miR-379-5p regulates the proliferation, cell cycle, and cisplatin resistance of oral squamous cell carcinoma cells by targeting ROR1

Abstract

Objective: To analyze the regulatory mechanism microRNA miR-379-5p in oral squamous cell carcinoma (OSCC).

Methods: We collected serum samples from patients with OSCC and examined the expression of miR-379-5p and receptor tyrosine kinase-like orphan receptor 1 (ROR1) by real-time polymerase chain reaction and western blot. OSCC cells were purchased for molecular research, cell multiplication was tested using the BrdU assay, cell cycle was tested using flow cytometry, and resistance to cisplatin (DDP) was assessed using the MTT assay.

Results: miR-379-5p expression was downregulated and ROR1 expression was upregulated in the serum of OSCC patients, and the area under the curve for OSCC identified by miR-379-5p and ROR1 was not less than 0.800. In the cell function test, overexpression of miR-379-5p could suppress the proliferation, cell cycle, and DDP resistance of OSCC cells. miR-379-5p could negatively regulate ROR1. Inhibition of ROR1 expression had a similar effect after the re-expression of miR-379-5p. Co-overexpression of miR-379-5p and ROR1 counteracted the above inhibitory effects on the proliferation, cell cycle, and DDP resistance of OSCC cells.

Conclusion: miR-379-5p in OSCC regulates the proliferation, cell cycle, and DDP resistance of tumor cells by targeting ROR1.

Keywords: DDP resistance; Oral squamous cell carcinoma; ROR1; miR-379-5p.

Figure 1

Expression of miR-379-5p and ROR1 in OSCC. A, B. Expressions of miR-379-5p and ROR1 in the serum of the OSCC group (n = 58) and control group (n = 50) were analyzed using RT-PCR. C. Pearson’s correlation coefficient was used to analyze the correlation between miR-379-5p and ROR1 expression. D. ROC curve was used to analyze the ability of miR-379-5p and ROR1 to identify OSCC. Note: ***P < 0.001. OSSC, Oral Squamous Cell Carcinoma; RT-PCR, Real-Time Polymerase Chain Reaction; ROC, Receiver Operating Characteristic.

Figure 2

Effect of miR-379-5p expression on OSCC cell proliferation and cell cycle. A. The expression of miR-379-5p in four OSCC cell lines (HSC-3, HSC-4, CAL-27, and UPCI-SCC-040) was analyzed using RT-PCR. B. RT-PCR was performed to evaluate the transfection efficiency of miR-379-5p in HSC-3 and CAL-27 cells. C, D. Effect of upregulation of miR-379-5p expression on the proliferation of HSC-3 and CAL-27 cells was analyzed using the CCK-8 assay. E, F. The effect of upregulation of miR-379-5p expression on the cell cycles of HSC-3 and CAL-27 cells was assessed using flow cytometry. Note: Compared to miR-NC, *P < 0.05, **P < 0.01. OSSC, Oral Squamous Cell Carcinoma; RT-PCR, Real-Time Polymerase Chain Reaction.

Figure 3

Targeted relationship between miR-379-5p and ROR1. A. Biological analysis was used to analyze the targeted binding sites of miR-379-5P with ROR1. B, C. The effects of miR-379-5p on the luciferase activities of ROR1-WT and ROR1-Mut were analyzed using the dual-luciferase gene reporter assay. D. Western blot was performed to analyze the effect of miR-379-5p on ROR1 protein level. E. Western blot images of ROR1 protein level. Note: Compared to miR-NC, **P < 0.01.

Figure 4

Effect of the inhibition of ROR1 expression on OSCC cell proliferation and cycle. A. The expression of ROR1 in four OSCC cell lines (HSC-3, HSC-4, CAL-27 and UPCI-SCC-040) was analyzed using western blot. B. The transfection efficiency of miR-379-5p in HSC-3 and CAL-27 cells was analyzed using western blot. C, D. The CCK-8 assay was performed to test the effect of downregulation of ROR1 on the proliferation of HSC-3 and CAL-27 cells. E, F. Flow cytometry was performed to test the effect of downregulation of ROR1 on the cell cycle of HSC-3 and CAL-27 cells. Note: Compared to si-NC, *P < 0.05, **P < 0.01. OSSC, Oral Squamous Cell Carcinoma.

Figure 5

Effect of miR-379-5p targeting ROR1 on the DDP resistance of OSCC cells. A. RT-PCR was performed to determine the expression of miR-379-5p in HSC-3/DDP and HSC-3 cells. B. The CCK-8 assay was performed to determine the effect of DDP stimulation on the proliferative ability of HSC-3/DDP and HSC-3 cells. C. Flow cytometry was performed to determine the effect of DDP stimulation on the apoptosis of HSC-3 cells. D, E. The effects of miR-379-5p targeting ROR1 on the DDP resistance of HSC-3 cells was analyzed using the CCK-8 assay. Note: Compared to HSC-3/DDP, *P < 0.05, **P < 0.01. Compared to DDP+miR-379-5p, ^aP < 0.01. OSSC, Oral Squamous Cell Carcinoma; RT-PCR, Real-Time Polymerase Chain Reaction.

Figure 6

Effects of miR-379-5p and ROR1 co-overexpression on OSCC cell proliferation and cell cycle. A, B. The effect of miR-379-5p and ROR1 co-overexpression on the proliferation of HSC-3 and CAL-27 cells was determined using the CCK-8 assay. C, D. The effect of miR-379-5p and ROR1 co-overexpression on the cell cycle of HSC-3 and CAL-27 cells was determined using flow cytometry. Note: Compared to miR-NC, *P < 0.05, **P < 0.01. Compared to miR-379-5p, ^aP < 0.01. OSSC, Oral Squamous Cell Carcinoma.

Figure 7

Schematic diagram of the regulatory mechanism of miR-379-5p in OSCC. OSSC, Oral Squamous Cell Carcinoma.