核糖体蛋白L34在骨肉瘤中的表达及对人骨肉瘤细胞增殖的影响

发布时间：2017-12-26 16:12

本文关键词：核糖体蛋白L34在骨肉瘤中的表达及对人骨肉瘤细胞增殖的影响　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:骨肉瘤是对儿童和青少年的健康危害极大的恶性骨肿瘤。但是,骨肉瘤的发病率低,肿瘤异质性较高,因此,近二十年来,研究者在提高骨肉瘤治疗的效果和改善骨肉瘤患者的预后上取得的进展甚微。核糖体蛋白L34(ribosomal protein L34,RPL34)被认为除了参与核糖体生物合成外,还具有多种核糖体外功能。早期研究发现RPL34参与了原核生物的细胞增殖调控,近年来研究发现RPL34在部分恶性肿瘤的增殖中扮演了重要角色,但在人骨肉瘤中的功能尚未明确,并且RPL34介导恶性肿瘤细胞增殖调控的分子机制仍不清楚。目的:明确RPL34在人骨肉瘤组织及骨肉瘤细胞株中的表达情况,以及对人骨肉瘤细胞增殖和骨肉瘤患者预后的影响;初步探讨RPL34介导人骨肉瘤细胞增殖调控的分子机制;通过本研究为骨肉瘤的靶向治疗提供新的思路。方法:1、采用Real time-PCR检测11例骨肉瘤患者的癌和癌旁组织样本中RPL34 m RNA的表达水平。2、采用免疫组织化学方法检测95例骨肉瘤患者的癌组织和60例正常骨组织中RPL34蛋白的表达水平。3、计算上述95例骨肉瘤组织样本免疫组织化学染色得分的均值,并把95例样本划分为RPL34高表达组和低表达组;采用Kaplan-Meier法估计RPL34高表达组和低表达组患者的生存率,采用log-rank检验比较两组患者的生存曲线是否有差别。4、采用Real time-PCR检测人成骨细胞h FOB1.19及骨肉瘤细胞株(U2OS、MG-63、HOS和Saos-2)中RPL34 m RNA的表达水平。5、设计和筛选针对RPL34基因的RNA干扰靶点,构建RNA干扰慢病毒并转染人骨肉瘤细胞Saos-2,采用Real time-PCR和Western blot验证RPL34 RNA干扰靶点的敲减效率。6、采用Cellomics细胞计数检测、MTT检测、细胞周期检测、Annexin V-APC凋亡检测及克隆形成实验等来评估RPL34敲减对Saos-2细胞生物学行为的影响。7、分别基于UCSC数据库和STING数据库进行RPL34基因的转录调控因子筛查和蛋白-蛋白互作网络构建,然后对整合关系网络中的基因进行GO和KEGG通路富集分析,获得调控RPL34基因表达的转录因子和介导RPL34调控人骨肉瘤细胞增殖的下游相互作用蛋白。结果:1、11例骨肉瘤患者的癌和癌旁组织样本中:7例(7/11,63.64%)患者的骨肉瘤组织中RPL34的表达显著高于癌旁组织;3例(3/11,27.27%)患者的骨肉瘤组织和癌旁组织中RPL34的表达无差异;1例(1/11,9.09%)患者的骨肉瘤组织中RPL34表达低于癌旁组织。2、免疫组织化学检测表明:75例(78.95%,75/95)骨肉瘤患者的癌组织染色为强阳性,20例(21.05%,20/95)患者的癌组织染色为弱阳性,而60例正常骨组织染色均为弱阳性;骨肉瘤组织中RPL34蛋白的表达水平明显高于正常骨组织。3、对上述95例骨肉瘤患者的生存分析显示:RPL34蛋白高表达患者的3年生存率为35.42%(17/48),RPL34蛋白低表达患者的3年生存率为61.70%(29/47),RPL34蛋白高表达患者的预后明显较差。4、RPL34 m RNA在4株骨肉瘤细胞(U2OS、MG-63、HOS和Saos-2)中的表达均高于人成骨细胞h FOB1.19。5、RPL34表达敲减使Saos-2细胞的增殖速率受到显著抑制,细胞的集落形成数目减少,并且诱导了Saos-2细胞凋亡和G2/M期阻滞。6、生物信息学分析获得了参与RPL34基因转录调控的11个因子,即MYC及MYC相关因子X(MYC associated factor X,MAX)、CEBPB、E2F6、GABP、NFKB、NRSF、SPI1、TAF1、TBP、YY1,以及RPL34下游与其相互作用的100个蛋白;进一步的GO和KEGG通路富集分析提示:RPL34基因的转录表达受原癌基因蛋白MYC调控,并且真核翻译起始因子3的亚基(EIF3A、EIF3F或EIF3G)可能介导了RPL34对人骨肉瘤细胞的增殖调控。结论:1、RPL34在骨肉瘤中高表达,并促进人骨肉瘤细胞的增殖。2、RPL34高表达的骨肉瘤患者临床预后较差。3、RPL34可能通过“MYC/RPL34/EIF3(EIF3A、EIF3F或EIF3G)”信号轴而参与了人骨肉瘤细胞的增殖调控。
[Abstract]:Background: osteosarcoma is a malignant bone tumor that is very harmful to the health of children and adolescents. However, the incidence of osteosarcoma is low and the tumor heterogeneity is high. Therefore, in recent twenty years, researchers have made little progress in improving the therapeutic effect of osteosarcoma and improving the prognosis of patients with osteosarcoma. Ribosomal protein L34 (ribosomal protein L34, RPL34) is believed to have a variety of ribose in vitro function besides participating in the biosynthesis of ribosome. Early studies found that RPL34 participates in the regulation of cell proliferation in prokaryotes, recent studies have found that RPL34 plays an important role in the proliferation of some malignant tumors, but in human osteosarcoma function is not clear, and the molecular mechanism of RPL34 mediated tumor cell proliferation remains unclear. Objective: To investigate the expression of RPL34 in osteosarcoma tissue and osteosarcoma cell lines, as well as the impact on human osteosarcoma cell proliferation and prognosis of osteosarcoma patients; to investigate the molecular mechanism of RPL34 mediated proliferation of human osteosarcoma cell regulation; through this research for osteosarcoma targeted therapy to provide new ideas. Methods: 1. The expression level of RPL34 m RNA in the samples of 11 cases of osteosarcoma and the tissue samples from the para cancerous tissue was detected by Real time-PCR. 2. Immunohistochemistry was used to detect the expression of RPL34 protein in 95 cases of osteosarcoma and 60 cases of normal bone tissue. 3, the calculation of the above 95 cases of osteosarcoma tissue samples by immunohistochemical staining and the average scores, the 95 samples were divided into RPL34 high expression group and low expression group; estimation of the RPL34 high expression group and low expression group of patients survival rate by Kaplan-Meier method, using log-rank test the survival curves were compared between the two groups if there is a difference. 4. Real time-PCR was used to detect the expression level of RPL34 m RNA in human osteoblast h FOB1.19 and osteosarcoma cell lines (U2OS, MG-63, HOS and Saos-2). 5, we designed and screened RNA interference targets for RPL34 gene, constructed RNA interference lentivirus and transfected human osteosarcoma cell Saos-2, and used Real time-PCR and Western blot to verify RPL34 RNA interference target knockdown efficiency. 6, we used Cellomics cell count test, MTT detection, cell cycle detection, Annexin V-APC apoptosis assay and clonogenic assay to evaluate the effect of RPL34 knockdown on Saos-2 cell biological behavior. 7, respectively, UCSC database and STING database based on RPL34 gene transcription factor screening and protein - protein interaction network construction, and to integrate the relationship network of genes GO and KEGG pathway enrichment analysis, protein transcription factor and RPL34 mediated regulation of human osteosarcoma cell proliferation downstream of the interaction and regulation of RPL34 gene the expression of. Results: 1 cases of bone sarcoma, 11 patients with cancer and adjacent tissue samples in 7 cases: (7/11,63.64%) RPL34 expression in osteosarcoma tissues were significantly higher than that in paracancerous tissues; 3 cases (3/11,27.27%) expression of RPL34 in osteosarcoma tissues and cancer tissues; 1 cases (1/ 11,9.09%) lower than that of adjacent tissues. The expression of RPL34 in osteosarcoma patients. 2, immunohistochemistry showed that 75 cases (78.95%, 75/95) cancer tissues of patients with osteosarcoma were strongly positive, 20 cases (21.05%, 20/95) cancer patients was weakly positive, and 60 cases of normal bone tissue staining were weakly positive; the expression level of RPL34 protein in osteosarcoma was higher than in normal bone tissues. 3, survival analysis of 95 cases of osteosarcoma showed that the 3 year survival rate of patients with high expression of RPL34 protein was 35.42% (17/48). The 3 year survival rate of patients with low expression of RPL34 protein was 61.70% (29/47), and the prognosis of patients with high expression of RPL34 protein was significantly poorer. The expression of 4 and RPL34 m RNA in 4 osteosarcoma cells (U2OS, MG-63, HOS and Saos-2) was higher than that of human osteoblast h FOB1.19. 5, knockdown of RPL34 expression significantly inhibited the proliferation rate of Saos-2 cells, reduced the number of colony forming cells, and induced Saos-2 cell apoptosis and G2/M phase arrest. 6, bioinformatics analysis obtained 11 factors involved in the transcriptional regulation of the RPL34 gene, namely MYC and MYC (MYC associated factor X factor X, MAX, CEBPB), E2F6, GABP, NFKB, NRSF, SPI1, TAF1, TBP, YY1, and 100 proteins downstream of RPL34 interacted with GO; further enrichment and KEGG pathway analysis showed that the transcription of RPL34 gene expression by the proto oncogene protein MYC regulation and subunit of the eukaryotic translation initiation factor 3 (EIF3A, EIF3F or EIF3G) may mediate the regulation of RPL34 on the proliferation of human osteosarcoma cells. Conclusion: 1, RPL34 is highly expressed in osteosarcoma and promotes the proliferation of human osteosarcoma cells. 2, RPL34 high expression of osteosarcoma patients have poor clinical prognosis. 3, RPL34 may be involved in the proliferation regulation of human osteosarcoma cells through the "MYC/RPL34/EIF3 (EIF3A, EIF3F or EIF3G)" signal axis.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738.1

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