Long Non-coding RNA LINC00114 Facilitates Colorectal Cancer Development Through EZH2/DNMT1-Induced miR-133b Suppression

Abstract

This study aimed to identify the roles of the long non-coding RNA LINC00114 in colorectal cancer (CRC) development. The expression levels of LINC00114 and miR-133b in CRC were determined by reverse transcription (RT)-polymerase chain reaction (PCR) and the functions of LINC00114 in CRC were evaluated in vitro and in vivo. Methylation-specific PCR assay was performed to detect the miR-133b promoter methylation in CRC cells. Bioinformatics analysis, RNA immunoprecipitation, dual luciferase assay, RNA pull-down, co-immunoprecipitation (IP), and chromatin IP (ChIP) assays were used to elucidate whether LINC00114 could recruit EZH2/DNMT1 and bind to the miR-133b promoter region, leading to dysregulated methylation and the depression of miR-133b. The expression levels of DNA methyltransferases (DNMTs), EZH2, and nucleoporin 214(NUP214) were analyzed by western blotting. Data showed that LINC00114 was highly expressed, whereas miR-133b was downregulated in the CRC tissues and cells. In vitro, silencing LINC00114 inhibited cell proliferation and impeded cell cycle at the G1/S phase by upregulating miR-133b. In vivo, LINC00114 knockdown reduced tumor growth. Further analysis showed that the methylation in miR-133b promoter region was increased in the CRC and silencing LINC00114 increased miR-133b expression through depressing methylation of its promoter region. ChIP-PCR experiments demonstrated that EZH2 and DNMT1 could bind to the miR-133b promoter region and it was abolished by LINC00114 knockdown. sh-EZH2 reversed the overexpression of DNMTs and CRC cell cycle progression induced by the LINC00114 upregulation. LINC00114 could regulate the NUP214 protein expression by sponging miR-133b. These results demonstrated that LINC00114 suppressed miR-133b expression via EZH2/DNMT1-mediated methylation of its promoter region, indicating that LINC00114 might be a potential novel target for CRC diagnosis and treatment.

Keywords: DNMT1; EZH2; LINC00114; colorectal cancer; methylation; miR-133b.

Figure 1

LINC00114 is upregulated in CRC. (A) Highly expressed LINC00114 is predicted in the GEPIA database. (B) LINC00114 is upregulated in the CRC samples compared to the adjacent normal tissue samples, determined via RT-PCR assay. (C) RT-PCR result shows that expression of miR-133b expression is reduced in the CRC samples. (D) LINC00114 expression is higher in the CRC as measured by in situ hybridization (200X). (E) There is an inverse correlation between LINC00114 and miR-133b expression in the CRC tissue samples. (F,G) The expression levels of LINC00114 and miR-133b in the CRC cells are determined by RT-PCR. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

LINC00114 interference facilitates apoptosis and represses cell cycle progression in vitro. (A–C) sh-LINC00114 inhibits cells proliferation. (D) Apoptosis of CRC cells transfected with the sh-LINC00114 or the control plasmid for 48 h, analyzed by flow cytometry. (E) Cell cycle analysis of HCT116 and LoVo cells transfected with the LINC00114 or the control plasmid for 48 h using flow cytometry. ***P < 0.001. Con, the group only treated with PBS; Mock, the cells transfected with the empty vector plasmid.

Figure 3

LINC00114 interference inhibits the development of CRC in vivo. (A) Tumor formation in nude mice. (B) Tumor body in nude mice. (C) Tumor growth curve. (D) Expression of miR-133b in tumor tissues. (E) Ki67 expression determined by immunohistochemistry. **P < 0.01; ***P < 0.001.

Figure 4

LINC00114 regulates DNA methylation of the miR-133b promoter region. (A) Expression levels of LINC00114 in the nucleus and cytoplasm of HCT116 and LoVo cells. (B) Luciferase activity of cells cotransfected with miR-133b overexpression and control plasmids after LINC00114 overexpression or mutation. (C) RIP assay was used to analyse the binding of LINC00114 to miR-133b. (D) The schematic diagram showed the binding site of miR-133b with LINC00114 and NUP214 mRNA. (E) Methylation detection of miR-133b promoter region in cells. (F) LINC00114 interference and RG108 treatment reduced the methylation levels of miR-133b promoter region in HCT116 and LoVo cells. (G) miR-133b levels after LINC00114 interference and RG108 treatment reduced the expression of miR-133b in cells. (H,I) Western blotting result showed that DNMT1 and DNMT3B were reduced in LINC00114-silenced or RG108-treated cells. *P < 0.05; **P < 0.01; ***P < 0.001. M, methylation. U, non-methylation.

Figure 5

LINC00114 binds to EZH2 protein. (A) Correlation analysis of LINC00114 and EZH2 using the GEPIA database, also see it in method section. (B) EZH2 interference plasmids were transfected into HCT116 and LoVo cells and then H3K27me3, DNMT1, and EZH2 were detected using western blotting assay. (C) miR-133b levels were detected by RT-PCR in cells transfected with sh-EZH2. (D) Co-IP analysis showed that EZH2 could bind to DNMT1 and H3K27me3. (E) RNA pull-down assay was performed to detect the binding of LINC00114 with EZH2 and DNMT1. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

LINC00114 promotes the methylation of miR-133b promoter region by recruiting EZH2 and DNMT1. (A) LINC00114 affects EZH2, DNMT1, and H3K27me3 binding with the miR-133b promoter region in CRC cells, as detected by ChIP assay. (B) EZH2-sh reversed the LINC00114-induced inhibition of miR-133b in cells. (C,D) MTT and TUNEL assays result showed that EZH2-sh reversed the LINC00114-incduced proliferation promotion of cells. (E) EZH2-sh reversed the upregulation of DNMT1 induced by LINC00114 overexpression. oe, overexpression. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

LINC00114 regulates cell proliferation via sponging miR-133b. (A) Expression of miR-133b detection in LINC00114 silenced cells. (B,C) The expression of NUP214 was determined by RT-PCR and western blotting, respectively. (D,E) Inhibition of miR-133b maintained proliferation promotion of cells in LINC00114 silenced cells, as shown in MTT and TUNEL assays, respectively. *P < 0.05; **P < 0.01; ***P < 0.001.