發(fā)布時間：2017-12-26 21:41

  本文關鍵詞：MiR-29c在骨肉瘤中的表達及對骨肉瘤細胞系SOSP9607增殖、轉(zhuǎn)移能力的影響　出處：《第四軍醫(yī)大學》2015年碩士論文　論文類型：學位論文

  更多相關文章： 骨肉瘤 增殖 侵襲、遷移 miRNA miR-29c SOSP9607

【摘要】：骨肉瘤是一種少見的骨原發(fā)惡性腫瘤,早期肺轉(zhuǎn)移、臨床預后差是骨肉瘤的特點,也是影響患者生存率的重要因素。microRNA是一種具有轉(zhuǎn)錄后調(diào)控作用的小分子RNA,具有多種生理功能,參與多種惡性腫瘤增殖、轉(zhuǎn)移的調(diào)控。研究發(fā)現(xiàn),miR-29家族中的miR-29c在多種惡性腫瘤中表達下調(diào),并驗證其對多種惡性腫瘤的轉(zhuǎn)移和增殖起到抑制作用,前期實驗證實mi R-29c在骨肉瘤組織和細胞系中異常表達,提示其可能參與了骨肉瘤的增殖和轉(zhuǎn)移。目的:驗證miR-29c在骨肉瘤組織中的異常表達和不同侵襲能力骨肉瘤細胞系中的差異表達;探究miR-29c對骨肉瘤細胞系增殖、侵襲和遷移能力的影響,為骨肉瘤的分子靶向治療提供理論基礎。方法:1.采用實時定量PCR檢測68例骨肉瘤、正常組織標本miR-29c的表達;用此法檢測SOSP-9607骨肉瘤細胞系的兩組侵襲性不同的細胞亞系F5M2和F4中miR-29c的表達。2.采用Lipofectamine2000脂質(zhì)體法分別將miR-29c mimics和inhibitor瞬時轉(zhuǎn)染入sosp-9607的兩個細胞亞系,構建mir-29c高表達、低表達骨肉瘤細胞模型。3.對所構建的細胞模型進行腫瘤細胞功能實驗,包括transwell侵襲和遷移實驗、mtt法檢測腫瘤細胞增殖曲線;流式細胞儀法檢測腫瘤細胞周期和凋亡比例。結果:1.骨肉瘤組織mir-29c表達明顯低于瘤旁正常組織;高侵襲性f5m2骨肉瘤細胞系mir-29c表達明顯低于低侵襲性f4骨肉瘤細胞系。2.用lipofectamine2000脂質(zhì)體分別將mir-29cmimics和inhibitor成功轉(zhuǎn)入f5m2骨肉瘤細胞系和f4骨肉瘤細胞系。rt-pcr結果驗證f5m2/29cmimics細胞中mir-29c表達增加;f4/29cinhibitor細胞通過rt-pcr間接驗證其靶基因的表達,證明f4骨肉瘤細胞mir-29c下調(diào)成功。3.transwell細胞侵襲和遷移實驗顯示,mir-29c上調(diào)的骨肉瘤細胞f5m2侵襲和遷移能力較對照組均明顯下降;mir-29c下調(diào)的骨肉瘤細胞f4侵襲和遷移能力較對照組明顯提升。mtt細胞增殖實驗顯示,mir-29c上調(diào)的骨肉瘤細胞f5m2增殖能力較對照組減弱,細胞增殖曲線下移;mir-29c下調(diào)的骨肉瘤細胞f4增殖能力較對照組增強,細胞增殖曲線上移。4.流式細胞儀細胞周期和細胞凋亡實驗顯示,mir-29c上調(diào)的骨肉瘤細胞f5m2與對照組相比,各周期腫瘤細胞比例無明顯差異,凋亡細胞比例也無明顯差異;mir-29c下調(diào)的骨肉瘤細胞f4與對照組相比,各周期腫瘤細胞比例亦無明顯差異,凋亡細胞比例無統(tǒng)計學差異。結論:1.mir-29c骨肉瘤組織中表達含量下調(diào),且高侵襲性骨肉瘤細胞mir-29c表達含量低于低侵襲性骨肉瘤細胞系,提示mir-29c與腫瘤侵襲能力呈負相關,mir-29c可能參與了骨肉瘤細胞的增殖和轉(zhuǎn)移能力的調(diào)控。2.通過構建mir-29c上、下調(diào)腫瘤細胞模型,驗證了mir-29c對骨肉瘤細胞增殖、侵襲及遷移能力的抑制作用,為下一步研究mir-29c抑制骨肉瘤細胞增殖和轉(zhuǎn)移的分子機制研究打下基礎3.mir-29c對骨肉瘤細胞系sosp9607的細胞周期和凋亡無明顯影響,mir-29c對腫瘤細胞周期和凋亡的作用可能具有組織特異性。
[Abstract]:Osteosarcoma is a rare primary malignant tumor of bone. Early pulmonary metastasis and poor clinical prognosis are the characteristics of osteosarcoma and also an important factor affecting the survival rate of the patients. MicroRNA, a small molecule RNA with post transcriptional regulation, has many physiological functions and participates in the regulation of multiple malignant tumor proliferation and metastasis. The study found that the expression of miR-29 family miR-29c in a variety of malignant tumors is down regulated, and verifies its effect on malignant tumor metastasis and proliferation play an inhibitory effect, previous experiments confirmed that the abnormal expression of MI and R-29c in osteosarcoma tissues and cell lines, suggesting that it may be involved in the proliferation and metastasis of osteosarcoma. Objective: to verify the abnormal expression of miR-29c in osteosarcoma and the differential expression of different invasiveness in osteosarcoma cell lines, and to explore the effect of miR-29c on the proliferation, invasion and migration of osteosarcoma cell lines, so as to provide a theoretical basis for molecular targeted therapy of osteosarcoma. Methods: 1.. Real time quantitative PCR was used to detect the expression of miR-29c in 68 cases of osteosarcoma and normal tissue. The expression of miR-29c in two groups of SOSP-9607 osteosarcoma cell lines and two subsets of invasive F5M2 and F4 were detected by this method. 2., Lipofectamine2000 liposome method was used to transfect miR-29c mimics and inhibitor into two subsets of sosp-9607 respectively, and the mir-29c expression model with high expression and low expression in osteosarcoma cells was constructed. 3., we conducted tumor cell function experiments on the constructed cell models, including Transwell invasion and migration experiments, MTT assay for tumor cell proliferation curve, and flow cytometry to detect tumor cell cycle and apoptosis ratio. Results: the expression of mir-29c in 1. osteosarcoma tissues was significantly lower than that in adjacent normal tissues. The expression of mir-29c in highly invasive f5m2 osteosarcoma cell lines was significantly lower than that in low invasive F4 osteosarcoma cell lines. 2. mir-29cmimics and inhibitor were successfully transferred into f5m2 osteosarcoma cell line and F4 osteosarcoma cell line with lipofectamine2000 liposomes. RT-PCR results showed that the expression of mir-29c increased in f5m2/29cmimics cells. F4/29cinhibitor cells indirectly verified the expression of target genes by RT-PCR, which showed that mir-29c of F4 osteosarcoma cells was down regulated successfully. 3.transwell cell invasion and migration experiments showed that mir-29c up-regulated f5m2 invasion and migration ability of osteosarcoma cells were significantly lower than that of the control group; mir-29c down regulated the invasion and migration ability of osteosarcoma cell F4, which was significantly higher than that of the control group. MTT cell proliferation assay showed that the proliferation ability of osteosarcoma cell f5m2 increased with the increase of mir-29c, and the cell proliferation curve decreased. Compared with the control group, the cell proliferation curve of mir-29c decreased, and the proliferation ability of osteosarcoma cells increased. 4. flow cytometry cell cycle and cell apoptosis in experimental mir-29c, upregulation of f5m2 osteosarcoma cells compared with the control group, no significant difference in the proportion of the tumor cells in each cycle, and no significant difference in the proportion of apoptotic cells; mir-29c downregulation of F4 osteosarcoma cells compared with the control group, no significant differences in the proportion of the tumor cells in each cycle. There was no significant difference in the proportion of apoptotic cells. Conclusion: the content of expression of 1.mir-29c in osteosarcoma, and invasion of osteosarcoma cell mir-29c expression was lower than that of low invasive osteosarcoma cell lines, suggesting that mir-29c was negatively related to invasion and tumor, the regulation of mir-29c may be involved in the proliferation and metastasis of osteosarcoma cells. 2. through the construction of mir-29c, down-regulation of tumor cell model, verified the inhibitory effect of mir-29c on the proliferation of osteosarcoma cell invasion and migration, the next step for the study of molecular mechanism of inhibitory effect of mir-29c on proliferation and metastasis of osteosarcoma cells lay the foundation of 3.mir-29c has no obvious effect on osteosarcoma cell line sosp9607 cell cycle and apoptosis and the effect of mir-29c on tumor cell cycle and apoptosis may have tissue specificity.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R738.1

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本文編號：1338954