本文選題：miR-224-3p + HPV��； 參考：《天津醫(yī)科大學(xué)》2017年博士論文

【摘要】：【目的】微小RNA(mi RNA)是約21-25個(gè)核苷酸組成的非編碼小RNA,作用是能夠作為基因表達(dá)的轉(zhuǎn)錄后調(diào)節(jié)劑。已經(jīng)發(fā)現(xiàn)這些小分子調(diào)節(jié)參與不同細(xì)胞生理過程:如調(diào)控細(xì)胞的增殖,發(fā)育,分化,凋亡等。宮頸癌是惡性實(shí)體瘤,其是女性惡性腫瘤中高發(fā)病率、死亡率的主要原因之一。高危人乳頭狀瘤病毒(hr HPV)持續(xù)感染與子宮頸癌密切相關(guān),自噬被認(rèn)為可抑制病毒感染。Mi RNA可以通過調(diào)節(jié)其靶基因從而參與細(xì)胞自噬的調(diào)控,發(fā)揮類似癌基因或抑癌基因的功能。本研究的目的是探尋mi R-224-3p基因簇是否參與了HPV感染引起的宮頸癌細(xì)胞自噬的調(diào)控過程,以及預(yù)測(cè)和確定mi R-224-3p直接作用的靶基因及靶蛋白,從而闡明mi R-224-3p在宮頸癌發(fā)生和發(fā)展過程中的分子機(jī)制�！痉椒ā吭谶@項(xiàng)研究中,我們對(duì)宮頸病變組織進(jìn)行了mi RNA基因芯片分析,發(fā)現(xiàn)大量的mi RNA在HPV感染的組織中具有差異表達(dá)。通過差異性分析確定mi R-224-3p作為選擇性上調(diào)在HPV感染的組織和細(xì)胞系中的候選mi RNA。應(yīng)用RT-PCR在宮頸病變組織中進(jìn)行驗(yàn)證。應(yīng)用Western-blotting驗(yàn)證宮頸組織中自噬相關(guān)蛋白P62、LC3表達(dá)。通過在不同HPV感染的細(xì)胞系中轉(zhuǎn)染mi R-224-3p質(zhì)粒及其抑制劑驗(yàn)證其調(diào)節(jié)自噬機(jī)制。并應(yīng)用免疫熒光檢驗(yàn)自噬相關(guān)蛋白P62在上述轉(zhuǎn)染后細(xì)胞株中表達(dá)水平。通過Target Scan7.0預(yù)測(cè)mi R-224-3p靶向基因,并在細(xì)胞系中應(yīng)用雙因素?zé)晒饷笇?shí)驗(yàn)驗(yàn)證,預(yù)測(cè)并篩選了mi R-224-3p候選靶基因FIP200。通過RT-PCR和Western-blotting方法檢測(cè)了在宮頸癌細(xì)胞系中mi R-224-3p對(duì)FIP200的m RNA及蛋白水平的影響。在宮頸癌細(xì)胞系中過表達(dá)或敲降mi R-224-3p后,用上述相同的方法檢測(cè)了mi R-224-3p對(duì)FIP200表達(dá)水平的影響�！窘Y(jié)果】基因芯片分析HPV(+)宮頸組織中mi R-224-3p表達(dá)明顯高于HPV(-)宮頸組織。不同病變階段的宮頸組織中mi R-224-3p隨病變級(jí)別的升高明顯表達(dá)增加。HPV(+)宮頸組織中的自噬相關(guān)蛋白LC3表達(dá)明顯下降,P62蛋白表達(dá)明顯升高,說明HPV(+)組織中細(xì)胞自噬受到抑制。在HPV(-)、HPV16(+)、HPV18(+)細(xì)胞系中,轉(zhuǎn)染mi R-224-3p后P62蛋白表達(dá)明顯升高,以HPV18(+)細(xì)胞系最為明顯,轉(zhuǎn)染mi R-224-3p-inhibitor后P62蛋白表達(dá)明顯下降,以HPV18(+)細(xì)胞系最為明顯,免疫熒光結(jié)果同Western-blotting結(jié)果。軟件預(yù)測(cè)結(jié)果提示FIP200為mi R-224-3p候選靶基因,轉(zhuǎn)染mi R-224-3p后FIP200表達(dá)明顯下降,轉(zhuǎn)染mi R-224-3p-inhibitor后FIP200表達(dá)明顯升高。上述結(jié)果經(jīng)統(tǒng)計(jì)學(xué)分析差異均具有統(tǒng)計(jì)學(xué)意義,即P0.05�！窘Y(jié)論】HPV感染能夠引起mi R-224-3p表達(dá)增加及抑制宮頸病變組織中的細(xì)胞自噬活性,相同結(jié)果在不同細(xì)胞系中得到驗(yàn)證。mi R-224-3p的過表達(dá)抑制HPV感染的細(xì)胞中的自噬,但敲低內(nèi)源性mi R-224-3p增加相同細(xì)胞中的自噬活性。上述結(jié)果的出現(xiàn)是mi R-224-3p通過抑制FIP200的表達(dá)實(shí)現(xiàn)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.33

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