miR-762通过靶向调控NCOR1表达对人舌鳞状细胞癌细胞增殖和凋亡的影响

doi:10.13481/j.1671-587X.20210504

摘要/Abstract

摘要： 目的

探讨miR-762与核受体辅助抑制因子1（NCOR1）的靶向关系及其对人舌鳞状细胞癌（TSCC）细胞增殖和凋亡的影响，为TSCC临床诊断和治疗提供新的分子标志物和靶点。

方法

采用实时荧光定量PCR（RT-qPCR）法检测48例TSCC患者癌组织及其癌旁正常组织中miR-762和NCOR1 mRNA表达水平，并通过Pearson相关分析法分析两者表达的相关性。采用RT-qPCR法和Western blotting法检测人正常舌上皮细胞Hacat及人TSCC细胞（Tca-8113、CAL-27、SCC-15和SCC-9）中miR-762和NCOR1 mRNA及蛋白表达水平。将miR-762 inhibitor 或si-NCOR1分别或同时转染至CAL-27细胞中，实验分为空白对照组、inhibitor-NC组、miR-762 inhibitor组、si-NC组、si-NCOR1组和miR-762 inhibitor+si-NCOR1组，采用RT-qPCR法检测CAL-27细胞中miR-762和NCOR1 mRNA表达水平，MTT法检测各组细胞增殖活性，EdU法检测各组EdU染色阳性细胞率，流式细胞术检测各组细胞凋亡率，Western blotting法检测各组细胞中NCOR1、活化半胱氨酸天冬氨酸蛋白酶3（cleaved-Caspase-3）、B细胞淋巴瘤2（Bcl-2）和Bcl-2相关X蛋白（Bax）表达水平，荧光素酶报告实验验证miR-762与NCOR1的靶向调控关系。

结果

与癌旁正常组织和正常上皮细胞Hacat比较，TSCC组织和TSCC细胞中miR-762表达水平明显升高（P<0.01），而NCOR1 mRNA表达水平明显降低（P<0.01），且在TSCC组织中miR-762与NCOR1 mRNA表达水平呈负相关关系（r=－0.711，P=0.003）。荧光素酶报告实验证实NCOR1是miR-762的靶基因。与空白对照组和inhibitor-NC组比较，miR-762 inhibitor组细胞增殖活性、EdU阳性细胞率和细胞中Bcl-2蛋白表达水平明显降低（P<0.01），细胞凋亡率和细胞中cleaved-Caspase-3及Bax蛋白表达水平明显升高（P<0.01）；与miR-762 inhibitor组比较，miR-762 inhibitor+si-NCOR1组细胞增殖活性、EdU阳性细胞率和细胞中Bcl-2蛋白表达水平明显升高（P<0.01），细胞凋亡率和细胞中cleaved-Caspase-3及Bax蛋白表达水平明显降低（P<0.01）。

结论

抑制miR-762表达能通过靶向上调NCOR1表达抑制TSCC细胞的增殖，并促进细胞凋亡。

关键词: 舌鳞状细胞癌, miR-762, 核受体辅助抑制因子1, 细胞增殖, 细胞凋亡

Abstract:

Objective： To investigate the targeting relationship between miR-762 and nuclear receptor corepressor 1（NCOR1）and its effects on the proliferation and apoptosis of human tongue squamous cell carcinoma（TSCC） cells， and to provide new molecular markers and targets for the clinical diagnosis and treatment of TSCC.

Methods

The expression levels of miR-762 and NCOR1 mRNA in cancer tissue and paracancerous normal tissue of 48 patients with TSCC were detected by Real-time fluorescence quantitative PCR（RT-qPCR）， and the correlation between the their expressions was analyzed by Pearson correlation analysis. The expression levels of miR-762 and NCOR1 mRNA and proteins in human normal tongue epithelial cells Hacat and TSCC cells （TCA-8113， CAL-27， SCC-15 and SCC-9） were detected by RT-qPCR and Western blotting methods.miR -762 inhibitor or si-NCOR1 was transfected into the CAL-27 cells separately or simultaneously；the experiment was divided into blank control group， inhibitor-NC group， miR-762 inhibitor group， si-NC group， si-NCOR1 group and miR-762 inhibitor+si-NCOR1 group. The expression levels of miR-762 and NCOR1 mRNA in the CAL-27 cells were detected by RT-qPCR method. The cell proliferation activities in various groups were detected by MTT assay； the proportions of EdU positive staining cells in various groups were detected by EdU assay， and the apoptotic rates were detected by flow cytometry. The expression levels of NCOR1， cleaved-Caspase-3， Bax and Bcl-2 proteins in the cells in various groups were detected by Western blotting method. The targeted regulatory relationship between miR-762 and NCOR1 was verified by luciferase report assay.

Results

Compared with the paracancerous normal tissue and the normal human tongue epithelial cells Hacat，the expression levels of miR-762 in TSCC tissue and TSCC cells were significantly increased（P<0.01）， while the expression levels of NCOR1 were significantly decreased（P<0.01）；there was a negative correlation between their expressions in TSCC tissue（r=－0.711，P=0.003）. Luciferase reporting assay confirmed that NCOR1 was the target gene of miR-762. Compared with blank control and inhibitor-NC groups， the proliferation activity of cells， the proportion of EdU positive cells and the expression level of Bcl-2 protein in miR-762 inhibitor group were significantly decreased （P<0.01），and the apoptotic rate and the expression levels of cleaved-Caspase-3 and Bax proteins were significantly increased （P<0.01）；compared with miR-762 inhibitor group， the proliferation activity， the proportion of EdU positive cells and the expression level of Bcl-2 protein in miR-762 inhibitor+si-NCOR1 group were significantly increased （P<0.01），and the apoptotic rate and the expression levels of cleaved-Caspase-3 and Bax proteins were significantly decreased（P<0.01）.

Conclusion

Inhibition of miR-762 expression can inhibit the proliferation and promote the apoptosis of TSCC cells by targeting and upregulating the expression of NCOR1.

Key words: tongue squamous cell carcinoma, miR-762, nuclear receptor corepressor 1, cell proliferation, apoptosis

中图分类号:

R739.86

唐乃高,郑根建. miR-762通过靶向调控NCOR1表达对人舌鳞状细胞癌细胞增殖和凋亡的影响[J]. 吉林大学学报(医学版), 2021, 47(5): 1099-1107.

Naigao TANG,Genjian ZHENG. Effects of miR-762 on proliferation and apoptosis of human tongue squamous cell carcinoma cells by targeting NCOR1 expression[J]. Journal of Jilin University(Medicine Edition), 2021, 47(5): 1099-1107.

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Group	miR-762	NCOR1
Group	miR-762	mRNA	protein
Hacat	1.00±0.08	1.02±0.09	1.14±0.12
Tca-8113	2.02±0.15^*	0.68±0.05^*	0.52±0.05^*
CAL-27	3.23±0.13^*	0.46±0.03^*	0.30±0.03^*
SCC-15	2.10±0.11^*	0.29±0.02^*	0.14±0.03^*
SCC-9	4.11±0.17^*	0.84±0.09^*	0.87±0.06^*

Group	miR-762	NCOR1
Group	miR-762	mRNA	protein
Blank control	1.00±0.05	1.00±0.17	0.30±0.03
Inhibitor-NC	0.99±0.06	1.01±0.16	0.28±0.02
miR-762 inhibitor	0.26±0.03^*△	5.69±0.30^*△	0.89±0.07^*△

Group	NCOR1
Group	mRNA	protein
Blank control	1.00±0.09	0.84±0.06
si-NC	1.02±0.07	0.82±0.08
si-NCOR1	0.22±0.06^*△	0.22±0.05^*△

Group	Proliferation activity
Group	(t/h) 12	24	48	72
Blank control	0.16±0.02	0.33±0.03	0.67±0.06	0.94±0.03
Inhibitor-NC	0.14±0.03	0.30±0.02	0.66±0.05	0.96±0.04
miR-762 inhibitor	0.15±0.03	0.23±0.06^*△	0.42±0.07^*△	0.62±0.04^*△
si-NC	0.17±0.04	0.31±0.02	0.67±0.03	0.95±0.08
si-NCOR1	0.16±0.02	0.44±0.05^*#	0.89±0.10^*#	1.22±0.12^*#
miR-762 Inhibitor+si-NCOR1	0.16±0.03	0.38±0.06^▲	0.75±0.04^▲	1.00±0.05^▲

Group	Cleaved-Caspase-3	Bax	Bcl-2
Blank control	0.28±0.02	0.32±0.03	0.45±0.02
Inhibitor-NC	0.29±0.03	0.33±0.02	0.42±0.03
miR-762 inhibitor	0.93±0.07^*△	0.92±0.06^*△	0.17±0.01^*△
si-NC	0.30±0.04	0.34±0.03	0.43±0.03
si-NCOR1	0.12±0.03^*#	0.15±0.02^*#	0.97±0.05^*#
miR-762 Inhibitor+si-NCOR1	0.67±0.06^▲	0.54±0.05^▲	0.64±0.04^▲