The prognostic value of LINC01296 in pan-cancers and the molecular regulatory mechanism in hepatocellular carcinoma: a comprehensive study based on data mining, bioinformatics, and in vitro validation

Abstract

Background and aims: This study aimed to clarify the prognostic role of LINC01296 in various cancers, and to evaluate its effect on proliferation, metastasis, and the cell cycle in hepatocellular carcinoma (HCC) by data mining, bioinformatics, and in vitro validation.

Methods: The prognostic role of LINC01296 in cancer patients was assessed by searching the PubMed, Embase, Web of Science, and Gene Expression Omnibus databases and calculating pooled hazard ratios (HRs) with 95% confidence intervals (CIs); this prognostic role was also evaluated using The Cancer Genome Atlas (TCGA). We detected LINC01296 expression in HCC cell lines, and lentivirus-mediated small interfering RNAs were used to silence LINC01296 in MHCC97H and Hep3B cells to explore the role of LINC01296 in cell proliferation, metastasis, and cell cycle progression with in vitro validation and bioinformatics.

Results: The results indicated that LINC01296 overexpression was associated with poor overall survival (OS) and disease-free survival (DFS) in various cancers; however, LINC01296 expression was not associated with recurrence-free survival (RFS). Similar results were found with TCGA, which showed that LINC01296 expression was associated with the pathologic stage, tumor size, and differentiation in Asian cancer patients. Additionally, bioinformatics analysis revealed expression of 394 related genes, which indicated that LINC01296 could be involved in the tumorigenesis and progression of HCC. In vitro gene silencing experiments indicated that LINC01296 downregulation repressed cell proliferation, cell cycle progression, and the metastatic potential of HCC through the regulation of BUB1, CCNA2, and CDK1 expression.

Conclusion: This study demonstrated that LINC01296 expression is related to poor OS and DFS in a variety of cancer types and that LINC01296 has an oncogenic role in HCC.

Keywords: HCC; LINC01296; bioinformatics; cancers; lncRNA.

Figure 1

Flow diagram indicating the study selection process.

Figure 2

A forest plot of studies that evaluates the relationship between LINC01296 expression and overall survival (OS).

Figure 3

Forest plots of the overall survival (OS) subgroup analysis: subgroup analysis by (A) sample size, (B) tumor type, (C) land region, and (D) source.

Figure 4

(A) A Begg’s publication bias plot and (B) sensitivity analysis for overall survival.

Figure 5

Forest plots, sensitivity, and Begg’s publication bias plots regarding studies where the relationship between LINC01296 expression and (A, C, E) disease-free survival (DFS) rates, and (B, D, F) recurrence-free survival were investigated.

Figure 6

Validation of LINC01296 expression in The Cancer Genome Atlas (TCGA) cohort. (A). LINC01296 expression in bladder urothelial carcinoma (BLCA), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNBC), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), kidney renal clear cell carcinoma (KIRC), lung squamous cell carcinoma (LUSC), stomach adenocarcinoma (STAD), and uterine corpus endometrial carcinoma (UCEC). “*” indicates a log2FC value >1 and a P-value <0.01 in the TCGA cohort. (B) Overall survival plots of LINC01296 expression in the TCGA cohort (n=9502, log-rank P<0.01). (C) Disease-free survival plots of LINC01296 expression in the TCGA cohort (n=9502, log-rank P<0.01).

Figure 7

The enriched annotation pathway analysis of potential genes targeted by LINC01296 in HCC. (A) A Venn diagram that shows overlap between the number of predicted target genes using the Multi Experiment Matrix (MEM) and lncRNA-related protein-coding genes (corlncRNA) and the R software package. (B) The significantly enriched annotation of the Gene Ontology (GO) categories. (C) The significantly enriched annotation of the KEGG pathway analysis. (D) The KEGG pathway map illustrating the cell cycle signaling pathway in humans as determined with the DAVID 6.8 database (https://david.ncifc rf.gov/).

Figure 8

The selected LINC01296 hub-related genes.

Figure 9

Correlation analysis between LINC01296 expression and the hub genes using Pearson’s correlation analysis in The Cancer Genome Altas (TCGA). (A) ASPM, (B) BUB1, (C) CCNA2, (D) CDK1, (E) CEP55, and (F) DLGAP5.

Figure 10

(A) LINC01296 expression in different HCC cell lines and the normal liver cell line, LO-2, was measured using qRT-PCR, (B) MHCC97H and Hep 3B cells were transfected with siRNA(si)-negative control (NC), si-LINC-01296–1#, and si-LINC01296-2#, and the CCK-8 assay was performed in (C) MHCC97H and (D) Hep 3B cells. Notes: *P<0.05, **P<0.01.

Figure 11

LINC01296 knockdown inhibited the migration and invasion of MHCC97H and Hep 3B cells. (A) Cell migration was determined by wound healing assays in MHCC97H and Hep 3B cells transfected with Si-NC, Si-LINC01296-1, and Si-LINC01296-2. (B) Cell invasion was determined in MHCC97H and Hep 3B cells transfected with siRNA (si)-negative control (NC), si-LINC01296-1# and si-LINC01296-2# by Transwell invasion assays. The data are presented as the mean ± standard deviation. *P<0.05.

Figure 12

LINC01296 knockdown promoted cellular apoptosis. (A) Cellular apoptosis was measured in MHCC97H and Hep3B cells transfected with SiRNA (Si)-negative control (NC), si-LINC01296-1# or si-LINC01296-2# by staining with Annexin V/PI. (B) The cell cycle distribution was determined with flow cytometry. The data are presented as the mean ± standard deviation. *P<0.05, **P<0.01, and ***P<0.001 vs Si-NC.

Figure 13

LINC01296 downregulation suppressed CDK-1, BUB1, and CCNA-2 expression. (A) CDK1, BUB1, CCNA2, and GAPDH expression in MHCC97H and Hep 3B cells, whose gene expression was silenced with siRNA (si)-negative control (NC), si-LINC01296-1, and si-LINC01296-2, was measured using Western blot. (B and C). The ratio of each protein to GAPDH in MHCC97H and Hep 3B cells is shown. **P < 0.01