Mir-675-5p通过UBQLN1-ZEB1-mir-200通路调控胰腺癌细胞上皮间质转化的机制研究

发布时间：2018-06-29 07:52

本文选题：Mir-675-5p + ZEB1　；参考：《江苏大学》2017年硕士论文

【摘要】：研究目的:分析mir-675-5p在不同人胰腺癌细胞中的表达,及其在癌组织中的表达量与患者生存预后的关系,并探究mir-675-5p对胰腺癌细胞增殖、凋亡、迁移、侵袭等方面的影响和可能的作用机制。研究方法:1.运用SPSS Statistics 20软件及Graphpad Prism5软件对从TCGA数据库中获得的mir-675相关胰腺癌临床样本数据进行统计学分析,获得mir-675与胰腺癌患者生存时间、最大肿瘤直径及肿瘤TNM分期的关系。2.运用qRT-PCR检测不同胰腺癌细胞中mir-675-5p的表达水平。利用RNA干扰技术将mir-675-5p mimics转入相对低表达mir-675-5p的胰腺癌细胞株,将mir-675-5p Inhibitor转入相对高表达mir-675-5p的胰腺癌细胞株。3.分别运用CCK-8法、平板克隆形成试验、流式细胞术、Transwell迁移和侵袭实验检测mir-675-5p对胰腺癌细胞增殖、细胞克隆形成能力、细胞凋亡能力及细胞周期、细胞迁移和侵袭能力的影响,以探讨mir-675-5p对胰腺癌细胞功能学方面的影响。4.运用qRT-PCR检测细胞周期相关基因RB1及细胞侵袭相关基因ZEB1、mir-200家族的表达量变化,Western blotting检测细胞增殖凋亡及侵袭相关指标PCNA、Bcl-2、Bax、cleaved-caspase3、MMP2、MMP9和上皮间质转化相关指标E-cadherin、N-cadherin、ZEB1、Snail、Slug、Vimentin的变化。5.运用RNA干扰技术将UBQLN1 si RNAs转染入胰腺癌细胞使其低表达UBQLN1,并运用qRT-PCR法和Western Blotting法检测UBQLN1 si RNAs的干扰效率;将UBQLN1si RNAs和mir-675-5p mimics共转染入Patu8988细胞,将UBQLN1 si RNAs和mir-675-5p Inhibitor共转染入SW1990细胞,检测共转染后ZEB1的m RNA和蛋白水平的变化。研究结果:1.Mir-675的高表达可使胰腺癌患者获得相对长的生存时间及相对小的最大肿瘤直径,而Mir-675的表达与胰腺癌患者肿瘤TNM分期无相关关系。2.Mir-675-5p差异性表达于4种胰腺癌细胞株(Patu8988Panc-1Bxpc-3SW1990),在Patu8988细胞株中表达量最低,在SW1990细胞株中表达量最高。3.上调mir-675-5p在胰腺癌细胞株Patu8988中的表达能够抑制细胞增殖,降低细胞克隆形成能力,促进细胞凋亡,引起细胞G1/S期阻滞。降低PCNA蛋白水平,同时能诱导活化的Caspase3表达,降低Bcl-2/Bax比值。上调mir-675-5p能够抑制细胞侵袭转移,下调侵袭相关蛋白MMP2及MMP9的蛋白水平,EMT相关指标N-cadherin、ZEB1、Snail、Slug、Vimentin的表达量减少,E-cadherin的表达量增加;而下调mir-675-5p在胰腺癌细胞株SW1990中的表达则能够促进细胞增殖,提高细胞克隆形成能力,促进细胞侵袭转移。升高PCNA蛋白水平,同时能抑制cleaved-caspase3表达,升高Bcl-2/Bax比值。侵袭相关蛋白MMP2及MMP9的蛋白水平上升,EMT相关指标N-cadherin、ZEB1、Snail、Slug、Vimentin的表达量增加,E-cadherin的表达量下降。4.上调mir-675-5p在胰腺癌细胞株Patu8988中的表达,可以使RB1的m RNA水平升高,ZEB1的m RNA转录上调,mir-200家族表达量下降;下调mir-675-5p在胰腺癌细胞株SW1990中的表达,可以使RB1的m RNA水平下降,ZEB1的m RNA转录下调,mir-200家族表达量上升。5.上调mir-675-5p在胰腺癌细胞株Patu8988中的表达,能够使泛素化相关基因UBQLN1的m RNA表达及蛋白表达均升高;下调mir-675-5p在胰腺癌细胞株SW1990中的表达,能够使泛素化相关基因UBQLN1的m RNA转录及蛋白表达均下降。6.下调UBQLN1在已上调mir-675-5p表达的Patu8988细胞株中的表达,可以使ZEB1 m RNA转录水平上升的趋势得到逆转,使ZEB1蛋白表达水平下降的趋势得到逆转;下调UBQLN1在已下调mir-675-5p表达的SW1990细胞株中的表达,可以使ZEB1m RNA表达水平下降的趋势得以维持,使ZEB1蛋白表达水平上升的趋势得以维持。研究结论:1.Mir-675的表达量越高,胰腺癌患者的生存预后相对越好。2.Mir-675-5p具有抑制胰腺癌细胞增殖、促进凋亡,抑制细胞迁移及侵袭等生物学功能。3.Mir-675-5p可引起ZEB1转录后水平的改变,进而引起ZEB1转录水平的反向改变。4.Mir-675-5p可以促进泛素化相关基因UBQLN1的表达,而UBQLN1可以在蛋白水平下调ZEB1蛋白的表达,引起ZEB1的m RNA水平反射性升高。Mir-675-5p可通过中间基因ZEB1而引起mir-200家族的变化,提示两个mi RNA之间也可以互相影响,并且Mir-675-5p可以通过UBQLN1-ZEB1-mir-200环路影响胰腺癌细胞上皮间质转化的过程。
[Abstract]:Objective: to analyze the expression of mir-675-5p in different human pancreatic cancer cells, the relationship between the expression in the cancer tissues and the survival prognosis of the patients, and to explore the effects and possible mechanisms of mir-675-5p on the proliferation, apoptosis, migration and invasion of pancreatic cancer cells. Research methods: 1. using the SPSS Statistics 20 software and Graphpad Pr Ism5 software was used to analyze the data of mir-675 related pancreatic cancer clinical samples obtained from the TCGA database to obtain the relationship between mir-675 and the survival time of the patients with pancreatic cancer, the maximum tumor diameter and the TNM stage of the tumor..2. was used to detect the expression of mir-675-5p in different pancreatic cancer cells by qRT-PCR. RNA interference technique was used to detect mir-675-5p. Mimics was transferred into the pancreatic cancer cell line with relatively low expression of mir-675-5p, and mir-675-5p Inhibitor was transferred to the pancreatic cancer cell line with relatively high mir-675-5p expression,.3., CCK-8 method, plate clone formation test, flow cytometry, Transwell migration and invasion test to detect the proliferation of pancreatic cancer cells and cell clone formation ability. The effect of apoptosis and cell cycle, cell migration and invasiveness in order to explore the effect of mir-675-5p on the functional aspects of pancreatic cancer cells.4. use qRT-PCR to detect cell cycle related genes RB1 and cell invasion related genes ZEB1, miR-200 family expression changes, Western blotting detection of cell proliferation and apoptosis and invasion correlation Indexes such as PCNA, Bcl-2, Bax, cleaved-caspase3, MMP2, MMP9, and epithelial mesenchymal transition related indicators E-cadherin, N-cadherin, ZEB1, Snail, Slug. Co transfection of UBQLN1si RNAs and mir-675-5p mimics into Patu8988 cells and co transfection of UBQLN1 Si RNAs and mir-675-5p Inhibitor into SW1990 cells. There is no correlation between the expression of Mir-675 and the TNM staging of pancreatic cancer patients.2.Mir-675-5p differentially expressed in 4 kinds of pancreatic cancer cell lines (Patu8988Panc-1Bxpc-3SW1990), and the lowest expression in Patu8988 cell lines. The expression of the highest expression of.3. in the SW1990 cell line can be suppressed by the expression of mir-675-5p in the pancreatic cancer cell strain. Cell proliferation, cell clone formation, cell apoptosis, cell G1/S phase block, PCNA protein level, activated Caspase3 expression and Bcl-2/Bax ratio can be induced. Up regulation of mir-675-5p can inhibit cell invasion and metastasis, decrease the protein level of invasion related protein MMP2 and MMP9, EMT related index N-cadherin The expression of ZEB1, Snail, Slug, Vimentin was reduced and the expression of E-cadherin increased, while the expression of mir-675-5p in the pancreatic cancer cell line SW1990 could promote cell proliferation, improve cell cloning and formation, promote cell invasion and metastasis, increase the level of PCNA protein, and inhibit the expression of cleaved-caspase3 and increase the ratio of Bcl-2/Bax. The protein levels of the related proteins MMP2 and MMP9 increased, and the expression of EMT related indicators N-cadherin, ZEB1, Snail, Slug, Vimentin increased, and the expression of E-cadherin expression decreased.4. up mir-675-5p in the pancreatic cancer cell Patu8988. The expression of mir-675-5p in the pancreatic cancer cell line SW1990 can decrease the m RNA level of RB1, the m RNA transcript of ZEB1, and the miR-200 family expression increase.5. increase in the expression of mir-675-5p in the pancreatic cancer cell Patu8988. The expression in the adenocarcinoma cell line SW1990 can decrease the m RNA transcriptional and protein expression of the ubiquitination related gene UBQLN1, and decrease the expression of.6. down UBQLN1 in the Patu8988 cell line that has increased the mir-675-5p expression. The trend of ZEB1 m RNA transcriptional level is reversed and the downward trend of the ZEB1 protein expression level is reversed. The expression of UBQLN1 in the SW1990 cell lines that have reduced the expression of mir-675-5p can maintain the tendency to decrease the expression level of ZEB1m RNA and maintain the trend of the increase in the expression level of ZEB1 protein. The higher the expression of 1.Mir-675, the better the survival of the patients with pancreatic cancer, the better the better.2.Mir-675-5p has the inhibition of the pancreatic cancer. Cell proliferation, promoting apoptosis, inhibiting cell migration and invasion and other biological functions.3.Mir-675-5p can cause changes in ZEB1 post transcriptional level, and then cause the reverse change of ZEB1 transcriptional level.4.Mir-675-5p to promote the expression of ubiquitination related gene UBQLN1, and UBQLN1 can downregulate the expression of ZEB1 protein at the egg white level, causing m RNA of ZEB1. .Mir-675-5p can cause changes in the miR-200 family through the intermediate gene ZEB1, suggesting that the two mi RNA can also interact with each other, and Mir-675-5p can affect the process of the epithelial mesenchymal transition of pancreatic cancer cells through the UBQLN1-ZEB1-mir-200 loop.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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