Identification of LINC02310 as an enhancer in lung adenocarcinoma and investigation of its regulatory network via comprehensive analyses

Abstract

Background: Lung adenocarcinoma (LADC) is a major subtype of non-small cell lung cancer and has one of the highest mortality rates. An increasing number of long non-coding RNAs (LncRNAs) were reported to be associated with the occurrence and progression of LADC. Thus, it is necessary and reasonable to find new prognostic biomarkers for LADC among LncRNAs.

Methods: Differential expression analysis, survival analysis, PCR experiments and clinical feature analysis were performed to screen out the LncRNA which was significantly related to LADC. Its role in LADC was verified by CCK-8 assay and colony. Furthermore, competing endogenous RNA (ceRNA) regulatory network construction, enrichment analysis and protein-protein interaction (PPI) network construction were performed to investigate the downstream regulatory network of the selected LncRNA.

Results: A total of 2431 differentially expressed LncRNAs (DELncRNAs) and 2227 differentially expressed mRNAs (DEmRNAs) were from The Cancer Genome Atlas database. Survival analysis results indicated that lnc-YARS2-5, lnc-NPR3-2 and LINC02310 were significantly related to overall survival. Their overexpression indicated poor prognostic. PCR experiments and clinical feature analysis suggested that LINC02310 was significantly correlated with TNM-stage and T-stage. CCK-8 assay and colony formation assay demonstrated that LINC02310 acted as an enhancer in LADC. In addition, 3 targeted miRNAs of LINC02310 and 414 downstream DEmRNAs were predicted. The downstream DEmRNAs were then enriched in 405 Gene Ontology terms and 11 Kyoto Encyclopedia of Genes and Genomes pathways, which revealed their potential functions and mechanisms. The PPI network showed the interactions among the downstream DEmRNAs.

Conclusions: This study verified LINC02310 as an enhancer in LADC and performed comprehensive analyses on its downstream regulatory network, which might benefit LADC prognoses and therapies.

Keywords: LADC; LncRNA; PPI; TCGA; ceRNA regulatory network.

Fig. 1

Volcano plots of LncRNAs and mRNAs. a LncRNAs: the red dots represent the LncRNAs with LFC > 1.5 and p < 0.05; the blue dots represent the LncRNAs with LFC < -1.5 and p < 0.05; the gray dots are the remaining LncRNAs; b mRNAs: the red dots represent the mRNAs with LFC > 2 and p < 0.01; the blue dots represent the mRNAs with LFC < -2 and p < 0.01; the gray dots are the remaining mRNAs

Fig. 2

Heatmaps of the top-50 DELncRNAs and DEmRNAs. a DELncRNAs; b DEmRNAs. The samples are divided into two groups: LADC and adjacent normal samples. The ‘n’ in ‘log10(n + 0.1)’ is the normalized read count (expression quantity), and adding 0.1 is to avoid meaningless calculation when n = 0

Fig. 3

KM survival plots of the top-3 significant DELncRNAs. a lnc-YARS2-5 (Symbol ID: AC087588.2, p = 6.9774e−7); b lnc-NPR3-2 (Symbol ID: AC034223.1, p = 5.2233e−6); c LINC02310 (p = 1.0380e−5)

Fig. 4

PCR expression quantification in LADC tissues. a lnc-YARS2-5; b lnc-NPR3-2; c LINC02310

Fig. 5

CCK-8 assays. LINC02310 promoted the cell proliferation of H1299 and PC-9. a H1299 OE/NC; b H1299 si-02310/si-NC; c PC-9 OE/NC; d PC-9 si-02310/si-NC. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Colony formation assays. LINC02310 promoted the clone formation of H1299 cells. a H1299 si-02310/si-NC; b H1299 OE/NC

Fig. 7

Binding sites of miRNAs with LINC02310. The red blocks and light blue blocks represent targeted miRNAs with RPM ≥ 1 and RPM < 1, respectively

Fig. 8

CeRNA (LINC02310-targeted miRNAs-downstream DEmRNAs) regulatory network. Red dot: LINC02310; yellow dots: targeted miRNAs; green dots: downstream DEmRNAs. downstream DEmRNAs connected to more than one targeted miRNA were placed together at the lower right in a circle

Fig. 9

The top-10 significant in GO and KEGG pathway analysis of the downstream DEmRNAs. a BPs; b MFs; c CCs; d KEGG pathway

Fig. 10

PPI network. The size and color of the nodes represent the degree, which is the number of nodes with which one node is interacted among the total 1081 interactions. Each line represents an interaction