CircRNA_036186 mediates HNSCC progression by regulating 14-3-3ζ

Abstract

Introduction: Head and neck squamous cell carcinoma (HNSCC) is a prevalent and lethal malignancy, accounting for 95% of head and neck cancers. Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein ZETA (14-3-3ζ) is central to various signalling pathways and is pivotal in tumour progression.

Methods: Cancerous and corresponding non-cancerous tissue samples were collected from five patients diagnosed with HNSCC. circRNA and mRNA expression profiles were analyzed using high-throughput sequencing techniques. Potential circRNA-microRNA (miRNA)-mRNA interactions were predicted using bioinformatics tools.

Results: The study found that CircRNA_036186 regulates the expression of 14-3-3ζ in HNSCC through miR-193b-5p.

Discussion: These findings suggest that CircRNA_036186 has the potential to be a biomarker and therapeutic target for HNSCC and provide some theoretical basis for further research on the role of circRNA in HNSCC.

Keywords: 14-3-3ζ; CircRNA; MiRNA; bioinformatics analysis; head and neck squamous cell carcinoma; high throughput sequencing technology.

Figure 1

14-3-3ζ is highly expressed in HNSCC tissues and cell lines. (A) The heatmap illustrates the expression of 1,053 differentially expressed mRNAs, of which 377 are upregulated and 676 are downregulated. The colouration gradually transitions from blue to red, indicating increased expression. (B) Volcano plot for the visualisation of the screened differentially expressed mRNAs. (C) RT-PCR was conducted to ascertain the expression of 14-3-3ζ in HNSCC cell lines. (D) Immunohistochemistry was employed to detect the expression of 14-3-3ζ in HNSCC tissues. Red arrows, 14-3-3ζ positive, Blue arrows: 14-3-3ζ negative. (E) RT-PCR was employed to detect the expression of 14-3-3ζ in HNSCC tissues. Ca, cancerous tissue; P, paracancerous tissue. (F, G) Western blotting was employed to detect the expression of 14-3-3ζ in HNSCC tissues. Ca, cancerous tissue; P, paracancerous tissue. Ca, cancerous tissue; P, paracancerous tissue. (n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 2

14-3-3ζ is significantly associated with the development and prognosis of HNSCC. (A) A pan-cancer analysis revealed that 14-3-3ζ expression was markedly elevated in 11 malignant tumours relative to normal tissues. (B) Data from the TCGA database indicated that 14-3-3ζ expression was relatively elevated in HNSCC. (C, D) The selected differential mRNAs were subjected to GO analysis and KEGG enrichment. (E) The infiltration of immune cells in HNSCC in the context of 14-3-3ζ. The relationship coefficient values are represented by dots, with the smallest values labelled from 0 to 0.1 and the most significant values labelled from 0.1 to 0.3. The p-value values are represented by a colour gradient, with the lowest p-values in blue and the highest p-values displayed in red. (F) Effect of 14-3-3ζ on the prognosis of HNSCC. Red represents a high expression of 14-3-3ζ, while green represents a low expression of 14-3-3ζ. (No significance(ns)>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 3

Identification of circRNA_036186-miR-193b-3p-14-3-3ζ-regulatory axis. (A) The heatmap illustrates the expression profiles of 287 circRNAs, of which 146 exhibited increased expression and 141 exhibited decreased expression. The colouration gradually transitions from blue to red, indicating increased expression. (B) The five most highly ranked circRNAs and their target miRNAs, each corresponding to a node, are shown with two interacting genes based on base sequence pairing connected by a solid line. (C) The intersection results of the HNSCC occurrence and development group, the prognosis group, and the competing endogenous RNA networks in OncomiR. (D) The predicted target mRNAs from three databases—Wayne’s map, miR-193b-3pmicroT-CDS, Tarbase, and TargetScan—were taken as intersections. (E) A search of the Tarbase database revealed that miR-193b-3p has a base sequence that binds and interacts with the 14-3-3ζ target. (F, G) The results of the RT-PCR experiments indicated that circRNA_036186 and miR-193b-3p exhibited elevated expression in HNSCC tissues relative to paracancerous tissues in five pairs of HNSCC and paracancerous tissue samples. (H, I) The results of RT-PCR experiments indicated that the mRNA expression of circRNA_036186 and miR-193b-3p was markedly elevated in all three HNSCC cell lines relative to HOK. (n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 4

Effect of down-regulation of circRNA_0361863p and miR-193b-3p on 14-3-3ζ in HSC2. (A) The results of the RT-PCR experiment indicated that si-circ_0036186 had been successfully transfected into HSC2. (B) The results of the RT-PCR experiments indicated that miR-193b-3p had been successfully transfected into HSC2. (C) Reverse transcription polymerase chain reaction (RT-PCR) experiments were conducted to ascertain the impact of transfection on 14-3-3ζ mRNA expression. (D, E) Western blotting experiments were conducted to ascertain the impact of transfection on 14-3-3ζ protein expression. (n=3, *p<0.05, ***p<0.001, ****p<0.0001).

Figure 5

Effect of down-regulation of circRNA_0361863p and miR-193b-3p on the proliferation, migration, invasion, and scratch healing rate of HSC2 cells. (A–D) The Transwell assay assessed the impact of circRNA_0361863p and miR-193b-3p down-regulation on cell migration and invasion of HSC2. (E, F) The Scratch test assessed the scratch healing rate of HSC2 after the down-regulation of circRNA_0361863p and miR-193b-3p. (G) The cell activity of HSC2 was evaluated after the down-regulation of circRNA_0361863p and miR-193b-3p through CCK-8 experiments. (n=3, ****p<0.0001).