摘要
目的
1.研究雷公藤内酯醇(TL)对人纤维肉瘤HT-1080细胞基质金属蛋白酶-9(MMP-9)表达的影响;
2.研究TL调控MMP-9表达的意义;
3.探讨TL调控MMP-9表达的机制。
方法
1.选择人纤维肉瘤细胞系HT-1080为研究对象,常规培养、传代,药物的3个工作浓度分别为6nM、12nM和18nM,处理时间均为72小时;
2.采用MMT法检测TL对HT-1080细胞增殖能力的影响;
3.用流式细胞仪检测TL致HT-1080细胞凋亡情况;
4.运用RT-PCR检测TL对9个基因mRNA表达的影响,这些基因是:MMP-9和-2,基质金属蛋白酶组织抑制物(TIMP)-1、-2、-3和-4,DNA甲基转移酶(DNMT)-1、-3A和-3B;
5.用明胶酶谱实验检测TL对HT-1080细胞MMP-9和-2活性的影响;
6.用Transwell侵袭实验检测TL对HT-1080细胞体外侵袭能力的影响;
7.用甲基化特异性PCR(MSP)检测TL对MMP-9基因甲基化水平的影响;
8.用Western blotting检测TL对DNMT-1、-3A和-3B蛋白表达的影响。
结果
1.TL作用72小时,随药物浓度的增加,HT-1080细胞的增殖逐渐受到抑制;其中,半数抑制浓度约为25nM;
2.浓度分别为6 nM、12 nM和18 nM的TL处理72小时后,HT-1080细胞的凋亡率分别为16.0%、17.3%和18.8%,对照组凋亡率为17.7%;
3.18 nM TL处理HT-1080细胞72小时后,MMP-9 mRNA的表达明显下调(P＜0.05),但MMP-2、TIMP-1/-2/-3/-4 mRNA的表达无明显变化(P均＞0.05);6 nM和12 nM TL处理组上述基因的mRNA表达均无明显变化(P均＞0.05);
4.12 nM TL处理HT-1080细胞72小时后,在细胞无血清培养基上清中检测到的MMP-9的活性明显下调(P＜0.05),18 nM TL处理组无血清培养基上清中则基本检测不到MMP-9(P＜0.01);3个药物处理组MMP-2的活性均无明显变化(P均＞0.05);
5.Transwell侵袭实验发现,18nM TL处理72小时后,穿过人工基底膜的细胞数明显减少,约相当于对照组的65%(P＜0.05);抗MMP-9单克隆抗体处理组穿过人工基底膜的细胞数亦明显减少,约相当于对照组的50%(P＜0.05);6nM和12nM TL处理组穿过人工基底膜的细胞数均无明显变化(P均＞0.05);
6.18 nM TL处理HT-1080细胞72小时后,MMP-9基因甲基化水平明显上调(P＜0.05);6nM和12 nM TL处理组MMP-9基因甲基化水平均无明显变化(P均＞0.05);
7.18 nM TL处理HT-1080细胞72小时后,DNMT-1和-3A mRNA及蛋白表达明显上调(P均＜0.05),DNMT-3B mRNA及蛋白表达无明显变化(P＞0.05);6nM和12nM TL处理组DNMT-1、-3A和-3BmRNA及蛋白表达均无明显变化(P均＞0.05)。
结论
1.TL抑制HT-1080细胞的增殖;
2.TL可能通过抑制MMP-9的表达及活性而抑制肿瘤细胞体外侵袭能力;
3.首次证实TL上调MMP-9基因甲基化水平;
4.首次证实TL上调DNMT-1和-3A的表达。
Objective
1.To investigate the effects of triptolide on the expression and activities of matrix metalloproteinase-9(MMP-9)in human fibrosarcoma HT-1080 cells in vitro.
2.To investigate the effects of triptolide on HT-1080 cells migration in Matrigel Matrix-coated Cell Culture Inserts in vitro.
3.To identify the mechanism by which triptolide regulates the expression of MMP-9 in HT-1080 cells.
Methods
1.The antiproliferative effect of triptolide on HT-1080 cells was performed by MTr assay.
2.Flow cytometry(FCM)was used to analyse triptolide-induced apoptosis in HT-1080 cells after 72 hours of treatment.
3.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the expression of 9 genes at mRNA level in HT-1080 cells after 72 hours of treatment with triptolide.The 9 genes were MMP-9/-2, TIMP-1/-2/-3/-4,and DNMT-1/-3A/-3B.
4.Western blotting was used to identify the expression of DNMT-1, -3A and-3B at protein level in HT-1080 cells after 72 hours of treatment with triptolide.
5.Gelatin zymography was employed to determine the expression and activities of MMP-9 and MMP-2 in triptolide treated HT-1080 cells.
6.In vitro migration assays were performed in Matrigel Matrix-coated Cell Culture Inserts to examine the ability of triptolide-treated HT-1080 cells to migrate through Matrigel Matrix(artificial extracellular matrix).
7.Methylation-specific PCR(MSP)was applied to assess the methylation status of MMP-9 promoter in HT-1080 cells after 72 hours of treatment with triptolide.
Results
1.The proliferation of HT-1080 cells was inhibited in a dose-dependent manner after 72 hours of treatment with triptolide.And the triptolide IC_(50)for HT-1080 cells was 25nM.
2.The percentages of apoptotic HT-1080 cells were 16.0%,17.3% and 18.8%after 72 hours of treatment with 6nM,12nM or 18nM triptolide,respectively.And the countpart of control group was 17.7%.
3.The expression of MMP-9 at mRNA level was significantly down-regulated in HT-1080 cells after 72 hours of treatment with 18nM triptolide(P＜0.05)However,the counterparts of MMP-2 and TIMP-1/-2/-3/-4 were not significantly changed(P＞0.05)And no significant change was identified in the expression of the above genes at mRNA level after 72 hours of treatment with 6nM or 12nM triptolide (P＞0.05).
4.Following 72 hours of treatment with 12nM or 18 nM triptolide, gelatinolytic activity of MMP-9 in serum-free media conditioned by HT-1080 cells was statistically reduced,P＜0.05,and P＜0.01,respectively. However,triptolide did not influence the gelatinolytic activity of MMP-2 in HT-1080 cells(P＞0.05).
5.The number of HT-1080 cells migrating through the artificial extracellular matrix was significantly reduced to 65%or 50%after 72 hours of incubation with 18nM triptolide or 3μg/ ml anti-MMP-9 antibody, respectively,relative to the control group(P＜0.05).
6.The methylation level of the MMP-9 promoter was elevated in HT-1080 cells after 72 hours of treatment with 18 nM triptolide(P＜0.05).
7.The expression of DNMT-1 and -3A at both mRNA and protein level was significantly up-regulated in HT-1080 cells after 72 hours of treatment with 18nM triptolide(P＜0.05).However,18nM triptolide did not modulate DNMT-3B Expression in HT-1080 cells(P＞0.05).And no significant change was detected in the expression of the above genes in HT-1080 cells after 72 hours of treatment with 6nM or 12nM triptolide(P＞0.05)
Conclusion
1.Triptolide decreases the proliferation of HT-1080 cells.
2.We detected triptolide significantly inhibits HT-1080 cell invasion in vitro,which may be related to inhibition of the expression and activities of MMP-9 by triptolide.
3.For the first time,we demonstrated that triptolide statistically modulates gene expression via elevated DNA methylation.
4.For the first time,our research suggested that triptolide significantly elevates the expression of DNMT-1 and -3A.
引文
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