miR-100调控PLK1基因在膀胱癌中的生物学功能及膀胱癌尿液miRNA标志物的筛选研究
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摘要
膀胱癌是人类最常见的恶性肿瘤之一,其发病机制复杂,涉及大量基因表达、功能异常及多种信号通路的改变。目前,膀胱癌发生发展的分子遗传学机制还很不清楚。近年来,microRNA (miRNA)在膀胱癌发生发展中的作用及意义受到了越来越多的关注。本论文探讨了miR-100及下游的靶基因PLK1在膀胱癌发生发展及侵袭转移中的生物学功能,对其相关的分子作用机制进行了较为深入的研究。此外,我们也筛选出了膀胱癌的尿液miRNA生物标志物,对发展新的膀胱癌临床无创性诊断的方法具有一定的参考价值。
首先,我们通过膀胱癌组织及癌旁正常粘膜的miRNA表达谱芯片分析,发现miRNA下调可能是膀胱癌发生发展的重要机制。通过进一步的筛选和验证,我们揭示了miR-100在膀胱癌中普遍下调,其表达水平与膀胱癌肿瘤分期、肿瘤分级和淋巴结转移相关。我们通过在体外过表达miR-100,发现其能够抑制膀胱癌细胞的增殖,迁移和侵袭能力,并能调控膀胱癌的细胞周期,使细胞周期阻滞在G2/M期,并能促进细胞凋亡。通过进一步构建miR-100慢病毒过表达载体,我们发现其能在体内抑制肿瘤的生长,这个新发现阐释了膀胱癌发生发展侵袭转移的分子机制,为临床治疗膀胱癌提供了新的思路。
其次,我们通过生物信息学方法预测了miR-100的靶基因,然后再经过生物学实验方法,我们确定了PLK1是膀胱癌中miR-100的靶基因。通过荧光定量RT-PCR,Western Blotting,免疫组化技术和荧光素酶报告基因的方法,我们发现了miR-100通过与PLK1基因mRNA3’UTR序列的不完全互补结合,抑制PLKl的翻译,从而在转录后水平调控PLK1蛋白的表达水平,膀胱癌组织中miR-100表达与PLK1蛋白表达呈现负相关。我们进一步使用PLK1抑制剂(BI2536),发现PLK1直接参与了miR-100诱导的膀胱癌细胞增殖,迁移和侵袭过程,为BI2536针对PLK1靶点治疗膀胱癌提供了切实的理论依据。
最后,我们建立了简单、易行、重复性好的尿液细胞外miRNA提取流程。通过膀胱癌及对照正常人群的尿液miRNA表达谱分析,筛选出了一些在膀胱癌患者尿液中高表达的miRNA,并发现U6可以作为尿液miRNA荧光定量RT-PCR的内参基因。通过验证20个尿液miRNA标志物,我们发现尿液miR-509-5p/miR-124比值显示了较高的准确性,可能是膀胱癌诊断的无创性生物标志物。
综上所述,我们发现了miR-100在膀胱癌中普遍下调,其表达水平与膀胱癌肿瘤分期、肿瘤分级和淋巴结转移相关,过表达miR-100能够抑制膀胱癌细胞的增殖,迁移和侵袭能力,并能调控细胞周期,促进细胞凋亡和抑制膀胱肿瘤的生长。PLK1基因直接参与了miR一100诱导的膀胱癌细胞增殖,迁移和侵袭过程。尿液miR一509-5p/miR-124比值可能是膀胱癌诊断的无创性生物标志物。这些发现为膀胱癌的分子遗传学提供了一个新的视野,也为膀胱癌的临床诊断和治疗提供了新的途径。
Bladder cancer is one of the most common human malignancies, and its pathogenesis is complex, involving a large number of genes'expression, aberrant functions and changes in multiple signaling pathways. At present, the molecular genetics of development and progression of bladder cancer is still unclear. In recent years, the pathological relevance and significance of microRNAs (miRNAs) in bladder cancer have attracted more and more attention. In the present study, we comprehensively studied the expression, biological function and molecular mechanisms of miR-100and its downstream target gene, PLK1, in the pathogenesis of bladder cancer. In addition, we also screened urine based miRNA biomarkers to develop a new clinical non-invasive diagnostic method in bladder cancer.
Firstly, we used miRNA microarray in bladder cancer tissues and adjacent normal mucosa, and found miRNAs'down-regulation was likely an important mechanism of bladder cancer development. After further screening and validation, we revealed miR-100was generally down-regulated and its expression level, was correlated to tumor stage, tumor grade and lymph node metastasis in bladder cancer. The gain of function study showed enhanced expression of miR-100could not only inhibit cell proliferation, migration, invasion, arrest cell cycle in G2/M phase and promote apoptosis in vitro but also inhibit tumor growth in vivo. These discoveries illustrated new molecular mechanisms of development, invasion and metastasis of bladder cancer, and provided a new therapeutic target for clinical treatment in bladder cancer.
Secondly, we predicted target genes of miR-100by bioinformatics, then identified PLK1was a target gene of miR-100in bladder cancer through biological experimental methods. By quantitative RT-PCR, Western Blotting,
immunohistochemical techniques and luciferase reporter gene method, we found
miR-100was partially complement to PLK1mRNA3' UTR sequence and inhibited the
translation of PLK1mRNA, which regulated PLK1in the post-transcriptional
level. The expression level of miR-100was negatively correlated to PLK1
protein expression in bladder cancer. In addition, we used one of PLK1
inhibitors (BI2536) and found PLK1was directly involved in miR-100induced
cell proliferation, migration and invasion in bladder cancer. This could
provide a practical theoretical basis for BI2536in the clinical treatment
of bladder cancer. Finally, we established a simple, easy and reproducible urine based
extracellular miRNA extraction process. By urine miRNA microarray, we filtered
out a series of high expression miRNA in the urine of patients with bladder
cancer. We also found U6can be used as a reference gene in urine miRNA qRT-PCR.
After validation of20urine miRNA markers, we found miR-509-5p/miR-124ratio
showed high accuracy, and therefore could be a non-invasive urine biomarker
for the diagnosis of bladder cancer. In summary, we found that miR-100was generally down-regulated and its
expression level was correlated to tumor stage, tumor grade and lymph node
metastasis in bladder cancer. The gain of function study showed enhanced
expression of miR-100could not only inhibit cell proliferation, migration,
invasion, arrest cell cycle in G2/M phase and promote apoptosis in vitro but
also inhibit tumor growth in vivo. PLK1was directly involved in miR-100induced
cell proliferation, migration and invasion in bladder cancer. miR-509-5p/
miR-124ratio could be a non-invasive urine biomarker for the diagnosis of
bladder cancer. These findings provided a new perspective for the molecular
genetics of bladder cancer pathogenesis and also provided new targets and
methods for the clinical diagnosis and treatment of bladder cancer.
首先,我们通过膀胱癌组织及癌旁正常粘膜的miRNA表达谱芯片分析,发现miRNA下调可能是膀胱癌发生发展的重要机制。通过进一步的筛选和验证,我们揭示了miR-100在膀胱癌中普遍下调,其表达水平与膀胱癌肿瘤分期、肿瘤分级和淋巴结转移相关。我们通过在体外过表达miR-100,发现其能够抑制膀胱癌细胞的增殖,迁移和侵袭能力,并能调控膀胱癌的细胞周期,使细胞周期阻滞在G2/M期,并能促进细胞凋亡。通过进一步构建miR-100慢病毒过表达载体,我们发现其能在体内抑制肿瘤的生长,这个新发现阐释了膀胱癌发生发展侵袭转移的分子机制,为临床治疗膀胱癌提供了新的思路。
其次,我们通过生物信息学方法预测了miR-100的靶基因,然后再经过生物学实验方法,我们确定了PLK1是膀胱癌中miR-100的靶基因。通过荧光定量RT-PCR,Western Blotting,免疫组化技术和荧光素酶报告基因的方法,我们发现了miR-100通过与PLK1基因mRNA3’UTR序列的不完全互补结合,抑制PLKl的翻译,从而在转录后水平调控PLK1蛋白的表达水平,膀胱癌组织中miR-100表达与PLK1蛋白表达呈现负相关。我们进一步使用PLK1抑制剂(BI2536),发现PLK1直接参与了miR-100诱导的膀胱癌细胞增殖,迁移和侵袭过程,为BI2536针对PLK1靶点治疗膀胱癌提供了切实的理论依据。
最后,我们建立了简单、易行、重复性好的尿液细胞外miRNA提取流程。通过膀胱癌及对照正常人群的尿液miRNA表达谱分析,筛选出了一些在膀胱癌患者尿液中高表达的miRNA,并发现U6可以作为尿液miRNA荧光定量RT-PCR的内参基因。通过验证20个尿液miRNA标志物,我们发现尿液miR-509-5p/miR-124比值显示了较高的准确性,可能是膀胱癌诊断的无创性生物标志物。
综上所述,我们发现了miR-100在膀胱癌中普遍下调,其表达水平与膀胱癌肿瘤分期、肿瘤分级和淋巴结转移相关,过表达miR-100能够抑制膀胱癌细胞的增殖,迁移和侵袭能力,并能调控细胞周期,促进细胞凋亡和抑制膀胱肿瘤的生长。PLK1基因直接参与了miR一100诱导的膀胱癌细胞增殖,迁移和侵袭过程。尿液miR一509-5p/miR-124比值可能是膀胱癌诊断的无创性生物标志物。这些发现为膀胱癌的分子遗传学提供了一个新的视野,也为膀胱癌的临床诊断和治疗提供了新的途径。
Bladder cancer is one of the most common human malignancies, and its pathogenesis is complex, involving a large number of genes'expression, aberrant functions and changes in multiple signaling pathways. At present, the molecular genetics of development and progression of bladder cancer is still unclear. In recent years, the pathological relevance and significance of microRNAs (miRNAs) in bladder cancer have attracted more and more attention. In the present study, we comprehensively studied the expression, biological function and molecular mechanisms of miR-100and its downstream target gene, PLK1, in the pathogenesis of bladder cancer. In addition, we also screened urine based miRNA biomarkers to develop a new clinical non-invasive diagnostic method in bladder cancer.
Firstly, we used miRNA microarray in bladder cancer tissues and adjacent normal mucosa, and found miRNAs'down-regulation was likely an important mechanism of bladder cancer development. After further screening and validation, we revealed miR-100was generally down-regulated and its expression level, was correlated to tumor stage, tumor grade and lymph node metastasis in bladder cancer. The gain of function study showed enhanced expression of miR-100could not only inhibit cell proliferation, migration, invasion, arrest cell cycle in G2/M phase and promote apoptosis in vitro but also inhibit tumor growth in vivo. These discoveries illustrated new molecular mechanisms of development, invasion and metastasis of bladder cancer, and provided a new therapeutic target for clinical treatment in bladder cancer.
Secondly, we predicted target genes of miR-100by bioinformatics, then identified PLK1was a target gene of miR-100in bladder cancer through biological experimental methods. By quantitative RT-PCR, Western Blotting,
immunohistochemical techniques and luciferase reporter gene method, we found
miR-100was partially complement to PLK1mRNA3' UTR sequence and inhibited the
translation of PLK1mRNA, which regulated PLK1in the post-transcriptional
level. The expression level of miR-100was negatively correlated to PLK1
protein expression in bladder cancer. In addition, we used one of PLK1
inhibitors (BI2536) and found PLK1was directly involved in miR-100induced
cell proliferation, migration and invasion in bladder cancer. This could
provide a practical theoretical basis for BI2536in the clinical treatment
of bladder cancer. Finally, we established a simple, easy and reproducible urine based
extracellular miRNA extraction process. By urine miRNA microarray, we filtered
out a series of high expression miRNA in the urine of patients with bladder
cancer. We also found U6can be used as a reference gene in urine miRNA qRT-PCR.
After validation of20urine miRNA markers, we found miR-509-5p/miR-124ratio
showed high accuracy, and therefore could be a non-invasive urine biomarker
for the diagnosis of bladder cancer. In summary, we found that miR-100was generally down-regulated and its
expression level was correlated to tumor stage, tumor grade and lymph node
metastasis in bladder cancer. The gain of function study showed enhanced
expression of miR-100could not only inhibit cell proliferation, migration,
invasion, arrest cell cycle in G2/M phase and promote apoptosis in vitro but
also inhibit tumor growth in vivo. PLK1was directly involved in miR-100induced
cell proliferation, migration and invasion in bladder cancer. miR-509-5p/
miR-124ratio could be a non-invasive urine biomarker for the diagnosis of
bladder cancer. These findings provided a new perspective for the molecular
genetics of bladder cancer pathogenesis and also provided new targets and
methods for the clinical diagnosis and treatment of bladder cancer.
引文
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