<strong>circPUM1</strong>靶向调控<strong>miR-144-3p</strong>对宫颈癌细胞放射抵抗的影响

doi:10.3724/zdxbyxb-2022-0021

浙江大学学报（医学版）

2022, Vol. 51

Issue (2): 215-224 DOI: 10.3724/zdxbyxb-2022-0021

原著

circPUM1靶向调控miR-144-3p对宫颈癌细胞放射抵抗的影响

胡滨¹,袁金金^2,*(

)

1.郑州大学第二附属医院妇科，河南郑州 450000
2.郑州大学第二附属医院放疗科，河南郑州 450000

Effect of circPUM1 on radioresistance of cervical cancer cells through targeting miR-144-3p

HU Bin¹,YUAN Jinjin^2,*(

)

1. Department of Gynecology, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China;
2. Department of Radiotherapy, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China

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摘要：

目的：探讨环状RNA?pumilio?RNA结合家族成员（circPUM）1对宫颈癌细胞放射抵抗的影响及其可能的作用机制。方法：收集2019年8月至2020年2月郑州大学第二附属医院收治的47例宫颈癌患者的癌组织及其相应癌旁组织（距离癌组织5?cm处正常组织）标本。定量反转录聚合酶链反应法检测宫颈癌组织与癌旁组织中circPUM1、miR-144-3p的表达量；Pearson法分析宫颈癌组织中circPUM1与miR-144-3p表达量的相关性。将circPUM1慢病毒短发夹RNA（sh-circPUM1）及其阴性对照（sh-NC）、miR-144-3p寡核苷酸模拟物（miR-144-3p?mimic）及其阴性对照（miR-NC）、sh-circPUM1与miR-144-3p抑制物（anti-miR）、sh-circPUM1与anti-miR阴性对照（anti-miR-NC）分别转染至人宫颈癌细胞SiHa，并通过0、4?Gy照射剂量照射，采用细胞计数试剂盒（CCK-8法）、平板克隆形成实验、流式细胞术、Transwell实验分别检测细胞增殖、集落形成、凋亡、迁移及侵袭能力；蛋白质印迹法检测活化的胱天蛋白酶3（cleaved-caspase3）蛋白表达量；Starbase平台分析及细胞实验检测circPUM1与miR-144-3p的靶向关系。结果：与癌旁组织比较，宫颈癌组织中circPUM1的表达量增加（P<0.05），miR-144-3p的表达量减少（P<0.05）；circPUM1与miR-144-3p呈负相关（r=–0.9282，P<0.01）；转染sh-circPUM1或miR-144-3p?mimic后，细胞增殖抑制率、细胞凋亡率和cleaved-caspase3蛋白表达水平升高（均P<0.05），集落形成数、迁移及侵袭细胞数减少（均P<0.05）；circPUM1可靶向结合miR-144-3p；共转染sh-circPUM1与anti-miR后，细胞增殖抑制率、细胞凋亡率和cleaved-caspase3蛋白表达水平降低（均P<0.05），集落形成数、迁移及侵袭细胞数增多（均P<0.05）。结论：沉默circPUM1可通过靶向调控miR-144-3p表达而抑制宫颈癌细胞增殖、集落形成、迁移、侵袭能力及诱导细胞凋亡，从而减弱细胞放射抵抗性。

关键词： 宫颈癌; SiHa细胞株；环状RNA pumilio RNA结合家族成员1; miR-144-3p; 细胞增殖; 迁移; 侵袭; 凋亡; 放射抵抗

Abstract:

Objective: To investigate the effect of circular RNA pumilio RNA binding family member (circPUM) 1 on radioresistance of cervical cancer cells and its mechanism. Methods: Cancer tissue and corresponding paricancerous tissue samples were collected from 47 patients with cervical cancer who underwent surgical treatment in the Second Affiliated Hospital of Zhengzhou University from August 2019 to February 2020. The expression levels of circPUM1 and miR-144-3p in cervical cancer tissues and paricancerous tissues were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The Pearson method was used to analyze the correlation between circPUM1 and miR-144-3p expression in cervical cancer tissues. circPUM1 lentiviral short hairpin RNA (sh-circPUM1) and its negative control (sh-NC), miR-144-3p oligonucleotide mimic (miR-144-3p mimic) and its negative control (miR-NC), sh-circPUM1 and miR-144-3p inhibitor (anti-miR), and sh-circPUM1 and anti-miR negative control (anti-miR-NC) were transfected into human cervical carcinoma SiHa cells, respectively, and the cells were irradiated with 0 and 4?Gy irradiation doses. Cell proliferation, colony formation, apoptosis, migration and invasion were detected by cell counting kit (CCK-8 method), plate colony formation assay, flow cytometry and Transwell assay, respectively. The protein expression of cleaved-caspase3 was detected by Western blotting. The targeting relationship between circPUM1 and miR-144-3p was analyzed with Starbase platform. Results: Compared with adjacent tissue, the expression of circPUM1 in cervical cancer tissue was significantly increased (P<0.05), while the expression of miR-144-3p was decreased (P<0.05). The circPUM1 was negatively correlated with miR-144-3p (r=–0.9282, P<0.01). After transfection with sh-circPUM1 or miR-144-3p mimic, the inhibition rate of cell proliferation, the rate of apoptosis and the expression level of cleaved-caspase3 protein increased (allP<0.05), while the number of colonies formed, migrated and invaded cells decreased (allP<0.05). CircPUM1 could targeted to miR-144-3p. After co-transfection of sh-circPUM1 and anti-miR, the inhibition rate of cell proliferation, the rate of apoptosis and the expression level of cleaved-caspase3 protein significantly decreased (allP<0.05), while the number of colonies formed, migrated and invaded cells increased (allP<0.05).Conclusion: Silencing circPUM1 may inhibit the proliferation, colony formation, migration, invasion and induce apoptosis of cervical cancer cells through targeting and regulating the expression of miR-144-3p.

Key words: Cervical cancer SiHa cells; CircPUM1 MiR-144-3p Cell proliferation Migration Invasion Apoptosis Radioresistance

收稿日期: 2022-01-27 出版日期: 2022-08-02

CLC:

R737.31

基金资助: 河南省医学科技攻关项目（LHGJ20200393，LHGJ20210420）；河南省重点研发与推广专项（222102310419）

通讯作者: 袁金金 E-mail: yuanjinjin1103@163.com

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引用本文:

胡滨,袁金金. circPUM1靶向调控miR-144-3p对宫颈癌细胞放射抵抗的影响[J]. 浙江大学学报（医学版）, 2022, 51(2): 215-224.

HU Bin,YUAN Jinjin. Effect of circPUM1 on radioresistance of cervical cancer cells through targeting miR-144-3p. J Zhejiang Univ (Med Sci), 2022, 51(2): 215-224.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2022-0021 或 https://www.zjujournals.com/med/CN/Y2022/V51/I2/215

图 1 宫颈癌组织中circPUM1与miR-144-3p表达的相关性circPUM：环状RNA pumilio RNA 结合家族成员；miR：微小RNA.

图 2 沉默circPUM1后宫颈癌细胞增殖、凋亡、迁移和侵袭情况A：sh-circPUM1组集落形成数较其阴性对照（sh-NC）组减少，标尺=20 mm；B：sh-circPUM1组细胞凋亡较sh-NC组增加；C：sh-circPUM1组迁移及侵袭细胞数较sh-NC组减少，标尺=200 μm. circPUM：环状RNA pumilio RNA 结合家族成员；sh-circPUM1：circPUM1慢病毒短发夹RNA.

图 3 cleaved-caspase3蛋白表达电泳图A：sh-circPUM1组及其阴性对照（sh-NC）组的电泳图；B：miR-144-3p mimic组及其阴性对照（miR-NC）组的电泳图；C：sh-circPUM1+anti-miR与sh-circPUM1+anti-miR阴性对照（anti-miR-NC）组的电泳图. circPUM：环状RNA pumilio RNA 结合家族成员；sh-circPUM1：circPUM1慢病毒短发夹RNA；miR-144-3p mimic：miR-144-3p寡核苷酸模拟物；anti-miR：miR-144-3p抑制物；cleaved-caspase：活化的胱天蛋白酶.

表 1 沉默circPUM1宫颈癌细胞增殖抑制率和集落形成、凋亡、迁移、侵袭能力变化

图 4 miR-144-3p过表达后宫颈癌细胞增殖、凋亡、迁移和侵袭情况A：miR-144-3p mimic组集落形成数较其阴性对照（miR-NC）组减少，标尺=20 mm；B：miR-144-3p mimic组细胞凋亡较miR-NC组增加；C：miR-144-3p mimic组迁移及侵袭细胞数较miR-NC组减少，标尺=200 μm. miR：微小RNA；miR-144-3p mimic：miR-144-3p寡核苷酸模拟物.

表 2 miR-144-3p过表达与对照宫颈癌细胞增殖抑制率、集落形成、凋亡、迁移和侵袭情况比较

图 5 circPUM1和miR-144-3p的互补序列circPUM：环状RNA pumilio RNA 结合家族成员；miR：微小RNA.

图 6 抑制miR-144-3p表达后沉默circPUM1的宫颈癌细胞增殖、凋亡、迁移和侵袭情况A：sh-circPUM1+anti-miR组集落形成数较sh-circPUM1+anti-miR-NC组增多，标尺=20 mm；B：sh-circPUM1+anti-miR组细胞凋亡较sh-circPUM1+anti-miR-NC组减少；C：sh-circPUM1+anti-miR组迁移及侵袭细胞数较sh-circPUM1+anti-miR-NC组增多，标尺=200 μm. circPUM：环状RNA pumilio RNA 结合家族成员；sh-circPUM1：circPUM1慢病毒短发夹RNA；anti-miR：miR-144-3p抑制物.

表 3 抑制miR-144-3p表达对沉默circPUM1的宫颈癌细胞增殖抑制率、集落形成、凋亡、迁移和侵袭的影响

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