m6A识别蛋白HuR调控lncRNA TRG-AS1抑制结直肠癌生长的机制研究

doi:10.11958/20220960

天津医药 ›› 2023, Vol. 51 ›› Issue (2): 124-130.doi: 10.11958/20220960

m6A识别蛋白HuR调控lncRNA TRG-AS1抑制结直肠癌生长的机制研究

柴小兵(), 张利, 褚菲菲, 吴慧丽^△()

郑州大学附属郑州中心医院消化内科（邮编450000）

收稿日期:2022-06-21 修回日期:2022-07-13 出版日期:2023-02-15 发布日期:2023-02-24
通讯作者: ^△E-mail：wuhuili616161@126.com
作者简介:柴小兵（1979），男，副主任医师，主要从事胃肠道肿瘤方面研究。E-mail：bingbingcX27@163.com
基金资助:
河南省医学科技攻关计划联合共建项目(LHGJ20210763)

The mechanism of m6A recognition protein HuR inhibiting the growth of colorectal cancer by regulating lncRNA TRG-AS1

CHAI Xiaobing(), ZHANG Li, CHU Feifei, WU Huili^△()

Department of Gastroenterology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450000, China

Received:2022-06-21 Revised:2022-07-13 Published:2023-02-15 Online:2023-02-24
Contact: ^△E-mail：wuhuili616161@126.com

柴小兵, 张利, 褚菲菲, 吴慧丽. m6A识别蛋白HuR调控lncRNA TRG-AS1抑制结直肠癌生长的机制研究[J]. 天津医药, 2023, 51(2): 124-130.

CHAI Xiaobing, ZHANG Li, CHU Feifei, WU Huili. The mechanism of m6A recognition protein HuR inhibiting the growth of colorectal cancer by regulating lncRNA TRG-AS1[J]. Tianjin Medical Journal, 2023, 51(2): 124-130.

摘要/Abstract

摘要：

目的探讨N6-甲基腺苷（m6A）识别蛋白人类抗原R（HuR）调控长链非编码RNA T细胞受体γ位点反义RNA 1（lncRNA TRG-AS1）对结直肠癌（CRC）生长的影响。方法比色法检测CRC患者的癌组织、癌旁组织及正常结肠上皮细胞NCM460及CRC细胞HCT116、SW480、LOVO中m6A含量；实时荧光定量PCR（qPCR）检测TRG-AS1表达；Western blot检测HuR蛋白表达。将HCT116细胞分为Ct组、OE-NC组、OE-HuR组、si-NC组、si-HuR组、si-HuR+pcDNA组、si-HuR+pcDNA-TRG-AS1组，CCK-8法检测细胞增殖；平板克隆实验检测细胞克隆形成能力；流式细胞术检测细胞凋亡率；划痕愈合实验检测细胞迁移；Transwell检测细胞侵袭；裸鼠体内移植瘤实验观察肿瘤生长情况；采用甲基化RNA免疫共沉淀（MeRIP）检测TRG-AS1上是否存在m6A位点；RNA pull-down实验和RNA免疫共沉淀（RIP）检测TRG-AS1与HuR蛋白的相互作用。结果在CRC组织和细胞中，HuR蛋白、TRG-AS1高表达，m6A含量降低，且在HCT116细胞中HuR蛋白、TRG-AS1表达最高，m6A含量最低（P<0.05），选择HCT116细胞为研究对象。与si-NC组比较，si-HuR组HuR蛋白、TRG-AS1表达降低，m6A含量升高（P<0.05）；与OE-NC组比较，OE-HuR组HuR蛋白、TRG-AS1表达升高，m6A含量降低（P<0.05）；与si-HuR组、si-HuR+pcDNA组比较，si-HuR+pcDNA-TRG-AS1组HuR蛋白、m6A含量变化差异无统计学意义，TRG-AS1表达升高（P<0.05）；下调HuR可抑制HCT116细胞增殖、迁移、侵袭及体内移植瘤的生长，促进细胞凋亡，而上调HuR则呈相反趋势；过表达TRG-AS1减弱了沉默HuR对HCT116细胞增殖、划痕愈合率、侵袭、体内移植瘤生长的抑制作用以及对细胞凋亡的促进作用；TRG-AS1上存在m6A位点，且TRG-AS1能与HuR蛋白相互作用。结论沉默m6A识别蛋白HuR可通过抑制TRG-AS1表达进而抑制HCT116细胞增殖、迁移与侵袭，促进细胞凋亡。

关键词: 结直肠肿瘤, 甲基化, RNA, 长链非编码, 细胞增殖, 人类抗原R, N6-甲基腺苷, T细胞受体γ位点反义RNA 1

Abstract:

Objective To investigate the influence of N6-methyladenosine (m6A) recognition protein human antigen R (HuR) on the growth of colorectal cancer (CRC) by regulating long non-coding RNA T cell receptor gamma locus antisense RNA 1 (lncRNA TRG-AS1). Methods The content of m6A in cancer tissue, paracancer tissue and normal colon epithelial cells NCM460 and CRC cells HCT116, SW480 and LOVO were detected by colorimetric method. The expression of TRG-AS1 was detected by real-time fluorescence quantitative PCR (qPCR). The expression of HuR protein was detected by Western blot assay. HCT116 cells were divided into the Ct group, the OE-NC group, the OE-HuR group, the si-NC group, the si-HuR group, the si-HuR + pcDNA group and the si-HuR+ pcDNA-TRG-AS1 group. CCK-8 assay was applied to detect cell proliferation. Plate cloning assay was used to detect the ability of cells to form clones. Flow cytometry was applied to detect apoptosis. Scratch-healing assay was applied to detect cell migration. Transwell assay was used to detect cell invasion. In vivo tumor xenograft experiment was used to observe tumor growth in nude mice. Methylated RNA immunoprecipitation (MeRIP) was applied to detect the presence of m6A sites on TRG-AS1. RNA co-immunoprecipitation (RIP) and RNA pull-down experiments were used to demonstrate the interaction of TRG-AS1 with HuR protein. Results In CRC tissue and cells, HuR protein and TRG-AS1 were highly expressed, and m6A content was decreased. In HCT116 cells, the expression levels of HuR protein and TRG-AS1 were the highest, and the m6A content was the lowest (P<0.05), therefore, HCT116 cells were selected as the research object. Compared with the si-NC group, the expression levels of HuR protein and TRG-AS1 decreased in the si-HuR group, and the m6A content increased (P<0.05). Compared with the OE-NC group, expression levels of HuR protein and TRG-AS1 increased in the OE-HuR group, and the m6A content decreased (P<0.05). Compared with the si-HuR group and the si-HuR+pcDNA group, there were no significant differences in changes of HuR protein and m6A content in the si-HuR+pcDNA-TRG-AS1 group, but the expression of TRG-AS1 increased (P<0.05). The down-regulation of HuR could inhibit HCT116 cell proliferation, migration, invasion and growth of transplanted tumor in vivo, and promote cell apoptosis, while up-regulation of HuR showed the opposite trends. The overexpression of TRG-AS1 attenuated the inhibitory effect of silencing HuR on HCT116 cell proliferation, migration, invasion, in vivo xenograft growth, and the promotion of apoptosis. There was m6A site on TRG-AS1, and TRG-AS1 could interact with HuR protein. Conclusion Silencing the m6A recognition protein HuR can inhibit the proliferation, migration, invasion of HCT116 cells and promote cell apoptosis by inhibiting the expression of TRG-AS1.

Key words: colorectal neoplasms, methylation, RNA, long noncoding, cell proliferation, human antigen R, N6-methyladenosine, T cell receptor gamma locus antisense RNA 1

中图分类号:

R735.3

图/表 16

表1 qPCR引物序列

Tab.1 qPCR primer sequences

基因名称	引物序列（5→3?）	产物大小（bp）
TRG-AS1	上游：GGAGTCTGCTCTAAGAGCTG 下游：CAGAGCAAAGATGCTCTGC	145
GAPDH	上游：TGACTTCAACAGCGACACCCA 下游：CACCCTGTTGCTGTAGCCAAA	126

图1 Western blot检测组织中HuR蛋白表达

Fig.1 Western blot detection of HuR protein expression in tissue

表2 Comparison of HuR protein, m6A content and TRG-AS1 expression in cancer and adjacent tissues

Tab.2 癌组织和癌旁组织HuR蛋白、m6A含量和TRG-AS1表达比较（n=19，$\bar{x}±s$）

组别	HuR蛋白	m6A含量（%）	TRG-AS1
癌旁组织	0.42±0.03	0.92±0.08	1.00±0.00
癌组织	1.35±0.21	0.31±0.02	2.22±0.21
t	19.110^＊＊	32.244^＊＊	25.323^＊＊

图2 Western blot检测细胞中HuR蛋白表达

Fig.2 Western blot detection of HuR protein expression in cells

表3 Comparison of m6A content, HuR protein and TRG-AS1 expression between normal colonic epithelial cells and CRC cells

Tab.3 正常结肠上皮细胞及CRC细胞m6A含量、HuR蛋白、TRG-AS1表达比较（n=6，$\bar{x}±s$）

组别	HuR	m6A含量（%）	TRG-AS1
NCM460	0.36±0.03	1.23±0.11	1.00±0.00
HCT116	1.48±0.23^a	0.27±0.02^a	2.58±0.24^a
SW480	1.21±0.20^ab	0.38±0.03^ab	2.27±0.22^ab
LOVO	1.01±0.09^ab	0.46±0.04^ab	2.04±0.23^ab
F	53.645^＊＊	305.547^＊＊	70.881^＊＊

图3 Western blot检测HCT116细胞中HuR蛋白表达 A：Ct组；B：OE-NC组；C：OE-HuR组；D：si-NC组；E：si-HuR组；F：si-HuR+pcDNA组；G：si-HuR+pcDNA-TRG-AS1组。

Fig.3 Western blot detection of HuR protein expression in HCT116 cells

表4 Comparison of HuR protein, m6A content and TRG-AS1 expression in HCT116 cells between the seven groups

Tab.4 各组HCT116细胞中的HuR蛋白、m6A含量、TRG-AS1表达比较（n=6，$\bar{x}±s$）

组别	HuR	m6A含量（%）	TRG-AS1
Ct组	1.46±0.21	0.28±0.03	1.00±0.00
OE-NC组	1.47±0.22	0.26±0.02	1.02±0.08
OE-HuR组	1.98±0.25^ab	0.10±0.01^ab	2.34±0.22^ab
si-NC组	1.45±0.22	0.27±0.01	1.01±0.09
si-HuR组	0.35±0.03^ac	1.12±0.08^ac	0.71±0.12^ac
si-HuR+pcDNA组	0.35±0.04	1.14±0.09	0.69±0.13
si-HuR+pcDNA-TRG-AS1组	0.36±0.03	1.13±0.10	0.94±0.11^de
F	95.617^＊＊	381.869^＊＊	125.637^＊＊

图4 沉默或过表达HuR对HCT116细胞克隆形成的影响

Fig.4 Effects of silencing or overexpression of HuR on HCT116 cell clone formation

图5 Annexin V-FITC/PI双重染色检测沉默或过表达HuR对HCT116细胞凋亡的影响

Fig.5 Effects of silencing or overexpression of HuR on apoptosis of HCT116 cells detected by Annexin V-FITC/PI double staining

表5 Comparison of OD450 value, clone formation rate and apoptosis rate between different groups of HCT116 cells

Tab.5 各组HCT116细胞OD450值、克隆形成率、细胞凋亡率比较（n=6，$\bar{x}±s$）

组别	OD₄₅₀值	克隆形成率（%）	细胞凋亡率（%）
Ct组	0.82±0.08	62.23±5.14	41.27±3.68
OE-NC组	0.83±0.09	63.15±5.09	42.26±3.71
OE-HuR组	1.25±0.12^ab	83.36±6.05^ab	24.48±2.39^ab
si-NC组	0.84±0.08	62.78±5.16	42.33±5.08
si-HuR组	0.32±0.02^ac	26.69±2.44^ac	65.54±5.62^ac
si-HuR+pcDNA组	0.33±0.03	26.75±2.38	64.57±5.41
si-HuR+pcDNA-TRG-AS1组	0.69±0.05^de	48.87±4.12^de	46.67±3.08^de
F	112.818^＊＊	126.394^＊＊	67.001^＊＊

表6 Comparison of wound healing rate and the number of invasive cells between different groups of HCT116 cells

Tab.6 各组HCT116细胞划痕愈合率、侵袭细胞数目比较（n=6，$\bar{x}±s$）

组别	划痕愈合率（%）	侵袭细胞数目（个）
Ct组	51.17±4.66	74.43±6.25
OE-NC组	51.28±5.02	74.58±6.12
OE-HuR组	69.44±6.24^ab	113.35±10.04^ab
si-NC组	51.34±5.13	73.96±6.11
si-HuR组	21.25±2.06^ac	27.75±2.16^ac
si-HuR+pcDNA组	21.36±2.09	28.14±2.02
si-HuR+pcDNA-TRG-AS1组	36.67±3.24^de	51.14±4.33^de
F	100.512^＊＊	159.505^＊＊

图6 沉默或过表达HuR对HCT116细胞迁移的影响 A：Ct组；B：OE-NC组；C：OE-HuR组；D：si-NC组；E：si-HuR组；F：si-HuR+pcDNA组；G：si-HuR+pcDNA-TRG-AS1组。

Fig.6 The effect of silencing or overexpressing HuR on the migration of HCT116 cells

图7 沉默或过表达HuR对HCT116细胞侵袭的影响（结晶紫染色，×200）

Fig.7 The effect of silencing or overexpressing HuR on the invasion of HCT116 cells (crystal violet dyeing, ×200)

图8 各组移植瘤质量比较

Fig.8 Comparison of tumor quality in each group

图9 RMVar数据库显示TRG-AS1上存在潜在的m6A位点

Fig.9 RMVar database showing potential m6A sites on TRG-AS1

图10 RNA下拉实验表明HuR与TRG-AS1结合

Fig.10 RNA pull-down experiments showing HuR binds to TRG-AS1

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m6A识别蛋白HuR调控lncRNA TRG-AS1抑制结直肠癌生长的机制研究

The mechanism of m6A recognition protein HuR inhibiting the growth of colorectal cancer by regulating lncRNA TRG-AS1

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