Expression of miRNA-130b in Osteosarcoma Tissues and Its Impact on Proliferation and Apoptosis of Osteosarcoma Cells
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摘要:目的
探讨骨肉瘤组织中miRNA-130b的表达及其对骨肉瘤细胞增殖、细胞周期以及凋亡的影响。
方法用Real-time PCR法检测48例骨肉瘤患者肿瘤组织与癌旁组织中miRNA-130b的表达水平。用脂质体法将miRNA-130b inhibitor瞬时转染入U2人骨肉瘤细胞，实时定量RT-PCR检测U2细胞miRNA-130b的表达，CCK8增殖实验检测各组细胞的增殖情况，流式细胞仪检测各组细胞周期及细胞凋亡率，荧光素酶基因报告实验检测miRNA-130b与RUNX3之间的调控机制、荧光素酶的相对活性和转染后RUNX3及miRNA-130b的表达水平。
结果骨肉瘤组织中miRNA-130b的表达明显高于癌旁组织，转染miRNA-130b inhibitor后U2细胞miRNA-130b的表达水平明显受抑制，人骨肉瘤细胞U2转染miRNA-130b inhibitor的细胞增殖速度明显下降，细胞周期阻滞在G0/G1期，凋亡率增高，细胞中RUNX3蛋白及mRNA表达水平明显升高；miRNA-130b显著抑制野生型RUNX3-3’UTR表达载体的荧光素酶活性；抑制miRNA-130b表达后，RUNX3表达明显升高；抑制RUNX3表达后，miRNA-130的表达无明显变化。
结论miRNA-130b在骨肉瘤中呈高表达水平，抑制miRNA-130b可能通过调控RUNX3抑制骨肉瘤细胞的增殖，并促进细胞凋亡。
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关键词:
- miRNA-130b /
- RUNX3 /
- 骨肉瘤 /
- 增殖 /
- 凋亡
Abstract:ObjectiveTo detect the expression of miRNA-130b in osteosarcoma tissues and investigate the effect of miRNA-130b on the proliferation, cell cycle and apoptosis of human osteosarcoma cell line U2.
MethodsReal-time PCR was used to detect the expression level of miRNA-130b in osteosarcoma tissues and corresponding paratumorous tissues collected from 48 patients.The expression level of miRNA-130b in osteosarcoma cell line U2 was measured by qRT-PCR after transfected with miRNA-130b inhibitor. The cell proliferation was determined by CCK8 assay.Flow cytometry was used to monitor cell cycle and apoptosis. Luciferase gene reporter assay was used to detect the regulation mechanism of miRNA-130b and RUNX3. The change of RUNX3 and miRNA-130b of U2 cells after transfection was detected.
ResultsmiRNA-130b was highly expressed in osteosarcoma tissues, compared with the adjacent normal tissues. The miRNA-130b expression of U2 cells after transfection was significantly down-regulated. The proliferation activity of U2 cells was inhibited, the cell cycle was arrested in G0/G1 phase, and the cell apoptosis was increased after transfected with miRNA-130b inhibitor. We found that miRNA-130b reduced wide-type RUNX3 3′-UTR luciferase expression intensity. The relative expression of RUNX3 in miRNA-130b inhibitor transfection group was significantly higher than those in negative control group. The relative expression of miRNA-130b in RUNX3 siRNA transfection group had no significant difference with those in negative control group.
ConclusionmiRNA-130b is highly expressed in human osteosarcoma tissue. miRNA-130b may inhibit the proliferation and induce the apoptosis of osteosarcoma cell line U2 via inhibiting the RUNX3 expression in vitro.
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Key words:
- miRNA-130b /
- RUNX3 /
- Osteosarcoma /
- Proliferation /
- Apoptosis
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图 1 Real-time PCR检测骨肉瘤及癌旁组织中miRNA-130b (A) 及RUNX3 mRNA (B) 的表达
Figure 1 The expressions of miRNA-130b (A) and RUNX3 mRNA (B) in osteosarcoma and adjacent normal tissues detected by real-time PCR
*: P < 0.05, compared with adjacent normal tissues
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图 2 miRNA-130b inhibitor转染U2细胞后miRNA-130b表达水平
Figure 2 The expression level of miRNA-130b in U2 cells after transfected with miRNA-130b inhibitor
*: P < 0.05, compared with Blank or miR-NC group
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图 3 CCK8检测转染后不同时间U2细胞的增殖能力
Figure 3 The proliferation of U2 cells after transfected with miRNA-130b inhibitor detected by CCK8 assay
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图 4 流式细胞术检测转染miRNA-130b inhibitor后各组细胞的凋亡率
Figure 4 The apoptosis rate of U2 cells after transfected with miRNA-130b inhibitor detected by FCM
*: P < 0.05, compared with Blank or miR-NC group
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图 5 流式细胞术检测转染miRNA-130b inhibitor后各组细胞周期的分布
Figure 5 Cell cycles of U2 cells after transfected with miRNA-130b inhibitor detected by FCM
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图 6 miRNA-130b靶向RUNX3的野生型3′-UTR及突变型RUNX3-3′ UTR示意图
Figure 6 The predicted miRNA-130b binding site with RUNX3 3′-UTR and its mutated version by site mutagenesis
*: mutated nucleotides
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图 7 荧光素酶基因报告实验检测miRNA-130b inhibitor转染U2的相对荧光素酶活性
Figure 7 Relative luciferase activity of U2 cells transfected by miRNA-130b inhibitor detected by luciferase gene reporter assay
*: P < 0.05, compared with miRNA-130b inhibitor group
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图 8 Real-time PCR检测miRNA-130b inhibitor转染后RUNX3 mRNA表达水平
Figure 8 The expressions of RUNX3 mRNA detected by real-time PCR after transfected by miRNA-130b inhibitor
*: P < 0.05, compared with Blank or miR-NC group
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图 9 Western blot检测miRNA-130b inhibitor转染后RUNX3蛋白表达水平
Figure 9 The expression of RUNX3 protein detected by Western blot after transfected by miRNA-130b inhibitor
*: P < 0.05, compared with Blank or miR-NC group
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