Biological Effect Of LTBP4 In Migration And Invasion Of Malignant Melanoma Cells

Objective:1. Inhibit the expression of LTBP4 in malignant melanoma A375 cells and A875 gene in the application of RNAi technology, malignant melanoma constructed LTBP4 deletion;2. Through CCK-8, Transwell, flow cytometry were used to detect malignant melanoma cell proliferation, migration, invasion and apoptosis, To investigate the effect of LTBP4 on the migration and invasion of malignant melanoma cells and its molecular mechanismMethods:1. Melanoma cell line LTBP4 deletion establishmentMalignant melanoma cell lines A375 and A875 were divided into 3 groups,namely experimental transfection group (siR-LTBP4), negative control group(siR-NC), control group (Control). The transfection group with LTBP4 specific shRNA expression vector plvx-shRNA2-LTBP4 by LipofectamineTM2000 mediated transfection of the two malignant melanoma cell lines; The negative control group with plvx-shRNA2-NC via LipofectamineTM2000 mediated transfection of two cells;the control group of two groups of cell lines were without any treatment. Observe the two cell transfection efficiency by fluorescence microscope. By using fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expression level of LTBP4, understanding the silencing efficiency.2. Effect of LTBP4 silencing on malignant melanoma cellsMalignant melanoma of the first part of the LTBP4 deletion constructs, the proliferation of CCK-8 cells; Transwell assay cell migration; The Transwell (PU adhesive) invasion detection cell; Cell apoptosis were measured by flow cytometry;To investigate the effect of LTBP4 on malignant melanoma cell migration and invasion.3. The effect of LTBP4 on BMP/TGF-4 signaling pathwayTo detect the changes of TGF- beta activity in malignant melanoma A375, and to detect the effect of LTBP4 on different forms of TGF- beta by Western blotResults:1. The successful construction of melanoma cell line LTBP4 silencingThe construction of LTBP4 silencing plasmid, and successfully transfected to two strains of melanoma cell lines. Observed under the fluorescence microscope, the transfection efficiency reached more than 80%.Quantitative PCR results showed that amplification, melting curve showed a specific peak, no nonspecific amplification, to obtain accurate and reliable data. The relative expression of each sample by 2- Delta Ct to carry on the preliminary statistics.Quantitative PCR results showed that LTBP4 siRNA can effectively inhibit the LTBP4 expression level of mRNA, compared with control group or siR-NC group,the difference was significant (P < 0.05). While in siR-NC group, LTBP4 mRNA expression level compared with the control group, no significant difference (P >0.05).Western blot test results show that: siR-LTBP4 can effectively inhibit the expression of LTBP4 in melanoma cell lines A375 and A875, compared with control group and siR-NC group, the difference was significant. Control group and siR-NC group compared, no significant difference in the expression level of LTBP4. It is worth noting that the same siRNA silencing efficiency of the A875 and LTBP4 in the far A375 high expression characteristics and between cells which may be related to the need for further research.2. Effect on melanoma cells after silencing of LTBP4CCK-8 24h and 48h siR-LTBP4 were found in the group of A875 cells and A3 75 cells were lower than siR-NC group and control group, the proliferation was markedly reduced, the difference was statistically significant (P<0.05); SiR-NC group and control group in 24h and 48h was no significant difference (P>0.05); At the same time,A375 and A875 in different time, the same group the difference was statistically significant (P<0.05)Transwell migration experiment group siR-LTBP4 migrated to the lower chamber of the Transwell A875 cells and A375 cells were lower than siR-NC group and control group were significantly reduced, the difference was statistically significant(P<0.05); SiR-NC group and control group, the number of migrated cells was no significant difference (P>0.05).Transwell invasion experiment group siR-LTBP4 invasion to Transwell the lower chamber of the A875 and A375 cells were lower than siR-NC group and control group were significantly reduced, the difference was statistically significant (P<0.05);SiR-NC group and control group compared to the number of invasive cells showed no significant difference (P>0.05).The detection results of flow cytometry showed that the transfection of siR-LTBP4 could significantly increase the apoptosis of melanoma cell lines A375 and A875, compared with control group and siR-NC group, the difference was significant. Control group and siR-NC group compared, there was no significant difference in the two cells.3. LTBP4 silencing can regulate the TGF- beta /BMP signaling pathwayThe results showed that the content of TGF- beta in the cell culture medium was significantly decreased after interference with LTBP4, and the content of total TGF-was significantly increased. The results of Western blot showed no significant change in the expression level of TGF- 1, while the expression level of TGF- beta 2 and TGF-beta protein increased significantly in.Conclusions:1. RNAi can effectively inhibit the expression of LTBP4 in melanoma cells A375 and A875, and the migration, invasion and value-added ability of siR-LTBP4 group were decreased, and the apoptosis was enhanced, LTBP4 gene may be related to migration, invasion, proliferation and apoptosis of malignant melanoma.2. After LTBP4 silencing, the content of TGF- beta in the activation form was decreased, while the content of total TGF- was significantly increased. There was no significant change in the expression level of TGF- 1, while the expression level of TGF- beta 2 and TGF- beta protein was significantly increased in, Therefore, LTBP4 silencing can regulate the TGF- beta /BMP signaling pathway.3. LTBP4 gene may be a new therapeutic target for malignant melanoma, which provides a theoretical basis for the development of targeted therapy of malignant melanoma.