| ObjectiveThe mortality of melanoma is the highest among all superficial malignant tumors. It is thought that its high invasion, metastasis and resistance to conventional chemotherapy are involved in lacking of therapeutic targets and poor prognosis for melanoma.As a molecular switch to control tumor metastasis, RhoC, which is expected to be an important target for novel therapeutic option, has been identified to be correlated with tumor progression, especially tumor invasion and metastasis by a variety of mechanisms. RNA interference, as a rapid and effective post-transcriptional gene silencing, has been widely used in gene research and therapy.Despite RNA interference has been confirmed to controll cell activities by manipulating gene expression, while, at present little is studied about construction of RNA interference vector targeting RhoC with RNA interference technique, as well as its impacts on biological behavior and drug resistance of human melanoma cells A375.In this study, based on successful construction of pLenti6.3-EGFP-453 targeting RhoC, we investigated the effects of RhoC silencing on biological activities of A375 cells including cell proliferation, apoptosis, adhesion and invasion, expression of matrix metalloproteinase 2 (MMP-2) and E-cadherin, as well as the reversibility of resistance of melanoma to dacarbazine (DTIC) and its mechanism with RNA interference, which to further assess the role of RhoC in melanoma progression, the feasibility of RhoC as gene therapy target to the metastasis and reversing the resistance of chemotherapy in melanoma. The results might provide a new way to treat malignant tumor with RNA interference.Methods1 According to RhoC gene encoding information, three pairs of pGPU6/GFP/Neo-shRNA vector were constructed and delivered into A375 cells. The silencing effects of mRNA and protein expression of RhoC were detected by Real-time PCR and Western blotting methods, and then the most effective target sequence was selected.2 pcDNA6.2-EmGFP-453-miR vector was constructed by using the most effective sequence, and then was packaged in the lentivirus and constructed pLenti6.3-EGFP-453 lentiviral vector targeting RhoC. After transfected into A375 cells, the silencing effects of mRNA and protein expression of RhoC was detected by Real-time PCR and Western blotting methods.3 The mRNA and protein expression of MMP-2, E-cadherin after RhoC silencing were detected by Real-time PCR and Western blotting methods when A375 cells was transfected by pLenti6.3-EGFP-453 lentiviral vector in vitro. The invasive capacity of A375 cell was examined with transwell chamber test. The adhesion of laminin and fibronectin was tested with cell adhesion experiment. The cell proliferation was detected by the tetrazolium dye colorimetric test (MTT test) and the apoptosis of the cells was tested by flow cytometer. The reversal rate of the resistance to DTIC was detected with MTT test. At the same time, the controls were detected with the same assays. All data were analyzed by SPSS 15.0 statistical software package. The level of significant difference is a=0.05.Results1 RhoC-shRNA vectors constructed were pGPU6/GFP/Neo-shRNA336, pGPU6/GFP/Neo-shRNA453 and pGPU6/GFP/Neo-shRNA680. The mRNA and protein expression of RhoC in the group of transfected cells were 1.47±0.26, 1.13±0.16,1.39±0.11 and 70.98±9.21,50.67±6.06,65.77±4.06. Among three pairs of pGPU6/GFP/Neo-shRNA vector, pGPU6/GFP/Neo-shRNA453 was the most effective sequence.2 pLenti6.3-EGFP-453 lentiviral vector targeting RhoC was constructed successfully and its sequence was proved to be accurate. After transfected into A375 cells, the mRNA and protein expression of RhoC in the group of transfected cells, negative lentivirus and normal cells were 1.05±0.05,62.04±15.86; 4.21±0.24,220.86±24.07 and 4.63±0.32,257.39±12.30 respectively. The expression of RhoC in the group of transfected cells significantly lower than those in the groups of normal cells and cells with negative lentivirus (P< 0.05)3 In vitro, the mRNA and protein expressions of MMP-2 in the group of transfected cells, negative lentivirus and normal cells were1.17±0.16,66.40±14.51; 2.28±0.31, 112.59±23.27 and 2.75±0.90,135.88±26.16 respectively. The mRNA and protein expressions of E-cadherin in the group of transfected cells, negative lentivirus and normal cells were 2.95±0.71,125.17±19.88; 1.80±0.47,77.39±13.38 and 0.97±0.54, 47.91±18.69. There was a significant difference between the group of transfected cells and two control groups (P<0.05) 4 In vitro, the results of transwell chamber cell invasion assay showed the penetrating cells in the group of transfected cells was 40.41±8.88, which were significantly less than those in the groups of normal cells and cells with negative lentivirus (P<0.05). However, there was no significant difference between the group of normal cells and the group of cells with negative lentivirus (P>0.05). Cell adhesion experiments by crystal violet staining showed that OD value of Ln and Fn in the group of transfected cells were 0.467±0.005,0.321±0.007, which were lower than those in the groups of normal cells and cells with negative lentivirus (P<0.05). There was no significant difference between the group of normal cells and the group of cells with negative lentivirus (P>0.05)5 In vitro, the results of cell proliferation detected by MTT assay showed that OD value in the group of transfected cells at 24h was 0.319±0.004, Compared with two control groups, there was no significant difference (P>0.05). At 72h,120h,168h, OD values in the group of transfected cells were 0.337±0.004,0.369±0.004 and 0.397±0.005, which were significantly less than those in cells with negative lentivirus and the groups of normal cells (P<0.05). The drug resistance to DTIC in A375 cell was detected by MTT assay. The inhibition rates in the the group of RNA interference combined DTIC of different concentrations(0.01?g/ml,0.1?g/ml, 1.0?g/ml, 10?g/ml) were 8.82%,14.01%,23.15%and 33.51%, and the value of IC50 was 162.41?g/ml. However, inhibition rates in the group of using DTIC alone were 1.97%,7.79%,13.57%and 20.37%, and the value of IC50 was 254.99?g/ml. The reversal rate of resistance to DTIC was 1.57. Flow cytometry analysis demonstrated that apoptosis rate in the group of transfected cells was 13.36±0.39%, which was higher than that in both normal cells and cells with negative lentivirus (P<0.05). There was no significant difference between the group of normal cells and the group of cells with negative lentivirus (P>0.05) Conclusions:1 pLenti6.3-EGFP-453 lentiviral vector targeting RhoC could be transfected into A375 cells effectively, and the expression of RhoC mRNA and protein were inhibited obviously.2 The expression of MMP-2 decreased and E-cadherin increased after silencing RhoC. The invasion and adhesion were apparently inhibited. RhoC silencing inhibitted the invasion and metastasis of tumor, probably by regulating tumor metastasis-related genes.3 When RhoC was silenced, the proliferation of A375 cells was suppressed and the apoptosis increased, the sencitivity of A375 cells to DTIC were enhanced. The resistance of A375 cells to DTIC could be reversed, probably via inducing the apoptosis of tumor cells by RNA interference. |
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