MiR-224-3p靶向FIP200抑制宫颈癌细胞自噬活性

发布时间：2018-06-15 11:25

本文选题：miR-224-3p + HPV　；参考：《天津医科大学》2017年博士论文

【摘要】：【目的】微小RNA(mi RNA)是约21-25个核苷酸组成的非编码小RNA,作用是能够作为基因表达的转录后调节剂。已经发现这些小分子调节参与不同细胞生理过程:如调控细胞的增殖,发育,分化,凋亡等。宫颈癌是恶性实体瘤,其是女性恶性肿瘤中高发病率、死亡率的主要原因之一。高危人乳头状瘤病毒(hr HPV)持续感染与子宫颈癌密切相关,自噬被认为可抑制病毒感染。Mi RNA可以通过调节其靶基因从而参与细胞自噬的调控,发挥类似癌基因或抑癌基因的功能。本研究的目的是探寻mi R-224-3p基因簇是否参与了HPV感染引起的宫颈癌细胞自噬的调控过程,以及预测和确定mi R-224-3p直接作用的靶基因及靶蛋白,从而阐明mi R-224-3p在宫颈癌发生和发展过程中的分子机制。【方法】在这项研究中,我们对宫颈病变组织进行了mi RNA基因芯片分析,发现大量的mi RNA在HPV感染的组织中具有差异表达。通过差异性分析确定mi R-224-3p作为选择性上调在HPV感染的组织和细胞系中的候选mi RNA。应用RT-PCR在宫颈病变组织中进行验证。应用Western-blotting验证宫颈组织中自噬相关蛋白P62、LC3表达。通过在不同HPV感染的细胞系中转染mi R-224-3p质粒及其抑制剂验证其调节自噬机制。并应用免疫荧光检验自噬相关蛋白P62在上述转染后细胞株中表达水平。通过Target Scan7.0预测mi R-224-3p靶向基因,并在细胞系中应用双因素荧光酶实验验证,预测并筛选了mi R-224-3p候选靶基因FIP200。通过RT-PCR和Western-blotting方法检测了在宫颈癌细胞系中mi R-224-3p对FIP200的m RNA及蛋白水平的影响。在宫颈癌细胞系中过表达或敲降mi R-224-3p后,用上述相同的方法检测了mi R-224-3p对FIP200表达水平的影响。【结果】基因芯片分析HPV(+)宫颈组织中mi R-224-3p表达明显高于HPV(-)宫颈组织。不同病变阶段的宫颈组织中mi R-224-3p随病变级别的升高明显表达增加。HPV(+)宫颈组织中的自噬相关蛋白LC3表达明显下降,P62蛋白表达明显升高,说明HPV(+)组织中细胞自噬受到抑制。在HPV(-)、HPV16(+)、HPV18(+)细胞系中,转染mi R-224-3p后P62蛋白表达明显升高,以HPV18(+)细胞系最为明显,转染mi R-224-3p-inhibitor后P62蛋白表达明显下降,以HPV18(+)细胞系最为明显,免疫荧光结果同Western-blotting结果。软件预测结果提示FIP200为mi R-224-3p候选靶基因,转染mi R-224-3p后FIP200表达明显下降,转染mi R-224-3p-inhibitor后FIP200表达明显升高。上述结果经统计学分析差异均具有统计学意义,即P0.05。【结论】HPV感染能够引起mi R-224-3p表达增加及抑制宫颈病变组织中的细胞自噬活性,相同结果在不同细胞系中得到验证。mi R-224-3p的过表达抑制HPV感染的细胞中的自噬,但敲低内源性mi R-224-3p增加相同细胞中的自噬活性。上述结果的出现是mi R-224-3p通过抑制FIP200的表达实现的。
[Abstract]:[objective] small RNAi RNAs are noncoding small RNAs consisting of about 21-25 nucleotides and can be used as posttranscriptional modulators for gene expression. It has been found that these small molecules are involved in different cellular physiological processes, such as regulating cell proliferation, development, differentiation, apoptosis and so on. Cervical cancer is a malignant solid tumor, which is one of the main causes of high morbidity and mortality in female malignant tumors. The persistent infection of high risk human papillomavirus (HPV) is closely related to cervical cancer. Autophagy is thought to inhibit viral infection. Mi RNA can play the role of oncogene or tumor suppressor gene by regulating its target gene and participating in the regulation of autophagy. The aim of this study was to investigate whether the miR-224-3p gene cluster is involved in the regulation of autophagy induced by HPV infection, and to predict and determine the target genes and target proteins directly acting on the MIR-224-3p. In order to elucidate the molecular mechanism of mi R-224-3p in the carcinogenesis and development of cervical cancer. [methods] in this study, we carried out the mi RNA gene chip analysis of cervical lesions. A large number of miRNAs were found to be differentially expressed in HPV infected tissues. Mi R-224-3p was identified as a candidate for selective upregulation of mi RNAin HPV infected tissues and cell lines by differential analysis. RT-PCR was used to verify the pathological changes of cervix. The expression of autophagy associated protein (P62) LC3 in cervical tissues was detected by Western-blotting. The mechanism of regulating autophagy was verified by transfection of mi R-224-3p plasmid and its inhibitor in different HPV infected cell lines. The expression of autophagy associated protein P62 was detected by immunofluorescence. The target gene of mi R-224-3p was predicted by Target Scan7.0, and the candidate gene FIP200was predicted and screened by double-factor fluorescence enzyme assay. The effects of miR-224-3p on the mRNA and protein levels of FIP200 in cervical cancer cell line were detected by RT-PCR and Western-blotting. After overexpression or knockdown of mi R-224-3p in cervical cancer cell line, the effect of mi R-224-3p on the expression of FIP200 was detected by the same method mentioned above. [results] the expression of mi R-224-3p in cervical tissue of HPV-224-3p was significantly higher than that in HPV-HPV-cervix tissue. The expression of autophagy associated protein LC3 in cervical tissues increased with the increase of pathological grade. The expression of autophagy associated protein LC3 in cervical tissues decreased significantly, indicating that autophagy was inhibited in HPVs. The expression of P62 protein was significantly increased after transfection of mi R-224-3p, especially in HPV18 () cell line. The expression of P62 protein decreased significantly after transfection of mi R-224-3p-inhibitor, especially in HPV18 (HPV18) cell line, and immunofluorescence was the same as Western-blotting. The results of software prediction indicated that FIP200 was a candidate gene for mi R-224-3p. The expression of FIP200 decreased significantly after transfection of mi R-224-3p, and increased after transfection of mi R-224-3p-inhibitor. The results were statistically significant, that is, P0.05. [conclusion] HPV infection can increase the expression of mi R-224-3p and inhibit the autophagy activity in cervical lesions. The same results were found in different cell lines. Overexpression of .mi R-224-3p inhibited autophagy in HPV-infected cells, but knocked down endogenous mi R-224-3p to increase autophagy activity in the same cells. The above results were achieved by inhibiting the expression of FIP200 in mi R-224-3 p.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33

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