长链非编码RNA01139靶向调控微小RNA-300对口腔鳞癌顺铂耐药细胞增殖、迁移和侵袭的影响

邓琳; 谢陆莉; 李鲲

doi:10.7619/jcmp.20214197

长链非编码RNA01139靶向调控微小RNA-300对口腔鳞癌顺铂耐药细胞增殖、迁移和侵袭的影响

四川省成都市龙泉驿区第一人民医院口腔科, 四川成都, 610100

详细信息

通讯作者:
李鲲, E-mail: 1745620905@qq.com

中图分类号: R739.8;R730.2
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出版历程
- 收稿日期: 2021-10-25
- 网络出版日期: 2022-03-28
- 发布日期: 2022-03-14

Effects of long non-coding RNA LINC01139 in regulating proliferation, migration and invasion of oral squamous cell carcinoma cisplatin-resistant cells by targeting microRNA-300

Department of Stomatology, the First People's Hospital of Longquanyi District of Chengdu in Sichuan Province, Chengdu, Sichuan, 610100

摘要

摘要:
目的探讨长链非编码RNA01139 (LINC01139)调控微小RNA-300(miR-300)对口腔鳞癌顺铂(DDP)耐药细胞(CAL-27/DDP)增殖、迁移和侵袭的影响。
方法采用实时荧光定量聚合酶链反应(qRT-PCR)检测CAL-27/DDP及其亲本细胞株CAL-27中LINC01139和miR-300的表达水平。将CAL-27/DDP分为DDP+si-NC组、DDP+si-LINC01139组、DDP+miR-NC组、DDP+miR-300组、DDP+si-LINC01139+ anti-miR-NC组、DDP+si-LINC01139+anti-miR-300组。采用噻唑兰(MTT)法检测细胞增殖抑制率; Transwell实验检测迁移和侵袭细胞数。采用双荧光素酶报告实验和qRT-PCR确定LINC01139与miR-300的靶向关系。
结果与CAL-27细胞比较, CAL-27/DDP中LINC01139的表达升高, miR-300的表达降低，差异有统计学意义(P＜0.05)。与DDP+si-NC组比较，DDP+si-LINC01139组CAL-27/DDP增殖抑制率升高，迁移和侵袭细胞数减少，差异有统计学意义(P＜0.05)。与DDP+miR-NC组比较, DDP+miR-300组CAL-27/DDP增殖抑制率升高，迁移和侵袭细胞数减少，差异有统计学意义(P＜0.05)。与DDP+si-LINC01139+anti-miR-NC组比较, DDP+si-LINC01139+ anti-miR-300组CAL-27/DDP增殖抑制率降低，迁移和侵袭细胞数增加，差异有统计学意义(P＜0.05)。
结论抑制LINC01139可通过上调miR-300抑制CAL-27/DDP的增殖、迁移和侵袭。
- 长链非编码RNA01139 /
- 微小RNA-300 /
- 口腔鳞癌顺铂耐药细胞 /
- 增殖 /
- 迁移 /
- 侵袭
Abstract:
Objective To investigate the effect of long intergenic non-coding RNA LINC01139 (LINC01139) in regulating the proliferation, migration and invasion of oral squamous cell carcinoma cisplatin(DDP)-resistant cells(CAL-27/DDP) by regulating microRNA-300 (miR-300).
Methods Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of LINC01139 and miR-300 in CAL-27/DDP and its parental cell line CAL-27. CAL-27/DDP was divided into DDP+si-NC, DDP+si-LINC01139, DDP+miR-NC, DDP+miR-300, DDP+ si-LINC01139 +anti-miR-NC, DDP+si-LINC01139+anti-miR-300 groups. The methyl thiazolyl tetrazolium (MTT) method was used to detect the cell proliferation inhibition rate; Transwell assay was used to detect the number of migrating and invading cells. The dual luciferase reporting experiment and qRT-PCR were used to detect the targeting relationship between LINC01139 and miR-300.
Results Compared with CAL-27 cells, the expression of LINC01139 in CAL-27/DDP was increased, while the expression of miR-300 was decreased (P < 0.05). Compared with the DDP+si-NC group, the proliferation inhibition rate of CAL-27/DDP in the DDP+si-LINC01139 group was increased, and the migrating and invading cells were reduced (P < 0.05). Compared with the DDP+miR-NC group, the proliferation inhibition rate of CAL-27/DDP in the DDP+miR-300 group was increased, and the number of migrating and invading cells was reduced (P < 0.05). Compared with the DDP+ si-LINC01139+anti-miR-NC group, the proliferation inhibition rate of CAL-27/DDP in the DDP+si-LINC01139+anti-miR-300 group was decreased, the migrating and invading cells were increased (P < 0.05).
Conclusion Inhibition of LINC01139 inhibits the proliferation, migration and invasion of oral squamous cell carcinoma cisplatin-resistant cells by up-regulating miR-300.
- long non-coding RNA LINC01139 /
- microRNA-300 /
- oral squamous cell carcinoma cisplatin-resistant cells /
- proliferation /
- migration /
- invasion

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图 1 增殖、迁移和侵袭相关蛋白表达

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图 2 增殖、迁移和侵袭相关蛋白表达

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图 3 LINC01139序列中含有与miR-300互补的核苷酸序列

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图 4 4组增殖、迁移和侵袭相关蛋白表达

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表 1 顺铂对CAL-27细胞和CAL-27/DDP增殖抑制率的影响(x±s)

细胞	DDP浓度							IC₅₀/(μg/mL)
细胞	0.125 μg/mL	0.25 μg/mL	0.5 μg/mL	1 μg/mL	2 μg/mL	4 μg/mL	8 μg/mL	IC₅₀/(μg/mL)
CAL-27(n=9)	11.32±1.14	22.45±2.27	34.62±3.55	45.86±4.31	59.33±5.12	73.21±7.56	86.22±8.13	1.17±0.19
CAL-27/DDP(n=9)	3.41±0.35^*	7.11±0.72^*	13.24±1.25^*	19.83±1.74^*	31.02±3.17^*	39.65±3.88^*	53.14±5.28^*	6.65±0.51^*
CAL-27: 口腔鳞癌细胞; CAL-27/DDP: 口腔鳞癌顺铂耐药细胞; DDP: 顺铂。与CAL-27细胞比较, * P＜0.05。

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表 2 LINC01139和miR-300在CAL-27细胞和CAL-27/DDP细胞中的表达(x±s)

细胞	LINC01139	miR-300
CAL-27(n=9)	1.00±0.06	1.00±0.05
CAL-27/DDP(n=9)	2.93±0.27^*	0.34±0.03^*
LINC01139: 长链非编码RNA01139; miR-300: 微小RNA-300; CAL-27: 口腔鳞癌细胞; CAL-27/DDP: 口腔鳞癌顺铂耐药细胞。与CAL-27细胞比较, * P＜0.05。

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表 3 抑制LINC01139表达联合DDP对CAL-27/DDP细胞增殖、迁移和侵袭的影响(x±s)

组别	LINC01139	抑制率/%	迁移细胞数/个	侵袭细胞数/个	CyclinD1蛋白	p21蛋白	MMP-2蛋白	MMP-9蛋白
DDP+si-NC组(n=9)	1.00±0.08	6.41±0.63	105.23±10.22	88.14±8.42	0.61±0.06	0.33±0.03	0.72±0.07	0.66±0.06
DDP+si-LINC01139组(n=9)	0.51±0.05^*	42.15±4.13^*	56.39±5.21^*	41.65±4.55^*	0.20±0.02^*	0.79±0.07^*	0.30±0.03^*	0.27±0.03^*
LINC01139: 长链非编码RNA01139; CyclinD1: 兔源细胞周期素D1; MMP-2: 基质金属蛋白酶-2; MMP-9: 基质金属蛋白酶-9。与DDP+si-NC组比较, * P＜0.05。

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表 4 miR-300过表达联合DDP对CAL-27/DDP细胞增殖、迁移和侵袭的影响(x±s)

组别	miR-300	抑制率/%	迁移细胞数/个	侵袭细胞数/个	CyclinD1蛋白	p21蛋白	MMP-2蛋白	MMP-9蛋白
DDP+miR-NC组(n=9)	1.00±0.06	7.12±0.77	101.25±9.84	86.14±8.63	0.63±0.06	0.32±0.03	0.71±0.07	0.68±0.06
DDP+miR-300组(n=9)	2.57±0.24^*	35.48±3.55^*	60.55±6.31^*	53.47±5.33^*	0.28±0.03^*	0.75±0.07^*	0.37±0.03^*	0.30±0.03^*
miR-300: 微小RNA-300; CyclinD1: 兔源细胞周期素D1; MMP-2: 基质金属蛋白酶-2; MMP-9: 基质金属蛋白酶-9。与DDP+miR-NC组比较, * P＜0.05。

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表 5 双荧光素酶报告实验(x±s)

组别	WT-LINC01139	MUT-LINC01139
miR-NC组(n=9)	1.00±0.06	1.02±0.08
miR-300组(n=9)	0.57±0.05^*	0.99±0.07
与miR-NC组比较, * P＜0.05。

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表 6 LINC01139调控miR-300的表达(x±s)

组别	miR-300
pcDNA组(n=9)	1.00±0.07
pcDNA-LINC01139组(n=9)	0.53±0.05^*
si-NC组(n=9)	1.02±0.06
si-LINC01139组(n=9)	2.68±0.27^#
F	381.236
P	＜0.001
与pcDNA组比较, * P＜0.05; 与si-NC组比较, #P＜0.05。

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表 7 干扰miR-300表达逆转了抑制LINC01139表达对CAL-27/DDP细胞DDP耐药性的作用(x±s)

组别	miR-300	抑制率/%	迁移细胞数/个	侵袭细胞数/个	CyclinD1蛋白	p21蛋白	MMP-2蛋白	MMP-9蛋白
DDP+si-NC组(n=9)	1.00±0.08	7.06±0.71	106.24±9.56	85.14±8.36	0.62±0.06	0.31±0.03	0.73±0.07	0.67±0.06
DDP+si-LINC01139组(n=9)	2.84±0.27^*	43.25±4.31^*	57.14±5.33^*	43.51±4.33^*	0.22±0.03^*	0.77±0.07^*	0.33±0.03^*	0.29±0.03^*
DDP+si-LINC01139+anti-miR-NC组(n=9)	2.86±0.28	44.69±4.52	55.86±5.52	42.69±4.74	0.21±0.02	0.78±0.06	0.32±0.03	0.28±0.02
DDP+si-LINC01139+anti-miR-300组(n=9)	1.53±0.15^#	21.36±2.54^#	83.47±8.35^#	69.71±6.33^#	0.51±0.05^#	0.40±0.04^#	0.64±0.06^#	0.56±0.05^#
miR-300: 微小RNA-300; CyclinD1: 兔源细胞周期素D1; MMP-2: 基质金属蛋白酶-2; MMP-9: 基质金属蛋白酶-9。与DDP+si-NC组比较, * P＜0.05; 与DDP+si-LINC01139+anti-miR-NC组比较, #P＜0.05。

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施引文献(2)

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