circHIPK2 promotes malignant progression of laryngeal squamous cell carcinoma through the miR-889-3p/MCTS1/IL-6 axis

Abstract

Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor of the head and neck with a poor prognosis. The role of circRNAs in LSCC remains largely unknown. In this study, quantitative real-time PCR (qRT-PCR), Sanger sequencing and fluorescence in situ hybridization were undertaken to detect the expression, localization, and clinical significance of circHIPK2 in LSCC tissues and TU686 and TU212 cells. The functions of circHIPK2 in LSCC were explored through proliferation analysis, EdU staining, colony formation assay, wound healing assay, and Transwell assay. The regulatory mechanisms underpinning circHIPK2, miR-889-3p, and MCTS1 were investigated using luciferase assay, Western blotting, and qRT-PCR. We found that LSCC tissues and cells demonstrated high expression of circHIPK2 that was closely associated with the malignant progression and poor prognosis of LSCC. Knockdown of circHIPK2 inhibited the proliferation and migration of LSCC cells in vitro. Mechanistic studies showed that circHIPK2 competitively bound to miR-889-3p, elevated MCTS1 level, promoted IL-6 secretion, and ultimately accelerated the malignant progression of LSCC. In conclusion, an axis involving circHIPK2, miR-889-3p, MCTS1 and IL-6 regulates the malignant progression of LSCC. circHIPK2 expression may serve as a novel diagnostic and prognostic biomarker for LSCC.

Keywords: IL-6; Laryngeal squamous cell carcinoma; MCTS1; Non-coding RNA; Tumor microenvironment; circHIPK2; miR-889–3p.

Fig. 1

Expression profiles of circHIPK2 in LSCC tissues and cells, knockdown circHIPK2 inhibits the proliferation and migration of LSCC cells. a, b: circHIPK2 expression levels in 24 pairs of LSCC and ANM samples were detected by qRT-PCR. c-e: Correlation analysis between circHIPK2 expression level and LSCC clinical parameters. High circHIPK2 expression was significantly correlated with N stage (c), T stage (d), and pathological differentiation (e). f: Expression levels of circHIPK2 in normal control HNEpC cells and LSCC cells TU686 and TU212 were detected by qRT-PCR. g: The splicing site of circHIPK2 was confirmed by Sanger sequencing. h: The localization of circHIPK2 in TU686 and TU212 cells was determined by FISH method with the scale = 50 μm. i: circHIPK2 siRNA was transfected into TU212 and TU686 cells, and the expression level of circHIPK2 was detected by qRT-PCR. j, k: circHIPK2 knockdown inhibited colony-forming abilities of TU212 and TU686 cells. l, m: sicircHIPK2 was transfected into TU212 and TU686 cells, and the cell proliferation capacity was detected by CCK8 at a specified time point. n, o: Cell proliferation after circHIPK2 knockdown was detected by EdU staining. p, q: Knocking down circHIPK2 inhibited TU212 and TU686 migration. r, s: Wound healing time of TU212 and TU686 cells after circHIPK2 knockdown was detected. t: After knocking down circHIPK2, the expression levels of migration marker proteins were detected by Western blotting. *P < 0.05, ^⁎⁎P < 0.01,^⁎⁎⁎P < 0.001.

Fig. 2

Overexpression of circHIPK2 promotes the proliferation and migration of LSCC cells. a: circHIPK2 overexpression plasmids were transfected into TU212 and TU686 cells, and the expression level of circHIPK2 was detected by qRT-PCR. b, c: Overexpression of circHIPK2 promoted colony-forming abilities of TU212 and TU686 cells. d, e: circHIPK2 was overexpressed, and cell proliferation was detected by CCK8 at a specified time point. f, g: Cell proliferation after circHIPK2 overexpression was detected by EdU staining. h, i: Overexpression of circHIPK2 improved the migration ability of TU212 and TU686 cells. j, k: Wound healing time of TU212 and TU686 cells after circHIPK2 overexpression was detected. l: In vivo experiment, a subcutaneous tumor formation model of nude mice was constructed with circHIPK2 overexpression and control cells. m, n: Comparison of tumor weight and volume between circHIPK2 overexpression and control cells. o: After circHIPK2 overexpression, the expression levels of migration marker proteins were detected by Western blotting. *P < 0.05, ^⁎⁎P < 0.01,^⁎⁎⁎P < 0.001.

Fig. 3

circHIPK2 acts as a miRNA sponge for miR-889–3p in LSCC cells and miR-889–3p inhibits the proliferation and migration of LSCC cells. a: Bioinformatic analysis was used to predict the potential miRNAs targeted by circHIPK2. b: circHIPK2 was knocked down and the enrichment of miRNAs was detected by qRT-PCR. c: circHIPK2 was overexpressed, and miRNAs enrichment was detected by qRT-PCR. d: The localization of circHIPK2 and miR-889–3p in TU212 and TU686 cells was detected by FISH assay with the scale = 50 μm. e, f: HEK293T cells were co-transfected with miR-889–3p mimics and wild-type or mutant circHIPK2 luciferase reporter vectors to detect luciferase reporter activity. g: The expression levels of miR-889–3p in HNEpC cells and LSCC TU212 and TU686 cells were detected by qRT-PCR. h, i: The expression level of miR-889–3p in 24 pairs of LSCC and ANM samples was detected by qRT-PCR. j: The expression level of circHIPK2 in 14-pair LSCC tissues was negatively correlated with that of miR-889–3p. k: TU212 and TU686 cells were transfected with miR-889–3p inhibitor, and the expression level of miR-889–3p was detected by qRT-PCR. l, m: The expression of miR-889–3p was inhibited, and cell viability was detected by CCK8 at a specified time point. n, o: Cell proliferation after inhibition of miR-889–3p were detected by EdU staining. p, q: Wound healing time of TU212 and TU686 cells after miR-889–3p inhibition was detected. r, s: After inhibiting miR-889–3p, the migrative ability of TU212 and TU686 cells was improved. t: After miR-889–3p was inhibited, the expression levels of migration marker proteins were detected by Western blotting. *P < 0.05, ^⁎⁎P < 0.01,^⁎⁎⁎P < 0.001.

Fig. 4

miR-889–3p reverses the tumor-promoting effects of circHIPK2 on LSCC cells. a: TU686 cells were transfected with sicircHIPK2 or co-transfected with sicircHIPK2 and miR-889–3p inhibitors. circHIPK2 and miR-889–3p expression was detected by qRT-PCR. b: TU686 cell was transfected with sicircHIPK2 or co-transfected with sicircHIPK2 and miR-889–3p inhibitor. Cell proliferation was determined by CCK8 assay. c, d: Colony formation assay was performed to evaluate the proliferative ability of TU686 cell transfected with sicircHIPK2 or co-transfected with sicircHIPK2 and miR-889–3p inhibitor. e, f: Effects of sicircHIPK2 and miR-889–3p inhibitor on the migration of TU686 cells was evaluated by Transwell migration assays. g, h: TU686 cells were transfected with sicircHIPK2 or co-transfected with sicircHIPK2 and miR-889–3p inhibitor. Cell wound healing time was determined. *P < 0.05, ^⁎⁎P < 0.01,^⁎⁎⁎P < 0.001.

Fig. 5

MCTS1 is a direct target of miR-889–3p and highly expressed in LSCC. a: Bioinformatic analysis to predict the target genes of miR-889–3p based on miRDB and TargetScan, then mapped with HNSCC survival in the GEPIA database. b, c: The relationship between MCTS1 expression and prognosis of HNSCC was analyzed by GEPIA. d: The expression level of MCTS1 in pancarcinoma in TCGA database was analyzed by UNLCAN. e: The expression level of MCTS1 in HNSCC in TCGA database was analyzed by UNLCAN. f, g: The expression of MCTS1 in LSCC and ANM was detected by qRT-PCR. h: Tissue immunofluorescence assay was used to detect the expression of MCTS1 in LSCC and negative control. i, j: After transfecting miR-889–3p inhibitor with TU212 and TU686 cells, the mRNA and protein levels of MCTS1 were detected by qRT-PCR and Western blotting. k, l: miR-889–3p mimics and MCTS1 3′UTR WT or Mut luciferase reporter plasmids were co-transfected into HEK293T cells to detect luciferase activity. *P < 0.05, ^⁎⁎P < 0.01,^⁎⁎⁎P < 0.001.

Fig. 6

circHIPK2 promotes malignant progression of LSCC by targeting MCTS1/IL-6. a, b: si-MCTS1 was transfected into TU686 and TU212 cells, and the levels of MCTS1 mRNA and protein were detected by qRT-PCR and Western blotting. c, d: TU686 and TU212 cells were transfected with si-MCTS1, and CCK8 assay was used to detect cell proliferation at specified time points. e, f: Wound healing time after transfection of TU686 and TU212 cells with si-MCTS1 was detected. g, h: si-MCTS1 was transfected into TU686 and TU212 cells, and the migrative ability of the cells was detected by Transwell migration assay. i, j: circHIPK2 overexpression plasmids were transfected into TU686 and TU212 cells alone or together with si-MCTS1, and MCTS1 mRNA and protein levels were detected by qRT-PCR and Western blotting. k, l: si-MCTS1 were transfected into TU686 and TU212 cells, and the expression and concentration of IL-6 were detected by qRT-PCR and ELISA. m, n: circHIPK2 overexpression plasmids were transfected into TU686 and TU212 cells alone or together with si-MCTS1, and the expression and concentration of IL-6 were detected by qRT-PCR and ELISA. *P < 0.05, ^⁎⁎P < 0.01,^⁎⁎⁎P < 0.001.

Fig. 7

Schematic illustration of the regulatory mechanism of the circHIPK2/miR-889–3p/MCTS1 axis in LSCC malignant progression. circHIPK2 promoted the proliferation and migration of LSCC cells, and bound to miR-889–3p to attenuate its inhibitory effect on target gene MCTS1, eliciting the overexpression of MCTS1 and promoting the proliferation and migration of LSCC cells.