Effect of circular RNA hsa_circ_0008898 on oral squamous cell carcinoma and its mechanism

Abstract

Objective: To investigate the effect and molecular mechanism of hsa_circ_0008898 on the cell proliferation, migration, invasion and tumor formation of oral squamous cell carcinoma (OSCC). Methods: Quantitative real-time PCR (qPCR) and Western blotting were used to detect the expression of hsa_circ_0008898, miR-197-5p and ras homolog gene family member A (RHOA) in OSCC tissues, adjacent tissues, OSCC cells and human normal oral keratinocytes (NOK). CAL27 and SCC-25 cells were transfected with si-hsa_circ_0008898#1 (knockdown group 1), si-hsa_circ_0008898#2 (knockdown group 2), hsa_circ_0008898 (circ overexpression group) and blank plasmid (circ blank group), respectively. Then miR-197-5p inhibitor (inhibition group) and blank plasmid (inhibition control group) were transfected into hsa_circ_0008898 knockdown cells (knockdown group 1). CAL27 and SCC-25 cells were transfected with miR-197-5p mimics (miR overexpression group) and blank plasmid (miR blank group), and then transfected hsa_circ_0008898 vector (co-transfection group 1), RHOA vector (co-transfection group 2) and blank plasmid (co-transfection control group) in cells overexpressing miR-197-5p. Cell counting kit 8 (CCK-8), colony formation, Transwell and scratch test were used to detect cell proliferation, cloning ability, cell cycle distribution, cell invasion and migration ability. Ten nude mice were equally divided into two groups, with 5 mice in each group. SCC-25 cells transfected with blank plasmid (control group) and SCC-25 cells transfected with sh-hsa_circ_0008898 (knockout group) were subcutaneously injected into the armpit. The volume and mass of the tumor were measured. Results: The expressions of hsa_circ_0008898 (2.89±0.72) and RHOA (2.62±0.21) in OSCC tissues were significantly higher than those in para-carcinoma tissues (1.00±0.48, 1.00±0.11, respectively), while the expression of miR-197-5p in OSCC tissues （0.46±0.24） was significantly lower than that in para-carcinoma tissues (1.00±0.42) (P<0.05). Compared with NOK, the expression of hsa_circ_0008898 and RHOA in CAL27 and SCC-25 cells increased significantly, while the expression of miR-197-5p decreased (P<0.05). Compared with circ blank group, the cell viability, colony formation, scratch healing rate and invasive cell number of CAL27 and SCC-25 cells in knockdown group 1 and group 2 were significantly decreased, while the proportion of cells in G1 phase was significantly increased (P<0.05). Compared with inhibition control group, the cell viability, colony formation, scratch healing rate and invasive cell number of CAL27 and SCC-25 in inhibition group were significantly increased, while the proportion of cells in G1 phase was significantly decreased in inhibition group (P<0.05). Compared with miR blank group, the cell viability, colony formation, scratch healing rate and invasive cell number of CAL27 and SCC-25 in miR overexpression group were significantly decreased, while the proportion of cells in G1 phase was significantly increased in miR overexpression group (P<0.05). Compared with co-transfection control group, the cell viability, colony formation, migration area and invasive cell number of CAL27 and SCC-25 in co-transfection group2 were significantly increased, while the proportion of cells in G1 phase was significantly decreased in co-transfection group 2 (P<0.05). The volume and mass of transplanted tumor in knockout group ［(660.4±67.8) mm(3 )and (0.60±0.06) g, respectively］ were significantly lower than those in control group ［(1 210.4±198.9) mm(3) and (1.00±0.12) g, respectively］. Conclusions: Knockdown of hsa_circ_0008898 inhibited OSCC cells proliferation, cloning, migration and invasion and induced cell cycle arrest in vitro by regulating the miR-197-5p/RHOA. Additionally, Knockdown of hsa_circ_0008898 also inhibited tumor formation of OSCC cells in vivo.