LOC401317, a p53-regulated long non-coding RNA, inhibits cell proliferation and induces apoptosis in the nasopharyngeal carcinoma cell line HNE2

Abstract

Recent studies have revealed that long non-coding RNAs participate in all steps of cancer initiation and progression by regulating protein-coding genes at the epigenetic, transcriptional, and post-transcriptional levels. Long non-coding RNAs are in turn regulated by other genes, forming a complex regulatory network. The regulation networks between the p53 tumor suppressor and these RNAs in nasopharyngeal carcinoma remains unclear. The aims of this study were to investigate the regulatory roles of the TP53 gene in regulating long non-coding RNA expression profiles and to study the function of a TP53-regulated long non-coding RNA (LOC401317) in the nasopharyngeal carcinoma cell line HNE2. Long non-coding RNA expression profiling indicated that 133 long non-coding RNAs were upregulated in the human NPC cell line HNE2 cells following TP53 overexpression, while 1057 were downregulated. Among these aberrantly expressed long non-coding RNAs, LOC401317 was the most significantly upregulated one. Further studies indicated that LOC401317 is directly regulated by p53 and that ectopic expression of LOC401317 inhibits HNE2 cell proliferation in vitro and in vivo by inducing cell cycle arrest and apoptosis. LOC401317 inhibited cell cycle progression by increasing p21 expression and decreasing cyclin D1 and cyclin E1 expression and promoted apoptosis through the induction of poly(ADP-ribose) polymerase and caspase-3 cleavage. Collectively, these results suggest that LOC401317 is directly regulated by p53 and exerts antitumor effects in HNE2 nasopharyngeal carcinoma cells.

Figure 1. TP53 overexpression in HNE2 cells.

HNE2 cells were transfected with a TP53 expression vector (the pCMV-p53 plasmid). Levels of (A) TP53 mRNA transcripts and (B) p53 protein expression were determined at 0–48 h post-transfection by qRT-PCR and western blotting, respectively. (C) To measure p53 transcriptional activity, the pCMV-p53 and the pp53-TA-luc plasmids were cotransfected into HNE2 cells, and transcriptional activity of p53 from 0–48 h post-transfection was determined by luciferase assays. Similarly, induction of (D) MDM2 mRNA transcripts and (E) MDM2 protein expression in HNE2 were also determined in HNE2 pCMV-p53 transfectants by qRT-PCR and western blotting. GAPDH protein expression was detected as a loading control for p53 and MDM2 western blots. Data shown are representative of 3 independent experiments. Bar graphs show mean ± S.D. **P<0.01, ***P<0.001.

Figure 2. lncRNAs that are dysregulated by TP53 overexpression.

(A) A total of 133 lncRNAs were upregulated in HNE2 cells by more than 2.0-fold in at least one time point (12, 24, or 48 h post-pCMV-p53 transfection), while (B) 1057 lncRNAs were downregulated by more than 2.0-fold in at least 1 time point in HNE2 cells. (C) Detailed expression profiles of the top 30 lncRNAs that were the most significantly upregulated by TP53 transgene expression. (D) Validation of 5 of the top 30 most-upregulated lncRNAs in HNE2 by qRT-PCR. Data shown reflect the means of 3 independent experiments ± S.D. *P<0.05, **P<0.01, ***P<0.001. Expression data are exhibited as normalized log ratios to control time points (0 h) for each lncRNA. (E) Validation of LOC401317 expression in HNE1 and CNE2 cells following TP53 transfection by qRT-PCR. Data shown reflect the mean of 3 independent experiments ± S.D. **P<0.01, ***P<0.001. Expression data were normalized to control time points (0 h).

Figure 3. p53 transcriptionally upregulates LOC401317 through a p53-binding site upstream of the LOC401317 transcription start site.

(A) Schematic representation of the potential promoter and p53-binding site upstream of LOC401317 transcription start site. Wild-type and mutated versions of the putative p53-binding site are indicated. (B) Transactivation of the putative LOC401317 promoter by p53. An 820-bp sequence, −570 bp to +249 bp to the transcript start site of LOC401317, containing the potential promoter and p53-binding site was inserted into the pGL3 luciferase reporter vector (LOC-promoter). The empty pGL3 vector served as a negative control (pGL3-EV). Luciferase vectors were cotransfected with pCMV-p53 or the parental vector (Vector) into HNE2 cells, as indicated. Cells were then harvested for luciferase assays 24 h after transfection. (C) Activation of the potential p53-binding site adjacent to the LOC401317 promoter by p53. Synthetic oligonucleotides containing 3 tandem wild-type p53-binding sites (LOC-WT) or mutant p53-binding sites (LOC-MT, negative control) were inserted into the luciferase reporter vector. The pp53-TA-luc vector, which contains conserved p53-binding sites, was used as a positive control. Luciferase vectors were cotransfected with pCMV-p53 or the parental vector into HNE2 cells, as shown. Cells were harvested for luciferase assays 24 h after transfection. The results showed that p53 significantly induces luciferase activity (>2.76-fold) via the wild-type p53-binding site. Data shown are the means of 3 independent experiments ± S.D. **P<0.01.

Figure 4. LOC401317 inhibits HNE2 cell growth by promoting cell cycle arrest and cellular apoptosis.

(A) Measurement of growth curves of HNE2 cells stably transfected with LOC401317 or an empty vector by MTT assays. LOC401317 overexpression inhibits HNE2 cell growth. Cell cycle distribution (B) and apoptosis (C) were determined by flow cytometry. LOC401317 arrested HNE2 cell cycling at the G0/G1 phase and induced HNE2 cell apoptosis. Data are expressed as means of 3 independent experiments ± S.D. *P<0.05, **P<0.01, ***P<0.001, relative to control cells.

Figure 5. Overexpression of LOC401317 inhibits HNE2 cell growth in xenografted tumors.

Untreated HNE2 cells (mock) as well as HNE2 cells that were stably transfected with empty vector (vector) or an LOC401317 overexpression plasmid (LOC401317) were injected subcutaneously into the right flanks of nude mice. (A) Xenograft tumor sizes were monitored by measuring every 5 days, between days 10–35 following injection (n = 10 mice per group). Error bars represent SEM, **P<0.01. (B) Mice were sacrificed at 35 days post-injection. (C) Xenografted tumors were separated and their sizes were measured. Formalin-fixed, paraffin-embedded tissues were prepared from xenografted tumors for subsequent in situ hybridization (ISH) or immunohistochemistry (IHC) staining.

Figure 6. Downstream effector molecules regulated by LOC401317 overexpression in HNE2 cells.

Overexpression of LOC401317 increased p21, cleaved PARP, and caspase-3 protein expression, while inhibiting cyclin D1 and cyclin E1 expression. The expression of α-tubulin or GAPDH was detected for protein loading controls.

Figure 7. The expression of LOC401317 and downstream effector molecules was validated in xenograft tumor tissues.

Xenograft tumor tissues shown in figure 5C were formalin fixed and paraffin embedded. LOC401317 expression was detected by in situ hybridization (ISH) using LOC401317-specific probes, while the expression of p21, cyclin E1, cyclin D1, and cleaved caspase-3 were detected by immunohistochemical (IHC) staining. LOC401317 upregulated p21, downregulated cyclin E1 and cyclin D1, and induced caspase-3 cleavage. (magnification: ×400, scale bars: 20 µm).