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ENGLISH ABSTRACT
CASC19调控miR-449b-5p表达对子宫颈癌细胞增殖、凋亡和辐射敏感性的影响
刘艳杰
郭瑞霞
韩丽萍
顾浩
刘明珠
作者及单位信息
·
DOI： 10.3760/cma.j.issn.0529-567X.2020.01.007
Effect of CASC19 on proliferation, apoptosis and radiation sensitivity of cervical cancer cells by regulating miR-449b-5p expression
Liu Yanjie
Guo Ruixia
Han Liping
Gu Hao
Liu Mingzhu
Authors Info & Affiliations
Liu Yanjie
Department of Gynecology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
Guo Ruixia
Department of Gynecology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
Han Liping
Department of Gynecology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
Gu Hao
Department of Radiology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
Liu Mingzhu
Department of Traditional Chinese Medicine and Gynecology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
·
DOI： 10.3760/cma.j.issn.0529-567X.2020.01.007
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摘要

目的探讨癌易感性候选基因19（CASC19）调控微小RNA-449b-5p（miR-449b-5p）的表达后对子宫颈癌细胞增殖、凋亡和辐射敏感性的影响。

方法（1）培养子宫颈癌细胞系HeLa细胞，予不同剂量（分别为0、2、4、6、8 Gy）的X线照射，实时荧光定量PCR技术检测HeLa细胞中CASC19 mRNA和miR-449b-5p的表达水平。（2）不同条件处理后HeLa细胞增殖、凋亡、辐射敏感性及其相关蛋白表达的检测，其中，不同处理条件包括沉默CASC19表达、过度表达miR-449b-5p、下调miR-449b-5p并沉默CASC19表达，相关蛋白包括细胞周期蛋白D1（cyclin D1）、活化的半胱氨酸天冬氨酸蛋白酶3（cleaved-caspase-3）和组蛋白变异体H2AX（γ-H2AX）；采用四甲基偶氮唑蓝（MTT）比色法检测细胞增殖情况，流式细胞仪检测细胞凋亡情况，细胞克隆形成实验检测细胞的辐射敏感性（以存活分数表示）。（3）采用双荧光素酶报告基因实验（结果以荧光素酶活性表示）和实时荧光定量PCR技术验证CASC19和miR-449b-5p之间的靶向调控关系。

结果（1）随着X线照射剂量（0、2、4、6、8 Gy）的增加，HeLa细胞中CASC19 mRNA的表达水平逐渐升高（ F=502.681， P=0.000），miR-449b-5p的表达水平逐渐降低（ F=202.936， P=0.000）。（2）沉默CASC19表达、过度表达miR-449b-5p后，HeLa细胞的存活率显著降低（ P<0.05），凋亡率显著升高（ P<0.05），存活分数显著降低（ P<0.05），cyclin D1蛋白的表达水平显著降低（ P<0.05），cleaved-caspase-3和γ-H2AX蛋白的表达水平显著升高（ P<0.05）；下调miR-449b-5p并沉默CASC19表达后，HeLa细胞的存活率显著升高（ P<0.05），凋亡率显著降低（ P<0.05），存活分数显著升高（ P<0.05），cyclin D1、γ-H2AX蛋白的表达水平显著升高（ P<0.05），cleaved-caspase-3的表达水平显著降低（ P<0.05）。（3）与对照比较，过度表达miR-449b-5p可显著降低CASC19基因野生型HeLa细胞的荧光素酶活性（分别为1.00±0.09、0.37±0.05， P<0.01），而对CASC19基因突变型HeLa细胞的荧光素酶活性无显著影响（分别为0.92±0.07、0.94±0.05， P>0.05）。与对照比较，沉默CASC19表达后HeLa细胞中miR-449b-5p的表达水平显著升高（分别为1.00±0.12、4.84±0.49， P<0.05），过度表达CASC19后HeLa细胞中miR-449b-5p的表达水平显著降低（分别为1.00±0.09、0.38±0.04， P<0.05）。

结论HeLa细胞中CASC19可负调控miR-449b-5p的表达，下调miR-449b-5p表达可部分逆转沉默CASC19对HeLa细胞增殖、凋亡及辐射敏感性的影响。

宫颈肿瘤;RNA，长链非编码;微RNAs;细胞增殖;细胞凋亡
ABSTRACT

ObjectiveTo investigate the effects of cancer susceptibility candidate gene 19 (CASC19) regulating the expression of microRNA-449b-5p (miR-449b-5p) on the proliferation, apoptosis and radiation sensitivity of cervical cancer cells.

Methods(1) HeLa cells of cervical cancer cell line were cultured. HeLa cells were irradiated with X-ray at different doses (0, 2, 4, 6, 8 Gy, respectively), then the expression level of CASC19 mRNA and miR-449b-5p were detected by real-time quantitative PCR. (2) HeLa cell proliferation, apoptosis, radiation sensitivity (expressed as a survival fraction) and its related protein expression included cyclin D1, cleaved-caspase-3, and histone variant H2AX (γ-H2AX) were examined after different treatment including silencing CASC19 expression, over-expressing miR-449b-5p, down-regulating miR-449b-5p and silencing CASC19 expression. (3) The dual luciferase reporter gene experiment and real-time quantitative PCR technology were used to verify the targeting relationship between CASC19 and miR-449b-5p.

Results(1) With the increase of X-ray irradiation different dose (0, 2, 4, 6, 8 Gy), the expression level of CASC19 mRNA in HeLa cells gradually increased ( F=502.681, P=0.000), and the expression level of miR-449b-5p gradually decreased ( F=202.936, P=0.000).(2) After silencing CASC19 expression or over-expressing miR-449b-5p, the survival rate of HeLa cells was significantly reduced ( P<0.05), the apoptosis rate was significantly increased ( P<0.05), the survival fraction was significantly reduced ( P<0.05), the expression level of cyclin D1 protein was significantly reduced ( P<0.05), and the expression levels of cleaved-caspase-3 and γ-H2AX protein were significantly increased ( P<0.05). After down-regulating miR-449b-5p and silencing CASC19 expression, the survival rate of HeLa cells was significantly reduced ( P<0.05), the apoptosis rate was significantly increased ( P<0.05), the survival fraction was significantly reduced ( P<0.05), the expression levels of cyclin D1 and γ-H2AX protein were significantly increased ( P<0.05), and the expression level of cleaved-caspase-3 was significantly decreased ( P<0.05). (3) Over expression of miR-449b-5p could significantly reduce the luciferase activity of CASC19 wild type (1.00±0.09 versus 0.37±0.05, P<0.01), but there were no significant effect on the luciferase activity of CASC19 mutant type (0.92±0.07 versus 0.94±0.05, P>0.05). After the expression of CASC19 was silenced, the expression level of miR-449b-5p in HeLa cells increased significantly (1.00±0.12 versus 4.84±0.49, P<0.05). After overexpression of CASC19, the expression level of miR-449b-5p in HeLa cells was significantly reduced (1.00±0.09 versus 0.38±0.04, P<0.05).

ConclusionCASC19 in HeLa cells negatively regulates the expression of miR-449b-5p, and down-regulating the expression of miR-449b-5p could partially reverse the effects of silencing CASC19 on HeLa cell proliferation, apoptosis and radiation sensitivity.

Uterine cervical neoplasms;RNA, long noncoding;MicroRNAs;Cell proliferation;Apoptosis
Liu Yanjie, Email: mocdef.qabq3168058
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引用本文

刘艳杰,郭瑞霞,韩丽萍,等. CASC19调控miR-449b-5p表达对子宫颈癌细胞增殖、凋亡和辐射敏感性的影响[J]. 中华妇产科杂志,2020,55(01)：36-44.

DOI:10.3760/cma.j.issn.0529-567X.2020.01.007

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子宫颈癌是女性常见的恶性肿瘤之一，严重威胁女性生命健康。子宫颈癌的治疗主要包括早期患者的根治性手术治疗和晚期患者的放疗，但是晚期患者的治疗效果不佳 [ 1 ]。研究显示，约有一半的晚期患者对放疗不敏感，易发生肿瘤转移 [ 2 ]。放疗抵抗是晚期子宫颈癌患者治疗失败的主要原因之一 [ 3 ]。目前，产生放疗抵抗的机制仍不清楚，探讨影响子宫颈癌细胞辐射敏感性的分子机制有助于提高放疗的疗效。长链非编码RNA（long non- coding RNA，lncRNA）在真核生物中广泛存在，其长度超过200个核苷酸。研究显示，多种lncRNA参与肿瘤细胞的增殖、凋亡、迁移和侵袭等恶性生物学行为，其在调控放疗抵抗方面的作用是当前的研究热点之一 [ 4 , 5 , 6 ]。癌易感性候选基因19（CASC19）是1种lncRNA，在多种肿瘤组织中表达异常，参与肿瘤的发生、发展。如CASC19在结直肠癌组织中的表达升高，其高表达与肿瘤转移密切相关，沉默CASC19的表达可抑制结直肠癌细胞的迁移能力 [ 7 ]；CASC19可影响结肠癌细胞对化疗药物的耐药性 [ 8 ]。为探讨CASC19对子宫颈癌细胞增殖、凋亡及辐射敏感性的影响，本研究在沉默CASC19 mRNA表达、过度表达微小RNA-449b-5p（miR-449b-5p）、下调miR-449b-5p并沉默CASC19表达后，采用四甲基偶氮唑蓝（MTT）比色法、流式细胞仪、细胞克隆形成实验分别检测HeLa细胞增殖、凋亡、辐射敏感性的变化，并探讨其可能的作用机制，以期为子宫颈癌的靶向治疗提供新靶点。
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