抑制MTDH基因对前列腺癌DU145细胞有氧糖酵解影响的研究
发布时间：2018-10-14 10:48
【摘要】：前列腺癌是常见的男性泌尿系肿瘤,在美国其发病率已经超过肺癌,成为第一位危害男性健康的肿瘤。晚期前列腺癌常因发生远处转移并容易产生化学耐药和雄激素抵抗而发展为去势抵抗性前列腺癌。异黏蛋白(metadherin,MTDH)是一个与肿瘤发生、发展密切相关的癌基因,参与调节肿瘤细胞增殖、侵袭转移、血管生成、抵抗凋亡和化学耐药等生物学行为,其在前列腺癌组织中的表达明显高于前列腺增生和正常前列腺组织。肿瘤中不仅存在基因异常,同时还具有能量代谢异常,偏好有氧糖酵解方式产生能量是肿瘤的一个重要特征。但在前列腺癌中异常表达的MTDH是否与肿瘤细胞糖酵解有关尚不是十分清楚。因此,本研究拟利用药物抑制MTDH的表达和小干扰RNA技术(small interfering RNA,si RNA)下调MTDH的表达,观察其对前列腺癌细胞糖酵解的影响及其分子作用机制,为MTDH作为前列腺癌的潜在治疗靶点提供理论和实验依据。隐丹参酮抑制MTDH表达对前列腺癌DU145细胞中糖酵解的影响目的:探讨隐丹参酮抑制MTDH表达对前列腺癌DU145细胞中糖酵解的影响。方法:不同浓度隐丹参酮作用DU145细胞不同时间后,MTT检测各组细胞的增殖情况;葡萄糖和乳酸检测试剂盒分别检测各组细胞葡萄糖消耗值及乳酸分泌值;Western blot和逆转录PCR(RT-PCR)检测各组细胞中MTDH和丙酮酸激酶M2(PKM2)的蛋白和m RNA表达情况。结果:隐丹参酮能够抑制DU145细胞的增殖,并且呈明显时间和剂量依赖性(P0.05)。随着隐丹参酮浓度的增加,DU145细胞对葡萄糖的消耗值减少,乳酸分泌值也下降(P0.05)。MTDH和PKM2蛋白的表达可随隐丹参酮浓度的增加及作用时间的延长逐渐下调,而且随着作用时间的延长,MTDH和PKM2 m RNA的表达量也逐渐下降(P0.05)。结论:隐丹参酮能够抑制前列腺癌DU145细胞的增殖及其糖酵解水平,其可能机制是通过下调MTDH和PKM2的表达,进而抑制细胞的糖酵解途径,最终抑制肿瘤细胞的增殖。sh RNA下调MTDH表达对前列腺癌DU145细胞中糖酵解的影响目的:探讨利用shRNA下调MTDH表达后对前列腺癌DU145细胞中糖酵解的影响。方法:建立稳定沉默MTDH的前列腺癌细胞株和空载体细胞株,倒置显微镜下观察细胞形态的改变,细胞免疫组化、Western blot和逆转录PCR技术检测干扰效果;MTT检测MTDH基因下调对DU145细胞增殖的影响;葡萄糖和乳酸检测试剂盒分别检测MTDH基因下调后细胞葡萄糖消耗值和乳酸分泌值;Western blot检测MTDH基因下调后细胞中AKT、p-AKT、PKM2蛋白的表达情况,逆转录PCR检测PKM2 m RNA的表达水平。结果:MTDH基因下调后,细胞形态变圆、间距变大,细胞的增殖能力显著下降(P0.05)。细胞对葡萄糖消耗和乳酸分泌也明显减少(P0.05)。P-AKT和PKM2蛋白的表达下降,而AKT蛋白的表达无明显改变,且PKM2 m RNA的表达也相对下调(P0.05)。结论:MTDH基因表达下调可抑制前列腺癌DU145细胞的增殖和糖酵解水平,并且我们发现下调MTDH基因表达可以抑制PI3K/AKT信号通路,下调PKM2的表达,从而抑制前列腺癌DU145的糖酵解途径。
[Abstract]:Prostate cancer is a common male urinary tract tumor, whose incidence has exceeded lung cancer in the United States, becoming the first tumor to risk male health. Advanced prostate cancer is often developed as castration-resistant prostate cancer due to distant metastasis and susceptibility to chemical resistance and androgen resistance. metadherin (mtdh) is an oncogene closely related to tumorigenesis and development, and is involved in regulating the biological behaviors of tumor cell proliferation, invasion and metastasis, angiogenesis, resistance to apoptosis and chemical resistance, Its expression in prostate cancer tissue is significantly higher than that of prostate hyperplasia and normal prostate tissue. There is not only gene abnormality in the tumor, but also the abnormal metabolism of energy metabolism. It is an important feature of the tumor to generate energy by preference for aerobic glycolysis. However, it is not very clear whether MTDH abnormally expressed in prostate cancer is related to tumor cell glycolysis. Therefore, it is proposed to use drugs to inhibit MTDH expression and small interfering RNA (si RNA) down-regulate the expression of MTDH, observe its effect on the glycolysis of prostate cancer cells and its molecular mechanism, and provide theoretical and experimental basis for the potential therapeutic targets of MTDH as prostate cancer. Objective: To investigate the effect of cryptotanshinone on glycolysis in DU145 cells of prostate cancer by inhibiting the expression of MTDH in prostate cancer DU145 cells. Methods: After different concentrations of cryptotanshinone on DU145 cells, MTT was used to detect the proliferation of each group, and glucose and lactic acid detection kits were used to detect glucose consumption and lactic acid secretion in each group. Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of MTDH and pyruvate kinase M2 (PKM2) in each group. Results: Cryptotanshinone could inhibit the proliferation of DU145 cells, and showed significant time and dose-dependent (P <0.05). With the increase of cryptotanshinone concentration, the depletion of glucose in DU145 cells decreased and the secretion of lactic acid decreased (P0.05). The expression of MTDH and PKM2 protein could decrease gradually with the increase of cryptotanshinone concentration and the prolongation of action time, and with the prolongation of action time, The expression of MTDH and PKM2 mRNA decreased gradually (P 0.05). Conclusion: cryptotanshinone can inhibit the proliferation of prostate cancer DU145 cells and its sugar level. The possible mechanism is to decrease the expression of MTDH and PKM2, so as to inhibit the glycolytic pathway of the cells and finally inhibit the proliferation of tumor cells. Effect of sh RNA down-regulation of MTDH expression on glycolysis in prostate cancer DU145 cells: To investigate the effect of MTDH expression on glycolysis in prostate cancer DU145 cells. Methods: To establish a stable silencing MTDH prostate cancer cell line and an empty vector cell line, observe the changes of cell morphology, cell immunohistochemistry, Western blot and reverse transcription polymerase chain reaction (RT-PCR) under an inverted microscope, and detect the effect of MTDH gene down-regulation on the proliferation of DU145 cells. The expression of AKT, p-AKT and PKM2 protein was detected by Western blot, and the expression level of PKM2 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Results: After the down-regulation of the MTDH gene, the cell morphology became round, the interval became big, and the proliferation ability of the cells decreased significantly (P0.05). There was no significant change in the expression of AKT and PKM2 protein, but the expression of PKM2 mRNA was also decreased (P <0.05). Conclusion: The down-regulation of MTDH gene can inhibit the proliferation and glycolysis of prostate cancer DU145 cells, and we find that the down-regulation of MTDH gene expression can inhibit the expression of p38/ AKT signaling pathway and down-regulate the expression of PKM2, so as to inhibit the glycolysis pathway of prostate cancer DU145.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.25
本文编号：2270223
[Abstract]:Prostate cancer is a common male urinary tract tumor, whose incidence has exceeded lung cancer in the United States, becoming the first tumor to risk male health. Advanced prostate cancer is often developed as castration-resistant prostate cancer due to distant metastasis and susceptibility to chemical resistance and androgen resistance. metadherin (mtdh) is an oncogene closely related to tumorigenesis and development, and is involved in regulating the biological behaviors of tumor cell proliferation, invasion and metastasis, angiogenesis, resistance to apoptosis and chemical resistance, Its expression in prostate cancer tissue is significantly higher than that of prostate hyperplasia and normal prostate tissue. There is not only gene abnormality in the tumor, but also the abnormal metabolism of energy metabolism. It is an important feature of the tumor to generate energy by preference for aerobic glycolysis. However, it is not very clear whether MTDH abnormally expressed in prostate cancer is related to tumor cell glycolysis. Therefore, it is proposed to use drugs to inhibit MTDH expression and small interfering RNA (si RNA) down-regulate the expression of MTDH, observe its effect on the glycolysis of prostate cancer cells and its molecular mechanism, and provide theoretical and experimental basis for the potential therapeutic targets of MTDH as prostate cancer. Objective: To investigate the effect of cryptotanshinone on glycolysis in DU145 cells of prostate cancer by inhibiting the expression of MTDH in prostate cancer DU145 cells. Methods: After different concentrations of cryptotanshinone on DU145 cells, MTT was used to detect the proliferation of each group, and glucose and lactic acid detection kits were used to detect glucose consumption and lactic acid secretion in each group. Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of MTDH and pyruvate kinase M2 (PKM2) in each group. Results: Cryptotanshinone could inhibit the proliferation of DU145 cells, and showed significant time and dose-dependent (P <0.05). With the increase of cryptotanshinone concentration, the depletion of glucose in DU145 cells decreased and the secretion of lactic acid decreased (P0.05). The expression of MTDH and PKM2 protein could decrease gradually with the increase of cryptotanshinone concentration and the prolongation of action time, and with the prolongation of action time, The expression of MTDH and PKM2 mRNA decreased gradually (P 0.05). Conclusion: cryptotanshinone can inhibit the proliferation of prostate cancer DU145 cells and its sugar level. The possible mechanism is to decrease the expression of MTDH and PKM2, so as to inhibit the glycolytic pathway of the cells and finally inhibit the proliferation of tumor cells. Effect of sh RNA down-regulation of MTDH expression on glycolysis in prostate cancer DU145 cells: To investigate the effect of MTDH expression on glycolysis in prostate cancer DU145 cells. Methods: To establish a stable silencing MTDH prostate cancer cell line and an empty vector cell line, observe the changes of cell morphology, cell immunohistochemistry, Western blot and reverse transcription polymerase chain reaction (RT-PCR) under an inverted microscope, and detect the effect of MTDH gene down-regulation on the proliferation of DU145 cells. The expression of AKT, p-AKT and PKM2 protein was detected by Western blot, and the expression level of PKM2 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Results: After the down-regulation of the MTDH gene, the cell morphology became round, the interval became big, and the proliferation ability of the cells decreased significantly (P0.05). There was no significant change in the expression of AKT and PKM2 protein, but the expression of PKM2 mRNA was also decreased (P <0.05). Conclusion: The down-regulation of MTDH gene can inhibit the proliferation and glycolysis of prostate cancer DU145 cells, and we find that the down-regulation of MTDH gene expression can inhibit the expression of p38/ AKT signaling pathway and down-regulate the expression of PKM2, so as to inhibit the glycolysis pathway of prostate cancer DU145.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.25
【参考文献】
相关期刊论文 前1条
1 邓年丰;冯云枝;;舌鳞状细胞癌组织中EphA7和MTDH表达及其临床病理意义[J];中南大学学报(医学版);2011年12期
，本文编号：2270223
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