骨肉瘤組織內(nèi)miR-29b作用于CDK6抑制腫瘤細胞增殖和遷移
發(fā)布時間：2018-07-25 20:12
【摘要】：[目的]文獻研究表明,miR-29b具有抑制腫瘤細胞增殖和遷移的作用,而CDK6可以促進腫瘤細胞增殖和遷移。研究骨肉瘤細胞中,miR-29b是否通過作用于CDK6抑制骨肉瘤細胞增殖和遷移,為骨肉瘤的臨床治療進展提供理論依據(jù),探尋骨肉瘤治療新方向。[方法]臨床中選取無合并其他腫瘤、非復(fù)發(fā)、未放化療的骨肉瘤患者,留取手術(shù)切除的骨肉瘤組織和瘤旁正常組織。通過Western blotting和qRT-PCR的方法測定骨肉瘤組織內(nèi)CDK6蛋白及其mRNA,同時測定細胞內(nèi)miR-29b水平,尋找miR-29b與CDK6相關(guān)性。使用網(wǎng)絡(luò)共享的miRNA靶點預(yù)測軟件,預(yù)測miR-29b的靶點為CDK6 mRNA 3'-UTR。通過pre-miR-29b重組質(zhì)粒轉(zhuǎn)染細胞,測定轉(zhuǎn)染后細胞內(nèi)CDK6蛋白水平,然后誘導(dǎo)細胞內(nèi)CDK6mRNA3'-UTR突變,消除miR-29b的預(yù)測結(jié)合位點,接著再使用pre-miR-29b重組質(zhì)粒轉(zhuǎn)染,測定轉(zhuǎn)染后細胞內(nèi)CDK6蛋白水平,驗證miR-29b靶位點是否為CDK6 mRNA3'-UTR。最后,將 pre-miR-29b 重組質(zhì)粒、pre-miR-control 重組質(zhì)粒、CDK6質(zhì)粒及control質(zhì)粒四種物質(zhì)按照對照的原則組合,經(jīng)脂質(zhì)體包裹后進行基因轉(zhuǎn)染體外培養(yǎng)MG-63細胞系,通過CCK8檢測分析轉(zhuǎn)染細胞的增殖能力,Transwell試驗檢測轉(zhuǎn)染細胞的遷移能力。[結(jié)果](1)骨肉瘤組織中表達上調(diào)的是CDK6,而不是CDK6mRNA水平;(2)骨肉瘤組織內(nèi)miR-29b與CDK6蛋白水平負相關(guān);(3)確定CDK6 mRNA3'-UTR是miR-29b的作用位點;(4)miR-29b作用于CDK6抑制骨肉瘤細胞增殖和遷移。[結(jié)論]在骨肉瘤細胞中,miR-29b通過作用于CDK6抑制腫瘤細胞增殖和遷移,為臨床中骨肉瘤的治療可能打開了新的思路。
[Abstract]:[objective] it has been shown that miR-29b can inhibit the proliferation and migration of tumor cells, while CDK6 can promote the proliferation and migration of tumor cells. To study whether miR-29b inhibits the proliferation and migration of osteosarcoma cells by acting on CDK6 in order to provide theoretical basis for the progress of clinical treatment of osteosarcoma and explore a new direction for the treatment of osteosarcoma. [methods] Osteosarcoma patients with no tumor, no recurrence, no radiotherapy and chemotherapy were selected, and the resected osteosarcoma tissues and adjacent normal tissues were retained. The CDK6 protein and its mRNAs in osteosarcoma tissues were determined by Western blotting and qRT-PCR, and the intracellular miR-29b levels were measured to find the correlation between miR-29b and CDK6. Using the miRNA target prediction software shared by the network, the target of miR-29b prediction is CDK6 mRNA 3U -UTR. After transfection with pre-miR-29b recombinant plasmid, the level of CDK6 protein in the transfected cells was measured, and then the CDK6mRNA3'-UTR mutation was induced to eliminate the predicted binding site of miR-29b. Then, the CDK6 protein level of the transfected cells was determined by using pre-miR-29b recombinant plasmid transfection. To verify whether the miR-29b target site is CDK6 mRNA3-UTR. Finally, the pre-miR-29b recombinant plasmid pre-miR-control recombinant plasmid CDK6 and control plasmid were combined according to the control principle, and then the MG-63 cell line was transfected with liposome. The proliferation ability of transfected cells was analyzed by CCK8 and the migration ability of transfected cells was detected by Transwell test. [results] (1) the expression of CDK6 was up-regulated in osteosarcoma, not the level of CDK6mRNA; (2) miR-29b was negatively correlated with CDK6 protein in osteosarcoma; (3) CDK6 mRNA3'-UTR was identified as the action site of miR-29b; (4) miR-29b inhibited proliferation and migration of osteosarcoma cells induced by CDK6. [conclusion] in osteosarcoma cells, miR-29b inhibits the proliferation and migration of osteosarcoma cells by acting on CDK6, which may open a new way for the treatment of osteosarcoma.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R738.1
，
本文編號：2144950
[Abstract]:[objective] it has been shown that miR-29b can inhibit the proliferation and migration of tumor cells, while CDK6 can promote the proliferation and migration of tumor cells. To study whether miR-29b inhibits the proliferation and migration of osteosarcoma cells by acting on CDK6 in order to provide theoretical basis for the progress of clinical treatment of osteosarcoma and explore a new direction for the treatment of osteosarcoma. [methods] Osteosarcoma patients with no tumor, no recurrence, no radiotherapy and chemotherapy were selected, and the resected osteosarcoma tissues and adjacent normal tissues were retained. The CDK6 protein and its mRNAs in osteosarcoma tissues were determined by Western blotting and qRT-PCR, and the intracellular miR-29b levels were measured to find the correlation between miR-29b and CDK6. Using the miRNA target prediction software shared by the network, the target of miR-29b prediction is CDK6 mRNA 3U -UTR. After transfection with pre-miR-29b recombinant plasmid, the level of CDK6 protein in the transfected cells was measured, and then the CDK6mRNA3'-UTR mutation was induced to eliminate the predicted binding site of miR-29b. Then, the CDK6 protein level of the transfected cells was determined by using pre-miR-29b recombinant plasmid transfection. To verify whether the miR-29b target site is CDK6 mRNA3-UTR. Finally, the pre-miR-29b recombinant plasmid pre-miR-control recombinant plasmid CDK6 and control plasmid were combined according to the control principle, and then the MG-63 cell line was transfected with liposome. The proliferation ability of transfected cells was analyzed by CCK8 and the migration ability of transfected cells was detected by Transwell test. [results] (1) the expression of CDK6 was up-regulated in osteosarcoma, not the level of CDK6mRNA; (2) miR-29b was negatively correlated with CDK6 protein in osteosarcoma; (3) CDK6 mRNA3'-UTR was identified as the action site of miR-29b; (4) miR-29b inhibited proliferation and migration of osteosarcoma cells induced by CDK6. [conclusion] in osteosarcoma cells, miR-29b inhibits the proliferation and migration of osteosarcoma cells by acting on CDK6, which may open a new way for the treatment of osteosarcoma.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R738.1
，
本文編號：2144950
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