[Effect and mechanism of circular RNA BICD2 on the biological behavior of oral squamous cell carcinoma cells].

Abstract

Objective: To investigate the effects of circular RNA (circRNA) BICD2 (circ-BICD2) on glutamine metabolism, cell proliferation, migration, invasion and apoptosis of oral squamous cell carcinoma (OSCC) cells and to further explore the possible mechanism. Methods: OSCC cells were purchased from Shanghai Institute of Cellular Cells, Chinese Academy of Sciences. Samples of OSCC tissues and corresponding adjacent tissues were collected from 35 OSCC patients who underwent surgical resection at the First Affiliated Hospital of Zhengzhou University from January 2016 to January 2018. Real-time quantitative PCR (RT-qPCR) and western blot were performed to detect the expression levels of circ-BICD2, miR-296-5p and transgelin2 (TAGLN2) in OSCC tissues and cells. Bioinformatics, dual luciferase report experiment and RNA immunoprecipitation (RIP) experiment were used to determine the targeting relationship between circ-BICD2 and miR-296-5p, miR-296-5p and TAGLN2. According to different transfection oligonucleotides or plasmids, CAL27 and SCC9 cells were divided into si-NC group (transfection si-NC), si-circ-BICD2 group (transfection si-circ-BICD2), si-circ-BICD2+anti-miR-NC group (transfected with si-circ-BICD2 and anti-miR-NC), si-circ-BICD2+anti-miR-296-5p group (transfected with si-circ-BICD2 and anti-miR-296-5p), miR-NC group (transfected with miR-NC), miR-296-5p group (transfected with miR-296-5p mimic), miR-296-5p+pcDNA (transfected with miR-296-5p mimic and pcDNA) and miR-296-5p+TAGLN2 group (transfected with miR-296-5p mimic and pcDNA-TAGLN2). Cell counting kit 8(CCK-8), colony formation experiment, flow cytometry, Scratch healing test and Transwell experiment were applied to detect OSCC cell viability, number of colonies, cycle distribution, apoptosis rate, migration rate and invasive cell numbers. Glutamine (gln) consumption, α-ketoglutaric acid (α-KG) production and adenosine triphosphate (ATP) concentration were also detected by the kit. The expression of cyclinD1 and Glutamine hydrolase (GLS1) proteins of CAL27 and SCC9 cells in each of the groups were detected by western blot. Twelve four-week-old clean BALB/c female nude mice were injected with a single-cell suspension of SCC9 cells into the axillary skin to establish a transplanted tumor model. Twelve transplanted tumor model mice were divided into sh-circ-BICD2 group and sh-NC group. Mice were sacrificed by cervical dislocation at 32 days after injection, the tumors were removed and the tumor weight was tested. Results: The expressions of circ-BICD2 (2.54±0.74) and TAGLN2 (1.86±0.15) were increased (P<0.05), while the expression of MiR-296-5p was decreased in OSCC tissues (P<0.05). Cell viability, clone formation numbers, migration rate, invasive cell numbers, S phase cell ratio, glu consumption, α-KG production, ATP concentration, expression of cyclinD1 and GLS1 proteins of OSCC cells were significantly reduced after interference with circ-BICD2 expression. The apoptosis rate, the proportion of cells in G0-G1 phase and the expression of miR-296-5p were significantly increased (P<0.05). Inhibiting the expression of miR-296-5p coulld reverse the effect of interfering with circ-BICD2 on OSCC cell proliferation, migration, invasion, apoptosis and glutamine metabolism (P<0.05). After overexpression of miR-296-5p, cell viability, clone formation red number, migration rate, number of invasive cells, S phase cell ratio, glu consumption, α-KG production, ATP concentration and expressions of cyclinD1, GLS1 and TAGLN2 proteins in OSCC cells were significant decrease. The rate of apoptosis and the proportion of cells in G0-G1 phase were significantly increased (P<0.05). Compared with overexpression of miR-296-5p, cell viability, clone formation red number, migration rate, number of invasive cells, S phase cell ratio, glu consumption, α-KG production, ATP concentration, cyclinD1, GLS1 and TAGLN2 proteins in OSCC cells after overexpression of miR-296-5p and TAGLN2 were significantly increased, and the apoptosis rate and the proportion of cells in the G0-G1 phase were significantly decreased (P<0.05). Compared with the sh-NC group, the tumor weights of mice in the sh-circ-BICD2 group were significantly reduced (P<0.05). Conclusions: Circ-BICD2 was highly expressed in OSCC cells. Interfering with circ-BICD2 could inhibit the proliferation, migration and invasion of OSCC cells, glutamine metabolism and tumor growth and promote cell apoptosis, which might be relate to the regulation of miR-296-5p/TAGLN2 molecular axis.