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目的:研究miR-7是否介导加味四君子汤含药血清对恶性黑色素瘤细胞B16迁移侵袭及其相关机制的研究。方法:37℃、5%CO2的恒温培养箱中培养B16恶性黑色素瘤细胞,收集对数期细胞,等效剂量为9.22 g/kg的加味四君子汤终浓度10%的小鼠含药血清,作用24 h。实验分为空白对照组、血清对照组、转染阴性对照组、miR-7模拟物组和miR-7抑制物组,每组含5个样本数,应用Real-time PCR法检测miR-7模拟物和miR-7抑制物的转染效率、应用Transwell实验检测B16细胞侵袭能力,应用Realtime PCR和Western Blot方法检测基质金属蛋白酶(metalloprotease,MMP)2和9基因的mRNA和蛋白的表达。结果:miR-7模拟物和miR-7抑制物成功转染到B16恶性黑色素瘤细胞中,与空白对照组、血清对照组和阴性对照组相比,miR-7模拟物组(Relative Conc值为8.47±0.28)和miR-7抑制物组(Relative Conc值为0.19±0.01)分别显著升高和显著降低miR-7表达;与空白对照组和血清对照组及阴性对照组相比,miR-7模拟物组的细胞的侵袭能力显著降低,miR-7抑制物组的细胞的迁移侵袭能力显著升高;与空白对照组和血清对照组及阴性对照组相比,miR-7模拟物组MMP2和MMP9mRNA和蛋白表达水平显著降低,miR-7抑制物组MMP2和MMP9mRNA和蛋白表达水平显著升高。结论:miR-7介导加味四君子汤含药血清抑制B16恶性黑色素瘤细胞迁移侵袭过程,并且能够显著降低与B16恶性黑色素瘤细胞侵袭相关的蛋白MMP2和MMP9表达,提示miR-7可能是加味四君子汤抗恶性黑色素瘤的重要分子之一。
AIM: To investigate whether miR-7 mediates the invasion and metastasis of malignant melanoma B16 cells and the related mechanisms by modified Sijunzi Decoction-containing serum. Methods: B16 malignant melanoma cells were cultured in a constant temperature incubator at 37 ℃ and 5% CO2. The logarithmic phase cells were collected. The effective dose of the modified Sijunzi Decoction 10% mouse serum containing the equivalent dose of 9.22 g / kg 24 h. The experiment was divided into blank control group, serum control group, transfected negative control group, miR-7 mimics group and miR-7 inhibitor group, each containing 5 samples. The Real-time PCR method was used to detect the miR-7 mimics Transfection assay was used to detect the invasion ability of B16 cells. Realtime PCR and Western Blot were used to detect the expression of mRNA and protein of matrix metalloprotease (MMP) 2 and 9 genes. Results: The miR-7 mimics and miR-7 inhibitors were successfully transfected into B16 malignant melanoma cells. Compared with the blank control group, the serum control group and the negative control group, the miR-7 mimics group (Relative Conc value 8.47 ± 0.28) and miR-7 inhibitor group (Relative Conc value 0.19 ± 0.01), respectively. Compared with the blank control group and the serum control group and the negative control group, miR-7 Mimic group significantly reduced the invasive ability of cells, miR-7 inhibitor group of cells migration and invasion was significantly increased; compared with the blank control group and serum control group and negative control group, miR-7 mimics group MMP2 and MMP9 mRNA and protein expression levels were significantly reduced, miR-7 inhibitor group MMP2 and MMP9 mRNA and protein expression levels were significantly increased. Conclusion: miR-7-mediated Jiajun Sijunzi decoction serum can inhibit the migration and invasion of B16 malignant melanoma cells, and can significantly reduce the expression of MMP2 and MMP9 proteins associated with invasion of B16 malignant melanoma cells, suggesting that miR-7 may be the flavored Sijunzi Soup anti-malignant melanoma one of the important molecules.