[circ-WHSC1 affects the growth, metastasis and radiotherapy sensitivity of nasopharyngeal carcinoma cells by targeting miR-338-3p/ELAVL1 axis]
- PMID: 36380666
- DOI: 10.3760/cma.j.cn112152-20201120-01005
[circ-WHSC1 affects the growth, metastasis and radiotherapy sensitivity of nasopharyngeal carcinoma cells by targeting miR-338-3p/ELAVL1 axis]
Abstract
Objective: To study the effect of circ-WHSC1 on the growth, metastasis and radiosensitivity of nasopharyngeal carcinoma cells and its molecular mechanism. Methods: Cancerous tissues and adjacent tissues were collected from 23 patients with nasopharyngeal carcinoma, and real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression levels of circ-WHSC1, miR-338-3p, and ELAVL1 mRNA. Western blot was used to detect the expression of ELAVL1 protein. Nasopharyngeal carcinoma cells 5-8F and SUNE1 were divided into si-NC group, si-circ-WHSC1 group, pCD5-ciR group, circ-WHSC1 group, anti-miR-NC group, anti-miR-338-3p group, miR-NC group, miR-338-3p group, si-circ-WHSC1+ anti-miR-NC group, si-circ-WHSC1+ anti-miR-338-3p group, miR-338-3p+ pcDNA group, miR-338-3p+ ELAVL1 group. Tetramethylazolium salt colorimetric method (MTT) was used to detect cell viability. Clone formation test was used to detect cell clone formation and cell radiosensitivity. Flow cytometry was used to detect cell apoptosis. Transwell was used to detect cell migration and invasion. Dual luciferase assay was used to detect the targeting relationship between circ-WHSC1 and miR-338-3p, miR-338-3p and ELAVL1. The SUNE1 cells stably transfected with sh-circ-WHSC1 were injected into nude mice and irradiated with radiation, and then the tumor volume and weight of mice were detected. Results: The expressions of circ-WHSC1 (1.57±0.94 vs 3.78±1.18, 1.00±0.10 vs 1.64±0.14/2.00±0.21/2.81±0.26/3.36±0.34) and ELAVL1 (1.28±0.74 vs 3.36±0.77, 1.00±0.08 vs 2.51±0.19/3.27±0.27) in nasopharyngeal carcinoma tissues and cells were increased, and the expression of miR-338-3p (3.13±0.96 vs 1.37±0.98, 1.00±0.08 vs 0.48±0.08/0.38±0.07) was decreased (P<0.05). After knockdown of circ-WHSC1, the activity of nasopharyngeal carcinoma cells was decreased [(100.00±8.00)% vs (51.33±8.62)%, (100.00±10.10)% vs (41.02±7.31)%], the number of clone-forming cells was decreased (101.00±8.54 vs 50.33±8.02, 114.00±14.10 vs 42.33±10.01), the rate of apoptosis was increased [(5.37±1.20)% vs (18.3±1.01)%, (6.5±1.18)% vs (22.43±1.40)%], and the numbers of migration (136.00±13.00 vs 72.33±9.50, 154.00±14.10 vs 62.67±11.50) and invasion (113.67±11.59 vs 60.67±9.07, 124.33±15.57 vs 50.33±9.01) were decreased; after different doses of radiation, the cell survival score was decreased (0.23±0.04 vs 0.06±0.01, 0.32±0.07 vs 0.05±0.02) (P<0.05). Circ-WHSC1 targeted and negatively regulated miR-338-3p. Inhibition of miR-338-3p affected the effect of knockdown of circ-WHSC1 on the proliferation, apoptosis, migration, invasion and radiosensitivity of nasopharyngeal carcinoma cells. MiR-338-3p targeted and negatively regulated ELAVL1; ELAVL1 overexpression affected the effects of miR-338-3p on the proliferation, apoptosis, migration, invasion and radiosensitivity of nasopharyngeal carcinoma cells. After the cells stably transfected with sh-circ-WHSC1 were injected into nude mice, the tumor volume [(884.67±95.63)mm(3) vs (487.33±76.51)mm(3)] and weight [(899.01±88.54)mg vs (558.67±75.04) mg] of the nude mice were reduced; after further irradiation, the tumor volume [(395.00±73.50)mm(3) vs 243.13±42.51)mm(3)] and weight[ (452.33±67.30)mg vs (211.09±57.51)mg] of the nude mice were reduced (P<0.05). Circ-WHSC1 regulated the expression of ELAVL1 by targeting miR-382. Conclusion: Knockdown of circ-WHSC1 can inhibit the growth and metastasis of nasopharyngeal carcinoma cells by targeting miR-338-3p/ELAVL1 axis, and enhances the radiosensitivity of nasopharyngeal carcinoma cells.
目的： 研究环状Wolf-Hirschhorn综合征候选基因1(circ-WHSC1)对鼻咽癌细胞增殖、凋亡、迁移、侵袭和放射敏感性的影响及分子机制。 方法： 收集2017年1月至2020年6月于郑州大学附属郑州中心医院手术治疗的23例鼻咽癌患者的癌组织和癌旁组织，采用实时荧光定量聚合酶链反应检测circ-WHSC1、miR-338-3p、果蝇胚胎致死视力异常样蛋白1(ELAVL1)mRNA的表达水平，Western blot检测ELAVL1蛋白的表达水平。将鼻咽癌细胞5-8F、SUNE1分为si-NC组、si-circ-WHSC1组、pCD5-ciR组、circ-WHSC1组、anti-miR-NC组、anti-miR-338-3p组、miR-NC组、miR-338-3p组、si-circ-WHSC1+anti-miR-NC组、si-circ-WHSC1+anti-miR-338-3p组、miR-338-3p+pcDNA组、miR-338-3p+ELAVL1组，采用四甲基偶氮唑蓝法检测细胞活性，克隆形成实验检测细胞克隆形成数及细胞放射敏感性，流式细胞术检测细胞凋亡，Transwell实验检测细胞迁移和侵袭能力，Western blot检测蛋白表达。采用双荧光素酶实验检测circ-WHSC1与miR-338-3p、miR-338-3p与ELAVL1的靶向关系。将稳定转染sh-circ-WHSC1的SUNE1细胞接种于裸鼠皮下，每隔3 d给予5 Gy红外线照射，23 d后测定移植瘤的体积和重量。 结果： 鼻咽癌组织中circ-WHSC1和ELAVL1 mRNA的表达水平分别为3.78±1.18和3.36±0.77，高于癌旁组织(1.57±0.94和1.28±0.74，均P<0.001)；miR-338-3p mRNA的表达水平为1.37±0.98，低于癌旁组织(3.13±0.96，P<0.001)。鼻咽癌细胞C666-1、6-10B、5-8F和SUNE1中circ-WHSC1 mRNA的表达水平分别为1.64±0.14、2.00±0.21、2.81±0.26和3.36±0.34，均高于鼻咽上皮细胞NP69(1.00±0.10，均P<0.05)。鼻咽癌细胞5-8F和SUNE1中miR-338-3p mRNA的表达水平分别为0.48±0.08和0.38±0.07，均低于鼻咽上皮细胞NP69(1.00±0.08，均P<0.001)；ELAVL1蛋白的表达水平分别为2.51±0.19和3.27±0.27，均高于鼻咽上皮细胞NP69(1.00±0.08，均P<0.001)。与si-NC组比较，敲除circ-WHSC1后，鼻咽癌细胞的相对细胞活性降低[5-8F细胞：(51.33±8.62)%比(100.00±8.00)%；SUNE1细胞：(41.02±7.31)%比(100.00±10.10)%；均P<0.01]，克隆形成数减少[5-8F细胞：(50.33±8.02)个比(101.00±8.54)个；SUNE1细胞：(42.33±10.01)个比(114.00±14.10)个；均P<0.01]，细胞凋亡率升高[5-8F细胞：(18.3±1.01)%比(5.37±1.20)%；SUNE1细胞：(22.43±1.40)%比(6.50±1.18)%；均P<0.01]，迁移数减少[5-8F细胞：(72.33±9.50)个比(136.00±13.00)个；SUNE1细胞：(62.67±11.50)个比(154.00±14.10)个；均P<0.01]、侵袭数减少[5-8F细胞：(60.67±9.07)个比(113.67±11.59)个；SUNE1细胞：(50.33±9.01)个比(124.33±15.57)个；均P<0.01]；8 Gy射线照射后，细胞存活分数降低[5-8F细胞：0.06±0.01比0.23±0.04；SUNE1细胞：0.05±0.02比0.32±0.07；均P<0.05]。circ-WHSC1靶向负调控miR-338-3p的表达，抑制miR-338-3p可逆转敲除circ-WHSC1对鼻咽癌细胞增殖、凋亡、迁移、侵袭和放射敏感性的影响。miR-338-3p靶向负调控ELAVL1的表达，ELAVL1过表达可逆转上调miR-338-3p对鼻咽癌细胞增殖、凋亡、迁移、侵袭和放射敏感性的影响。裸鼠成瘤实验显示，与sh-NC组相比，细胞接种后23 d，sh-circ-WHSC1组小鼠肿瘤体积缩小[(487.33±76.51)mm(3)比(884.67±95.63)mm(3)，P<0.001]，肿瘤重量降低[(558.67±75.04)mg比(899.01±88.54)mg，P＝0.002)]；5 Gy红外线照射后，裸鼠肿瘤体积减小[(243.13±42.51)mm(3)比(395.00±73.50)mm(3)，P<0.001]，肿瘤重量降低[(211.09±57.51)mg比(452.33±67.30)mg，P＝0.015]。 结论： 鼻咽癌组织和细胞中circ-WHSC1高表达。circ-WHSC1通过靶向作用于miR-338-3p/ELAVL1轴影响鼻咽癌细胞的增殖、迁移、侵袭及放射敏感性。.
Keywords: Apoptosis; Circ-WHSC1; Embryonic lethal abnormal visionlike 1; Invasion; MiR-338-3p; Migration; Nasopharyngeal neoplasms; Proliferation; Radiosensitivity.
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