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中华细胞与干细胞杂志(电子版) ›› 2020, Vol. 10 ›› Issue (03) : 146 -154. doi: 10.3877/cma.j.issn.2095-1221.2020.03.003

所属专题: 文献

论著

lncRNA RPL34-AS1通过靶向调控miR-575表达对卵巢癌SKOV3细胞增殖和凋亡的影响
张敬1, 董杰1, 王秋桔1, 崔允巍1, 张辉2,()   
  1. 1. 053000 衡水,河北省衡水市妇幼保健院妇产科
    2. 050019 石家庄,河北医科大学第四医院妇产科
  • 收稿日期:2020-02-13 出版日期:2020-06-01
  • 通信作者: 张辉
  • 基金资助:
    河北省青年科技课题(20171228)

Effects of lncRNA RPL34-AS1 on the proliferation and apoptosis of ovarian cancer cells by targeted regulation of miR-575 expression

Jing Zhang1, Jie Dong1, Qiuju Wang1, Yunwei Cui1, Hui Zhang2,()   

  1. 1. Department of Obstetrics and Gynecology, Maternal and Child Health Hospital of Hengshui City, Hebei Province, Hengshui 053000, China
    2. Department of Obstetrics and Gynecology, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050019, China
  • Received:2020-02-13 Published:2020-06-01
  • Corresponding author: Hui Zhang
  • About author:
    Corresponding author: Zhang Hui, Email:
引用本文:

张敬, 董杰, 王秋桔, 崔允巍, 张辉. lncRNA RPL34-AS1通过靶向调控miR-575表达对卵巢癌SKOV3细胞增殖和凋亡的影响[J/OL]. 中华细胞与干细胞杂志(电子版), 2020, 10(03): 146-154.

Jing Zhang, Jie Dong, Qiuju Wang, Yunwei Cui, Hui Zhang. Effects of lncRNA RPL34-AS1 on the proliferation and apoptosis of ovarian cancer cells by targeted regulation of miR-575 expression[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2020, 10(03): 146-154.

目的

探究RPL34-AS1对卵巢癌细胞增殖、迁移的影响及其作用机制。

方法

取对数生长期SKOV3细胞用无血清培养基同步化12 h,将pcDNA、pcDNA-RPL34-AS1、si-NC、si-RPL34-AS1、anti-miR-NC、anti-miR-575转染至SKOV3细胞中,分别记为pcDNA组、pcDNA-RPL34-AS1组、si-NC组、si-RPL34-AS1组、anti-miR-NC组、anti-miR-575组;将pcDNA-RPL34-AS1与miR-NC、miR-575分别共转染至SKOV3细胞中,记为pcDNA-RPL34-AS1+miR-NC组、pcDNA-RPL34-AS1+miR-575组。实时荧光定量PCR (RT-qPCR)检测临床组织标本及转染后各组细胞中RPL34-AS1和miR-575的表达水平;双荧光素酶报告实验检测RPL34-AS1和miR-575的靶向关系;四甲基偶氮唑盐比色法(MTT)检测细胞存活率;流式细胞术检测细胞凋亡;蛋白质印迹(Western blot)法检测细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白依赖性激酶抑制剂1A (p21)、B细胞淋巴瘤/白血病-2 (Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达水平。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。

结果

与癌旁组织相比,卵巢癌组织中RPL34-AS1表达水平降低(1.00±0.08比0.47±0.05),miR-575表达水平升高(1.01±0.07比3.12±0.28)(P < 0.05)。转染si-RPL34-AS1后,细胞活性升高(48 h:0.68±0.06比0.55±0.05;72 h:0.99±0.08比0.71±0.06),G1期细胞所占比例降低(13.42±1.38比32.15±2.11),S期细胞所占比例升高(53.75±5.22比34.69±3.41),细胞凋亡率降低(4.31±0.42比9.25±0.91),CyclinD1、Bcl-2表达水平升高(0.92±0.08比0.71±0.07;0.86±0.07比0.61±0.06),p21、Bax表达水平降低(0.13±0.02比0.29±0.03;0.19±0.02比0.31±0.03) (P均< 0.05)。RPL34-AS1靶向调控miR-575,过表达RPL34-AS1或抑制miR-575后可抑制细胞活性,阻滞细胞周期和促进细胞凋亡。miR-575过表达逆转了RPL34-AS1过表达对卵巢癌SKOV3细胞增殖抑制和凋亡促进的作用。

结论

过表达RPL34-AS1可抑制卵巢癌SKOV3细胞增殖,促进细胞凋亡,其机制可能与下调miR-575有关。

Objective

To investigate the effect of RPL34-AS1 on the proliferation and apoptosis of ovarian cancer cells and whether its mechanism is related to miR-575.

Method

SKOV3 cells in logarithmic growth phase were synchronized with serum-free mediumfor 12 h and pcDNA, pcDNA-RPL34-AS1, si-NC, si-NC, si-RPL34-AS1, si-RPL34-AS1, anti-miR-NC, anti-anti-miR-NC, anti-miR-575 were transfected into SKOV3 cells, respectively labeled as pcDNA group, pcDNA-RPL34-AS1 group, si-NC group, si-NC group, si-RPL34-AS1 group, si-RPL34-AS1 group, anti-miR-NC group, anti-miR-NC group, anti-miR-575 group; PcDNA-RPL34-AS1 was co-transfected into SKOV3 cells with miR-NC and miR-575, respectively, labeled as pcDNA-RPL34-AS1+miR-NC group, pcDNA-RPL34-AS1+miR-575 group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression level of RPL34-AS1 and miR-575 in clinical tissue specimens and cells of each group after transfection; dual luciferase reporter assay was used to detect the targeting relationship between RPL34-AS1 and miR-575; tetramethylazozolium salt colorimetric method (MTT) was used to detect cell viability; flow cytometry was used to detect apoptosis; Western Blot was used to detect expression of Cyclin D1, Cyclin-dependent kinase inhibitor 1A (p21) , B-cell lymphoma/leukemia-2 (Bcl-2) , Bcl-2 related X protein (Bax) proteins.The comparison between the two groups was performed using the independent sample t test, and the comparison between multiple groups was performed using the one-way analysis of variance; LSD-t test was used for pairwise comparison between groups.

Results

Compared with adjacent tissues, the expression level of RPL34-AS1 was decreased in ovarian cancer tissues, the expression level ofmiR-575 was increased (1.01±0.07 vs 3.12±0.28) (P < 0.05) . After transfection with si-RPL34-AS1, cell viability was increased (48 h: 0.68±0.06 vs 0.55±0.05; 72 h: 0.99±0.08 vs 0.71±0.06) , and the proportion of G1 phase cells was decreased (13.42±1.38 vs 32.15±2.11) , the proportion of S-phase cells was increased (53.75±5.22 vs 34.69±3.41) , the apoptosis rate was decreased (4.31±0.42 vs 9.25±0.91) , and the expression level of CyclinD1 and Bcl-2 were increased (0.92±0.08 vs 0.71±0.07; 0.86±0.07 vs 0.61±0.06) , the expression level of p21 and Bax were decreased (0.13±0.02 vs 0.29±0.03; 0.19±0.02 vs 0.31±0.03) (all P < 0.05) . RPL34-AS1 targets and regulates miR-575. Overexpression of RPL34-AS1 or inhibition of miR-575 can inhibit cell activity, block the cell cycle and promote apoptosis. MiR-575 overexpression reversed RPL34-AS1 overexpression effect on ovarian cancer SKOV3 cell proliferation inhibition as well as its apoptosis promotion.

Conclusion

Overexpression of RPL34-AS1 can inhibit the proliferation of ovarian cancer SKOV3 cells and promote cell apoptosis, which may be related to the down-regulation of miR-575.

表1 引物序列信息
图1 lncRNA RPL34-AS1和miR-575在卵巢癌组织中的表达
图2 RPL34-AS1的序列中含有与miR-575互补的核苷酸序
表2 miR-NC组和miR-575组荧光素酶相对活性(±sn = 3)
图3 lncRNA RPL34-AS1过表达对卵巢癌SKOV3细胞增殖和凋亡的影响
表3 lncRNA RPL34-AS1过表达对卵巢癌SKOV3细胞增殖和凋亡的影响(±sn = 3)
表4 lncRNA RPL34-AS1过表达对卵巢癌SKOV3细胞增殖和凋亡相关蛋白表达的影响(±sn = 3)
图4 抑制RPL34-AS1表达对卵巢癌SKOV3细胞增殖和凋亡的影响
表5 抑制si-RPL34-AS1表达对卵巢癌SKOV3细胞增殖、周期和凋亡的影响(±sn = 3)
表6 抑制si-RPL34-AS1表达对卵巢癌SKOV3细胞增殖和凋亡相关蛋白表达的影响(±sn = 3)
图5 抑制miR-575表达对卵巢癌SKOV3细胞增殖和凋亡的影响
表7 抑制miR-575表达对卵巢癌SKOV3细胞增殖、周期和凋亡的影响(±sn = 3)
表8 抑制miR-575表达对卵巢癌SKOV3细胞增殖和凋亡相关蛋白表达的影响(±sn = 3)
图6 miR-575过表达逆转lncRNA RPL34-AS1过表达对卵巢癌SKOV3细胞和凋亡的作用
表9 miR-575过表达逆转了lncRNA RPL34-AS1过表达对卵巢癌SKOV3细胞增殖、周期和凋亡的作用(±sn = 3)
表10 miR-575过表达逆转了lncRNA RPL34-AS1过表达对卵巢癌SKOV3细胞增殖和凋亡相关蛋白表达的作用(±sn = 3)
9
郭坤蕾, 张国楠. 卵巢癌靶向治疗的研究现状[J]. 肿瘤预防与治疗, 2018, 31(6):75-80.
10
Zhao J, Liu Y, Zhang W, et al. Long non-coding RNA Linc00152 is involved in cell cycle arrest, apoptosis, epithelial to mesenchymal transition, cell migration and invasion in gastric cancer[J]. Cell Cycle, 2015, 14(19):3112-3123.
11
Ji L, Fan X, Zhou F, et al. lncRNA RPL34-AS1 inhibits cell proliferation and invasion while promoting apoptosis by competitively binding miR-3663-3p/RGS4 in papillary thyroid cancer[J]. J Cell Physiol, 2020, 235(4):3669-3678.
12
王明阳, 王光磊, 陈新亮, 等. MicroRNA的生物学功能及其与肿瘤诊断和治疗的研究进展[J]. 动物医学进展, 2018, 39(1):95-98.
13
Gray A N, Cui T, Geurts M, et al. Elucidation of the role of miR-575 on tumorigenesis in glioblastoma[J]. Cancer Res, 2017, 77(13):3423.
14
Zhao X, Yi Y, Meng C, et al. MiRNA-575 suppresses angiogenesis by targeting Rab5-MEK-ERK pathway in endothelial cells[J]. Biosci Rep, 2019, 39(1): BSR20181218.
1
导镰, 王金华. 卵巢癌的发病机制与分子靶向治疗研究进展[J]. 肿瘤研究与临床, 2018, 30(3):204-207.
2
韩萍, 翟东霞, 张丹英, 等. 耐药性复发卵巢癌治疗药物的研究进展[J]. 医学研究杂志, 2018, 47(1):171-174.
3
王晓彤, 谭文华, 刘巍. 抑制长链非编码RNA FAL1表达对上皮性卵巢癌细胞生物学行为的影响及其分子机制[J]. 国际妇产科学杂志, 2018, 45(2):221-225.
4
Shi DB, Zheng HT, Zhuo CH, et al. Low expression of novel lncRNA RP11-462C24.1 suggests a biomarker of poor prognosis in colorectal cancer[J]. Medical Oncol, 2014, 31(7):31.
5
Gong Z, Li J, Cang P, et al. RPL34-AS1 functions as tumor suppressive ncRNA in esophageal cancer[J]. Biomed Pharmacother, 2019, 120:109440.
6
高瑶, 邹霞, 高军. miRNA在卵巢癌诊治及预后预测中作用的研究进展[J]. 山东医药, 2019, 59(3):87-90.
15
Yan S, Tang Z, Chen K, et al. Long noncoding RNA MIR31HG inhibits hepatocellular carcinoma proliferation and metastasis by sponging microRNA-575 to modulate ST7L expression[J]. J Exp Clin Cancer Res, 2018, 37(1):214.
7
焉春华, 史晓东, 于方飞, 等. MicroRNA-575靶向抑制BLID促进非小细胞肺癌细胞的增殖和侵袭[J]. 现代生物医学进展, 2017, 17(23):42-46, 134.
8
Wang YN, Xu F, Zhang P, et al. MicroRNA-575 regulates development of gastric cancer by targeting PTEN[J]. Biomed Pharmacother, 2019, 113:108716.
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