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

doi:10.11958/20220960

Abstract

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

CLC Number:

R735.3

Figures/Tables 16

References 17

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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

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

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^＊＊

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^＊＊

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^＊＊