[1]侯立功,耿宪杰,张现伟,等.沉默长链非编码RNA Linc00839对神经母细胞瘤SH-SY5Y细胞增殖的影响[J].新乡医学院学报,2020,37(8):707-712.[doi:10.7683/xxyxyxb.2020.08.002]
　HOU Ligong,GENG Xianjie,ZHANG Xianwei,et al.Effect of silencing long non-coding RNA Linc00839 on the proliferation of SH-SY5Y cells in neuroblastoma[J].Journal of Xinxiang Medical University,2020,37(8):707-712.[doi:10.7683/xxyxyxb.2020.08.002]
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沉默长链非编码RNA Linc00839对神经母细胞瘤SH-SY5Y细胞增殖的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
37
期数:
2020年8
页码:
707-712
栏目:
基础研究
出版日期:
2020-08-05

文章信息/Info

Title:
Effect of silencing long non-coding RNA Linc00839 on the proliferation of SH-SY5Y cells in neuroblastoma
作者:
侯立功1; 耿宪杰1; 张现伟1; 侯广军1; 石贞玉2
（1.郑州大学附属儿童医院，河南省儿童医院，郑州儿童医院儿外科，河南　郑州　450018；2.河南大学研究生教育与科研办公室 ，河南　开封　475004）
Author(s):
HOU Ligong1; GENG Xianjie1; ZHANG Xianwei1; HOU Guangjun1; SHI Zhenyu2
(1.Department of Pediatric Surgery,Affiliated Children′s Hospital of Zhengzhou University,Henan Children′s Hospital,Zhengzhou Children′s Hospital,Zhengzhou 450018,Henan Province,China;2.Graduate Education and Research Office of Henan University,Kaifeng 475004,Henan Province,China )
关键词:
神经母细胞瘤; 长链非编码RNA; Linc00839; 细胞增殖
Keywords:
neuroblastoma; long non-coding RNA; Linc00839; cell proliferation
分类号:
R739.4
DOI:
10.7683/xxyxyxb.2020.08.002
文献标志码:
A
摘要:
目的　探讨长链非编码RNA（LncRNA）Linc00839对神经母细胞瘤（NB）SH-SY5Y细胞增殖的影响及其机制。方法　取对数生长期NB SH-SY5Y细胞，分为干扰组、空载组，采用脂质体转染法分别将Linc00839-小分子干扰RNA（siRNA）、阴性对照siRNA转染至干扰组、空载组SH-SY5Y细胞，另取不作处理的SH-SY5Y细胞设为空白组。采用荧光显微镜观察各组细胞转染效率，并采用实时荧光定量聚合酶链反应（qRT-PCR）法测定转染后空载组、干扰组细胞Linc00839表达情况；采用二甲基噻唑法测定各组细胞增殖情况；采用流式细胞术测定各组细胞周期分布情况；采用qRT-PCR法测定转染72 h后各组细胞β-catenin、c-myc、cyclin D1 mRNA相对表达量；采用Western blot法测定转染72 h后各组细胞c-myc、cyclin D1、β-catenin、p-β-catenin蛋白相对表达量，并比较p-β-catenin/β-catenin。结果　空载组、干扰组细胞转染效率分别为（82.45±8.63）％、（83.04±8.79）％，2组细胞转染效率比较差异无统计学意义（P>0.05）。转染后干扰组细胞Linc00839 mRNA相对表达量显著低于空白组、空载组（P<0.01）；空载组细胞Linc00839 mRNA相对表达量与空白组比较差异无统计学意义（P>0.05）。干扰组细胞培养32、56、80 h时的增殖能力显著弱于空白组和空载组（P<0.05）；空载组细胞培养32、56、80 h时的增殖能力与空白组比较差异均无统计学意义（P>0.05）。空白组、空载组、干扰组细胞培养56、80 h时的增殖能力显著强于32 h（P<0.05），培养80 h时的增殖能力显著强于56 h（P<0.05）。干扰组细胞G0/G1期细胞比例显著高于空白组、空载组（P<0.05），S、G2/M期细胞比例显著低于空白组、空载组（P<0.05）；空载组细胞G0/G1、S、G2/M期细胞比例与空白组比较差异均无统计学意义（P>0.05）。干扰组细胞c-myc、cyclin D1 mRNA相对表达量显著低于空白组、空载组（P<0.05）；空载组细胞c-myc、cyclin D1 mRNA相对表达量与空白组比较差异均无统计学意义（P<0.05）；3组细胞 β-catenin mRNA相对表达量组间比较差异无统计学意义（P>0.05）。3组细胞β-catenin蛋白相对表达量比较差异无统计学意义（P>0.05）。干扰组细胞c-myc、cyclin D1、p-β-catenin蛋白相对表达量及p-β-catenin/β-catenin低于空白组、空载组（P<0.05）；空载组细胞c-myc、cyclin D1、β-catenin蛋白相对表达量及p-β-catenin/β-catenin与空白组比较差异无统计学意义（P>0.05）。结论　 沉默Linc00839可抑制NB SH-SY5Y细胞增殖，其调控机制可能与抑制β-catenin磷酸化、下调c-myc、cyclin D1 mRNA及蛋白表达，从而抑制Wnt/β-catenin信号通路有关。
Abstract:
Objective　To explore the effect of long non-coding RNA (LncRNA) Linc00839 on the proliferation of neuroblastoma (NB) SH-SY5Y cells and its mechanism.Methods　NB SH-SY5Y cells at logarithmic stage were divided into interference group and empty group.Linc00859-siRNA and control-siRNA were respectively transfected into the SH-SY5Y cells of interference group and empty group by liposome transfection.SH-SY5Y cells that were not treated were set as blank group.The transfection efficiency in every group was observed with a fluorescence microscope.The expression of Linc00839 after transfection in the interference group and empty group was measured by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR).Dimethyl thiazole method was used to determine the cell proliferation in each group.Flow cytometry was used to determine the cell cycle distribution in each group.qRT-PCR was used to determine the relative expression levels of β-catenin，c-myc and cyclin D1 mRNA in each group after transfection for 72 h.Western blot was used to determine the relative expression of c-myc,cyclin D1,β-catenin and p-β-catenin protein in each group after transfection for 72 h.And the p-β-catenin/β-catenin was compared.Results　The transfection efficiency of the empty group was (82.45±8.63)% and that of the transfection group was (83.04±8.79)%，and there was no statistically significant difference in the transfection efficiency between the two groups (P>0.05).The relative expression level of Linc00839 mRNA in the interference group was lower than that in the blank group and the empty group (P<0.01).There was no significant difference in the relative expression of Linc00839 mRNA between the blank group and the empty group (P>0.05).The proliferation ability of the interference group at 32,56,and 80 h after transfection were weaker than those of the blank group and the empty group (P<0.05).There was no significant difference in the proliferation ability of cells cultured for 32,56 and 80 h between the empty group and the blank group (P>0.05).The proliferation ability of the blank group,empty group and interference group at 56 and 80 h was significantly higher than that at 24 h (P<0.05),and the proliferation ability at 80 h was significantly higher than that at 56 h (P<0.05).The proportion of cells at G0/G1 phase in the interference group was significantly higher than that in the blank group and the empty group (P<0.05),and the proportion of cells in S and G2/M phase was significantly lower than that in the blank group and the empty group (P<0.05).There was no significant difference in the proportion of cells in G0/G1，S，G2/M phase cells between the blank group and the empty group (P>0.05).The relative mRNA expression levels of c-myc and cyclin D1 in the interference group were lower than those in the blank group and the empty group (P<0.05).There was no significant difference in the relative mRNA expressions levels of c-myc and cyclin D1 between the blank group and the empty group (P>0.05).There was no significant difference in the relative expression of β-catenin mRNA among the 3 groups (P>0.05).There was no significant difference in the relative expression of β-catenin among the 3 groups (P>0.05).The relative expression levels of c-myc and cyclin D1,p-β-catenin proteins and p-β-catenin/β-catenin in the interference group were lower than those in the blank group and the empty group (P<0.05).There were no significant differences in the relative expression levels of c-myc and cyclin D1,β-catenin proteins and p-β-catenin/β-catenin between the blank group and the empty group(P>0.05).Conclusion　Knockdown of Linc00839 can inhibit the proliferation of SH-SY5Y cells，and its regulatory mechanism may be related to inhibition of β-catenin phosphorylation，down-regulation of c-myc，cyclin D1 mRNA and protein expression，and thereby inhibit Wnt/β-catenin signaling pathway.

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更新日期/Last Update: 2020-08-05