hsa-mir-143-3p通过靶基因MAGE-A9对喉癌生物学行为的影响

发布时间：2018-05-09 22:40

本文选题：mir-143-3p + MAGE-A9　；参考：《苏州大学》2016年博士论文

【摘要】：第一部分MAGE-A9在喉鳞状细胞癌中的表达与预后价值目的:黑色素瘤相关抗原(MAGE)家族基因已经被报道在人类癌症的发展中发挥了重要的作用。然而MAGE-A9的表达和临床病理参数之间的关系在人类喉癌中并不明确。本研究旨在考察MAGE-A9在喉癌中的表达水平,并评估其表达在人类喉鳞状细胞癌(LSCC)中的临床意义。方法:用实时荧光定量PCR(qPCR)和免疫组织化学法(IHC)对人类喉鳞状细胞癌(LSCC)组织和癌旁正常组织检测MAGE-A9的表达,并用Kaplan-meier法生存分析和Cox回归分析评估LSCC的预后。结果:MAGE-A9的表达在人类喉鳞状细胞癌(LSCC)组织中明显高于正常组织。123例人类喉鳞状细胞癌(LSCC)组织中有70例细胞质中表达MAGE-A9阳性(56.9%)。人类喉鳞状细胞癌(LSCC)组织中MAGE-A9的表达水平与病理学分级有关(P=0.024)。Kaplan-meier法生存分析和Cox回归分析显示:MAGE-A9表达水平与淋巴结转移是LSCC的独立预后因素,分别为(P=0.005;P=0.005)。结论:研究表明MAGE-A9表达是LSCC患者预后的生物标志物。高表达MAGE-A9患者生存期短于低MAGE-A9表达患者。第二部分hsa-mir-143-3p通过靶基因MAGE-A9对喉癌生物学行为的影响目的:研究hsa-mir-143-3p在喉癌中的表达情况,验证hsa-mir-143-3p与MAGE-A9基因的靶向关系,并探讨其在该疾病中的功能,为阐明喉癌发病机制,寻求有效的治疗途径提供理论和实验依据。材料和方法:通过qPCR和免疫蛋白印记(Western Blot)检测喉癌TU212和TU686细胞株中mage-a9mrna及蛋白的表达,选择了mage-a9在tu686高表达的细胞株。对喉癌tu686细胞株用qpcr和westernblot检测hsa-mir-31-5p组、hsa-mir-124-3p组、hsa-mir-138-5p组、hsa-mir-143-3p组、cel-mir-67组、normal组转染后对magea9基因表达的影响,其中hsa-mir-143-3p抑制magea9基因的表达最佳。应用生物信息学和分子生物学技术预测并构建携带hsa-mir-143-3pmimics与mage-a93'utr可特异性结合的报告基因载体。利用双荧光素酶报告基因实验验证hsa-mir-143-3p和magea9基因的靶向关系。mtt试验、细胞侵袭实验和细胞划痕实验来探讨hsa-mir-143-3p转染后对肿瘤细胞增殖、凋亡及侵袭和转移能力的影响。将转染hsa-mir-143-3p、cel-mir-67、空白对照的tu686喉癌细胞株建立裸鼠喉癌皮下移植瘤模型,观察肿瘤的重量、体积,并用qpcr、westernblot及ihc分别检测肿瘤组织中mage-a9的mrna和蛋白水平。tunnel染色法检测肿瘤组织中细胞凋亡的情况。收集15例行手术治疗的喉癌患者的肿瘤标本,qpcr及ihc检测hsa-mir-143-3p及mage-a9在肿瘤及癌旁组织中的表达水平,进一步验证hsa-mir-143-3p及mage-a9的关系。结果:1.用qpcr和westernblot检测tu686细胞株各mirna转染后对magea9基因表达的影响,发现hsa-mir-143-3p抑制magea9基因的表达最佳,差异有统计学意义(p0.05)。2.基因测序结果表明,将pgl3-wt3’utr和hsa-mir-143-3pmimics共转染可显著降低荧光素酶的活性(p0.001),此外,突变质粒pgl3-mu3’utr与pgl3-control共转染均不能改变荧光素酶的活性,无显著影响(p0.05)。3.mtt试验中,转染hsa-mir-143-3p后,对细胞增殖的抑制能力明显高于对照组,且差异有统计学意义(p0.05)。细胞体外侵袭实验显示,hsa-mir-143组细胞数明显低于空白对照组和cel-mir-67,差异有统计学意义(p0.05),而空白对照组和cel-mir-67组之间细胞数目无明显差异,提示hsa-mir-143表达能显著降低喉癌tu686细胞的侵袭能力。细胞划痕试验中,hsa-mir-143组细胞的划痕愈合速度较慢,未愈合。表明构建的hsa-mir-143有效的抑制细胞迁移能力,差异有统计学意义(p0.05)。4.经转染hsa-mir-143-3p、cel-mir-67、空白对照的tu686喉癌细胞株所形成的皮下移植瘤中hsa-mir-143-3p-tu686组裸鼠的瘤块重量与tu686组、cel-mir-67-tu686肿瘤重量比较有差异(p0.05)。hsa-mir-143-3p-tu686组裸鼠的瘤块出现瘤后18天的肿瘤体积与tu686组、cel-mir-67-tu686体积比较有差异(p0.05)。提示hsa-mir-143-3p对动物模型中移植瘤生长呈明显抑制。通过qPCR、Western Blot及免疫组化检测hsa-mir-143-3p-TU686组、TU686组、cel-mir-67-TU686组三组肿瘤组织中MAGE-A9mRNA及蛋白的表达,发现hsa-mir-143-3p-TU686组瘤块中MAGE-A9的表达下降(P0.05)Tunne染色法检测hsa-mir-143-3p-TU686组肿瘤组织中细胞凋亡最多。5.qRT-PCR检测发现hsa-mir-143-3p在人喉癌组织中中比癌旁组织中表达量低,免疫组化检测hsa-mir-143-3p的靶基因MAGE-A9在人喉癌组织中中比癌旁组织中表达量高。结论: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通过抑制MAGE-A9蛋白,抑制了细胞的增殖和侵袭能力。3.如何稳定而有效地上调喉癌中hsa-mir-143-3p的表达水平,以达到生物治疗效果有待进一步探讨。
[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.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.65

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