Overexpression of Long Noncoding RNA LBX2-AS1 Promotes the Proliferation of Colorectal Cancer

Abstract

Background: LBX2 antisense RNA 1 (LBX2-AS1), a long noncoding RNA, has been identified to be closely associated with the progression of various cancers. However, the role of LBX2-AS1 in colorectal cancer (CRC) is still poorly understood. In this study, we aimed to investigate the expression and function of LBX2-AS1 in CRC.

Material and methods: Expression data from the Gene Expression Omnibus (GEO) and Gene Expression Profiling Interactive Analysis (GEPIA) databases and results obtained from clinical samples/patients were used to determine the correlation between LBX2-AS1 expression and pathological stages, overall survival (OS). Furthermore, knockdown of LBX2-AS1 in CRC cells using the short interfering RNA (siRNA) technique, and observed its biological functions using western blotting, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), cell counting kit-8 (CCK-8) and flow cytometry assay in the CRC cell line.

Results: Our study demonstrated that the expression levels of LBX2-AS1 were higher in CRC cell lines than in normal colon mucosal cell lines. Bioinformatics analysis revealed that CRC patients with high LBX2-AS1 expression levels had poor OS. Furthermore, knockdown of LBX2-AS1 in CRC cells could attenuate the proliferative ability of CRC cells in vitro, which is associated with decreased expression of cyclin-dependent kinase (CDK) 3, CDK6, and CCND1 and enhanced expression of cyclin-dependent kinase inhibitor 1A.

Conclusions: LBX2-AS1 plays a crucial role in the tumorigenesis of CRC, providing a potential therapeutic target for CRC patients.

Keywords: LBX2 antisense RNA 1; colorectal cancer; long noncoding RNA; prognosis; therapy.

Figure 1.

LBX2 antisense RNA 1 (LBX2-AS1) is overexpressed in colorectal cancer (CRC). (A) LBX2-AS1 expression, as measured by Affymetrix microarray, was more upregulated in CRC tissues compared with normal colon mucosal tissues in #GSE41328 (containing 10 pairs of CRC tissues and corresponding normal colorectal tissues) from the Gene Expression Omnibus database. (B) The Gene Expression Profiling Interactive Analysis database was used to analyze the expression of LBX2-AS1 in CRC tissues using the display form of box plots. (C) LBX2-AS1 expression was significantly higher in CRC cell lines (HT29, LoVo, SW620, and HCT116) than in the normal colon mucosal cell line NCM460. (D) Nucleic and cytoplasmic RNA were analyzed using quantitative real-time polymerase chain reaction to detect the expression of LBX2-AS1 in HCT116 cells. U6 was used as a nucleic RNA control; glyceraldehyde 3-phosphate dehydrogenase and β-actin were used as cytoplasmic RNA controls. Data are shown as mean ± standard error of the mean. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 compared with control

Figure 2.

Upregulated LBX2 antisense RNA 1 (LBX2-AS1) is associated with the clinicopathological features of colorectal cancer (CRC). (A) The Gene Expression Profiling Interactive Analysis (GEPIA) database was used to analyze the association between LBX2-AS1 expression and the pathological stages of CRC. (B) The GEPIA database was used to analyze the clinical impact of LBX2-AS1 expression patterns on CRC patients’ survival in a CRC specimen expression profile dataset, group 1 = low expression of LBX2-AS1, n = 181; group 2 = high expression of LBX2-AS1, n = 181.

Figure 3.

LBX2 antisense RNA 1 (LBX2-AS1) knockdown suppressed the proliferative ability of colorectal cancer cells. (A) The interference efficiency of si-LBX2-AS1 was verified in HCT116 and LoVo cells. HCT116 and LoVo cells were transfected with either si-NC or si-LBX2-AS1 (1#, 2#, 1+2#) for 48 h, and subsequently, LBX2-AS1 expression was analyzed by quantitative real-time polymerase chain reaction. (B-C) Cell Counting Kit-8 proliferation assay and flow cytometry assay were used to detect the cell proliferative ability after transfection with si-NC or si-LBX2-AS1 for 48 h in HCT116 and LoVo cells. Data are shown as mean ± standard error of the mean. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 compared with control.

Figure 4.

LBX2 antisense RNA 1 (LBX2-AS1) knockdown inhibited the expression of the proliferation-associated markers in colorectal cancer (CRC) cells. After transfection with si-NC or si-LBX2-AS1 for 48 h in HCT116 cells, mRNA expression of CCND1, cyclin-dependent kinase 3 (CDK3), CDK6, and cyclin-dependent kinase inhibitor 1A (CDKN1A) in CRC cells were analyzed by quantitative real-time polymerase chain reaction (A); meanwhile, the protein expressions of CCND1, CDK3, CDK6, and CDKN1A in CRC cells were analyzed by western blotting and densitometry (B). Data are shown as mean ± standard error of the mean. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 compared with control.

Figure 5.

Scatter plots showing the statistical association between LBX2 antisense RNA 1 (LBX2-AS1) expression and proliferation marker expression. The Gene Expression Profiling Interactive Analysis database was used to analyze the association between LBX2-AS1 expression and the expression of CCND1, cyclin-dependent kinase 3 (CDK3), CDK6, and cyclin-dependent kinase inhibitor 1A in colorectal cancer.