长链非编码RNA LNC01309对人肝癌细胞增殖和迁移能力的影响及作用机制

刘红燕; 李婧姣; 路泽军; 刘咏真; 温居一

doi:10.3969/j.issn.1001-5256.2022.03.014

长链非编码RNA LNC01309对人肝癌细胞增殖和迁移能力的影响及作用机制

DOI: 10.3969/j.issn.1001-5256.2022.03.014

刘红燕¹,
李婧姣²,
路泽军²,
刘咏真²,
温居一^{1, 2, , ,}

1.
安徽医科大学海军临床学院，合肥 230032
2.
解放军总医院第六医学中心肿瘤内科，北京 100048

基金项目:

全军后勤科研重点课题 (BHJ14C008)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：刘红燕参与课题设计，完成试验，文章写作；李婧姣、路泽军、刘咏真参与试验、收集和分析数据及修改文章；温居一负责研究方案总体规划设计，指导撰写文章及论文修改。

详细信息

通信作者:
温居一，wenjuyi@126.com

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出版历程
- 收稿日期: 2021-07-21
- 录用日期: 2021-09-29
- 出版日期: 2022-03-20

Effect of long non-coding RNA LNC 01309 on proliferation and migration abilities of human hepatoma cells and its mechanism of action

1.
Naval Clinical College of Anhui Medical University, Hefei 230032, China
2.
Department of Oncology, The Sixth Medical Center of PLA General Hospital, Beijing 100048, China

Research funding:

The Key Logistics Science Research Project of PLA (BHJ14C008)

More Information

Corresponding author: WEN Juyi, wenjuyi@126.com(ORCID:0000-0002-7469-7811)

摘要

摘要: 目的探讨长链非编码RNA(lncRNA)LNC01309对人肝癌细胞增殖和迁移能力的影响及作用机制。方法收集2018年2月—2019年6月在解放军总医院第六医学中心手术治疗的12例肝细胞癌(以下简称肝癌)患者标本及对应的癌旁组织，采用荧光定量PCR方法检测LNC01309的相对表达量。利用荧光定量PCR法检测LNC01309在人肝癌细胞系(Hep G2、SNU-398和Hep 3B)和人永生化正常肝细胞系(THLE-2)中的表达水平。在肝癌细胞Hep G2中过表达LNC01309，分为质粒对照组(pEXP-control)和过表达组(pEXP-LNC01309)。采用CCK-8检测各组细胞增殖变化，采用划痕愈合试验和Transwell试验检测各组细胞迁移能力。利用RNA免疫共沉淀试验检测LNC01309与RBM38的相互作用(分组为IgG组和RBM38抗体组)。利用放线菌酮追踪分析检测LNC01309对于RBM38蛋白稳定性的影响。在肝癌细胞Hep G2中过表达RBM38进行回复试验，采用CCK-8试验、划痕愈合试验和Transwell试验，分别检测细胞增殖和迁移能力的变化。计量资料两组间比较采用t检验。结果 LNC01309在肝癌组织中的平均表达水平高于癌旁组织(4.225±2.285 vs 1.541±0.530, t=3.618，P=0.004)。LNC01309在肝癌细胞(Hep G2、SNU-398和Hep 3B)中的相对表达水平亦均高于正常肝细胞(THLE-2)(t值分别为4.231、6.489、14.480，P值均＜0.05)。过表达LNC01309的Hep G2细胞(t=9.172，P＜0.001)相对于对照组，细胞的生长速度明显上升(第96小时OD₄₅₀值：1.885±0.107 vs 2.527±0.234，t=4.330，P=0.012)，迁移能力上调(划痕愈合试验：11.65%±2.40% vs 35.66%±4.90%，t=9.837，P＜0.001；Transwell试验：100.00%±3.11% vs 161.00%±35.93%，t=4.399，P=0.005)；然而过表达LNC01309诱导的肝癌细胞增殖和迁移能力的上调效应，都能够被RBM38部分抑制(第96小时OD₄₅₀值：2.500±0.227 vs 1.913±0.282，t=2.812，P=0.048；168.00%±9.43% vs 117.20%±18.03%，t=6.622，P＜0.001)。相对于IgG对照组，RBM38抗体能够显著富集沉淀LNC01309(t=3.846，P=0.031)。放线菌酮试验结果显示，过表达LNC01309组细胞中RBM38蛋白稳定性显著下调(t=8.038，P=0.001)。结论新发现的LNC01309通过与RBM38的相互作用降低了RBM38蛋白稳定性，促进肝癌细胞的增殖与迁移。
- 癌, 肝细胞 /
- RNA, 长链非编码 /
- RNA结合蛋白质类
Abstract: Objective To investigate the effect of long non-coding RNA (lncRNA) LNC01309 on the proliferation and migration abilities of human hepatocellular carcinoma (HCC) cells and its mechanism of action. Methods HCC samples and corresponding adjacent tissue samples were collected from 12 patients with HCC who underwent surgical treatment in The Sixth Medical Center of PLA General Hospital from February 2018 to June 2019, and quantitative real-time PCR was used to measure the relative expression level of LNC01309. Quantitative real-time PCR was also used to measure the expression level of LNC01309 in human hepatoma cell lines (HepG2, SNU-398, and Hep3B) and the human immortalized normal liver cell line THLE-2. After LNC01309 was overexpressed in HepG2 cells, the cells were divided into plasmid control group (pEXP-control) and overexpression group (pEXP-LNC01309). CCK-8 assay was used to observe the change in cell proliferation, and wound healing assay and Transwell assay were used to observe migration ability. RNA co-immunoprecipitation was used to detect the interaction between LNC01309 with RBM38, with cells divided into IgG group and RBM38 antibody group, and cycloheximide chase assay was used to analyze the effect of LNC01309 on the stability of RBM38 protein. RBM38 was overexpressed in HepG2 cells to conduct the recovery experiment, and CCK-8 assay, wound healing assay, and Transwell assay were used to observe the changes in cell proliferation and migration abilities. The t-test was used for comparison of continuous data between two groups. Results The mean expression level of LNC01309 in HCC tissue was significantly higher than that in adjacent tissue (4.225±2.285 vs 1.541±0.530, t=3.618, P=0.004), and the relative expression level of LNC01309 in hepatoma cells (HepG2, SNU-398, and Hep3B) was also significantly higher than that in normal hepatocytes (THLE-2) (t=4.231、6.489、14.480, all P < 0.05). Compared with the control group, HepG2 cells with the overexpression of LNC01309 had significant increases in growth rate (OD₄₅₀ value at 96 hours: 1.885±0.107 vs 2.527±0.234, t=4.330, P=0.012) and migration ability (11.65%±2.40% vs 35.66%±4.90%, t=9.837, P < 0.001; 100.00%±3.11% vs 161.00%±35.93%, t=4.399, P=0.005); however, the upregulated proliferation and migration abilities of hepatoma cells induced by LNC01309 overexpression were partially inhibited by RBM38 (OD₄₅₀ value at 96 hours: 2.500±0.227 vs 1.913±0.282, t=2.812, P=0.048; 168.00%±9.43% vs 117.20%±18.03%, t=6.622, P < 0.001). Compared with the IgG control group, RBM38 antibody significantly enriched the precipitation of LNC01309 (t=3.846, P=0.031). The results of cycloheximide chase assay showed that the LNC01309 overexpression group had a significant reduction in the stability of RBM38 protein (t=8.038, P=0.001). Conclusion The newly identified LNC01309 reduces the stability of RBM38 protein through interaction with RBM38 and promotes the proliferation and migration of HCC cells.
- Carcinoma, Hepatocellular /
- RNA, Long Noncoding /
- RNA-Binding Proteins

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图 1 LNC01309在肝癌组织和细胞中的表达分析

注：a，PacBio测序分析获得的LNC01309序列；b，利用荧光定量PCR分析LNC01309在肝癌组织与癌旁组织中的表达差异；c，利用荧光定量PCR分析LNC01309在肝癌细胞和正常肝细胞中的表达差异。

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图 2 LNC01309与lncRNA MIR205HG的关系分析

注：a，LNC01309与lncRNA MIR205HG序列相似性，并根据序列差异性设计特异性检测引物；b，利用荧光定量PCR分析lncRNA MIR205HG在肝癌组织与癌旁组织的表达差异；c，利用荧光定量PCR分析lncRNA MIR205HG在肝癌细胞和正常肝细胞中的表达差异；d、e，Hep 3B细胞经转染siRNA(20 nmol/L)处理24 h后，利用荧光定量PCR分别检测lncRNA MIR205HG和LNC01309的相对表达含量；f、g，Hep G2细胞中转染过表达质粒24 h后，利用荧光定量PCR分别检测LNC01309和miR-205的相对表达含量。

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图 3 在线工具Locate-R预测LNC01309的细胞定位

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图 4 FISH试验检测LNC01309在肝癌细胞中的定位

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图 5 CCK-8检测转染LNC01309表达质粒的Hep G2细胞不同时间点的细胞活性

注：Hep G2细胞中转染LNC01309表达质粒以及对照质粒(24孔板中使用量为1 μg/孔)处理，于不同时间点利用CCK-8检测细胞活性。

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图 6 EdU染色试验检测转染LNC01309表达质粒的Hep G2细胞增殖

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图 7 细胞划痕试验检测转染LNC01309表达质粒的Hep G2细胞的迁移能力

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图 8 Transwell检测转染LNC01309表达质粒的Hep G2细胞的迁移能力

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图 9 Western Blot检测各组细胞中EMT标志蛋白的相对含量

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图 10 LNC01309与潜在RNA结合蛋白的相互作用

注：a，利用潜在RNA结合蛋白的特异性抗体进行RNA-IP试验，荧光定量PCR检测LNC01309的富集丰度；b，荧光定量PCR比较RBM38的抗体对野生型和突变型LNC01309的富集丰度差异。

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图 11 Western Blot检测转染LNC01309表达质粒的Hep G2细胞24 h后RBM38蛋白的相对表达含量

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图 12 Western Blot检测不同时间点经放线菌酮处理转染LNC01309表达质粒的Hep G2细胞中的RBM38蛋白相对含量

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图 13 不同时间点检测经放线菌酮处理转染LNC01309表达质粒的Hep G2细胞中RBM38蛋白相对含量

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图 14 CCK-8检测不同时间点各组转染的Hep G2细胞活性

注：Hep G2细胞中单独转染pEXP-LNC01309或者共转染pCMV-RBM38质粒24 h后，于不同时间点利用CCK-8检测各组细胞活性。

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图 15 细胞划痕愈合试验检测各组转染HepG2细胞的迁移能力

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图 16 Transwell检测各组转染Hep G2细胞的迁移能力

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表 1 利用NCBI BLAST比较LNC01309与lncRNA MIR205HG序列相似性

描述	种属	最高分	总分	查询覆盖值	E值	同源百分比	查询长度	登录号
Homo sapiens MIR205 host gene (MIR205HG), transcript variant 3, long non-coding RNA	Homo sapiens	516	516	1	5×10^-142	0.963	992	NR_145434.1
Homo sapiens MIR205 host gene (MIR205HG), transcript variant 1, long non-coding RNA	Homo sapiens	516	516	1	5×10^-142	0.963	895	NR_145437.1
Homo sapiens MIR205 host gene (MIR205HG), transcript variant 2, long non-coding RNA	Homo sapiens	516	516	1	5×10^-142	0.963	857	NR_145433.1
Homo sapiens MIR205 host gene (MIR205HG), transcript variant 4, long non-coding RNA	Homo sapiens	516	516	1	5×10^-142	0.963	940	NR_145435.1
Homo sapiens MIR205 host gene (MIR205HG), transcript variant 5, long non-coding RNA	Homo sapiens	516	516	1	5×10^-142	0.963	651	NR_145436.1

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表 2 利用RBPmap和catRAPID在线预测能够与LNC01309结合的潜在靶标蛋白

蛋白质	序列位置	基序	K-mer	Z评分	P值
CNOT4	129	gacaga	gagaga	3.554	1.90×10^-4
EIF4G2	50	cgccg	cgccg	4.390	5.67×10^-6
HNRNPA1	66	guaguagu	guagcagc	3.361	3.88×10^-4
MBNL1	70	gcgcagc	cagcagc	3.903	4.75×10^-5
RBM24	160	wgwgugd	ggagugu	3.403	3.33×10^-4
RBM38	163	kkguguk	gugugcg	3.333	4.30×10^-4
RBM6	37	ccacc	ccacc	4.228	1.18×10^-5
SRSF10	70	cagcag	cagcag	4.513	3.20×10^-6

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