miR-125b对卵巢癌SKOV3细胞增殖、迁移及侵袭的影响及其潜在靶基因的研究
发布时间：2018-08-27 19:35
【摘要】：目的 卵巢癌（Ovarian cancer）是女性生殖系统常见的三大恶性肿瘤之一，但其致死率居各类妇科恶性肿瘤之首。由于其发病隐匿，缺乏早期诊断手段，约70%的卵巢癌患者确诊时已属晚期。研究发现卵巢癌的发生与遗传因素、初潮和绝经年龄、生殖及内分泌等多种因素有关。近年来，一类非蛋白质编码microRNAs（miRNAs）的出现，为卵巢肿瘤的研究提供了新的思路。miRNAs是一类长度为20-22个核苷酸的非编码小分子RNA，在动物体内miRNA主要通过与靶基因3’-UTR区不完全配对，抑制靶基因mRNAs翻译，，参与调控个体发育、细胞凋亡、增殖及分化等生命活动。随着对miRNA研究的不断深入发现miRNA在多种恶性肿瘤中扮演着重要角色，miRNA可作为抑癌基因或癌基因参与肿瘤的发生发展、转移、耐药等进程。miR-125b是miRNA家族中的重要一员，它在多种肿瘤发生发展过程中发挥重要作用,特别是在卵巢癌的发生发展过程中。本研究拟通过在卵巢癌SKOV3细胞株中过表达miR-125b，检测miR-125b对卵巢癌SKOV3细胞增殖、迁移及侵袭能力的影响，及其所调控的潜在靶基因进行预测，以期明确miR-125b在卵巢癌发生发展中的作用。 方法 本研究通过过表达miR-125b慢病毒颗粒感染卵巢癌SKOV3细胞，嘌呤霉素筛选稳定感染细胞株，建立稳定过表达miR-125b的SKOV3细胞系。采用实时荧光定量PCR（qRT-PCR）检测miR-125b在SKVO3细胞中的表达；MTT实验观察miR-125b过表达后SKOV3细胞增殖能力的的改变；划痕实验和Transwell侵袭实验分别观察miR-125b过表达后SKOV3细胞迁移和侵袭能力的改变。采用iTRAQ多重化学标记串联质谱（iTRAQ-LC/MS/MS）实验定性及定量分析miR-125b过表达前后SKOV3细胞内蛋白质的变化，筛选出miR-125b可能调控的潜在靶基因。再进一步通过Western blot来验证筛选出的靶基因是否受miR-125b的影响。 结果 1. MTT实验显示，miR-125b过表达后SKOV3细胞的增殖能力减弱；划痕实验和Transwell侵袭实验显示，miR-125b过表达后SKOV3细胞的迁移和侵袭能力明显减弱。 2. iTQAR多重化学标记串联质谱(iTQAR-LC/MS/MS)实验，结合miRNA候选靶基因预测软件（TargetScan、Pictar等）分析筛选出61种可能受到miR-125b调控的差异表达蛋白质，其中16种蛋白质在表达水平上存在显著差异。通过生物信息学对差异表达的蛋白质进行功能分析（整合NCBI、KEGG PATHWAY、Swiss-Prot等多个数据库信息），我们选择ERBB2基因作为miR-125b的一个潜在靶基因进行进一步研究。 3. Western blot结果显示，miR-125b能抑制ERBB2基因的表达。 结论 本研究证明miR-125b能够抑制SKOV3细胞的增殖、迁移和侵袭，并可能通过降低潜在靶基因ERBB2的表达而实现。
[Abstract]:Objective Ovarian cancer (Ovarian cancer) is one of the three most common malignant tumors in the female reproductive system, but its mortality is the highest among all kinds of gynecologic malignancies. Because of its hidden incidence and lack of early diagnosis, about 70% of patients with ovarian cancer are advanced when diagnosed. Genetic factors, menarche and menopausal age, reproductive and endocrine factors were found in the occurrence of ovarian cancer. In recent years, the emergence of a class of non-protein encoded microRNAs (miRNAs) provides a new idea for the study of ovarian tumors. MiRNAs are a kind of non-coding small molecule RNA, with a length of 20-22 nucleotides, which is mainly paired with the 3'-UTR region of the target gene in animals. Inhibition of target gene mRNAs translation, regulation of ontogeny, apoptosis, proliferation and differentiation, and other vital activities. With the further study of miRNA, it has been found that miRNA plays an important role in many kinds of malignant tumors. As a tumor suppressor gene or oncogene, miR-125b is an important member of the miRNA family as tumor suppressor gene or oncogene is involved in tumorigenesis, metastasis, drug resistance and other processes. It plays an important role in the development of many kinds of tumors, especially in the development of ovarian cancer. The purpose of this study was to detect the effect of miR-125b on the proliferation, migration and invasion of ovarian cancer SKOV3 cells by overexpression of miR-125b, in ovarian cancer SKOV3 cell lines, and to predict the potential target genes regulated by miR-125b in order to clarify the role of miR-125b in the development and development of ovarian cancer. Methods in this study, ovarian cancer SKOV3 cells were infected by overexpression of miR-125b lentivirus particles, and stable infection cell lines were screened by purine mycin, and SKOV3 cell lines with stable expression of miR-125b were established. The expression of miR-125b in SKVO3 cells was detected by real-time fluorescence quantitative PCR (qRT-PCR). The proliferation of SKOV3 cells after miR-125b overexpression was observed by MTT assay, and the migration and invasion ability of SKOV3 cells after miR-125b overexpression was observed by scratch test and Transwell invasion assay, respectively. ITRAQ multiplex chemical labeling tandem mass spectrometry (iTRAQ-LC/MS/MS) assay was used to qualitatively and quantitatively analyze the changes of proteins in SKOV3 cells before and after miR-125b overexpression, and the potential target genes regulated by miR-125b were screened. Further, Western blot was used to verify whether the target gene was affected by miR-125b. Result 1. MTT assay showed that the proliferation ability of SKOV3 cells decreased after overexpression of miR-125b, the migration and invasion of SKOV3 cells were significantly weakened by scratch assay and Transwell invasion assay. 2. ITQAR multiple chemical labeling mass spectrometry (iTQAR-LC/MS/MS) assay. According to miRNA candidate target gene prediction software (TargetScan,Pictar et al), 61 differentially expressed proteins possibly regulated by miR-125b were screened, among which 16 proteins were significantly different in expression level. Using bioinformatics to analyze the function of differentially expressed proteins (integrating NCBI,KEGG PATHWAY,Swiss-Prot and other database information), we select the ERBB2 gene as a potential target gene of miR-125b for further study. 3. Western blot results showed that miR-125b could inhibit the expression of ERBB2 gene. Conclusion this study suggests that miR-125b can inhibit the proliferation, migration and invasion of SKOV3 cells, and may be achieved by reducing the expression of ERBB2, a potential target gene.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31
本文编号：2208240
[Abstract]:Objective Ovarian cancer (Ovarian cancer) is one of the three most common malignant tumors in the female reproductive system, but its mortality is the highest among all kinds of gynecologic malignancies. Because of its hidden incidence and lack of early diagnosis, about 70% of patients with ovarian cancer are advanced when diagnosed. Genetic factors, menarche and menopausal age, reproductive and endocrine factors were found in the occurrence of ovarian cancer. In recent years, the emergence of a class of non-protein encoded microRNAs (miRNAs) provides a new idea for the study of ovarian tumors. MiRNAs are a kind of non-coding small molecule RNA, with a length of 20-22 nucleotides, which is mainly paired with the 3'-UTR region of the target gene in animals. Inhibition of target gene mRNAs translation, regulation of ontogeny, apoptosis, proliferation and differentiation, and other vital activities. With the further study of miRNA, it has been found that miRNA plays an important role in many kinds of malignant tumors. As a tumor suppressor gene or oncogene, miR-125b is an important member of the miRNA family as tumor suppressor gene or oncogene is involved in tumorigenesis, metastasis, drug resistance and other processes. It plays an important role in the development of many kinds of tumors, especially in the development of ovarian cancer. The purpose of this study was to detect the effect of miR-125b on the proliferation, migration and invasion of ovarian cancer SKOV3 cells by overexpression of miR-125b, in ovarian cancer SKOV3 cell lines, and to predict the potential target genes regulated by miR-125b in order to clarify the role of miR-125b in the development and development of ovarian cancer. Methods in this study, ovarian cancer SKOV3 cells were infected by overexpression of miR-125b lentivirus particles, and stable infection cell lines were screened by purine mycin, and SKOV3 cell lines with stable expression of miR-125b were established. The expression of miR-125b in SKVO3 cells was detected by real-time fluorescence quantitative PCR (qRT-PCR). The proliferation of SKOV3 cells after miR-125b overexpression was observed by MTT assay, and the migration and invasion ability of SKOV3 cells after miR-125b overexpression was observed by scratch test and Transwell invasion assay, respectively. ITRAQ multiplex chemical labeling tandem mass spectrometry (iTRAQ-LC/MS/MS) assay was used to qualitatively and quantitatively analyze the changes of proteins in SKOV3 cells before and after miR-125b overexpression, and the potential target genes regulated by miR-125b were screened. Further, Western blot was used to verify whether the target gene was affected by miR-125b. Result 1. MTT assay showed that the proliferation ability of SKOV3 cells decreased after overexpression of miR-125b, the migration and invasion of SKOV3 cells were significantly weakened by scratch assay and Transwell invasion assay. 2. ITQAR multiple chemical labeling mass spectrometry (iTQAR-LC/MS/MS) assay. According to miRNA candidate target gene prediction software (TargetScan,Pictar et al), 61 differentially expressed proteins possibly regulated by miR-125b were screened, among which 16 proteins were significantly different in expression level. Using bioinformatics to analyze the function of differentially expressed proteins (integrating NCBI,KEGG PATHWAY,Swiss-Prot and other database information), we select the ERBB2 gene as a potential target gene of miR-125b for further study. 3. Western blot results showed that miR-125b could inhibit the expression of ERBB2 gene. Conclusion this study suggests that miR-125b can inhibit the proliferation, migration and invasion of SKOV3 cells, and may be achieved by reducing the expression of ERBB2, a potential target gene.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31
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相关期刊论文 前2条
1 郑莉,任加强,陈琦,张慧萍,朱虹光;HER2/neu过表达通过PI3K/Akt通路对乳腺癌细胞MCF7中p53基因表达及细胞生长和放疗敏感性的影响[J];中华肿瘤杂志;2004年10期
2 ;MicroRNA-125b Induces Cancer Cell Apoptosis Through Suppression of Bcl-2 Expression[J];遗传学报;2012年01期
本文编号：2208240
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