TYK2和突变型P53对乳腺癌MDA-MB-231细胞株生物学行为的影响

发布时间：2018-03-07 00:30

本文选题：TYK2　切入点：P53　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:三阴性乳腺癌(Triple-negative breast cancer,TNBC)不表达雌激素受体(ER)、孕激素受体(PR)和缺乏人类表皮生长因子受体2(HER2),这类肿瘤具有侵袭性表型和预后较差的特点,虽然部分患者对化疗敏感,但很快出现化疗抵抗。因此,该类型乳腺癌缺乏具体的针对性治疗方法。无受体酪氨酸激酶2(Non-receptor tyrosine kinase 2,TYK2)是JAK家族的一员,参与多种细胞因子信号通路,和人类癌症的发生关系密切。既往研究表明TYK2在三阴性乳腺癌细胞系中过表达,但TYK2在参与三阴性乳腺癌发生、发展中涉及的相关分子机制尚不明确。人类大多数肿瘤与P53基因变异有关,野生型P53是调节细胞周期进程的肿瘤抑制基因,在DNA损伤修复,细胞衰老,细胞凋亡中起重要作用。而突变型P53(mtP53)能够表现出促进肿瘤细胞的迁移、侵袭以及抗凋亡等恶性生物学行为。研究表明P53在三阴性乳腺癌MDA-MB-231细胞株中呈突变表达,且突变型P53和三阴性乳腺癌较差临床预后相关。近些年来,关于P53与JAK家族的多个成员相互作用关系的研究较多,但是探讨TYK2与突变型P53关系的相关报道较少。研究目的:明确TYK2对乳腺癌MDA-MB-231细胞株增殖、迁移、凋亡等生物学行为的影响。进一步明确在三阴性乳腺癌中TYK2和mtP53的相互作用。为三阴性乳腺癌的治疗提供新的思路及实验依据。方法:(1)利用siRNA瞬时转染技术,将TYK2-siRNA及P53-siRNA分别转染至三阴性乳腺癌细胞株MDA-MB-231中,同时将Negative control转染至MDA-MB-231细胞中作为阴性对照。采用Western blot检测三组细胞中TYK2、P53的蛋白表达情况。(2)采用MTT方法检测转染TYK2-siRNA和(或)P53-siRNA后MDA-MB-231细胞的增殖情况。(3)采用划痕实验方法检测TYK2及P53基因沉默后,乳腺癌MDA-MB-231细胞的迁移情况。(4)通过流式细胞仪对转染siRNA的细胞和NC组的细胞进行凋亡检测。结果:1.Western blot结果提示与NC组比较,在乳腺癌MDA-MB-231细胞中干扰P53基因后,P53基因被沉默,TYK2表达也下调。干扰TYK2基因后,TYK2基因被沉默,P53基因表达也随之下调。2.MTT结果提示:与NC组比较,联合或者单转染siTYK2、siP53后,乳腺癌MDA-MB-231细胞的增殖率均下降。3.划痕实验示:联合转染组及单转染siTYK2组和siP53组均较NC组细胞迁移能力下降。但联合组较单转染组迁移能力未见明显升高。4.流式细胞术示:联合或者分别转染siTYK2、siP53后,对比NC组,乳腺癌MDA-MB-231细胞凋亡率升高。结论:1.沉默TYK2基因的表达可以抑制乳腺癌MBA-MB-231细胞的P53基因的表达,而沉默P53基因对乳腺癌MBA-MB-231细胞的TYK2基因的表达也有抑制作用,提示TYK2和mtpP53在三阴性乳腺癌的发生和发展中具有一定的关联作用,两者可以相互通过某种信号通路或某种基因直接或间接地调控基因的转录和翻译。2.沉默TYK2基因能够导致乳腺癌MDA-MB-231细胞株凋亡的增加,增殖及迁移能力降低,提示TYK2基因能够通过某种机制影响MDA-MB-231细胞株的增殖、迁移及凋亡等生物学行为。3.沉默mtP53能够导致乳腺癌MDA-MB-231细胞株凋亡的增加,增殖及迁移能力降低,在三阴性乳腺癌的发展中起着重要作用。
[Abstract]:Background: three negative breast cancer (Triple-negative breast, cancer, TNBC) is the expression of estrogen receptor (ER), progesterone receptor (PR) and the lack of human epidermal growth factor receptor 2 (HER2), which has the characteristics of tumor invasive phenotype and poor prognosis, although part of the patients are sensitive to chemotherapy, but soon resistance to chemotherapy. Therefore, this type of breast cancer is lack of specific targeted therapies. Non receptor tyrosine kinase 2 (Non-receptor tyrosine 2 kinase, TYK2) is a member of the JAK family, is involved in a variety of cytokine signaling pathways, and human cancer are closely related. Previous study showed that overexpression of TYK2 in three negative breast cancer cells lines, but TYK2 in three negative breast cancer, the molecular mechanism involved in the development is not clear. The most human tumors with P53 gene mutation, wild type P53 is the regulation of cell cycle progression is swollen Tumor suppressor gene, DNA repair, cell senescence, play an important role in apoptosis. Mutant P53 (mtP53) can show to promote tumor cell migration, invasion and apoptosis of malignant biological behavior. The research indicated that the P53 was expressed in three mutation negative breast cancer cell line MDA-MB-231, and mutant P53 and poor clinical outcome three negative breast cancer. In recent years, more research about relationship between multiple members of the JAK family and P53 interaction, but related to the reported TYK2 and mutant P53 relationship less. Objective: To explore the TYK2 on the proliferation and migration of breast cancer cell line MDA-MB-231, the influence of biological behavior of apoptosis. To further clarify the interaction of TYK2 in three negative breast cancer and mtP53. Provide new ideas and experimental evidence for the treatment of three negative breast cancer. Methods: (1) using siRNA to instantaneous staining technique, TYK2-s IRNA and P53-siRNA were transfected with three negative breast cancer cell line MDA-MB-231, and will be used as negative control Negative control was transfected into MDA-MB-231 cells. Using Western blot detection of three groups of TYK2 cells, the expression of P53 protein. (2) transfection and TYK2-siRNA were detected by MTT method (or) the proliferation of MDA-MB-231 cells after P53-siRNA. (3) detected by silencing TYK2 and P53 gene scratch test method, the migration of breast cancer MDA-MB-231 cells. (4) were detected by cell apoptosis and NC group of transfected siRNA cells by flow cytometry. Results: 1.Western blot showed that compared with group NC, the interference of P53 gene in breast cancer MDA-MB-231 cells, P53 genes are silenced, TYK2 expression was down regulated. The interference of TYK2 gene, TYK2 gene was silenced, the expression of P53 gene with the down-regulation of.2.MTT results showed that compared with the NC group, the joint or single turn With siTYK2, siP53,.3. were decreased in wound healing rate of MDA-MB-231 breast cancer cell proliferation: the co transfection group and siTYK2 transfection group and siP53 group were lower than NC group cell migration ability decreased. But the combined group than single transfection group there was no significant increase in the migration ability of.4. flow cytometry showed that the transfection of siTYK2 or combined siP53, NC, contrast group, breast cancer MDA-MB-231 cell apoptosis rate increased. Conclusion: the expression of P53 1. gene expression silencing TYK2 gene can inhibit breast cancer MBA-MB-231 cells, and the expression of TYK2 gene silencing P53 gene on MBA-MB-231 breast cancer cells also inhibited, suggesting that TYK2 and mtpP53 have a certain correlation function in the occurrence and development of three negative breast cancer, both can be through the transcription and translation of.2. gene silence of TYK2 gene regulation a signaling pathway or directly or indirectly can lead to milk Increased apoptosis of adenocarcinoma MDA-MB-231 cells, decreased proliferation and migration, suggesting that the TYK2 gene can affect MDA-MB-231 through a mechanism of cell proliferation, migration and apoptosis of.3. and the biological behavior of mtP53 silencing can lead to increased apoptosis of breast cancer MDA-MB-231 cells, reduce the proliferation and migration ability, plays an important role in the development of the three negative breast cancer.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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