LncRNA PVT1 Enhances Proliferation and Cisplatin Resistance via Regulating miR-194-5p/HIF1a Axis in Oral Squamous Cell Carcinoma

Abstract

Background: Oral squamous cell carcinoma (OSCC) is the most frequent oral malignancy. Recent studies have revealed that long non-coding RNA (lncRNA) PVT1 plays important roles in the pathogenesis of various cancers. However, the functional roles of PVT1 in OSCC progression and cisplatin resistance have not been elucidated.

Material and methods: In this study, PVT1 expression level in cisplatin-sensitive and cisplatin-resistant OSCC tissues and cell lines was determined using qRT-PCR. Gain-of-function and loss-of-function assays were performed to explore the biological roles of PVT1 in OSCC cell proliferation and cisplatin resistance. Western blot, luciferase reporter assay and bioinformatics analysis were employed to investigate the underlying mechanism of PVT1 in OSCC progression.

Results: Here, we found that PVT1 was frequently up-regulated in cisplatin-resistant tissues and cell lines and strongly correlated with worse overall survival. Functional studies showed that PVT1 promoted OSCC cell proliferation and cisplatin resistance. Mechanistic investigation revealed that PVT1 could positively regulate HIF1a expression via its competing endogenous RNA (ceRNA) activity on miR-194-5p. In addition, miR-194-5p conversely correlated with PVT1 and HIF1a expression in OSCC samples. More importantly, HIF1a knock-down or miR-194-5p overexpression reversed PVT1-induced promotion of OSCC cell proliferation and cisplatin resistance.

Conclusion: Our results indicated that PVT1 functions as an oncogene involved in OSCC cell proliferation and cisplatin-resistance and may serve as a novel therapeutic target for OSCC treatment.

Keywords: HIF1a; cisplatin resistance; miR-194-5p; oral squamous cell carcinoma.

Figure 1

PVT1 is up-regulated and associated with poor prognosis and cisplatin resistance in OSCC. (A) The level of PVT1 in cisplatin-resistant OSCC tissues and cisplatin-sensitive OSCC tissues. (B) Kaplan-Meier analysis of overall survival in OSCC patients according to PVT1 level. (C) CCK-8 assay analysis of cisplatin-resistance for SCC9-R cells and its parallel SCC9 cells. (D) CCK-8 assay analysis of cisplatin-resistance for CAL27-R cells and its parallel CAL27 cells. (E) The level of PVT1 in SCC9-R and CAL27-R cells and their parallel cells. *P<0.05, **P<0.01.

Figure 2

PVT1 enhances OSCC cell proliferation and cisplatin resistance. (A) Effect of pcDNA-PVT1 on PVT1 level in SCC9-R and CAL27-R cells. (B) Effect of si-PVT1 on PVT1 level in SCC9-R and CAL27-R cells. (C and D) Cell survival rate of SCC9-R (C) and CAL27-R (D) cells transfected with pcDNA-NC, pcDNA-PVT1, si-NC or si-PVT1 simultaneously under cisplatin pressure examined with CCK-8 assay. (E and F) Colony formation ability of SCC9-R (E) and CAL27-R (F) cells transfected with pcDNA-NC, pcDNA-PVT1, si-NC or si-PVT1 simultaneously under cisplatin pressure examined with colony formation assay. (G and H) Cell proliferation ability of SCC9-R (G) and CAL27-R (H) cells transfected with pcDNA-NC, pcDNA-PVT1, si-NC or si-PVT1 simultaneously under cisplatin pressure examined with CCK-8 assay. *P<0.05, **P<0.01.

Figure 3

PVT1 positively correlates HIF1a. (A and B) The HIF1a mRNA level in SCC9-R (A) and CAL27-R (B) cells transfected with pcDNA-NC, pcDNA-PVT1, si-NC or si-PVT1 detected using qRT-PCR. (C) The HIF1a protein level in SCC9-R and CAL27-R cells transfected with pcDNA-NC, pcDNA-PVT1, si-NC or si-PVT1 detected using Western blot. (D) The HIF1a level in cisplatin-resistant OSCC tissues and cisplatin-sensitive OSCC tissues. (E) Pearson correlation analysis between PVT1 and HIF1a in OSCC tissues. (F) Kaplan-Meier analysis of overall survival in OSCC patients according to HIF1a level. **P<0.01.

Figure 4

PVT1 up-regulated HIF1a via interaction with miR-194-5p. (A) The putative binding sites of miR-194-5p in the PVT1 and HIF1a 3ʹUTR predicted with starBase v3.0. (B and C) Luciferase reporter assay in HEK-293T cells, co-transfected of WT or Mut of PVT1 (B) and HIF1a 3ʹUTR (C), with miR-194-5p mimics or miRNA-NC. (D) The miR-194-5p level in SCC9-R and CAL27-R cells transfected with pcDNA-PVT1 or pcDNA-NC determined using qRT-PCR. (E) The PVT1 level in SCC9-R and CAL27-R cells transfected with miR-194-5p mimics or miRNA-NC determined using qRT-PCR. (F) The HIF1a level in SCC9-R and CAL27-R cells transfected with miR-194-5p mimics or miRNA-NC determined using qRT-PCR. (G) The miR-194-5p level in cisplatin-resistant OSCC tissues and cisplatin-sensitive OSCC tissues. (H) Kaplan-Meier analysis of overall survival in OSCC patients according to miR-194-5p level. **P<0.01.

Figure 5

PVT1 promotes cell proliferation and cisplatin resistance via up-regulation of HIF1a. (A and B) The HIF1a mRNA level in SCC9-R (A) and CAL27-R (B) cells co-transfected pcDNA-NC or pcDNA-PVT1 with si-NC or si-HIF1a detected using qRT-PCR. (C and D) Cell survival rate of SCC9-R (C) and CAL27-R (D) cells co-transfected pcDNA-NC or pcDNA-PVT1 with si-NC or si-HIF1a examined with CCK-8 assay. (E and F) Colony formation ability of SCC9-R (E) and CAL27-R (F) cells co-transfected pcDNA-NC or pcDNA-PVT1 with si-NC or si-HIF1a examined with colony formation assay. (G and H) Cell proliferation ability of SCC9-R (G) and CAL27-R (H) cells co-transfected pcDNA-NC or pcDNA-PVT1 with si-NC or si-HIF1a simultaneously examined with CCK-8 assay. *P<0.05, **P<0.01.

Figure 6

miR-194-5p reverses PVT1-induced OSCC cell proliferation and cisplatin resistance. (A and B) Cell survival rate of SCC9-R (A) and CAL27-R (B) cells co-transfected pcDNA-NC or pcDNA-PVT1 with miRNA-NC or miR-194-5p mimics examined with CCK-8 assay. (C and D) Colony formation ability of SCC9-R (C) and CAL27-R (D) cells co-transfected pcDNA-NC or pcDNA-PVT1 with miRNA-NC or miR-194-5p mimics examined with colony formation assay. (E and F) Cell proliferation ability of SCC9-R (E) and CAL27-R (F) cells co-transfected pcDNA-NC or pcDNA-PVT1 with miRNA-NC or miR-194-5p mimics examined with CCK-8 assay. *P<0.05, **P<0.01.