Long non-coding RNA LINC01296 promotes progression of oral squamous cell carcinoma through activating the MAPK/ERK signaling pathway via the miR-485-5p/PAK4 axis

Abstract

Introduction: Long intergenic non-protein-coding RNA 1296 (LINC01296), a newly identified lncRNA, can function as an oncogenic driver to promote the development of multiple carcinomas. However, the effect of LINC01296 on oral squamous cell carcinoma (OSCC) is still unclear.

Material and methods: We determined the expression and role of LINC01296 in OSCC tissues and cell lines. The cell viability, migration and invasion were determined by MTT, wound healing assay and transwell assay, respectively. Flow cytometry was used for detecting cell cycle and apoptosis. The interaction and association between LINC01296, microRNA-485-5p (miR-485-5p) and p21 (RAC1) activated kinase 4 (PAK4) were analyzed by RNA immunoprecipitation (RIP) and luciferase reporter assays. The xenograft mouse model was established to detect the effect of LINC01296 on OSCC tumor growth.

Results: Our study showed that LINC01296 was over-expressed in OSCC tissues and cell lines. The level of LINC01296 was positively correlated with the patient's tumor node metastasis (TNM) stage and nodal invasion. Knockdown of LINC01296 effectively inhibits cell viability, migration and invasion but promotes cell apoptosis in vitro. The in vivo experiment showed that LINC01296 knockdown inhibited OSCC tumor growth. The following analysis indicated that LINC01296 acted as a ceRNA for miR-485-5p, and PAK4 was identified as a direct target of miR-485-5p. Furthermore, we found that the effects of LINC01296 on OSCC progression were through regulating the expression of PAK4/p-MEK/p-ERK via sponging miR-485-5p.

Conclusions: LINC01296 promote the cell cycle, proliferation, migration and invasion, and inhibit apoptosis of OSCC cells through activating the MAPK/ERK signaling pathway via sponging miR-485-5p to regulate PAK4 expression. These results suggested that the LINC01296/miR-485-5p/PAK4 axis was closely associated with OSCC progression. Our study provides a new insight into the molecular pathogenesis of OSCC, and may supply novel biomarkers for diagnosis and therapy of OSCC.

Keywords: LINC01296; MAPK/ERK signaling pathway; PAK4; miR-485-5p; oral squamous cell carcinoma.

Figure 1

Expression level of LINC01296 in oral squamous cell carcinoma (OSCC). A – qRT-PCR was performed to determine the level of LINC01296 in OSCC tissues. The level of LINC01296 was positively correlated with the patient’s tumor node metastasis (TNM) stage (B) and nodal invasion (C). D – Expression of LINC01296 in OSCC cell lines was detected by qRT-PCR ***p < 0.001, **p < 0.01, *p < 0.05 versus control groups.

Figure 2

The role of LINC01296 in OSCC cells. After HSC-2 and CAL-27 were transfected with siRNAs of LINC01296 (si-LINC01296-#1 and si-LINC01296-#2), si-control (si-NC) or blank, (A) the expression of LINC01296 was assessed by qRT-PCR; (B) cell viability was examined by MTT assay; (C) migration and (D) invasion of the cell lines were detected by wound healing assay and transwell assay, respectively; (E) cell cycle progression and (F) apoptosis were separately determined by flow cytometry ***p < 0.001, **p < 0.01 versus control group.

Figure 3

The effect of LINC01296 on tumor growth in OSCC mice. A – Relative expression of LINC01296 in HSC-2 and CAL-27 cell lines transfected with shRNA LINC01296 or shRNA control (sh-ctrl) was detected by qRT-PCR. B – Images of tumors obtaining from xenograft mice. C – Changes of tumor volume were recorded during the feeding period. D – Weight of OSCC tumors after treatment with shRNA LINC01296 or sh-ctrl ***p < 0.001, **p < 0.01 versus control group.

Figure 4

LINC01296 acts as a ceRNA for miR-485-5p. A – miRDB website was used to predict possible binding sites of LINC01296. B – Luciferase reporter assay and C – RIP assay were carried out to confirm the interaction between LINC01296 and miR-485-5p. D – Transfection of pcDNA LINC01296 was measured using qRT-PCR. Expression levels of miR-485-5p in (E) HSC-2 and CAL-27 cell lines with pcDNA LINC01296, pcDNA3.1, si-LINC01296 or si-NC transfection and in (F) OSCC tissues were determined by qRT-PCR. G – The correlation between LINC01296 and miR-485-5P was assessed by Pearson correlation analysis ***p < 0.001, **p < 0.01 versus control group.

Figure 5

PAK4 is identified as a target of miR-485-5p. A – Images of the potential binding sites between miR-485-5p and PAK4. B – The interaction between PAK4 and miR-485-5p was firstly determined via luciferase reporter assay. C – Transfection efficiency of miR-485-5p mimics or miR-485-5p inhibitor was evaluated by qRT-PCR. PAK4 expression level was measured by (D) qRT-PCR and (E) western blot, respectively. F – The level of PAK4 in OSCC tissues was assessed by qRT-PCR. Pearson correlation analysis suggested (G) a negative correlation between PAK4 and miR-485-5p and (H) a positive correlation with LINC01296

Figure 6

The oncogenic effect of LINC01296 on OSCC cells was through mediating the miR-485-5p/PAK4 axis. After HSC-2 and CAL-27 cell lines were separately transfected with inhibitor control (NC-inh) + siRNA control (si-NC), NC-inh + si-LINC01296, miR-485-5p inhibitor + si-NC, or si-LINC01296 + miR-485-5p inhibitor, (A) cell viability, (B) migration and (C) invasion were determined by MTT, wound healing assay and transwell assay, respectively. Flow cytometry detected the (D) cell cycle and (E) apoptosis. F – Protein level of PAK4, cyclin D1, CDK2, CDK4, CDK6, cyclin E1, cleaved caspase 3, cleaved caspase 9, E-cadherin, N-cadherin, p-MEK, MEK, p-ERK, ERK and GAPDH ***p < 0.001, **p < 0.01 versus control group.

Figure 6

The oncogenic effect of LINC01296 on OSCC cells was through mediating the miR-485-5p/PAK4 axis. After HSC-2 and CAL-27 cell lines were separately transfected with inhibitor control (NC-inh) + siRNA control (si-NC), NC-inh + si-LINC01296, miR-485-5p inhibitor + si-NC, or si-LINC01296 + miR-485-5p inhibitor, (A) cell viability, (B) migration and (C) invasion were determined by MTT, wound healing assay and transwell assay, respectively. Flow cytometry detected the (D) cell cycle and (E) apoptosis. F – Protein level of PAK4, cyclin D1, CDK2, CDK4, CDK6, cyclin E1, cleaved caspase 3, cleaved caspase 9, E-cadherin, N-cadherin, p-MEK, MEK, p-ERK, ERK and GAPDH ***p < 0.001, **p < 0.01 versus control group.