| Cutaneous malignant melanoma (CMM) is the most malignant skin tumor and has an increasing global incidence rate of 3%-7%. This disease is characterized by rapid progression, nonsensitivity to chemotherapy, metastasis, high recurrence rate, and poor prognosis, among others. Invasion and metastasis are the primary factors that influence patient survival. These factors are the significant obstacles to the treatment and the key factors for determining poor prognosis. However, the mechanism of invasion or metastasis has not been fully elucidated yet. Cytoskeletal reorganization is necessary for cell migration and invasion, in which the Rho GTPase family is the key regulatory factor.Therefore, three strains of human maglinant melanoma cells M14, A375, and MV3 and melanocyte(MC) were investigated. Relations between the expression of different members of the Rho GTPase family and the cytoskeleton, cell migration, and invasion are discussed. RhoD, which exhibits the most remarkable difference in expression between the tumor cell lines and MC in the family, is selected for further research. A375 cell lines that exhibit lentivirus-mediated high expression of RhoD were modeled to explore the effects and possible mechanisms of overexpressed RhoD on the biological functions of A375 cell lines, such as for cytoskeleton, migration, and invasion.Part I:Roles and Molecular Mechanisms of Rho GTPase Family in Melanoma Cell Migration and cytoskeletonObjective:Cancer cell migration and invasion is one of the critical factors affecting the survival of patients with melanoma. Cytoskeletal reorganization is necessary for cell migration and invasion, in which the Rho GTPase family is the key regulatory factor. This research aims to explore the molecular mechanisms of different Rho GTPases in filopodia, lamellipodia, stress fibers, and focal adhesions by regulating the cytoskeletal reorganization of melanoma. Consequently, this study will reveal the roles of the Rho GTPase family in the migration of melanoma cells.Methods:(1) Morphological differences among M14, A375, MV3 human melanoma cells,and human Melanocyte(MC) were observed under a microscope. Immunofluorescence staining was conducted to detect the differences in filopodia, lamellipodia, stress fibers, and focal adhesions among these four kinds of cells. (2) The migration abilities of the four cell strains were evaluated by Transwell migration assay. (3) Finally, the mRNA transcription of all members of the Rho GTPase family was identified through QPCR, and the expression of RhoD, Diaph2, cofilin, and P-cofilin was distinguished using Western blot (WB).Results:(1) Obvious differences in the filopodia, lamellipodia, stress fibers, and focal adhesions were observed among M14, A375, MV3, and MC. Among these four kinds of cells. MC contained no stress fibers and focal adhesions. By contrast, M14, A375, and MV3 contained stress fibers and focal adhesions, even though their morphologies were different. All these four kinds of cells contained very fine and short filopodia. MV3 was significantly different from M14 and A375 in terms of stress fibers; MV3 was less in quantity but thicker than the latter cell types.(2) Transwell migration assay showed that M14 demonstrated a migration ability similar to that presented by A375, with a high migration rate. The migration rate of MV3 was lower than those of M14 and A375. MC did not almost migrate to the other side of the small chamber. (3) The members of the four kinds of cells of Rho GTPases changed, which corresponded to the immunofluorescence shown in different QPCR results. The WB result showed that RhoD was not basically expressed in M14, A375, and MV3 melanoma cells. The downstream effect factor Diaph2 correspondingly changed. Cofilin protein, which is an important regulatory factor for microfilaments, did not significantly changed, but its phosphorylation level (P-cofilin) was significantly higher than that of MC.Conclusion:Rho GTPases affect migration and invasion of melanoma cells through its regulatory effect on the filopodia, lamellipodia, stress fibers, and focal adhesions in which microfilament was regarded as the main component. Cell migration and cytoskeleton of melanocytes and melanoma cells are implied to be the result of precise regulation from different members of the Rho GTPase family.Part Ⅱ:Construction and identification of RhoD protein overexpressing A375 cell lines via lentiviral vectorsObjective:To construct a lentiviral vector carrying human RhoD gene and to package and identify a virus particles, and transfect it into melanoma A375 cells so as to lay a foundation for further study on the role of RhoD in malignant melanoma.Methods:1、The RhoD lentiviral vector (pLV[Exp]-EGFP/Neo-CMV>hRhoD) was constructed by Gateway technology, and identified by PCR and gene sequencing. The lentiviral vector was mixed with helper vector pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5 by Lipofectamine 2000 to prepare DNA-LipofectamineTM 2000 complexes. The complexes were then added to transfect 293FT cells and package virus. The virus titers were determined by Quantitative PCR.2、The lentiviruses were transfected into A375.The expression of EGFP was observed and transfection efficiency was detected by Flow Cytometer. QPCR and Western blot analysis were carried out to confirm overexpression of RhoD in A375 cells.Results:1、RhoD lentiviral vector pLV[Exp]-EGFP/Neo-CMV>hRhoD was constructed successfully and identified by PCR and gene sequencing. Lentivirus with high-efficiency infection was produced by transfection to 293FT cells and the virus titers were (5.13±2) ×108TU/ml.2、The EGFP could be observed after the lentiviruses were transfected into A375 cells and the transfection efficiency was higher than 80%.RhoD mRNA and protein levels in RhoD overexpression cells (A375-RhoD) were significantly higher than that in cells infected by Lentiviral vector only expressing EGFP (A375-EGFP) and no infected cells (A375) (P<0.05)Conclusion:The recombinant lentiviral vector for over-expressing RhoD was successfully constructed by Gateway technology and transfected into melanoma A375 cells for further experiment.Part Ⅲ:Effect of overexpression of RhoD on the cytoskeleton and migration and invasion of human melanoma cell line A375Objective To investigate the role of RhoD in the cytoskeleton and migration and invasion of melanoma and its possible mechanisms.Methods Biological behaviors of untreated A375 cell, A375-EGFP and A375- RhoD were evaluated and compared with each other. Immunofluorescence staining with Rhodamine phalloidin was performed to observe the cytoskeleton. Transwell assay was conducted to investigate the migration and invasion abilities. Cell cycle was tested by Flow Cytometry Method. Western blot was carried out to measure the expressions of Diaph2, cofilin and P-cofilin.Results 1. Cytoskeleton staining showed that compared with A375 and A375-EGFP groups, A375-RhoD group increased cell volume, stress fibers got thinner, softer; the formation of focal adhesions and filopodias increased; the lamellipodia and ruffled border had no obvious changes.2.Transwell migration study showed the number of transmigrated cells of A375、A375-EGFP and A375-RhoD were (149.67±11.93) (152.67±11.23) and (72.67.25±5.03) respectively (P<0.05); The number of invasive cells of A375-RhoD decreased compared with A375 and A375-EGFP in Transwell invasion study ((83±7)、(78.33±12.34) and (9±1), respectively) (P<0.05);these results proved the significant suppression of cell motility,transmigration and invasion ability by RhoD overexpression.3. Flow cytometer showed not-significant difference in G1,S and G2 phases between these three groups (P>0.05).4.Western blot showed that RhoD might induce the expression of downstream signaling protein Diaph2 in A375-RhoD cells, but not in A375-EGFP and A375 cells (P<0.05). There was no significant difference in the expression of cofilin and P-cofilin proteins between these three groups.Conclusion:RhoD may play a certain role in the cytoskeleton, migration and invasion of A375 melanoma cells, and might serve as a target in the treatment of melanoma. |
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