Experiment Study Of Inhibitary Effect Of PcDNA3.1-IGFBP7 Gene Therapy On Murine Malignant Melanoma Growth

Part I:expression of IGFBP7, VEGF, Caspase-3 in malignant melanomaObjectives:To study the expression of IGFBP7, VEGF and Caspase-3 in the B16-F10 melanoma of tumor-bearing C57BL/6J mice, and the possibility of IGFBP7 to be a gene therapy target for malignant melanoma.Methods:B16-F10 mouse malignant melanoma cell lines were cultured, and then made into suspension, and injected into the backs of C57BL/6J bearing mice. After 5-week formation of B16-F10 malignant melanoma, we obtained the melanoma specimens by surgery, used immunohistochemistry to test the expression of IGFBP7, VEGF and Caspase-3 inside, and analysed the correlation of IGFBP7, VEGF and Caspase-3 expression.Results:The culture of B16-F10 mouse malignant melanoma cell lines were successful, and after 5 week B16-F10 malignant melanoma formed. Pathology report indicated spindle tumor cells, with obvious heteromorphism in cytoblast. Immunohistochemistry showed strong expression of S100 in the cytoplasm, they being proved to be malignant melanoma cells. There are significant differences between the expression in the C57BL/6J mouse skin and that in the melanoma tumor tissues of either IGFBP7, VEGF, or Caspase-3. Expression of IGFBP7 and Caspase-3 was higher in the C57BL/6J mouse skin, as (W=261.50, P<0.01) and (W= 341.0, P<0.05), respectively, while VEGF was expressed higher in melanoma tissues of tumor-bearing mouse (W= 237.0, P<0.01). IGFBP7 and Caspase-3 were expressed positively correlative in both tissues, with Spearman correlation analysis showing that rs(IGFBP7, Caspase-3=0.728, P<0.01). Contrarily, IGFBP7 and VEGF were expressed negatively correlative, with rs(IGFBP7, VEGF=-0.631, P<0.01.)Conclusions:In B16-F10 melanoma tissue, the loss of IGFBP7 expression is a key step in malignization, accompanied by high VEGF expression and low Caspase-3 expression. IGFBP7 were negatively correlated with VEGF, and positively with Caspase-3. Therefore, we assumed that it would be an ideal gene therapy target for malignant melanoma, by up-regulating IGFBP7 expression, to inhibit VEGF expression and increase Caspase-3, which suppress the growth of malignant melanoma in coordination, through apoptosis. Part II:the construction of the pcDNA3.1-IGFBP7 expression vectorObjectives:To construct pcDNA3.1-IGFBP7 eukaryotic expression vector, for malignant melanoma in-vivo and in-vitro gene therapy.Methods:pcDNA3.1 and pOTB7-IGFBP7 purified, and digested by DNA restriction enzymes EcoR I and Xho I. The obtained IGFBP7 cDNA were extracted by gel, and then linked to the digested linear pcDNA3.1 with T4DNA ligase, to construct pcDNA3.1-IGFBP7 eukaryotic expression vector. Then, pcDNA3.1-IGFBP7 was transformed into E.coli DH5a, after screened, and amplified pcDNA3.1-IGFBP7 by bacteria shaking. We tested the above plasmids by using EcoR I and Xho I enzyme digestion, then gel electrophoresis and sequencing, to identify the success of pcDNA3.1-IGFBP7 construction.Results:We digested pcDNA3.1-IGFBP7 with EcoR I and Xho I, and obtained fragments by gel electrophoresis, the size of fragment complex consistent with the purpose. Plasmid sequencing resulted that the pcDNA3.1-IGFBP7 plasmid conform to the target, with objective size of about 1100bp, and gene sequence of IGFBP7.Conclusions:The success of pcDNA3.1-IGFBP7 construction laid a foundation for in-vivo and in-vitro gene therapy for malignant melanoma with point mutation on the V600E site of BRAF gene. PartⅢ:inhibitory effect of in-vitro pcDNA3.1-IGFBP7 transfection on the tumor growthObjectives:To investigate whether in-vitro transfection of pcDNA3.1-IGFBP7 into B16-F10 can inhibit B16-F10 growth and promote apoptosis in murine malignant melanoma cell lines.Methods:We set three groups to investigate the effect of in-vitro pcDNA3.1-IGFBP7 transfection, as pcDNA3.1-IGFBP7 group, pcDNA3.1-CONTROL group and blank control group. We transfected the plasmids into B16-F10 melanoma cells by using Effectene. Then we used RT-PCR, CCK8, flow cytometry to detect IGFBP7 mRNA expression, proliferative and survival activity of malignant melanoma cells, and apoptosis of malignant melanoma cell, respectively in the transfected group and the controls.Results:The transfection of pcDNA3.1-IGFBP7 was uccessful, with transfection rate of 50%to 60%. In transfected 48 hours, expression of IGFBP7 mRNA was dose-dependent on the concentration of plasmid pcDNA3.1-IGFBP7. On 1,3,6,12 days after in-vitro plasmid transfection, IGFBP7 mRNA expression of pcDNA3.1-IGFBP7 group, increased by respectively 4,8,7,6-fold (P<0.01), compared to pcDNA3.1-CONTROL group and blank control group. While transfection of pcDNA3.1-CONTROL plasmid did not increase IGFBP7 mRNA expression. IGFBP7 mRNA,β-actin mRNA expression level of pcDNA3.1-CONTROL group did not differ from the blank control group (P>0.05). These experiment results suggested that pcDNA3.1-IGFBP7 plasmid could increase the IGFBP7 mRNA expression but not affectβ-actin mRNA.Proliferation of B16-F10 melanoma cells transfected with plasmid pcDNA3.1-IGFBP7 in 48 hours was significantly inhibited (P<0.01). The inhibitory effect enhanced as the concentration of plasmid pcDNA3.1-IGFBP7 increased, in a dose dependent manner. The max inhibitory effect appeared in 24 hours after pcDNA3.1-IGFBP7 transfection (plasmid amount of 1μg), while group transfected with plasmid pcDNA3.1-CONTROL and blank control group without any transfection had no significant difference in cell proliferation. It showed that the transfection of pcDNA3.1-IGFBP7 could increase the synthesis and secretion of IGFBP7 to inhibit proliferation of B16-F10 cells.Results of flow cytometry showed that 24 hours after transfection, the apoptosis rate of B16-F10 in pcDNA3.1-IGFBP7 group was 24.6%, while in pcDNA3.1-CONTROL group and blank control group, there was no significant differences in apoptosis rate between transfected and before. Cell apoptosis of pcDNA3.1-IGFBP7 group was distinctively higher than the two control groups, with significant differences (P<0.01).Conclusions:In-vitro pcDNA3.1-IGFBP7 transfection could effectively inhibit the grouth and promote the apoptosis of B16-F10 melanoma, which would be a basis for in-vivo inhibitory gene therapy for malignant melanoma. PartⅣ:inhibitory effect of in-vivo pcDNA3.1-IGFBP7 transfection on the tumor growthObjectives:To investigate whether intratumoral injection of pcDNA3.1-IGFBP7 can inhibit tumor growth of murine malignant melanoma B16-F10 and the corresponding mechanism.Methods:The 36 mice burden with B16-F10 melanoma were randomly divided into three:pcDNA3.1-IGFBP7 group, pcDNA3.1-CONTROL group, and B16-F10 group. pcDNA3.1-IGFBP7 group were intratumoral injected with pcDNA3.1-IGFBP7 plasmid chelation suspension by using in-vivo transfection reagent invivofectamine. pcDNA3.1-CONTROL group were intratumoral injected with pcDNA3.1-CONTROL plasmid chelation suspension and invivofectamine. While B16-F10 group were intratumoral injected with invivofectamine+DMEM. Tumor growth and survival rate of mice of the groups were observed during the treatment, and expression of IGFBP7 protein after transfection in each group was concurrently monitored by Western blotting. Besides, we detected the expression distribution of IGFBP7, VEGF and Caspase-3 in the malignant melanoma tissue of each group by immunohistochemistry and tumor apoptosis by TUNEL, and analyzed the correlation between IGFBP7 and VEGF, Caspase-3 and tumor apoptosis of each group.Results:Survival rates of pcDNA3.1-IGFBP7 group, pcDNA3.1-CONTROL group, and B16-F10 group were respectively 75%,50%,41.7%. The results suggested that in-vivo gene therapy with pcDNA3.1-IGFBP7 could significantly extend the life of tumor-bearing mice. We killed the tumor-bearing mice on the 25th day, at which time tumor volumes of B16-F10 group and pcDNA3.1-CONTROL group were respectively 587±35 mm3 and 566±34 mm3, while tumor volumes of mice injected with pcDNA3.1-IGFBP7 was 256±25 mm3. Intratumoral injection of pcDNA3.1-IGFBP7 could significantly delay tumor growth, compared with the control group with significant difference (F=256.1, P<0.0014). The Western blot results of IGFBP7 expression showed that pcDNA3.1-IGFBP7 group could significantly increase the expression of IGFBP7 by intratumoral injection of pcDNA3.1-IGFBP7 plasmid, while pcDNA3.1-CONTROL group and B16-F10 group expressed IGFBP7 at a low and consistent level. Morever, the expressions of P-actin of the three groups were of no significant difference, indicating that pcDNA3.1-IGFBP7 could specifically promote the expression of IGFBP7, while empty pcDNA3.1 vector could not promote IGFBP7 expression and not affect P-actin expression. Transfection rate of pcDNA3.1-IGFBP7 group was approximately 50%, the same to that of pcDNA3.1-CONTROL group. Immunohistochemistry results suggested that pcDNA3.1-IGFBP7 group expressed IGFBP7 more than pcDNA3.1-CONTROL and B16-F10 groups (P<0.01), while IGFBP7 expressions between pcDNA3.1-CONTROL and B16-F10 groups were of no significant difference (P>0.05). Expression of VEGF in the pcDNA3.1-IGFBP7 group was significantly lower than those of pcDNA3.1-CONTROL group and the blank group with significant differences(P<0.006). While expression of Caspase-3 in the tumors of pcDNA3.1-IGFBP7 group was higher (caspase-3 P<0.004), with significant differences, too. We found that IGFBP7 expression was positively correlated to Caspase-3 and cell apoptosis, with correlation coefficients as rs(IGFBP7, Caspase-3)=0.704 and rs(IGFBP7, apoptosis=0.806, P<0.01). While IGFBP7 expression was negatively correlated to the expression of VEGF, with rs(IGFBP7, VEGF=-0.564, P<0.01).Conclusions:In BRAFV600E positive malignant melanoma, gene therapy of pcDNA3.1-IGFBP7 intratumoral injection could promote IGFBP7 expression, and furthermore, reduce in vivo VEGF expression and induce tumor apoptosis, to approach the purpose of inhibiting malignant melanoma growth.