Hsa_circ_0060927 Is a Novel Tumor Biomarker by Sponging miR-195-5p in the Malignant Transformation of OLK to OSCC.

Siming Xu,Yanxiong Shao,Haiwen Zhou,Yuhan Song

doi:10.3389/fonc.2021.747086

Abstract

ObjectiveTo investigate the clinical significance of differentially expressed circRNAs and candidate circRNAs in the transformation of oral leukoplakia (OLK) to oral squamous cell carcinoma (OSCC).MethodsWe performed high-throughput circRNA sequencing in six cases of normal oral mucosal (NOM) tissues, six cases of OLK tissues, and six cases of OSCC tissues. Ten circRNAs with significant differential expression were verified by qRT-PCR. Enzyme tolerance assay and Sanger sequencing were performed on the screened target circRNA hsa_circ_0060927, and a qRT-PCR assay of hsa_circ_0060927 was performed in three tissues (24 cases in each group); this was followed by an ROC analysis. The ceRNA network was predicted using TargetScan and miRanda. MiR-195-5p and TRIM14 were selected as the downstream research objects of hsa_circ_0060927. The sponge mechanism of hsa_circ_0060927 was detected by AGO2 RIP. The interaction between hsa_circ_0060927 and miR-195-5p was verified by RNA pull-down assay and dual luciferase reporter gene assay. The expressions of hsa_circ_0060927, miR-195-5p, and TRIM14 were verified by normal oral epithelial primary cells and cell lines of LEUK1, SCC9, and SCC25. The hsa_circ_0060927 overexpressed plasmid and miR-195-5p mimics were constructed to transfection LEUK1 to detect the changes in cell proliferation, apoptosis, and migration.ResultsThe results of qRT-PCR validation were consistent with the sequencing results. Hsa_circ_0060927 is a true circRNA with trans-splicing sites. The expression of hsa_circ_0060927 increased in NOM, OLK, and OSCC. Overexpression of hsa_circ_0060927 enhanced the ability of cell proliferation and migration, and decreased cell apoptosis capacity. The prediction of ceRNA network suggested that hsa_circ_0060927 could regulate the target gene TRIM14 through sponging miR-195-5p. AGO2 RIP indicated that hsa_circ_0060927 had a sponge mechanism. RNA pull-down and dual luciferase reporter gene assay suggested that hsa_circ_0060927 interacted with miR-195-5p. Hsa_circ_0060927 was positively correlated with the expression of TRIM14, and could relieve the inhibition of miR-195-5p on TRIM14 to regulate cell proliferation, apoptosis, and migration of LEUK1 cells.ConclusionHsa_circ_0060927 acted as a potential key ceRNA to sponge downstream miR-195-5p and promote OLK carcinogenesis by upregulating TRIM14. Hsa_circ_0060927 was expected to be a molecular marker for the prevention and treatment of OLK carcinogenesis through the hsa_circ_0060927/miR-195-5p/TRIM14 axis.

Highlights

Oral squamous cell carcinoma (OSCC) is the most common cancer in the oral maxillofacial region, and the incidence rate of OSCC has remained on an upward trend [1]
The tissues used for qRT-PCR verification were obtained from 72 patients consisting of OSCC, Oral leukoplakia (OLK), and normal oral mucosa (NOM) groups
Since the carcinogenesis of OLK is mainly discussed in this manuscript, we compared the differentially expressed circRNAs between OLK and OSCC, and a volcanic plot was painted with statistical criteria determined by fold change (FC) and p-value (Figure 1B)

Summary

Introduction

Oral squamous cell carcinoma (OSCC) is the most common cancer in the oral maxillofacial region, and the incidence rate of OSCC has remained on an upward trend [1]. It is essential to find out the reliable OSCC biomarkers for early diagnosis. Oral leukoplakia (OLK) cannot be pathologically or clinically defined as any other diseases with white plaques or patches that cannot be rubbed off [5]. It is well known that OLK is the most common oral potentially malignant disease clinically and may transform into OSCC [6]. It has been reported that the rate of malignant transformation of OLK is between 0.13% and 34.0% [7, 8].

Methods

Results

Conclusion