本文選題：mir-143-3p + MAGE-A9　； 參考：《蘇州大學(xué)》2016年博士論文

【摘要】：第一部分MAGE-A9在喉鱗狀細(xì)胞癌中的表達(dá)與預(yù)后價(jià)值目的:黑色素瘤相關(guān)抗原(MAGE)家族基因已經(jīng)被報(bào)道在人類癌癥的發(fā)展中發(fā)揮了重要的作用。然而MAGE-A9的表達(dá)和臨床病理參數(shù)之間的關(guān)系在人類喉癌中并不明確。本研究旨在考察MAGE-A9在喉癌中的表達(dá)水平,并評(píng)估其表達(dá)在人類喉鱗狀細(xì)胞癌(LSCC)中的臨床意義。方法:用實(shí)時(shí)熒光定量PCR(qPCR)和免疫組織化學(xué)法(IHC)對(duì)人類喉鱗狀細(xì)胞癌(LSCC)組織和癌旁正常組織檢測MAGE-A9的表達(dá),并用Kaplan-meier法生存分析和Cox回歸分析評(píng)估LSCC的預(yù)后。結(jié)果:MAGE-A9的表達(dá)在人類喉鱗狀細(xì)胞癌(LSCC)組織中明顯高于正常組織。123例人類喉鱗狀細(xì)胞癌(LSCC)組織中有70例細(xì)胞質(zhì)中表達(dá)MAGE-A9陽性(56.9%)。人類喉鱗狀細(xì)胞癌(LSCC)組織中MAGE-A9的表達(dá)水平與病理學(xué)分級(jí)有關(guān)(P=0.024)。Kaplan-meier法生存分析和Cox回歸分析顯示:MAGE-A9表達(dá)水平與淋巴結(jié)轉(zhuǎn)移是LSCC的獨(dú)立預(yù)后因素,分別為(P=0.005;P=0.005)。結(jié)論:研究表明MAGE-A9表達(dá)是LSCC患者預(yù)后的生物標(biāo)志物。高表達(dá)MAGE-A9患者生存期短于低MAGE-A9表達(dá)患者。第二部分hsa-mir-143-3p通過靶基因MAGE-A9對(duì)喉癌生物學(xué)行為的影響目的:研究hsa-mir-143-3p在喉癌中的表達(dá)情況,驗(yàn)證hsa-mir-143-3p與MAGE-A9基因的靶向關(guān)系,并探討其在該疾病中的功能,為闡明喉癌發(fā)病機(jī)制,尋求有效的治療途徑提供理論和實(shí)驗(yàn)依據(jù)。材料和方法:通過qPCR和免疫蛋白印記(Western Blot)檢測喉癌TU212和TU686細(xì)胞株中mage-a9mrna及蛋白的表達(dá),選擇了mage-a9在tu686高表達(dá)的細(xì)胞株。對(duì)喉癌tu686細(xì)胞株用qpcr和westernblot檢測hsa-mir-31-5p組、hsa-mir-124-3p組、hsa-mir-138-5p組、hsa-mir-143-3p組、cel-mir-67組、normal組轉(zhuǎn)染后對(duì)magea9基因表達(dá)的影響,其中hsa-mir-143-3p抑制magea9基因的表達(dá)最佳。應(yīng)用生物信息學(xué)和分子生物學(xué)技術(shù)預(yù)測并構(gòu)建攜帶hsa-mir-143-3pmimics與mage-a93'utr可特異性結(jié)合的報(bào)告基因載體。利用雙熒光素酶報(bào)告基因?qū)嶒?yàn)驗(yàn)證hsa-mir-143-3p和magea9基因的靶向關(guān)系。mtt試驗(yàn)、細(xì)胞侵襲實(shí)驗(yàn)和細(xì)胞劃痕實(shí)驗(yàn)來探討hsa-mir-143-3p轉(zhuǎn)染后對(duì)腫瘤細(xì)胞增殖、凋亡及侵襲和轉(zhuǎn)移能力的影響。將轉(zhuǎn)染hsa-mir-143-3p、cel-mir-67、空白對(duì)照的tu686喉癌細(xì)胞株建立裸鼠喉癌皮下移植瘤模型,觀察腫瘤的重量、體積,并用qpcr、westernblot及ihc分別檢測腫瘤組織中mage-a9的mrna和蛋白水平。tunnel染色法檢測腫瘤組織中細(xì)胞凋亡的情況。收集15例行手術(shù)治療的喉癌患者的腫瘤標(biāo)本,qpcr及ihc檢測hsa-mir-143-3p及mage-a9在腫瘤及癌旁組織中的表達(dá)水平,進(jìn)一步驗(yàn)證hsa-mir-143-3p及mage-a9的關(guān)系。結(jié)果:1.用qpcr和westernblot檢測tu686細(xì)胞株各mirna轉(zhuǎn)染后對(duì)magea9基因表達(dá)的影響,發(fā)現(xiàn)hsa-mir-143-3p抑制magea9基因的表達(dá)最佳,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。2.基因測序結(jié)果表明,將pgl3-wt3’utr和hsa-mir-143-3pmimics共轉(zhuǎn)染可顯著降低熒光素酶的活性(p0.001),此外,突變質(zhì)粒pgl3-mu3’utr與pgl3-control共轉(zhuǎn)染均不能改變熒光素酶的活性,無顯著影響(p0.05)。3.mtt試驗(yàn)中,轉(zhuǎn)染hsa-mir-143-3p后,對(duì)細(xì)胞增殖的抑制能力明顯高于對(duì)照組,且差異有統(tǒng)計(jì)學(xué)意義(p0.05)。細(xì)胞體外侵襲實(shí)驗(yàn)顯示,hsa-mir-143組細(xì)胞數(shù)明顯低于空白對(duì)照組和cel-mir-67,差異有統(tǒng)計(jì)學(xué)意義(p0.05),而空白對(duì)照組和cel-mir-67組之間細(xì)胞數(shù)目無明顯差異,提示hsa-mir-143表達(dá)能顯著降低喉癌tu686細(xì)胞的侵襲能力。細(xì)胞劃痕試驗(yàn)中,hsa-mir-143組細(xì)胞的劃痕愈合速度較慢,未愈合。表明構(gòu)建的hsa-mir-143有效的抑制細(xì)胞遷移能力,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。4.經(jīng)轉(zhuǎn)染hsa-mir-143-3p、cel-mir-67、空白對(duì)照的tu686喉癌細(xì)胞株所形成的皮下移植瘤中hsa-mir-143-3p-tu686組裸鼠的瘤塊重量與tu686組、cel-mir-67-tu686腫瘤重量比較有差異(p0.05)。hsa-mir-143-3p-tu686組裸鼠的瘤塊出現(xiàn)瘤后18天的腫瘤體積與tu686組、cel-mir-67-tu686體積比較有差異(p0.05)。提示hsa-mir-143-3p對(duì)動(dòng)物模型中移植瘤生長呈明顯抑制。通過qPCR、Western Blot及免疫組化檢測hsa-mir-143-3p-TU686組、TU686組、cel-mir-67-TU686組三組腫瘤組織中MAGE-A9mRNA及蛋白的表達(dá),發(fā)現(xiàn)hsa-mir-143-3p-TU686組瘤塊中MAGE-A9的表達(dá)下降(P0.05)Tunne染色法檢測hsa-mir-143-3p-TU686組腫瘤組織中細(xì)胞凋亡最多。5.qRT-PCR檢測發(fā)現(xiàn)hsa-mir-143-3p在人喉癌組織中中比癌旁組織中表達(dá)量低,免疫組化檢測hsa-mir-143-3p的靶基因MAGE-A9在人喉癌組織中中比癌旁組織中表達(dá)量高。結(jié)論:1.hsa-mir-143-3p在喉癌中起抑癌作用,MAGE-A9是其靶基因之一。2.hsa-mir-143-3p通過抑制MAGE-A9蛋白,抑制了細(xì)胞的增殖和侵襲能力。3.如何穩(wěn)定而有效地上調(diào)喉癌中hsa-mir-143-3p的表達(dá)水平,以達(dá)到生物治療效果有待進(jìn)一步探討。
[Abstract]:The expression and prognostic value of MAGE-A9 in laryngeal squamous cell carcinoma: melanoma associated antigen (MAGE) gene has been reported to play an important role in the development of human cancer. However, the relationship between the expression of MAGE-A9 and the clinicopathological parameters is not clear in human laryngoma. This study aims to investigate MAGE-A 9 the expression level in larynx cancer and the clinical significance of its expression in human laryngeal squamous cell carcinoma (LSCC). Methods: the expression of MAGE-A9 in human laryngeal squamous cell carcinoma (LSCC) tissues and adjacent normal tissues was detected by real-time fluorescence quantitative PCR (qPCR) and immunohistochemistry (IHC), and Kaplan-meier method survival analysis and Cox regression analysis were used. The prognosis of LSCC was evaluated. Results: the expression of MAGE-A9 in human laryngeal squamous cell carcinoma (LSCC) tissues was significantly higher than that of normal tissues. The expression of MAGE-A9 was positive in 70 cases of human laryngeal squamous cell carcinoma (LSCC) tissue (LSCC). The expression of MAGE-A9 in human laryngeal squamous cell carcinoma (LSCC) group was associated with pathological grade (P=0.024).Ka (P=0.024).Ka. Plan-meier method survival analysis and Cox regression analysis showed that MAGE-A9 expression level and lymph node metastasis were independent prognostic factors of LSCC, respectively (P=0.005; P=0.005). Conclusion: the study showed that MAGE-A9 expression was a biomarker for the prognosis of LSCC patients. The survival time of the patients with high expression of MAGE-A9 was shorter than that of low MAGE-A9 expression. Second part hsa-mir-143-3p. The effect of target gene MAGE-A9 on the biological behavior of larynx cancer: To study the expression of hsa-mir-143-3p in larynx cancer, to verify the target relationship between hsa-mir-143-3p and MAGE-A9 gene, and to explore its function in the disease, and to provide theoretical and experimental basis for clarifying the pathogenesis of larynx cancer and seeking effective treatment methods. The expression of mage-a9mrna and protein in the TU212 and TU686 cell lines of larynx cancer was detected by qPCR and Western Blot. The cell lines with high expression of MAGE-A9 in tu686 were selected. The tu686 cell lines of the larynx cancer were detected by qPCR and Westernblot. The effect of the transfection on the expression of MAGEA9 gene, in which hsa-mir-143-3p inhibits the expression of MAGEA9 gene, and uses bioinformatics and molecular biology techniques to predict and construct a reporter gene carrier with the specific binding of hsa-mir-143-3pmimics and mage-a93'utr. Using the double fluorescent enzyme reporter gene test to verify hsa-mir-143-3p The target relationship with the MAGEA9 gene.Mtt test, cell invasion experiment and cell scratch test were used to explore the effect of hsa-mir-143-3p transfection on tumor cell proliferation, apoptosis and invasion and metastasis. Hsa-mir-143-3p, cel-mir-67, blank control tu686 larynx cell lines were constructed to build a nude mouse larynx subcutaneous transplant tumor model and observe the tumor. Weight, volume, and detection of apoptosis in tumor tissues by MAGE-A9 mRNA and protein level.Tunnel staining in tumor tissues using qPCR, Westernblot and IHC respectively. 15 cases of surgical treatment of larynx cancer were collected, and qPCR and IHC were used to detect the expression level of hsa-mir-143-3p and MAGE-A9 in tumor and para cancer tissues. The relationship between hsa-mir-143-3p and MAGE-A9 was further verified. Results: 1. the effect of miRNA on the expression of MAGEA9 gene in tu686 cell lines was detected by qPCR and Westernblot, and it was found that hsa-mir-143-3p inhibited the expression of MAGEA9 gene, and the difference was statistically significant (P0.05).2. gene sequencing results showed that pgl3-wt3 'and pgl3-wt3' were used. S co transfection could significantly reduce the activity of luciferase (p0.001). In addition, the mutation plasmid pgl3-mu3 'UTR and pgl3-control co transfection did not change the activity of luciferase, and there was no significant influence (P0.05).3.mtt test. After transfection of hsa-mir-143-3p, the inhibitory ability to cell proliferation was significantly higher than that of the control group, and the difference was statistically significant (P0.05). The cell invasion test in vitro showed that the number of cells in the hsa-mir-143 group was significantly lower than that of the blank control group and cel-mir-67, and the difference was statistically significant (P0.05), but there was no significant difference in the number of cells between the blank control group and the cel-mir-67 group, suggesting that the hsa-mir-143 expression could significantly reduce the invasion ability of the tu686 cells in the larynx cancer. In the cell scratch test, hsa-mir-143 The wound healing speed of the group cells was slow and not healed. It showed that the constructed hsa-mir-143 effectively inhibited the cell migration ability, and the difference was statistically significant (P0.05).4. was transfected with hsa-mir-143-3p, cel-mir-67, and tu686 larynx cell line of the blank control group, the weight of the tumor block of the nude mice in the hsa-mir-143-3p-tu686 group of the hsa-mir-143-3p-tu686 group and the tu686 group, CE L-mir-67-tu686 tumor weight was different (P0.05) the tumor volume of the tumor block in the nude mice of the.Hsa-mir-143-3p-tu686 group was compared with the tu686 group at 18 days after the tumor, and the cel-mir-67-tu686 volume was different (P0.05). It suggested that hsa-mir-143-3p was significantly inhibited in the growth of the transplanted tumor in the animal model. QPCR, Western Blot, and immunohistochemical detection hsa-mir-14. The expression of MAGE-A9mRNA and protein in three groups of tumor tissues in group 3-3p-TU686, group TU686 and group cel-mir-67-TU686, the expression of MAGE-A9 in the hsa-mir-143-3p-TU686 group was found to be decreased (P0.05) Tunne staining method was used to detect the maximum apoptotic.5.qRT-PCR detection in the tumor tissue of the hsa-mir-143-3p-TU686 group and hsa-mir-143-3p in the human larynx tissue was found. The expression of hsa-mir-143-3p in the para cancerous tissue is low, and the target gene MAGE-A9 of hsa-mir-143-3p is highly expressed in human larynx tissues. Conclusion: 1.hsa-mir-143-3p plays a role in inhibiting cancer in larynx cancer. MAGE-A9 is one of its target genes, which inhibits the proliferation and invasion ability of cell.3. by inhibiting the MAGE-A9 protein. How to effectively and effectively raise the expression level of hsa-mir-143-3p in laryngeal carcinoma to achieve the effect of biological therapy needs further study.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R739.65

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1 韓靚;hsa-mir-143-3p通過靶基因MAGE-A9對(duì)喉癌生物學(xué)行為的影響[D];蘇州大學(xué);2016年

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