PKM2与MST1相互作用抑制他莫昔芬诱导的人乳腺癌细胞凋亡

发布时间：2018-06-03 10:46

本文选题：他莫昔芬 + M2型丙酮酸激酶　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:探讨4-羟基他莫昔芬(4-hydroxytamoxifen,4-OHT)对M2型丙酮酸激酶(M2-pyruvate kinase,PKM2)表达的影响,研究PKM2与MST1(Mammalian Sterile20-like kinase 1,MST1)相互作用的机制及其对MST1介导的人乳腺癌细胞MCF-7、SKBR3凋亡的影响。方法:1.4-OHT处理人乳腺癌MCF-7、SKBR3细胞及人正常乳腺上皮MCF-10A细胞后,流式检测4-OHT对MCF-7、SKBR3和MCF-10A细胞凋亡的影响,Western blot检测细胞PKM2、Cleaved-Caspase3以及MST1的表达情况。用4-OHT浓度梯度法长期诱导人乳腺癌MCF-7细胞,获得他莫昔芬耐药细胞株MCF-7R,并用CCK-8检测MCF-7和MCF-7R对4-OHT的药物敏感性,Western blot检测PKM2的表达。2.Co-IP实验证明PKM2与MST1存在相互作用,并找出与PKM2作用的MST1的变构体,免疫荧光检测PKM2与MST1在细胞内定位情况。3.构建PKM2的shRNA慢病毒,感染乳腺癌MCF-7细胞株,用4-OHT诱导细胞凋亡,采用Western blot检测PKM2干扰后,乳腺癌细胞MCF-7和SKBR3中凋亡蛋白Cleaved-Caspase3和MST1蛋白的表达,核浆分离和免疫荧光实验检测细胞内MST1的分布情况。4.在4-OHT处理细胞的基础上,通过干扰或者过表达PKM2蛋白,同时过表达MST1,Western blot检测凋亡蛋白Cleaved-Caspase3的表达,流式检测细胞凋亡发生的比例。结果:1.流式和Western blot结果表明,4-OHT能诱导乳腺癌细胞SKBR3和MCF-7发生凋亡,对正常乳腺上皮细胞MCF-10A凋亡没有影响;随着浓度的增高,PKM2表达逐渐减少,cl-Casepase3和cl-MST1逐渐增多。他莫昔芬耐药细胞株MCF-7R的PKM2表达量明显升高。2.Co-IP结果显示MST1与PKM2存在相互作用,且与PKM2相互作用的MST1的变构体为cl-MST1,他莫昔芬能明显减弱此蛋白间的相互作用。免疫荧光实验显示,在MCF-7和SK-BR-3细胞中,PKM2与MST1主要共定位于细胞浆中。3.采用sh-PKM2慢病毒感染乳腺癌MCF-7细胞,Western blot实验结果表明PKM2的表达显著减少,cl-MST1增多,核浆蛋白分离实验和免疫荧光证实,PKM2干扰能够促进MST1剪切后入核,从而促进细胞的凋亡。4.Western blot结果表明,PKM2过表达能够抑制凋亡蛋白cl-Caspase3的表达,反之,PKM2干扰能够增强cl-Caspase3的表达。流式结果表明,PKM2过表达能够抑制MST1介导的细胞凋亡作用,反之,PKM2干扰能够增强MST1介导的细胞凋亡作用。结论:1.他莫昔芬促进乳腺癌MCF-7和SKBR3细胞凋亡,并且下调PKM2的表达,同时促进cl-MST1的表达。2.MST1与PKM2存在相互作用,与PKM2相互作用的MST1的变构体为cl-MST1.3.干扰PKM2能够上调cl-MST1并促进其入核。4.PKM2抑制MST1介导的细胞凋亡,PKM2可能是MST1上游的调控乳腺癌细胞凋亡的重要靶点。
[Abstract]:Aim: to investigate the effect of 4-hydroxytamoxifenn 4-OHT on the expression of M2-pyruvate kinase M2-pyruvate kinasePKM2, and to study the mechanism of interaction of PKM2 with MST1(Mammalian Sterile20-like kinase 1 MST1 and its effect on apoptosis of MCF-7SKBR3 cell line MCF-7SKBR3 mediated by MST1. Methods Human breast cancer MCF-7 SKBR3 cells and normal breast epithelial MCF-10A cells were treated with: 1.4-OHT. The effect of 4-OHT on apoptosis of MCF-7SKBR3 and MCF-10A cells was detected by flow cytometry. The expression of PKM2Cleaved-Caspase3 and MST1 in MCF-7SKBR3 and MCF-10A cells were detected by Western blot. Human breast cancer MCF-7 cells were induced by 4-OHT concentration gradient method for a long time, and tamoxifen resistant cell line MCF-7R was obtained. The expression of PKM2 was detected by MCF-7 and MCF-7R sensitivity to 4-OHT by CCK-8. 2. Co-IP experiments showed that PKM2 interacted with MST1. The mutagenesis of MST1 interacting with PKM2 was found, and the localization of PKM2 and MST1 in cells was detected by immunofluorescence. ShRNA lentivirus of PKM2 was constructed and infected with MCF-7 cell line of breast cancer. Apoptosis was induced by 4-OHT. Western blot was used to detect the expression of Cleaved-Caspase3 and MST1 proteins in MCF-7 and SKBR3 of breast cancer cells after PKM2 interference. Nuclear and cytoplasmic isolation and immunofluorescence assay were used to detect the distribution of MST1 in cells. On the basis of 4-OHT treatment, the expression of PKM2 protein was detected by interfering or overexpressing PKM2 protein, and the expression of apoptotic protein Cleaved-Caspase3 was detected by overexpression of MST1 blot, and the percentage of apoptosis was detected by flow cytometry. The result is 1: 1. The results of flow cytometry and Western blot showed that 4-OHT could induce apoptosis of SKBR3 and MCF-7 in breast cancer cells, but had no effect on MCF-10A apoptosis of normal breast epithelial cells, and decreased the expression of MCF-10A M2 and increased cl-MST1 gradually with the increase of concentration. The expression of PKM2 in tamoxifen resistant cell line MCF-7R was significantly increased. 2. The results of co-IP showed that MST1 interacted with PKM2, and the MST1 interacting with PKM2 was cl-MST1, and tamoxifen could attenuate the interaction between MST1 and PKM2. Immunofluorescence assay showed that PKM2 and MST1 were mainly located in cytoplasm of MCF-7 and SK-BR-3 cells. Western blot assay of breast cancer MCF-7 cells infected with sh-PKM2 lentivirus showed that the expression of PKM2 decreased significantly. The nucleoplasma protein separation assay and immunofluorescence confirmed that PKM2 interference could promote the entry of MST1 into the nucleus after shearing. The results of Western blot showed that the overexpression of PKM2 could inhibit the expression of apoptotic protein cl-Caspase3, whereas the interference of PKM2 could enhance the expression of cl-Caspase3. The results of flow cytometry showed that the overexpression of PKM2 could inhibit the apoptosis induced by MST1, whereas the interference of PKM2 could enhance the apoptosis induced by MST1. Conclusion 1. Tamoxifen promoted the apoptosis of MCF-7 and SKBR3 cells and down-regulated the expression of PKM2 in breast cancer. At the same time, it promoted the expression of cl-MST1. 2. MST1 interacted with PKM2, and the variant of MST1 interacting with PKM2 was cl-MST1.3. Interfering with PKM2 can up-regulate cl-MST1 and promote its entry. 4. PKM2 inhibits apoptosis mediated by MST1. PKM2 may be an important target of MST1 upstream in regulating apoptosis of breast cancer cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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