miR-203对HaCaT细胞p63和SOCS3表达调控初步研究
发布时间：2019-02-11 09:26
【摘要】：目的：银屑病(psoriasis)是严重影响人类身心健康的自身免疫性皮肤病。导致银屑病表皮细胞过度增殖和异常分化的确切原因尚未确定。miRNAs是内源性非蛋白编码单链小分子RNAs，主要功能为转录后水平抑制靶基因的表达。miRNAs对其靶基因的调节障碍可导致不同疾病的发生。miR-203具有高度皮肤特异性，在银屑病皮损中异常表达，，有报道推测p63和SOCS3是其进化保守的靶基因。miR-203对p63和SOCS3的调节及其在角质形成细胞增殖、分化和银屑病发病机制中的作用尚不明确。STAT3和Notch信号通路在银屑病的发病中起着关键的作用，而p63和SOCS3是上述信号通路的2个重要调节子。 本研究旨在通过将miR-203的模拟物和其抑制物转染人永生化角质形成细胞HaCaT细胞系，确定其对p63和SOCS3的调控机制、明确其与STAT3和Notch信号通路的关系及其在银屑病发病机制中的作用，确定它们的调控方式和相互关系，为其作为银屑病新的治疗靶标提供理论依据。 方法：用miR-203的模拟物和抑制物(miR-203mimic和miR-203inhibitor)定量转染人角质形成细胞系HaCaT细胞。通过qRT-PCR、Western blot、流式细胞术方法检测p63、SOCS3、p-STAT3和Notch1基因及蛋白的表达水平；并用含TNF-α (20ng/ml)和IL-6(20ng/ml)的培养液处理HaCaT细胞，并以qRT-PCR法对miR-203的表达水平进行检测，观察细胞因子TNF-α和IL-6对miR-203表达水平的影响。 结果：1. qRT-PCR检测p63、SOCS3、p-STAT3、Notch1的mRNA表达水平，其中转染miR-203mimic组与对照组相比SOCS3、Notch1、p-STAT3的mRNA表达上调，Notch1组间差异具有显著性(P 0.05)，p63mRNA表达水平无明显变化；转染miR-203inhibitor组与对照组相比SOCS3、Notch1、p-STAT3的mRNA表达下调，SOCS3和Notch1组间差异有显著性(P 0.05)，p63mRNA表达亦无明显变化。2. Western blot和流式细胞术方法检测蛋白表达，转染miR-203mimic的细胞，p63和SOCS3表达下降，p-STAT3和Notch1表达升高;转染miR-203inhibitor的细胞结果与之相反。3. HaCaT细胞经TNF-α和IL-6处理后，经qRT-PCR方法检测miR-203表达升高，差异具有显著性(P 0.05)。 结论：miR-203的过表达，可以抑制p63和SOCS3表达，从而使STAT3持续激活，STAT3的磷酸化水平增加，导致角质形成细胞过度增殖。本研究为探讨miR-203对p63、SOCS3的调控机制及其与STAT3信号转导通路的关系提供了初步的实验基础，有助于了解miR-203在银屑病发病机制中的作用。
[Abstract]:Objective: psoriatic (psoriasis) is an autoimmune dermatosis that seriously affects human physical and mental health. The exact cause of excessive proliferation and abnormal differentiation of psoriatic epidermal cells has not been determined. MiRNAs is an endogenous non-protein encoded single-stranded small molecule RNAs,. The main function of miR-203 is to inhibit the expression of target gene at post-transcriptional level. The regulation of target gene by miRNAs may lead to the occurrence of different diseases. MiR-203 has high skin specificity and abnormal expression in psoriatic lesions. It has been reported that p63 and SOCS3 are the conserved target genes of p63 and SOCS3. MiR-203 regulates p63 and SOCS3 and proliferates in keratinocytes. The role of differentiation and psoriasis pathogenesis is unclear. STAT3 and Notch signaling pathway play a key role in psoriasis pathogenesis, while p63 and SOCS3 are two important regulators of these signaling pathways. The aim of this study was to determine the regulatory mechanism of miR-203 on p63 and SOCS3 by transfection of immortalized human keratinocytes HaCaT cell line with miR-203 mimic and its inhibitor. To clarify its relationship with STAT3 and Notch signaling pathway and its role in the pathogenesis of psoriasis, to determine their regulatory mode and mutual relationship, to provide a theoretical basis for its new therapeutic target for psoriasis. Methods: human keratinocytes HaCaT cells were quantitatively transfected with miR-203 mimics and inhibitors (miR-203mimic and miR-203inhibitor). The expression of p-STAT3 and Notch1 genes and proteins were detected by qRT-PCR,Western blot, flow cytometry. HaCaT cells were treated with TNF- 伪 (20ng/ml) and IL-6 (20ng/ml), and the expression of miR-203 was detected by qRT-PCR method. The effects of cytokines TNF- 伪 and IL-6 on miR-203 expression were observed. Results: 1. QRT-PCR was used to detect the mRNA expression of p63- SOCS3pSTAT3pSTAT3ntch1. The expression of SOCS3,Notch1,p-STAT3 mRNA was up-regulated in the transfected miR-203mimic group compared with the control group. There was significant difference between the Notch1 group and the Notch1 group (P 0.05), but the p63mRNA expression level did not change significantly. Compared with the control group, the mRNA expression of SOCS3,Notch1,p-STAT3 in miR-203inhibitor transfected group was down-regulated, the difference between SOCS3 and Notch1 group was significant (P 0.05), and the expression of p63mRNA was not changed significantly. 2. Western blot and flow cytometry were used to detect the protein expression. The expression of p63 and SOCS3 decreased and the expression of p-STAT3 and Notch1 increased in the cells transfected with miR-203mimic, whereas the expression of p-STAT3 and Notch1 was increased in the transfected miR-203inhibitor cells. After HaCaT cells were treated with TNF- 伪 and IL-6, the expression of miR-203 was detected by qRT-PCR method, and the difference was significant (P 0.05). Conclusion: overexpression of miR-203 can inhibit the expression of p63 and SOCS3, which leads to the sustained activation of STAT3 and the increase of phosphorylation of STAT3, resulting in excessive proliferation of keratinocytes. This study provides a preliminary experimental basis for the study of the regulatory mechanism of miR-203 on p63 SOCS3 and its relationship with STAT3 signal transduction pathway, and helps to understand the role of miR-203 in the pathogenesis of psoriasis.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R758.63
本文编号：2419600
[Abstract]:Objective: psoriatic (psoriasis) is an autoimmune dermatosis that seriously affects human physical and mental health. The exact cause of excessive proliferation and abnormal differentiation of psoriatic epidermal cells has not been determined. MiRNAs is an endogenous non-protein encoded single-stranded small molecule RNAs,. The main function of miR-203 is to inhibit the expression of target gene at post-transcriptional level. The regulation of target gene by miRNAs may lead to the occurrence of different diseases. MiR-203 has high skin specificity and abnormal expression in psoriatic lesions. It has been reported that p63 and SOCS3 are the conserved target genes of p63 and SOCS3. MiR-203 regulates p63 and SOCS3 and proliferates in keratinocytes. The role of differentiation and psoriasis pathogenesis is unclear. STAT3 and Notch signaling pathway play a key role in psoriasis pathogenesis, while p63 and SOCS3 are two important regulators of these signaling pathways. The aim of this study was to determine the regulatory mechanism of miR-203 on p63 and SOCS3 by transfection of immortalized human keratinocytes HaCaT cell line with miR-203 mimic and its inhibitor. To clarify its relationship with STAT3 and Notch signaling pathway and its role in the pathogenesis of psoriasis, to determine their regulatory mode and mutual relationship, to provide a theoretical basis for its new therapeutic target for psoriasis. Methods: human keratinocytes HaCaT cells were quantitatively transfected with miR-203 mimics and inhibitors (miR-203mimic and miR-203inhibitor). The expression of p-STAT3 and Notch1 genes and proteins were detected by qRT-PCR,Western blot, flow cytometry. HaCaT cells were treated with TNF- 伪 (20ng/ml) and IL-6 (20ng/ml), and the expression of miR-203 was detected by qRT-PCR method. The effects of cytokines TNF- 伪 and IL-6 on miR-203 expression were observed. Results: 1. QRT-PCR was used to detect the mRNA expression of p63- SOCS3pSTAT3pSTAT3ntch1. The expression of SOCS3,Notch1,p-STAT3 mRNA was up-regulated in the transfected miR-203mimic group compared with the control group. There was significant difference between the Notch1 group and the Notch1 group (P 0.05), but the p63mRNA expression level did not change significantly. Compared with the control group, the mRNA expression of SOCS3,Notch1,p-STAT3 in miR-203inhibitor transfected group was down-regulated, the difference between SOCS3 and Notch1 group was significant (P 0.05), and the expression of p63mRNA was not changed significantly. 2. Western blot and flow cytometry were used to detect the protein expression. The expression of p63 and SOCS3 decreased and the expression of p-STAT3 and Notch1 increased in the cells transfected with miR-203mimic, whereas the expression of p-STAT3 and Notch1 was increased in the transfected miR-203inhibitor cells. After HaCaT cells were treated with TNF- 伪 and IL-6, the expression of miR-203 was detected by qRT-PCR method, and the difference was significant (P 0.05). Conclusion: overexpression of miR-203 can inhibit the expression of p63 and SOCS3, which leads to the sustained activation of STAT3 and the increase of phosphorylation of STAT3, resulting in excessive proliferation of keratinocytes. This study provides a preliminary experimental basis for the study of the regulatory mechanism of miR-203 on p63 SOCS3 and its relationship with STAT3 signal transduction pathway, and helps to understand the role of miR-203 in the pathogenesis of psoriasis.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R758.63
【参考文献】
相关期刊论文 前4条
1 汤仲明;;2011年美国FDA批准药物简介[J];国际药学研究杂志;2012年01期
2 杨建营;徐翔峰;向珍蛹;;NF-κB的研究进展[J];淮海医药;2011年01期
3 万永山;张开明;;角质形成细胞在银屑病发病中的再认识[J];中国麻风皮肤病杂志;2008年01期
4 邵长庚;我国银屑病的流行和防治现状[J];中华皮肤科杂志;1996年02期
本文编号：2419600
本文链接：https://www.wllwen.com/yixuelunwen/pifb/2419600.html
最近更新
教材专著
