LINC02154 promotes cell cycle and mitochondrial function in oral squamous cell carcinoma

Abstract

Long noncoding RNAs (lncRNAs) play pivotal roles in the development of human malignancies, though their involvement in oral squamous cell carcinoma (OSCC) remains incompletely understood. Using The Cancer Genome Atlas (TCGA) dataset, we analyzed expression of 7840 lncRNAs in primary head and neck squamous cell carcinoma (HNSCC) and found that upregulation of LINC02154 is associated with a poorer prognosis. LINC02154 knockdown in OSCC cell lines induced cell cycle arrest and apoptosis, and significantly attenuated tumor growth in vitro and in vivo. Notably, depletion of LINC02154 downregulated FOXM1, a master regulator of cell cycle-related genes. RNA pulldown and mass spectrometry analyses identified a series of proteins that could potentially interact with LINC02154, including HNRNPK and LRPPRC. HNRNPK stabilizes FOXM1 expression by interacting with the 3'-UTR of FOXM1 mRNA, which suggests LINC02154 and HNRNPK promote cell cycling by regulating FOXM1 expression. Additionally, LINC02154 positively regulates HNRNPK expression by inhibiting microRNAs targeting HNRPNK. Moreover, LINC02154 affects mitochondrial function by interacting with LRPPRC. Depletion of LINC02154 suppressed expression of mitochondrial genes, including MTCO1 and MTCO2, and inhibited mitochondrial respiratory function in OSCC cells. These results suggest that LINC02154 exerts its oncogenic effects by modulating the cell cycle and oxidative phosphorylation in OSCC, highlighting LINC02154 as a potential therapeutic target.

Keywords: RNA binding protein; cell cycle regulation; lncRNA; mitochondrial function; oral cancer.

FIGURE 1

Identification of LINC02154 as an oral squamous cell carcinoma (OSCC)‐related long noncoding RNA (lncRNA). (A) Workflow to screen for novel cancer‐related lncRNAs using The Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSC) dataset. (B) Heatmap showing expression of 24 lncRNAs upregulated in primary HNSCC tumors. (C) Kaplan–Meier curves showing the effect of LINC02154 expression on survival of HNSCC patients (n = 249). (D) LINC02154 expression in normal tissues (n = 44) and primary tumors (n = 500) in the TCGA‐HNSC dataset. (E) LINC02154 expression in normal tissues (n = 50) and primary laryngeal squamous cell carcinoma (n = 50) in the indicated dataset. (F) LINC02154 expression in normal tissues (n = 15) and primary oral squamous cell carcinoma tissues (n = 15) in the indicated dataset. (G) LINC02154 expression in normal tissues (n = 19) and primary OSCC tumors (n = 259) in the TCGA dataset. (H) Quantitative RT‐PCR analysis of LINC02154 in a normal tongue tissue and OSCC cell lines (n = 3). Error bars represent SD. ***p < 0.001. RNA‐seq, RNA sequencing; TPM, transcripts per million.

FIGURE 2

Functional analysis of LINC02154 in oral squamous cell carcinoma cells. (A) Quantitative RT‐PCR analysis of LINC02154 in Ca9‐22 cells transfected with control siRNA (siCtrl) or siRNAs targeting LINC02154 (n = 3). (B) Cell viability assays in Ca9‐22 cells transfected with the indicated siRNAs (n = 6). (C) Colony formation assays using Ca9‐22 cells transfected with inducible shRNAs and treated with or without doxycycline (Dox). Summarized results are shown on the right (n = 3). (D) Colony formation assays using KON cells with or without ectopic LINC02154 expression. Summarized results are shown on the right (n = 3). (E) Apoptosis and (F) cell cycle analyses in Ca9‐22 cells transfected with the indicated siRNA. Representative results are shown on the left, summarized results on the right (n = 3). (G) Tumor growth in mice injected with Ca9‐22 cells inducibly expressing the indicated shRNAs. (H) Resected tumors are shown on the left, tumor weights on the right (n = 4). Error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

LINC02154 regulates cell cycle‐related genes in oral squamous cell carcinoma (OSCC) cells. (A) Microarray analysis in KON cells transfected with the indicated siRNAs. Shown is a heatmap of genes altered (>2‐fold) by LINC02154 knockdown. Representative genes are indicated on the right. (B) Gene Set Enrichment Analysis of the indicated gene sets using the microarray data in (A). (C) Gene Ontology (GO) and (D) pathway analyses of genes downregulated by LINC02154 knockdown. (E) Quantitative RT‐PCR (qRT‐PCR) analysis of cell cycle‐related genes in OSCC cells transfected with the indicated siRNAs (n = 3). (F) Western blot analysis of cell cycle‐related proteins in OSCC cells transfected with the indicated siRNAs. (G) qRT‐PCR analysis of FOXM1 in KON cells with or without ectopic LINC02154 expression (n = 3). (H) Western blot analysis of FOXM1 in KON cells transfected with the indicated vectors. Error bars represent SD. ***p < 0.001. NES, normalized enrichment score; siCtrl, control siRNA.

FIGURE 4

Identification and functional analysis of proteins that interact with LINC02154. (A) Gene Ontology (GO) analysis of proteins potentially interacting with LINC02154 identified by mass spectrometry. (B) Interaction network among the proteins potentially interacting with LINC02154. (C) Results of RIP assays. HNRNPK was immunoprecipitated from Ca9‐22 cells, after which coprecipitated LINC02154 was detected with quantitative RT‐PCR (qRT‐PCR). IgG served as a negative control (n = 3). (D) qRT‐PCR analysis of HNPNPK in Ca9‐22 cells transfected with a control siRNA (siCtrl) or siRNAs targeting HNRNPK (n = 3). (E) Cell viability assays in Ca9‐22 cells transfected with the indicated siRNAs (n = 6). (F) qRT‐PCR analysis of cell cycle‐related genes in Ca9‐22 cells transfected with the indicated siRNAs (n = 3). (G) Western blot analysis of FOXM1 in Ca9‐22 cells transfected with the indicated siRNAs. (H) Correlations between expression levels of HNRNPK and those of the indicated genes in primary oral squamous cell carcinoma tumors in The Cancer Genome Atlas dataset. Pearson's correlation coefficients and p values are shown. (I) Results of RIP assays. HNRNPK was immunoprecipitated from Ca9‐22 cells, after which coprecipitated FOXM1 was detected with qRT‐PCR (n = 3). (J) HNRNPK CLIP‐seq data at the 3′‐UTR of FOXM1 in HeLa cells. (K) Results of FOXM1 3′‐UTR reporter assays in Ca9‐22 cells transfected with the indicated siRNAs (n = 4). Error bars represent SD. **p < 0.01, ***p < 0.001. FDR, false discovery rate.

FIGURE 5

Modulation of HNRNPK expression by LINC02154 in oral squamous cell carcinoma (OSCC) cells. (A) Correlation between expression levels of LINC02154 and those of HNRNPK in primary OSCC tumors in The Cancer Genome Atlas dataset. Pearson's correlation coefficients and p values are shown. (B) Quantitative RT‐PCR (qRT‐PCR) analysis of HNRNPK in the indicated OSCC cells transfected with the indicated siRNAs (n = 3). (C) Western blot analysis of HNRNPK in OSCC cells transfected with the indicated siRNAs. (D) Heatmap showing miRNAs upregulated (>1.3‐fold) by LINC02154 knockdown identified by small RNA sequencing in Ca9‐22 cells. (E) Venn diagram of miRNAs upregulated by LINC02154 knockdown and those that putatively target HNRNPK. (F) Putative microRNA (miR)‐17‐5p binding site in the 3′‐UTR of HNRNPK. (G) Western blot analyses of HNRNPK and FOXM1 in OSCC cells transfected with a miR‐17‐5p mimic or a negative control. (H) qRT‐PCR analysis of the indicated genes in OSCC cells transfected with the indicated miRNA mimics (n = 3). Error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001. RPM, reads per million; siCtrl, control siRNA.

FIGURE 6

Regulation of mitochondrial function by LINC02154 and LRPPRC in oral squamous cell carcinoma (OSCC) cells. (A) Results of RIP assays. LRPPRC was immunoprecipitated from Ca9‐22 cells, after which coprecipitated LINC02154 was detected with quantitative RT‐PCR (qRT‐PCR) (n = 3). (B) qRT‐PCR analysis of LRPPRC in KON cells transfected with a control siRNA or siRNAs targeting LRPPRC (n = 3). (C) qRT‐PCR analysis of mitochondrial genes in OSCC cells transfected with the indicated siRNAs (n = 3). (D) Cell viability assays in KON cells transfected with the indicated siRNAs (n = 6). (E) qRT‐PCR analysis of mitochondrial genes in OSCC cells transfected with a control siRNA or siRNAs targeting LINC02154 (n = 3). (F) qRT‐PCR analysis of mitochondrial genes in KON cells with or without ectopic LINC02154 expression (n = 3). (G) Results of mitochondrial respiration analysis in KOC cells transfected with the indicated siRNAs. Oxygen consumption rate (OCR) was measured using a Seahorse XFe96 Bioanalyzer at baseline and with sequential injections of oligomycin (Oligo), carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP), and a mixture of rotenone and antimycin A (R/A). (n = 6). (H) Key parameters of mitochondrial function in KON cells transfected with the indicated siRNAs. Error bars represent SD (A–F) and SEM (G, H). *p < 0.05, **p < 0.01, ***p < 0.001. siCtrl, control siRNA.

FIGURE 7

Oncogenic functions of LINC02154 in oral squamous cell carcinoma (OSCC) cells. LINC02154 regulates OSCC cell proliferation by regulating cell cycle‐related genes through the LINC02154/HNRNPK/FOXM1 axis and the modulation of mitochondrial metabolism by interacting with LINC02154.