【摘要】：皮膚鱗狀細(xì)胞癌(簡(jiǎn)稱(chēng)皮膚鱗癌)是我國(guó)常見(jiàn)的皮膚惡性腫瘤之一,容易發(fā)生轉(zhuǎn)移和復(fù)發(fā),是導(dǎo)致非黑色素瘤皮膚癌患者死亡的主要病因。因此,尋找其發(fā)病過(guò)程中發(fā)揮關(guān)鍵作用的分子對(duì)鱗癌的診斷和治療是必不可少的。 微小RNA (microRNA或miRNA)是一類(lèi)內(nèi)源性的單鏈非編碼小RNA分子,在轉(zhuǎn)錄后水平調(diào)控基因的表達(dá)。miRNA的異常表達(dá)與腫瘤的發(fā)生以及發(fā)展密不可分。本課題主要研究miR-125b對(duì)皮膚鱗癌細(xì)胞的作用及其機(jī)制,并對(duì)影響miR-125b表達(dá)的因素進(jìn)行初步探討。 本研究分為四部分進(jìn)行： 1.首先運(yùn)用低密度芯片檢測(cè)皮膚鱗癌組織中差異性表達(dá)的miRNAs,運(yùn)用實(shí)時(shí)定量PCR檢測(cè)miR-125b在皮膚鱗癌、光化性角化病和健康人皮膚組織以及不同分化程度的皮膚鱗癌組織中的表達(dá)。最后運(yùn)用原位雜交檢測(cè)皮膚鱗癌組織中miR-125b的表達(dá)水平。 2.運(yùn)用脂質(zhì)體介導(dǎo)方法將人工合成的miRNA前體轉(zhuǎn)染皮膚鱗癌UT-SCC-7和A431細(xì)胞系,升高細(xì)胞內(nèi)miR-125b的表達(dá)水平,觀察細(xì)胞生長(zhǎng)增殖、侵襲和遷移以及細(xì)胞凋亡的變化情況。 3.運(yùn)用芯片和生物信息學(xué)方法預(yù)測(cè)miR-125b的潛在靶基因,并運(yùn)用熒光素酶報(bào)告基因系統(tǒng)、實(shí)時(shí)定量PCR和western印跡實(shí)驗(yàn)對(duì)靶基因進(jìn)行驗(yàn)證。同時(shí)運(yùn)用實(shí)時(shí)定量PCR檢測(cè)皮膚鱗癌組織中靶基因的表達(dá),并分析靶基因與miR-125b表達(dá)水平的相關(guān)性。運(yùn)用RNA干擾技術(shù)敲降細(xì)胞內(nèi)靶基因的表達(dá),觀察細(xì)胞生長(zhǎng)增殖、侵襲和遷移以及細(xì)胞凋亡的變化情況。最后研究miR-125b下游調(diào)控可能涉及的分子機(jī)制,運(yùn)用實(shí)時(shí)定量PCR檢測(cè)miR-125b對(duì)下游基因的影響,闡述miR-125b在皮膚鱗癌細(xì)胞中可能參與的調(diào)控網(wǎng)絡(luò)。 4.通過(guò)生物信息學(xué)方法對(duì)miR-125b的啟動(dòng)子及其上游區(qū)域進(jìn)行分析,利用藥物5-aza對(duì)鱗癌細(xì)胞進(jìn)行處理,運(yùn)用實(shí)時(shí)定量PCR檢測(cè)藥物處理后細(xì)胞內(nèi)miR-125b的表達(dá),探討miR-125b的表觀遺傳學(xué)改變。此外,利用KGF(角質(zhì)細(xì)胞生長(zhǎng)因子)刺激細(xì)胞,檢測(cè)miR-125b的表達(dá)是否受影響。 通過(guò)對(duì)本課題的研究,可以得到以下幾個(gè)結(jié)論： 1. miR-125b在皮膚鱗癌組織中低表達(dá),但是miR-125b可能與腫瘤的分化程度無(wú)關(guān)。 2.細(xì)胞內(nèi)miR-125b的表達(dá)升高后,造成細(xì)胞克隆形成能力降低、細(xì)胞周期G1/S進(jìn)程阻滯、細(xì)胞增殖減慢以及侵襲和遷移能力均降低,而細(xì)胞凋亡增加。 3.MMP-13和MAP2K7是miR-125b的直接靶基因,miR-125b抑制MMP-13和MAP2K7的表達(dá)；同時(shí)MMP-13和MAP2K7與miR-125b的表達(dá)呈負(fù)相關(guān)。除此之外,TGFBR2、TP53INP1、MMP-7、cyclinD1和c-Jun等基因也受miR-125b的調(diào)控。敲降細(xì)胞內(nèi)MMP-13的表達(dá)后,細(xì)胞克隆形成、侵襲和遷移能力降低,但細(xì)胞的增殖和凋亡不受影響。敲降MAP2K7的表達(dá)后,細(xì)胞的增殖和克隆形成能力降低,細(xì)胞凋亡增加。通過(guò)對(duì)下游基因的分析,推測(cè)miR-125b可能參與TGF-β、NF-kappaB. JNK和Wnt信號(hào)通路的調(diào)控。 4. miR-125b的啟動(dòng)子區(qū)域呈現(xiàn)密集的CpG島,并且利用5-aza對(duì)鱗癌細(xì)胞進(jìn)行去甲基化處理后miR-125b的表達(dá)水平升高,說(shuō)明受到甲基化的調(diào)控。同時(shí)KGF刺激細(xì)胞后miR-125b的表達(dá)受抑制。
[Abstract]:Cutaneous squamous cell carcinoma (SCC) is one of the most common skin malignant tumors in China. It is easy to transfer and relapse. It is the main cause of the death of non-melanoma skin cancer patients. Therefore, it is necessary to find the molecules that play a key role in the diagnosis and treatment of squamous cell carcinoma. MicroRNA (microRNA or miRNA) is a kind of endogenous single-chain non-coding small RNA molecule, and the table of the gene is regulated after transcription. The abnormal expression of the miRNA and the occurrence of the tumor and the development of the miRNA are not The purpose of this study is to study the role of miR-125b on skin squamous cell carcinoma and its mechanism, and to investigate the factors that affect the expression of miR-125b. This study is divided into four parts. 1. First, using low-density chip to detect the differentially expressed miRNAs in skin squamous cell carcinoma (SCC), real-time quantitative PCR was used to detect the expression of miR-125b in the squamous cell carcinoma of the skin, the actinic keratosis and the skin tissue of healthy people and the different degree of differentiation of the squamous cell carcinoma of the skin. The expression of miR-125b in the tissue of skin squamous cell carcinoma was detected by in situ hybridization. 2. The expression level of miR-125b in the cell was increased by using the liposome-mediated method to transfect the human skin squamous cell carcinoma (UT-SCC-7) and the A431 cell line (A431), and the cell growth, proliferation, invasion and migration and the cell were observed. 3. The potential target genes of miR-125b were predicted by using the method of chip and bioinformatics, and the luciferase reporter gene system, real-time quantitative PCR and western blot were used. The target gene was verified by real-time quantitative PCR, and the target gene and miR-125 were analyzed. B. the expression of the target gene in the cell is knocked down by using the RNA interference technique, and the growth, the invasion and the migration of the cell are observed, and the expression of the target gene in the cell is observed by using the RNA interference technique. The effect of miR-125b on the downstream gene was detected by real-time quantitative PCR, and the expression of miR-125b in the skin squamous cell carcinoma was described. and 4. the promoter of the miR-125b and the upstream region of the miR-125b are analyzed by a bioinformatics method, the scale cancer cells are treated by using the drug 5-aza, and the expression of the miR-125b in the cells after the drug treatment is detected by the real-time quantitative PCR, and the miR-1 25b. In addition, the cells were stimulated with KGF (Keratinocyte Growth Factor) to detect miR-1. The expression of 25b is affected. By this subject The following conclusions can be obtained by the study:1. miR-125b is low-expressed in the skin squamous cell carcinoma tissue, but miR-1 25b may not be related to the degree of differentiation of the tumor.2. After the expression of miR-125b in the cell is increased, the cell clone formation ability is reduced, the cell cycle G1/ S process block, the cell proliferation is slowed down, and the invasion 3. MMP-13 and MAP2K7 are the direct target genes of miR-125b, and miR-125b inhibits the expression of MMP-13 and MAP2K7; and MMP-13 and MAP 2K7 was negatively correlated with the expression of miR-125b. In addition, TGFBR2, TP53INP1, MMP-7, cyclinD1 and c- The gene of Jun et al is also regulated by miR-125b. After the expression of MMP-13 in the cell, the cell clones form, invade and move. The cell proliferation and apoptosis are not affected. After the expression of MAP2K7, the cells The proliferation and clone formation ability of the cells is reduced, and the cell apoptosis is increased. By analyzing the downstream gene, it is presumed that the miR-125b May be involved in TGF-1, NF-ka Regulation of the ppaB. JNK and Wnt signaling pathways.4. The promoter region of miR-125b presents a dense CpG island and miR-1 is miR-1 after demethylation of the squamous cell with 5-aza. The expression level of 25b is increased and the regulation of methylation is indicated. At the same time K
【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】：2013
【分類(lèi)號(hào)】：R739.5

【共引文獻(xiàn)】

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