本文選題：lncRNA + ANRIL��； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：研究背景和目的骨肉瘤是骨外科常見的惡性腫瘤,其起源于骨髓間充質(zhì)干細(xì)胞,且好發(fā)青少年兒童。手術(shù)及放化療聯(lián)合應(yīng)用是本病目前治療的基本手段,伴隨手術(shù)方式的改進(jìn)和新型化療藥物的應(yīng)用使得本病的預(yù)后有了較為明顯的改善,但仍約有40%的患者治療后出現(xiàn)腫瘤轉(zhuǎn)移,五年生存率未有明顯提高,這預(yù)示仍需新的治療方法來改善患者愈后,發(fā)現(xiàn)新的基因?qū)侨饬龅挠绊懰坪跏且环N有效的手段。長鏈非編碼RNA(Long non-coding RNA,lncRNA)是長度大于200個(gè)核苷酸的非編碼RNA。做為遺傳學(xué)研究熱點(diǎn),許多研究表明,lncRNA在劑量補(bǔ)償效應(yīng)、表觀遺傳調(diào)控、細(xì)胞周期調(diào)控和細(xì)胞分化調(diào)控等眾多生命活動(dòng)中發(fā)揮重要作用。lncRNA表達(dá)量的變化或其功能異常與導(dǎo)致人類疾病的發(fā)生密切相關(guān),包括癌癥、退行性神經(jīng)疾病等在內(nèi)的許多對(duì)人類健康危害嚴(yán)重的重大疾病,具體表現(xiàn)為lncRNA在序列和空間結(jié)構(gòu)的異常、表達(dá)水平的異常、與結(jié)合蛋白相互作用的異常等。lncRNA ANRIL是細(xì)胞周期激酶抑制因子4b(INK4b)位點(diǎn)的反義非編碼RNA(antisense non-coding RNA in the INK4 Locus),其表達(dá)與INK4a的表觀遺傳學(xué)沉默相關(guān)。INK4b-ARF-INK4a位點(diǎn)對(duì)細(xì)胞周期、衰老和調(diào)亡的調(diào)控中具有重要的作用。然而,關(guān)于lncRNA ANRIL對(duì)骨肉瘤細(xì)胞生物學(xué)行為的影響尚未見報(bào)道。因此,探討lncRNA ANRIL對(duì)人骨肉瘤細(xì)胞生物學(xué)功能的影響,從而為骨肉瘤的臨床治療提供新的思路和策略。本實(shí)驗(yàn)通過收集骨肉瘤癌組織及癌旁組織的標(biāo)本,比較lncRNA ANRIL在癌組織及癌旁組織表達(dá)的差異。將小干擾RNA(siRNA)分子轉(zhuǎn)染進(jìn)入骨肉瘤細(xì)胞來抑制lncRNA ANRIL基因的表達(dá),觀察抑制lncRNA ANRIL的表達(dá)對(duì)骨肉瘤細(xì)胞增殖、凋亡及侵襲遷移能力的影響,為lncRNA ANRIL在骨肉瘤的防治中提供實(shí)驗(yàn)依據(jù)。方法1.在鄭州大學(xué)第一附屬醫(yī)院骨科手術(shù)室收集25例配對(duì)骨肉瘤患者的癌組織及癌旁組織標(biāo)本,保存于-80°C冰箱。使用熒光實(shí)時(shí)定量PCR(real-time quantitative PCR,RT-q PCR)方法檢測25例配對(duì)骨肉瘤癌組織及癌旁組織標(biāo)本中l(wèi)ncRNA ANRIL的表達(dá)水平。2.試劑公司人工合成lncRNA ANRIL的抑制物:siRNA-ANRIL。實(shí)驗(yàn)分為三組,實(shí)驗(yàn)組(siRNA-ANRIL):將siRNA-ANRIL轉(zhuǎn)染入骨肉瘤細(xì)胞MG63中,陰性對(duì)照組(Nagetive control,NC):轉(zhuǎn)染lncRNA ANRIL Nagetive control,空白組(Blank):僅轉(zhuǎn)染脂質(zhì)體。轉(zhuǎn)染后,將細(xì)胞至于CO2培養(yǎng)箱中,用于后續(xù)實(shí)驗(yàn)。3.轉(zhuǎn)染48h后,用RT-q PCR技術(shù)測量三組細(xì)胞內(nèi)lncRNA ANRIL基因的表達(dá)量,觀察siRNA-ANRIL對(duì)lncRNA ANRIL表達(dá)的抑制效果。4.利用CCK-8細(xì)胞計(jì)數(shù)試劑盒(Cell Counting Kit-8,CCK-8)在處理24h、48h、72h、96h后分別測量三組細(xì)胞的增殖能力,觀察抑制lncRNA ANRIL表達(dá)對(duì)骨肉瘤細(xì)胞增殖的影響。5.用流式細(xì)胞技術(shù)和caspase-3酶活性檢測試劑盒分別對(duì)三組細(xì)胞的凋亡進(jìn)行測量,觀察抑制lncRNA ANRIL表達(dá)后對(duì)骨肉瘤細(xì)胞凋亡的影響。6.Transwell侵襲實(shí)驗(yàn),抑制lncRNA ANRIL表達(dá)后在12h時(shí)觀察對(duì)骨肉瘤細(xì)胞侵襲能力的影響。7.劃痕實(shí)驗(yàn),對(duì)三組骨肉瘤細(xì)胞進(jìn)行劃痕處理,在處理12h后觀察抑制lncRNA ANRIL表達(dá)后對(duì)骨肉瘤細(xì)胞遷移能力的影響。8.利用蛋白質(zhì)印跡法(Western blot)分別檢測轉(zhuǎn)染后的三組骨肉瘤細(xì)胞中腫瘤轉(zhuǎn)移相關(guān)基因1蛋白(metastasis-associaled gene 1,MTA1),上皮細(xì)胞鈣黏蛋白(E-cadherin,E-cad),B淋巴細(xì)胞瘤-2蛋白(bcl-2)的表達(dá)量。結(jié)果1.與癌旁組織相比,骨肉瘤癌組織中l(wèi)ncRNA ANRIL基因的表達(dá)水平明顯增高,其差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。2.RT-q PCR結(jié)果顯示,轉(zhuǎn)染后siRNA-ANRIL組lncRNA ANRIL基因的表達(dá)水平明顯低于陰性對(duì)照組和空白組,其差異具有統(tǒng)計(jì)學(xué)意義(P0.05),陰性對(duì)照組和空白組lncRNA ANRIL表達(dá)量無明顯差異(P0.05)。3.CCK-8結(jié)果顯示,siRNA-ANRIL組的骨肉瘤細(xì)胞在OD450處的吸光度值明顯下降,且隨時(shí)間延長,吸光度值與陰性對(duì)照組和空白組相比差異越明顯,其差異具有統(tǒng)計(jì)學(xué)意義(P0.05),陰性對(duì)照組和空白組的吸光度值相近,且隨時(shí)間變化不明顯,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。4.流式細(xì)胞技術(shù)和caspase-3酶活性檢測試劑盒結(jié)果均表明,與陰性對(duì)照組和空白組相比,siRNA-ANRIL組的骨肉瘤細(xì)胞的凋亡率和Caspase-3酶活性均明顯升高,其差異具有統(tǒng)計(jì)學(xué)意義(P0.05),陰性對(duì)照組和空白組骨肉瘤細(xì)胞的凋亡率和Caspase-3酶活性無明顯差異(P0.05)。5.Transwell侵襲實(shí)驗(yàn)結(jié)果顯示,siRNA-ANRIL組骨肉瘤細(xì)胞穿過基底膜的數(shù)量明顯低于陰性對(duì)照組和空白組,其差異具有統(tǒng)計(jì)學(xué)意義(P0.05),而陰性對(duì)照組和空白組骨肉瘤細(xì)胞穿過基底膜的數(shù)量差異無統(tǒng)計(jì)學(xué)意義(P0.05)。6.對(duì)三組細(xì)胞進(jìn)行劃痕處理12h以后的結(jié)果提示,siRNA-ANRIL組骨肉瘤細(xì)胞的遷移能力與陰性對(duì)照組和空白組相比明顯降低,陰性對(duì)照組和空白組骨肉瘤細(xì)胞的遷移能力無明顯差異。7.Western blot結(jié)果顯示,siRNA-ANRIL組中MTA1表達(dá)量降低,E-cad表達(dá)量升高,bcl-2蛋白表達(dá)量降低,與陰性對(duì)照組和空白組相比較,其差異具有統(tǒng)計(jì)學(xué)意義(P0.05),而陰性對(duì)照組和空白組MTA1,E-cad和bcl-2蛋白的表達(dá)量相近,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論1.lncRNA ANRIL在骨肉瘤癌組織的表達(dá)高于癌旁組織,且可通過使用siRNA-ANRIL來抑制骨肉瘤細(xì)胞lncRNA ANRIL的表達(dá)。2.抑制lncRNA ANRIL基因的表達(dá)能夠促進(jìn)骨肉瘤細(xì)胞的凋亡,且能夠抑制骨肉瘤細(xì)胞的增殖、侵襲和遷移能力。
[Abstract]:Background and objective osteosarcoma is a common malignant tumor in bone surgery. It originates in bone marrow mesenchymal stem cells (MSCs) and develops young children well. Operation and radiotherapy combined with chemotherapy are the basic methods for the treatment of this disease. With the improvement of surgical methods and the use of new chemotherapeutic drugs, the prognosis of this disease has been improved significantly. However, about 40% of the patients still have tumor metastasis after treatment, and the five year survival rate has not been significantly improved. This indicates that a new treatment is still needed to improve the patient's recovery. It is found that the effect of the new gene on osteosarcoma seems to be an effective means. Long chain non coded RNA (Long non-coding RNA, lncRNA) is a non coding length greater than 200 nucleotides. RNA. is a hot topic in genetics. Many studies have shown that lncRNA plays an important role in many life activities such as dose compensation effect, epigenetic regulation, cell cycle regulation and cell differentiation regulation. The changes in the expression of.LncRNA or its function abnormality are closely related to the occurrence of human diseases, including cancer and degenerative neurological disease. Many serious diseases, such as lncRNA in sequence and space structure, abnormal expression level, abnormal interaction with binding proteins,.LncRNA ANRIL are antisense non coded RNA (antisense non-coding RNA in the INK4), and so on. CUS), its expression and epigenetic silencing of INK4a have an important role in the regulation of cell cycle, senescence and apoptosis. However, the effect of lncRNA ANRIL on the biological behavior of osteosarcoma cells has not yet been reported. Therefore, the effect of lncRNA ANRIL on the biological function of human osteosarcoma cells is discussed, thus the effect of lncRNA ANRIL on the biological function of human osteosarcoma cells is discussed. This experiment provides a new way of thinking and strategy for the clinical treatment of osteosarcoma. By collecting the specimens of osteosarcoma and para cancer tissue, the difference of expression of lncRNA ANRIL in cancer tissue and para cancer tissue was compared. Small interference RNA (siRNA) molecules were transfected into osteosarcoma cells to inhibit the expression of lncRNA ANRIL gene and to observe the inhibition of lncRNA ANRIL. The effects of expression on the proliferation, apoptosis and invasion and migration of osteosarcoma cells were provided for lncRNA ANRIL in the prevention and treatment of osteosarcoma. Methods 1. the specimens of 25 cases of paired osteosarcoma in the Department of orthopedics, the First Affiliated Hospital of Zhengzhou University, were collected and preserved in the -80 C refrigerator. The fluorescence real-time quantitative PCR (R) was used. The eal-time quantitative PCR, RT-q PCR) method was used to detect the expression of lncRNA ANRIL in 25 cases of paired osteosarcoma and paracarkoma tissue. The inhibitor of lncRNA ANRIL was synthesized by.2. reagent company. The siRNA-ANRIL. experiment was divided into three groups, and the experimental group (siRNA-ANRIL) was transfected into osteosarcoma cells and negative control group. Tive control, NC): transfection of lncRNA ANRIL Nagetive control, blank group (Blank): transfection only liposomes. After transfection, the cells were used in the CO2 incubator for the follow-up experimental.3. transfection 48h. The 8 cell counting Kit (Cell Counting Kit-8, CCK-8) measured the proliferation ability of three groups of cells after 24h, 48h, 72h and 96h, and observed the effect of the inhibition of lncRNA ANRIL expression on the proliferation of osteosarcoma cells.5. using flow cytometry and caspase-3 enzyme activity detection kit to measure the apoptosis of three groups of cells respectively. The effect of A ANRIL expression on osteosarcoma cell apoptosis,.6.Transwell invasion experiment, inhibition of lncRNA ANRIL expression on the invasion ability of osteosarcoma cells after 12h,.7. scratch test, three groups of osteosarcoma cells were scratched, and the migration ability of osteosarcoma cells after lncRNA ANRIL expression was observed after 12h treatment. The expression of tumor metastasis related gene 1 protein (metastasis-associaled gene 1, MTA1), epithelial calcic mucin (E-cadherin, E-cad) and B lymphocytic -2 protein (Bcl-2) expression in three groups of osteosarcoma cells after transfection were detected by Western blot (Western blot). Results 1. in the tissues of osteosarcoma carcinoma, LN.8. was compared with that of para cancerous tissue. The expression level of cRNA ANRIL gene was significantly higher, and the difference was statistically significant (P0.05).2.RT-q PCR results showed that the expression level of lncRNA ANRIL gene in the siRNA-ANRIL group was significantly lower than that of the negative control group and the blank group, and the difference was statistically significant (P0.05). There was no significant difference in the expression of lncRNA ANRIL in the negative control group and the blank group. P0.05.3.CCK-8 results showed that the absorbance value of osteosarcoma cells in group siRNA-ANRIL decreased obviously at OD450, and the difference of absorbance value was more obvious compared to negative control group and blank group with time, and the difference was statistically significant (P0.05). The absorbance value of negative control group and blank group was similar, and the change was not obvious with time. The difference was not statistically significant (P0.05).4. flow cytometry and caspase-3 enzyme activity detection kit showed that, compared with the negative control group and the blank group, the apoptosis rate and the Caspase-3 enzyme activity of the osteosarcoma cells in the siRNA-ANRIL group were significantly increased, and the difference was of the significance (P0.05), the negative control group and the blank group of osteosarcoma. The apoptosis rate and Caspase-3 enzyme activity of the cells were not significantly different (P0.05).5.Transwell invasion test results showed that the number of osteosarcoma cells passing through the basement membrane in siRNA-ANRIL group was significantly lower than that of the negative control group and the blank group, and the difference was statistically significant (P0.05), while the number of osteosarcoma cells in negative and blank groups passed through the basal membrane of the negative group and the blank group. The difference was not statistically significant (P0.05).6. after the scratch treatment of three groups of cells 12h results suggested that the migration of osteosarcoma cells in the siRNA-ANRIL group was significantly lower than that in the negative control group and the blank group. The migration ability of the osteosarcoma cells in the negative control group and the blank group was not significantly different from the.7.Western blot results, the siRNA-ANRIL group was shown in the siRNA-ANRIL group. The expression of MTA1 was reduced, the expression of E-cad increased and the expression of Bcl-2 protein decreased. Compared with the negative control group and the blank group, the difference was statistically significant (P0.05), while the expression of MTA1, E-cad and bcl-2 protein in the negative control group and the blank group was similar, and the difference was not statistically significant (P0.05). Conclusion 1.lncRNA ANRIL is in the osteosarcoma carcinoma tissue. The expression is higher than that of para cancerous tissue, and siRNA-ANRIL can be used to inhibit the expression of lncRNA ANRIL in osteosarcoma cells by.2. to inhibit the expression of lncRNA ANRIL gene, which can promote the apoptosis of osteosarcoma cells and inhibit the proliferation, invasion and migration of osteosarcoma cells.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R738.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Mohammadreza Hajjari;Abbas Salavaty;;HOTAIR:an oncogenic long non-coding RNA in different cancers[J];Cancer Biology & Medicine;2015年01期

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本文編號(hào)：1809270