本文關(guān)鍵詞：核糖體蛋白L34在骨肉瘤中的表達(dá)及對人骨肉瘤細(xì)胞增殖的影響　出處：《廣西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨肉瘤 RPL34 腫瘤增殖 MYC 真核翻譯起始因子3亞基

【摘要】：背景:骨肉瘤是對兒童和青少年的健康危害極大的惡性骨腫瘤。但是,骨肉瘤的發(fā)病率低,腫瘤異質(zhì)性較高,因此,近二十年來,研究者在提高骨肉瘤治療的效果和改善骨肉瘤患者的預(yù)后上取得的進(jìn)展甚微。核糖體蛋白L34(ribosomal protein L34,RPL34)被認(rèn)為除了參與核糖體生物合成外,還具有多種核糖體外功能。早期研究發(fā)現(xiàn)RPL34參與了原核生物的細(xì)胞增殖調(diào)控,近年來研究發(fā)現(xiàn)RPL34在部分惡性腫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確,并且RPL34介導(dǎo)惡性腫瘤細(xì)胞增殖調(diào)控的分子機(jī)制仍不清楚。目的:明確RPL34在人骨肉瘤組織及骨肉瘤細(xì)胞株中的表達(dá)情況,以及對人骨肉瘤細(xì)胞增殖和骨肉瘤患者預(yù)后的影響;初步探討RPL34介導(dǎo)人骨肉瘤細(xì)胞增殖調(diào)控的分子機(jī)制;通過本研究為骨肉瘤的靶向治療提供新的思路。方法:1、采用Real time-PCR檢測11例骨肉瘤患者的癌和癌旁組織樣本中RPL34 m RNA的表達(dá)水平。2、采用免疫組織化學(xué)方法檢測95例骨肉瘤患者的癌組織和60例正常骨組織中RPL34蛋白的表達(dá)水平。3、計算上述95例骨肉瘤組織樣本免疫組織化學(xué)染色得分的均值,并把95例樣本劃分為RPL34高表達(dá)組和低表達(dá)組;采用Kaplan-Meier法估計RPL34高表達(dá)組和低表達(dá)組患者的生存率,采用log-rank檢驗比較兩組患者的生存曲線是否有差別。4、采用Real time-PCR檢測人成骨細(xì)胞h FOB1.19及骨肉瘤細(xì)胞株(U2OS、MG-63、HOS和Saos-2)中RPL34 m RNA的表達(dá)水平。5、設(shè)計和篩選針對RPL34基因的RNA干擾靶點,構(gòu)建RNA干擾慢病毒并轉(zhuǎn)染人骨肉瘤細(xì)胞Saos-2,采用Real time-PCR和Western blot驗證RPL34 RNA干擾靶點的敲減效率。6、采用Cellomics細(xì)胞計數(shù)檢測、MTT檢測、細(xì)胞周期檢測、Annexin V-APC凋亡檢測及克隆形成實驗等來評估RPL34敲減對Saos-2細(xì)胞生物學(xué)行為的影響。7、分別基于UCSC數(shù)據(jù)庫和STING數(shù)據(jù)庫進(jìn)行RPL34基因的轉(zhuǎn)錄調(diào)控因子篩查和蛋白-蛋白互作網(wǎng)絡(luò)構(gòu)建,然后對整合關(guān)系網(wǎng)絡(luò)中的基因進(jìn)行GO和KEGG通路富集分析,獲得調(diào)控RPL34基因表達(dá)的轉(zhuǎn)錄因子和介導(dǎo)RPL34調(diào)控人骨肉瘤細(xì)胞增殖的下游相互作用蛋白。結(jié)果:1、11例骨肉瘤患者的癌和癌旁組織樣本中:7例(7/11,63.64%)患者的骨肉瘤組織中RPL34的表達(dá)顯著高于癌旁組織;3例(3/11,27.27%)患者的骨肉瘤組織和癌旁組織中RPL34的表達(dá)無差異;1例(1/11,9.09%)患者的骨肉瘤組織中RPL34表達(dá)低于癌旁組織。2、免疫組織化學(xué)檢測表明:75例(78.95%,75/95)骨肉瘤患者的癌組織染色為強(qiáng)陽性,20例(21.05%,20/95)患者的癌組織染色為弱陽性,而60例正常骨組織染色均為弱陽性;骨肉瘤組織中RPL34蛋白的表達(dá)水平明顯高于正常骨組織。3、對上述95例骨肉瘤患者的生存分析顯示:RPL34蛋白高表達(dá)患者的3年生存率為35.42%(17/48),RPL34蛋白低表達(dá)患者的3年生存率為61.70%(29/47),RPL34蛋白高表達(dá)患者的預(yù)后明顯較差。4、RPL34 m RNA在4株骨肉瘤細(xì)胞(U2OS、MG-63、HOS和Saos-2)中的表達(dá)均高于人成骨細(xì)胞h FOB1.19。5、RPL34表達(dá)敲減使Saos-2細(xì)胞的增殖速率受到顯著抑制,細(xì)胞的集落形成數(shù)目減少,并且誘導(dǎo)了Saos-2細(xì)胞凋亡和G2/M期阻滯。6、生物信息學(xué)分析獲得了參與RPL34基因轉(zhuǎn)錄調(diào)控的11個因子,即MYC及MYC相關(guān)因子X(MYC associated factor X,MAX)、CEBPB、E2F6、GABP、NFKB、NRSF、SPI1、TAF1、TBP、YY1,以及RPL34下游與其相互作用的100個蛋白;進(jìn)一步的GO和KEGG通路富集分析提示:RPL34基因的轉(zhuǎn)錄表達(dá)受原癌基因蛋白MYC調(diào)控,并且真核翻譯起始因子3的亞基(EIF3A、EIF3F或EIF3G)可能介導(dǎo)了RPL34對人骨肉瘤細(xì)胞的增殖調(diào)控。結(jié)論:1、RPL34在骨肉瘤中高表達(dá),并促進(jìn)人骨肉瘤細(xì)胞的增殖。2、RPL34高表達(dá)的骨肉瘤患者臨床預(yù)后較差。3、RPL34可能通過“MYC/RPL34/EIF3(EIF3A、EIF3F或EIF3G)”信號軸而參與了人骨肉瘤細(xì)胞的增殖調(diào)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R738.1

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