miR-7介导加味四君子汤含药血清抑制B16恶性黑色素瘤细胞侵袭和相关蛋白MMP2和MMP9表达的影响
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摘要
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。
Objective: To investigate whether miR-7 mediated the plus Sijunzi decoction containing serum inhibiting B16 malignant melanoma cell invasion and related protein MMP2 and MMP9 expression. Methods: Plus Sijunzi decoction was administrated by serum pharmacological method,which B16 melanoma cells were cultured in 37℃,5% CO2 thermostat incubator,and logarithmic phase cells were collected and exposed to final concentration 10% mice containing serum in 24 h. The experimental group were divided into blank control group,serum control group,negative control group,miR-7 mimic group,miR-7 inhibitor group,and each group contained 5 samples. MiR-7 mimic and miR-7 inhibitor were transfected into B16 malignant melanoma cells,respectively. Transfection efficiency of miR-7 mimic and miR-7 inhibitor were detected by Real-time PCR. Transwell assay were used to analyse miR-7 mediated Plus Si Jun Zi Decoction containing serum influence on B16 malignant melanoma invasion. The mRNA and proein of MMP2 and MMP9 expression were observed and analysed by Real-time PCR and Western Blot,respectively. Results: MiR-7 mimic and miR-7 inhibitor were successfully transfected into B16 malignant melanoma cell. The vaule of miR-7 Relative Conc in mimic and inhibitor group was( 8. 47 ± 0. 28) and( 0. 19 ± 0. 01) respectively. There was a significant increase and decrease compared to the control group,serum control group and negative control group. MiR-7 mimic group decrease significantly B16 malignant melanoma cell invasion and MMP2,MMP9 expression compared to the blank control group,serum control group and negative control group. The results of miR-7 inhibitor group were opposite to miR-7 mimic group. Conclusion: MiR-7 mediated the plus Sijunzi decoction containing serum inhibiting B16 malignant melanoma cell invasion and related protein MMP2 and MMP9 expression,which could be an important molecule of plus Sijunzi decoction antitumor.
Objective: To investigate whether miR-7 mediated the plus Sijunzi decoction containing serum inhibiting B16 malignant melanoma cell invasion and related protein MMP2 and MMP9 expression. Methods: Plus Sijunzi decoction was administrated by serum pharmacological method,which B16 melanoma cells were cultured in 37℃,5% CO2 thermostat incubator,and logarithmic phase cells were collected and exposed to final concentration 10% mice containing serum in 24 h. The experimental group were divided into blank control group,serum control group,negative control group,miR-7 mimic group,miR-7 inhibitor group,and each group contained 5 samples. MiR-7 mimic and miR-7 inhibitor were transfected into B16 malignant melanoma cells,respectively. Transfection efficiency of miR-7 mimic and miR-7 inhibitor were detected by Real-time PCR. Transwell assay were used to analyse miR-7 mediated Plus Si Jun Zi Decoction containing serum influence on B16 malignant melanoma invasion. The mRNA and proein of MMP2 and MMP9 expression were observed and analysed by Real-time PCR and Western Blot,respectively. Results: MiR-7 mimic and miR-7 inhibitor were successfully transfected into B16 malignant melanoma cell. The vaule of miR-7 Relative Conc in mimic and inhibitor group was( 8. 47 ± 0. 28) and( 0. 19 ± 0. 01) respectively. There was a significant increase and decrease compared to the control group,serum control group and negative control group. MiR-7 mimic group decrease significantly B16 malignant melanoma cell invasion and MMP2,MMP9 expression compared to the blank control group,serum control group and negative control group. The results of miR-7 inhibitor group were opposite to miR-7 mimic group. Conclusion: MiR-7 mediated the plus Sijunzi decoction containing serum inhibiting B16 malignant melanoma cell invasion and related protein MMP2 and MMP9 expression,which could be an important molecule of plus Sijunzi decoction antitumor.
引文
1周昕欣,王彩霞.加味四君子汤含药血清对B16恶性黑色素瘤细胞增殖和凋亡的影响.中国实验方剂学杂志,2013;19(10)∶175~179
2 周昕欣,杨关林.miR-7介导加味四君子汤含药血清抑制B16恶性黑色素瘤细胞增殖和促进凋亡的研究.中药药理与临床,2014;30(6)∶124~127
3 周昕欣,王彩霞.加味四君子汤含药血清对B16恶性黑色素瘤细胞侵袭和相关蛋白MMP2和MMP9表达的影响.中药药理与临床,2013;29 (1)∶2108~2110
4 马腾,刘丽波,黄波,等.人血管生成素-1的克隆和毕氏酵母表达载体p PIC9K/Ang-1的构建.解剖科学进展,2005;11(3)∶194~196,200
5 Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function.Cell,2004;116(2)∶281~297
6 Shengtao Qu,Yilong Yao,Chao Shang,et al.MicroRNA-330 Is an oncogenic factor in glioblastoma cells by regulating SH3GL2 gene.Plos One,2012;7(9)∶e46010
7 周昕欣,金龙,吴晓兰.加味四君子汤含药血清对B16恶性黑色素瘤细胞NF-κB(p65)信号分子活性和表达的影响.中药药理与临床,2013;29(6)∶115~120
8 Li G,Zhang Y,Qian Y,et al.Interleukin-17A promotes rheumatoid arthritis synoviocytes migration and invasion under hypoxia by increasing MMP2and MMP9 expression through NF-κB/HIF-1αpathway.Mol Immunol,2013;53(3)∶227~236
9 陈华江,王杰军.基质金属蛋白酶的结构及其调节机制.国外医学(肿瘤学分册),2001;28(1)∶20~23
10 虞桂,王阶.miRNA及其调控网络与中医治病求本机制研究.中华中医药杂志,2012;27(11)∶2789~2791
2 周昕欣,杨关林.miR-7介导加味四君子汤含药血清抑制B16恶性黑色素瘤细胞增殖和促进凋亡的研究.中药药理与临床,2014;30(6)∶124~127
3 周昕欣,王彩霞.加味四君子汤含药血清对B16恶性黑色素瘤细胞侵袭和相关蛋白MMP2和MMP9表达的影响.中药药理与临床,2013;29 (1)∶2108~2110
4 马腾,刘丽波,黄波,等.人血管生成素-1的克隆和毕氏酵母表达载体p PIC9K/Ang-1的构建.解剖科学进展,2005;11(3)∶194~196,200
5 Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function.Cell,2004;116(2)∶281~297
6 Shengtao Qu,Yilong Yao,Chao Shang,et al.MicroRNA-330 Is an oncogenic factor in glioblastoma cells by regulating SH3GL2 gene.Plos One,2012;7(9)∶e46010
7 周昕欣,金龙,吴晓兰.加味四君子汤含药血清对B16恶性黑色素瘤细胞NF-κB(p65)信号分子活性和表达的影响.中药药理与临床,2013;29(6)∶115~120
8 Li G,Zhang Y,Qian Y,et al.Interleukin-17A promotes rheumatoid arthritis synoviocytes migration and invasion under hypoxia by increasing MMP2and MMP9 expression through NF-κB/HIF-1αpathway.Mol Immunol,2013;53(3)∶227~236
9 陈华江,王杰军.基质金属蛋白酶的结构及其调节机制.国外医学(肿瘤学分册),2001;28(1)∶20~23
10 虞桂,王阶.miRNA及其调控网络与中医治病求本机制研究.中华中医药杂志,2012;27(11)∶2789~2791