Linc01234 promotes cell proliferation and metastasis in oral squamous cell carcinoma via miR-433/PAK4 axis

Abstract

Background: Increasing studies have demonstrated that long non-coding RNAs (lncRNAs) play an important role in tumor progression. However, the potential biological functions and clinical importance of Linc01234 in oral squamous cell carcinoma (OSCC) remain unclear.

Methods: We evaluated the expression profile and prognostic value of Linc01234 in OSCC tissues by RT-qPCR. Then, functional in vitro experiments were performed to investigate the effects of Linc01234 on tumor growth, migration and invasion in OSCC. Mechanistically, RT-qPCR, bioinformatic analysis and dual luciferase reporter assays were performed to identify a competitive endogenous RNA (ceRNA) mechanism involving Linc01234, miR-433-3p and PAK4.

Results: We found that Linc01234 was clearly upregulated in OSCC tissues and cell lines, and its level was positively associated with T stage, lymph node metastasis, differentiation and poor prognosis of patients with OSCC. Our results shown that Linc01234 inhibited cell proliferation and metastatic abilities in CAL27 and SCC25 cells following its knockdown. Mechanistic analysis indicated that Linc01234 may act as a ceRNA (competing endogenous RNA) of miR-433-3p to relieve the repressive effect of miR-433-3p on its target PAK4.

Conclusions: Our results indicated that Linc01234 promotes OSCC progression through the Linc01234/miR-433/PAK4 axis and might be a potential therapeutic target for OSCC.

Keywords: Linc01234; OSCC; PAK4; ceRNA; miR-433.

Fig. 1

Linc01234 was upregulated in OSCC tissues and cell lines. a Linc01234 expression was significantly increased in OSCC tissues, compared with in adjacent non-tumor tissues via StarBase analysis. b Linc01234 expression was examined in OSCC and NOK cells via RT-qPCR assays. c Linc01234 expression was significantly increased in OSCC tissues, compared with in adjacent non-tumor tissues analyzed by RT-qPCR. d Kaplan-Meier analysis for the effects of Linc01234 expression on the OS of HNSC patients based on StarBase database. **,P < 0.01;***,P < 0.001 vs control

Fig. 2

Decreased Linc01234 inhibits cell proliferation in vitro. a The relative expression of Linc01234 was detected with RT-qPCR when CAL27 and SCC25 cells were transfected with siNC, Linc01234 si#1 or Linc01234 si#2. b CCK8 assays were performed to detect the cell proliferation ability after CAL27 and SCC25 cells transfected with siNC, Linc01234 si#1 or Linc01234 si#2. c EDU assays were performed to detect the cell proliferation ability in CAL27 and SCC25 cells transfected with siNC, Linc01234 si#1 or Linc01234 si#2. *P < 0.05; **,P < 0.01 vs control

Fig. 3

Decreased Linc01234 inhibits migration and invasion in vitro. a, b The ability of cell migration and invasion in CAL27 and SCC25 cells with Linc01234 knockdown was detected by Transwell assays. c, d The migration ability of CAL27 and SCC25 cells with Linc01234 knockdown was detected by wound healing assays. * P < 0.05; **,P < 0.01 vs control

Fig. 4

Linc01234 could sponge miR-433-3p in OSCC. a The cellular localization of Linc01234 was determined by Subcellular fractionation and RT-qPCR assay. GAPDH: cytoplasmic control, U6: nuclear control. b Schematic illustration of the predicted binding sites between Linc01234 and miR-433-3p and mutation of potential miR-433-3p binding sequence in Linc01234. c miR-433-3p expression was examined in OSCC tissues using RT-qPCR and StarBase analysis. d Luciferase reporter assay indicated the direct bind between Linc01234 and miR-433-3p. e The mRNA level of miR-433-3p after Linc01234 knockdown was observed by RT-qPCR assay. *P < 0.05; **,P < 0.01 vs control

Fig. 5

PAK4 is a target gene of miR-433-3p in OSCC. a and b revealed the correlation between PAK4 expression and miR-433-3p expression or Linc01234 expression in HNSC tissues. c PAK4 expression in HNSC tissues and normal tissues was veritied by StarBase analysis. d The putative binding sites of miR-433-3p and PAK4 was shown. e The expression of PAK4 was detected by Western Blot in OSCC cells with miR-433-3p overexpression. f The direct interaction between miR-433-3p and PAK4 in OSCC cells was examined by luciferase reporter assay. *P < 0.05; **,P < 0.01 vs control