danming labeled phallus cytosine staining was observed under the ultrahigh resolution microscope. The difference between the actin filaments as the main components of the filamentous pseudo foot, the plate-like pseudo foot, the stress fiber and the plaque in the four cells was detected by.2. The migration tests of the four cells of M14, A375 and MV3 and MC were detected by the Transwell migration test, and the real-time fluorescence quantitative polymerase chain reaction (Quantitative Real-time Polymerase Chain Reaction) was detected. The mRNA transcriptional level of the members of the Rho GTPases family; Western blotting (WB) for the detection of RhoD, Diaph2 (diaphanous related formin2), the expression of filamentin Cofilin and phosphorylated silk cut proteins. Results: 1. The three melanoma cells of.MC, M14, A375, and MV3 were different in morphology, but they all contained stress fibers and adhesion spots, and the number of MV3 stress fibers was less than M14 and A375, but the latter two were coarsely. The four cells all had very thin and short filamentous pseudfoot.2, Transwell migration experimental results showed M14 and A375 cells. The transmembrane migration ability was similar and the migration rate was high; the migration rate of MV3 cells was lower than that of M14 and A375, and the migration ability of MC without transmembrane was.3. The QPCR results of the different members of the Rho GTPases family were different in the four cells, and the corresponding changes in the cytoskeleton immunofluorescence were presented; WB results showed that RhoD in the Rho GTPases family members was MV3 was basically not expressed, consistent with the change of QPCR level, and its downstream effect factor Diaph2 also changed. Although the important regulatory factor of actin filament cofilin protein did not change significantly, the phosphorylation level P-Cofilin was obviously higher in melanoma cells than black vegetarian cells. Conclusion: Rho GTPases is regulated by muscle movement. The main components of the filaments are filamentous pseudo foot, plate-like pseudo foot, stress fiber, and adhesion spots that affect the migration and invasion of melanoma cells; the cytoskeleton and cell migration of melanoma and melanoma are the result of the precise regulation of Rho GTPases family members. The second part of lentivirus mediated overexpression of RhoD human melanoma A375 cells Construction and identification of the plant: construct the lentivirus vector of human RhoD gene and carry out lentivirus packaging and identification, transfect human melanoma cell A375 in vitro, make it overexpress RhoD protein, and lay the foundation for the follow-up study of the role of RhoD in melanoma. Method: 1, using Gateway technique to construct RhoD with enhanced green fluorescent protein EGFP. The lentivirus vector was identified by PCR and gene sequencing, and DNA-Lipofectamine2000 complex was prepared by liposomes mixed with auxiliary plasmid pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5, and 293FT cells were transfected together to package lentivirus, produce corresponding lentivirus particles, determine the virus titer.2 by quantitative PCR, and use packaging. A good lentivirus was transfected into human melanoma A375 cells to establish a human melanoma A375 cell line overexpressed by RhoD, the fluorescence expression was observed under the fluorescence microscope, and the transfection efficiency was detected by flow cytometry. The experiment was divided into A375 (untreated control group), A375-EGFP (free virus control group without target gene) and A375-RhoD (containing RhoD gene virus group). Three groups, using real time fluorescence quantitative PCR (QPCR) and immune imprint (Western blot, WB) to verify the overexpression of RhoD in the A375-RhoD group cells. Results: 1, through PCR, gene sequencing confirmed that the Lentivirus Expression Vector pLV[Exp]-EGFP/Neo-CMVhRhoD construction was successful. The slow disease of the high effective infection force was issued with the adjuvant plasmids co transfected 293FT cells. The virus titer was (5.13 + 2) x 108TU/ml.2. After transfection of A375 cells, there was a clear green fluorescence expression on both cell and cell membrane surface, and the transfection efficiency of flow detection was more than 80%. The level of RhoD mRNA and protein in group A375-RhoD was significantly higher than that in A375-EGFP and A375 groups (P0.05). Conclusion: RhoD lentivirus was successfully constructed. The expression vector, packed with highly effective lentivirus particles and successfully transfected human melanoma A375 cells, provided an experimental basis for further study of the role of RhoD in melanoma. The third part overexpressed the effect of RhoD on the A375 cytoskeleton and migration and invasion of melanoma cells and the mechanism to explore the effect of RhoD on human melanin. The effect and mechanism of biological behavior such as cytoskeleton, migration and invasion ability of tumor cell line A375. Methods: the cytoskeleton was observed by Luo Danming phallus cyclin staining, cell migration and invasion ability was detected by Transwell chamber test, cell cycle was detected by flow cytometry, A375 in blank control group and negative control lentivirus were observed. The biological behavior changes in the transfected group of A375-EGFP and RhoD lentivirus vector transfected group A375-RhoD three were compared, and the expression of Diaph2, cofilin and P-cofilin in the three groups was compared by Western blot immunoblotting, and the possible mechanism of RhoD affecting the bone frame and movement of melanoma cells and the possible mechanism of invasion was analyzed. 1, cytoskeleton staining showed that, compared with A375 and A375-EGFP groups, the cell volume of the A375-RhoD group increased, the stress fiber became thinner, the soft weak, the adhesion plaque increased, the formation of filamentous pseudo foot increased, the folds margin and the plate like pseudo foot had no obvious changes of.2, and the Transwell chamber migration experiment showed that A375, A375-EGFP, and A375-RhoD groups averaged every film in each field of vision. The number of cells was (149.67 + 11.93), (152.67 + 11.23) and (72.67.25 + 5.03). The overexpression of RhoD could significantly inhibit the transmembrane movement and migration of A375 in melanoma cells (P0.05). The invasion test of artificial basement membrane in the Transwell compartment showed that the average number of membrane cells in each field of A375-EGFP and A375-RhoD was (83 + 7), respectively (78.33 + 12.34). And (9 + 1), RhoD overexpression could significantly inhibit the invasiveness of the melanoma cell A375 (P0.05).3. Flow cytometry analysis of cell cycle results showed that A375, A375-EGFP and A375-RhoD three groups had G1 phase, and there was no statistical difference in the percentage of S and G2 cells (P =0.685,0.138 and negative), over expression and negative control lentivirus The carrier had no effect on the cell cycle of.5, and the WB immunoblotting showed that A375-RhoD cells could activate the downstream signal effector Diaph2 and promote its expression under the stimulation of RhoD, while the control group failed to activate.RhoD overexpression and negative control lentivirus vector with no influence on cofilin and P-cofilin. Conclusion: RhoD is in the thinner A375 cell of melanoma. Cytoskeleton may play a role in motility, migration and invasion, and may become a target in melanoma therapy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.5
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