PGI通过HIF-1α影响乳腺癌MCF-7细胞增殖凋亡的机制研究

发布时间：2018-07-12 11:15

本文选题：乳腺癌 + MCF-7细胞　；参考：《重庆医科大学》2017年硕士论文

【摘要】：背景目的近年来,我国乳腺癌的发病率和死亡率均呈持续上升趋势,其中雌激素依赖型乳腺癌发病率约占50%~70%。内分泌治疗对雌激素依赖型乳腺癌具有良好疗效,但其治疗后期转移率高,导致治疗效果不佳。作为肿瘤细胞有氧糖酵解途径的关键酶,磷酸葡萄糖异构酶(phosphoglucose isomerase,PGI)在糖酵解代谢过程中调控葡萄糖-6-磷酸酶与果糖-6-磷酸酶之间的互相转化。PGI分泌到细胞外后被称为自分泌因子(autocrine motility factors,AMF),AMF与自分泌因子受体相结合,促进肿瘤的增殖、浸润和转移。在乳腺癌、肺癌等多种肿瘤中均可见PGI的异常表达,而PGI的异常表达可能还与肿瘤恶性程度密切相关。缺氧诱导因子(hypoxia inducible factor-1,HIF-1)是低氧条件下肿瘤细胞的中心调节因子,参与了肿瘤血管形成、细胞增殖和细胞粘附等一系列过程。HIF-1包括HIF-1α和HIF-1β,HIF-1α主要调控肿瘤的生长和转移,此外,还可以调控HKII、PGI、PKM2和LDHA等基因的转录和表达。新近研究表明:糖酵解关键酶中的PKM2可反向调控HIF-1α的表达。PGI是否也有类似的作用?PGI与HIF-1α二者相关性如何?PGI是怎样进行调控的?目前尚未见报道。因此,本研究对乳腺癌中的PGI和HIF-1α进行检测,探究两者在乳腺癌中的表达情况,通过RNAi技术干扰PGI的表达,进而检测干扰PGI对HIF-1α的表达以及对MCF-7细胞增殖、凋亡和糖酵解的影响,并对其相关调控机制进行初步探究。方法第一部分收集30例乳腺癌患者的癌组织及其相应癌旁组织,通过PCR、免疫组化检测癌组织与癌旁组织中PGI及HIF-1α的表达;用PCR和Western blot检测乳腺癌细胞MCF-7及正常乳腺细胞MCF-10A中PGI以及HIF-1α的表达。第二部分采用慢病毒质粒进行感染,构建干扰PGI的MCF-7细胞并筛选稳转株,采用免疫荧光、RT-PCR和Western blot检测感染效果,并采用Western blot技术检测干扰PGI对MCF-7细胞中HIF-1α蛋白表达的影响。光学显微镜下观察干扰PGI对细胞生长的影响;CCK-8法检测干扰PGI对细胞增殖的抑制作用;Hochest染色以及FCM检测干扰PGI对细胞凋亡的影响;同时通过FCM检测干扰PGI对MCF-7细胞周期的影响;随后,采用Western blot检测干扰PGI对细胞周期以及凋亡通路蛋白的影响。通过乳酸含量检测法检测干扰PGI对MCF-7细胞乳酸产量的影响,并采用Western blot检测干扰PGI对肿瘤糖酵解相关酶表达的影响。结果1.乳腺癌组织中的PGI和HIF-1α均明显高于癌旁组织。2.乳腺癌细胞MCF-7中PGI和HIF-1α的含量高于正常乳腺细胞MCF-10A。3.乳腺癌细胞MCF-7中干扰PGI后HIF-1α的表达量随之减少。4.干扰PGI明显抑制MCF-7细胞的增殖能力。5.干扰PGI可将MCF-7细胞的周期阻滞在G0/G1期,各组G0/G1期所占比例如下:Control组(43.98±0.45)%、Mock组的(45.57±1.16)%、PGI-si RNA组(66.12±1.12)%,差异均具有统计学意义(P0.01)。6.FCM检测结果显示:Control组、Mock组和PGI-si RNA组凋亡率分别为(3.45±0.12)%、(4.35±0.08)%和(11.89±0.65)%,PGI-si RNA组细胞凋亡率高于Control组和Mock组(P0.01)。7.Hochest 33258染色结果显示:Control组和Mock组细胞呈均匀的蓝色荧光;PGI-si RNA组细胞可见不同程度的染色质凝集、核浓缩且发出较强的蓝白色荧光等典型的凋亡形态学改变。8.乳酸含量检测结果显示:Control组、Mock组和PGI-si RNA组乳酸浓度分别为(3.801±0.346)mmol/L、(3.863±0.194)mmol/L和(1.133±0.194)mmol/L,PGI-si RNA组乳酸浓度低于Control组和Mock组(P0.01)。结果提示:干扰PGI可使MCF-7细胞的乳酸总产量降低。9.Western blot检测:干扰PGI后凋亡相关蛋白Bax和Cleaved Caspase-3表达增加,抑凋亡蛋白Bcl-2表达减少;细胞周期相关蛋白Cyclin D1和CDK4及糖酵解相关酶HKII、PKM2和LDHA的表达均减少;PI3K、AKT和m TOR蛋白的表达亦减少。结论1.乳腺癌中HIF-1α与PGI均异常增高。2.干扰PGI后HIF-1α的表达量减少,同时可以抑制MCF-7细胞的糖酵解和细胞增殖并促进细胞的凋亡,其相关调控机制可能与HIF-1α介导的PI3K/AKT/m TOR信号通路有关。
[Abstract]:Background objective in recent years, the incidence and mortality of breast cancer in China are on the upward trend, and the incidence of estrogen dependent breast cancer is about 50%~70%. endocrine therapy for estrogen dependent breast cancer, but its late stage of treatment is high, resulting in poor treatment effect. As a tumor cell aerobic glycolysis way The key enzyme, phosphoglucose isomerase, PGI, regulates the transformation of glucose -6- phosphatase from fructose -6- phosphatase between glucose -6- phosphatase and fructose -6- phosphatase during the secretion of.PGI to the cell and is called the autocrine (autocrine motility factors, AMF), and the AMF is combined with the autocrine receptor to promote the tumor. Proliferation, infiltration and metastasis. The abnormal expression of PGI in all kinds of tumors such as breast cancer and lung cancer, and the abnormal expression of PGI may also be closely related to the malignancy of the tumor. The hypoxia inducible factor (hypoxia inducible factor-1, HIF-1) is the central regulatory factor of the tumor cells under the hypoxic condition, and participates in the angiogenesis and cell proliferation of the tumor. A series of processes, such as cell adhesion,.HIF-1 including HIF-1 alpha and HIF-1 beta, HIF-1 alpha mainly regulate the growth and metastasis of tumor, in addition, it can regulate the transcription and expression of HKII, PGI, PKM2 and LDHA genes. What is the correlation of alpha two? How is PGI regulated? So far, there is no report on the expression of PGI and HIF-1 alpha in breast cancer. The expression of both in breast cancer and the expression of PGI are investigated by RNAi technique, and then the expression of HIF-1 alpha by interfering PGI and the proliferation, apoptosis and glycolysis of MCF-7 cells are detected. In the first part, we collected 30 cases of breast cancer tissues and their corresponding para cancerous tissues, and detected the expression of PGI and HIF-1 alpha in the cancer tissues and para cancerous tissues by PCR, and used PCR and Western blot to detect MCF-7 in mammary gland cancer cells and PGI in MCF-10A of normal breast cells. And the expression of HIF-1 alpha. The second part was infected by the lentivirus plasmid, constructed the MCF-7 cells that interfered with PGI and screened the stable transgenic plants. The effect of the infection was detected by immunofluorescence, RT-PCR and Western blot, and the effect of PGI on the expression of HIF-1 alpha protein in MCF-7 cells was detected by Western blot technique. The interference PGI was observed under the optical microscope. The effect of cell growth; CCK-8 assay was used to detect the inhibitory effect of interfering PGI on cell proliferation; Hochest staining and FCM detected the effect of PGI on cell apoptosis; meanwhile, the effect of PGI on the cell cycle of MCF-7 was detected by FCM; and then Western blot was used to detect the effect of interference PGI on the cell cycle and apoptosis pathway protein. The effect of interference PGI on the output of lactic acid in MCF-7 cells was detected by the content detection method, and the effects of PGI on the expression of glycolysis related enzymes were detected by Western blot. Results 1. the PGI and HIF-1 alpha in the breast cancer tissues were significantly higher than those of.2. breast cancer cells in the Para cancerous tissue, and the PGI and HIF-1 alpha levels were higher than those of normal mammary gland cell MCF-10A.3. milk. The expression of HIF-1 alpha in the adenocarcinoma cell MCF-7 decreased after the interference of the.4. interference PGI significantly inhibited the proliferation of MCF-7 cells and inhibited the MCF-7 cell proliferation ability.5. interference PGI could block the MCF-7 cell cycle in G0/G1 period. The proportion of G0/G1 phase in each group was as follows: Control group (43.98 + 0.45)%, (45.57 + 1.16)%, 66.12 + 1.12%, and the difference was all The results of P0.01.6.FCM detection showed that the apoptosis rate of group Control, Mock and PGI-si RNA group was (3.45 + 0.12)%, (4.35 + 0.08)% and (11.89 + 0.65)%, and the apoptosis rate of PGI-si RNA group was higher than that of Control group and Mock group (P0.01).7.Hochest 33258 staining results showed that the cells and cells showed uniform blue fluorescence. The group cells showed different degrees of chromatin agglutination, nuclear concentration and strong blue white fluorescence and other typical morphological changes of.8. lactic acid. The results of.8. lactic acid content in Control group, Mock group and PGI-si RNA group were (3.801 + 0.346) mmol/L, (3.863 + 0.194) mmol/L and (1.133 + 0.194) mmol/L, and PGI-si RNA group was strong lactic acid. The results were lower than that of group Control and group Mock (P0.01). The results suggest that interference of PGI can reduce the total output of lactic acid in MCF-7 cells by.9.Western blot detection: the expression of Bax and Cleaved Caspase-3, and the decrease of apoptotic protein Bcl-2 expression after PGI, decrease the expression of apoptosis protein Bcl-2 expression. The expression of PI3K, AKT and m TOR also decreased. Conclusion 1. breast cancer, HIF-1 alpha and PGI all increase the decrease of HIF-1 alpha expression after.2. interference PGI, and can inhibit the glycolysis and cell proliferation of MCF-7 cells and promote cell apoptosis. The related regulatory mechanism may be associated with HIF-1 alpha mediated PI3K/AKT/m signaling. The road is related.
【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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