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MiR-875-5p suppresses cervical cancer cell proliferation and metastasis via negative regulation of EGFR

Purpose: To explore miRNA-875-5p and epidermal growth factor receptor (EGFR) activities in tissues or cells from cervical cancer, and their underlying molecular mechanisms. Methods: Tissues were obtained from cervical cancer patients and their miR-875-5p expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Caski or HeLa cells were transfected with miR-875-5p mimics or miR-875-5p inhibitor to assess the effect of miR-8755p expression on cell viability, cell cycle, migration, and invasion using Cell Counting Kit-8 (CCK-8), flow cytometry, wound healing, and Transwell assays. Potential target genes of miR-875-5p were predicted and verified using a dual luciferase reporter assay. In addition, EGFR expression was evaluated by western blot. Results: MicroRNA-875-5p was expressed at low levels in cervical cancer tissues and was related to FIGO stage, lymph node metastasis, pathological grade, vascular involvement, and deep stromal invasion in patients with cervical cancer. MicroRNA-875-5p overexpression inhibited cell viability, migration, and invasion, and caused G0/G1 phase block of Caski and HeLa cells. Moreover, EGFR was the target gene of miR-875-5p and was negatively regulated by miR-875-5p. Reductions in cell viability, migration, invasion, and the number of G0/G1-phase cells were inhibited by EGFR overexpression. Conclusion: MiR-875-5p suppresses cervical cancer cell growth and metastasis by negatively regulating EGFR. Therefore, miR-875-5p can potentially be exploited for the management of cervical cancer.


INTRODUCTION
Cervical cancer is second to breast cancer as the most common gynecologic malignancy [1]. The incidence of cervical cancer has been increasing and tending toward younger ages over the past decade [2]. The course of cervical cancer generally involves cervical hyperplasia, early invasive carcinoma, and cervical cancer [2]. More than 50% of patients have progressed to invasive carcinoma by the time they are diagnosed, and at this stage invasion and metastasis greatly reduce their survival. Moreover, patients with cervical cancer usually have a poor prognosis and lower survival rate, and the underlying pathological mechanisms of cervical cancer are complex [3]. Therefore, it is important to identify biomarkers that influence the development of cervical cancer. MicroRNAs (miRNAs) regulate gene expression through the post-transcriptional regulation of the translation of control mRNAs [4]. In cervical cancer, miRNAs have also been reported to act as biomarkers, oncogenes, and tumor suppressors regulating cervical carcinogenesis, metastasis, or apoptosis [5][6][7]. Weng et al. found that miR-875-5p was highly expressed in renal clear cell carcinoma and at much higher levels than other miRNAs [4]. MicroRNA-875 and miR-3144 suppress the expression of E6 oncogene and HPV16-positive cells, which are common in cervical cancer, suggesting that miR-875 might have a tumorsuppressing effect [8]. However, the mechanism of action of miR-875-5p on cervical cancer cells is still unclear. Epidermal growth factor receptor (EGFR) is a cell membrane protein that is widely distributed in human tissues [9]. Epidermal growth factor receptor is overexpressed, amplified, or mutated in various epithelial tumor cells and is closely associated with tumor aggressiveness and resistance to treatment [10]. Overexpression of EGFR in malignant cells induces autocrine factors in tumor cells [10]. The abnormal expression of EGFR has also been shown to influence the progression of cervical cancer [11]. Nonetheless, the specific regulatory mechanisms involved in the effects of miR-875-5p and EGFR on cervical cancer are not known.
In this study, the biological functions of miR-875-5p and its potential molecular mechanisms in cervical cancer cells were investigated by in vitro cell experiments, with the aim of seeking new therapeutic avenues for cervical cancer.

Tissue collection
Thirty-seven patients admitted to Renmin Hospital of Wuhan University who had a postoperative histopathological diagnosis of cervical cancer were selected as the cancer group. Patients with other types of tumors or with cardiac, hepatic, renal, or other vital organ disease were excluded. The average age of the patients with cervical cancer was 46.29 ± 5.33 years, and other clinical information about these patients is shown in Table 1. In addition, 33 patients with suspected cervical cancer and a histopathological diagnosis of a normal cervix, average age 51.08 ± 6.21 years, were selected as the control group. Cervical tissue specimens were collected from the cancer and control groups for surgical resection or biopsy, and stored at -80 °C for subsequent experiments.
The experiment was approved by the Medical Ethics Committee of Renmin Hospital of Wuhan University (approval no. 2019113), and informed consent was obtained from patients or their families.

Cell viability assay
Caski or HeLa cells were treated with Cell Counting Kit-8 solution (CCK-8, Solarbio, Beijing, China) and co-incubated for 1 h at 37°C. The optical density (OD) was measured at 450 nm using a microplate reader (Shanghai Aucy Scientific Instrument, China).

Cell cycle assay
Transfected cells were digested with 0.25 % trypsin reagent (Sigma-Aldrich, St. Louis, MO, USA) and fixed in anhydrous ethanol at 4 °C for 30 min. Phosphate-buffered saline (PBS, Sigma-Aldrich) was used to wash fixed cells. The treated cells were incubated with 0.25 mg/ml RNAse (Thermo Fisher Scientific) at 37 °C for 30 min. Propidium iodide (PI) staining solution was added to cells for 30 min in the dark. The proportion of cells at different stages of the cell cycle was determined using flow cytometry (Beckman Coulter, USA).

Cell migration assay
After evenly scribing at least 5 lines per well on the back of a 6-well plate, transfected cells were added to plates and incubated overnight. After horizontal lines were scratched in the cell layer with a pipette tip held perpendicular to the plate, the cells were washed with PBS and incubated in serum-free medium for 24 h. The plates were photographed, and the relative wound width was calculated using Image J software (NIH Image, Bethesda, MD, USA).

Cell invasion assay
Matrigel (Sigma-Aldrich) was added to the upper chamber of the Transwell unit, and serumcontaining medium was added to the lower chamber. Transfected cells were incubated in the upper Transwell chamber overnight. The upper chambers were removed, and cells in the lower chamber were washed and stained with 0.1 % crystal violet (Beyotime, Shanghai, China). These invading cells were observed and counted using a microscope (Leica Microsystems, Weitzlar, Germany).

Reverse-transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cancer tissues or transfected cells, and PCR amplification was performed using Quant One Step qRT-PCR Kit (Probe, Bjbalb, Beijing, China). The conditions of PCR amplification were: pre-denaturation at 92 °C for 3 min; denature at 92 °C for 10 s, anneal at 50 °C for 20 s, extend at 68 °C for 20 s, repeated 35 times; extension at 68 °C for 8 min. The sequences of miR-875-5p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers are shown in Table 1.

Western blot
Cellular proteins were extracted from transfected Caski or HeLa cells using lysis buffer (Sigma-Aldrich). The protein concentrations were measured using the Bradford method. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membranes. The proteins were co-incubated with primary antibodies, anti-EGFR (1:2000, ab52894, Abcam, Cambridge, MA, USA) and anti-β-actin (1:1500, ab8227, Abcam), for 16 h at 4°C. After washing with PBS, the proteins were incubated with horseradish peroxidase (HP)labeled secondary antibody (ab191866, Abcam) for 1 h. The protein bands on NC membranes were visualized with color developing agent and recorded using an imaging system. The bands were analyzed using Quantity One software (NIH Image, USA).

Statistical analysis
The experimental data were calculated as mean ± standard deviation (SD) of at least three independent experiments, and correlation analyses were performed. The results were analyzed by analysis of variance (ANOVA) using SPSS 21.0 software (SPSS Inc, USA). Values of p < 0.05 were considered statistically significant.

MicroRNA-875-5p expression was downregulated in cervical cancer
In this study, tissues were obtained from 37 cervical cancer patients. Reverse-transcription polymerase chain reaction results in Figure 1 showed that miR-875-5p expression was reduced in the cancer group, compared with the normal group, in cervical cancer (p < 0.001). As shown in Table 2, the miR-875-5p level correlated with pathological grade, Federation International of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis, vascular involvement, and deep stromal invasion in cervical cancer patients (p < 0.05).

EGFR was a target gene of miR-875-5p
To better investigate the mechanism of the effect of miR-875-5p on cervical cancer cells, this study used the Targetscan website to predict target genes that could bind to miR-875-5p and found target sites between EGFR and miR-875-5p (Figure 4 A). Furthermore, the results of dual luciferase reporter assays (Figure 4 B) showed that luciferase intensity was significantly reduced in cells co-transfected with the miR-875-5p mimic and EGFR-WT (p < 0.01), while the intensity changed very little in cells co-transfected with EGFR-MUT. In addition, luciferase intensity was increased when cells were co-transfected with miR-875-5p inhibitor and EGFR-WT and was almost unchanged in EGFR-MUT co-transfected cells. Moreover, western blot results (Figure 4 C) showed that overexpression of miR-875-5p decreased the level of EGFR expression, whereas knockdown of miR-875-5p increased the level of EGFR expression (p < 0.01). The results of the correlation analysis showed that EGFR was negatively correlated with the expression of miR-875-5p (Figure 4 D).  The target relationship between miR-875-5p and EGFR was predicted using the Targetscan website (http://www.targetscan.org). (B) Relative luciferase was analyzed using dual luciferase reporter assays to verify the target relationship between miR-875-5p and EGFR. (C) The EGFR expression level was examined by western blot in cells transfected with miR-875-5p mimic or miR-875-5p inhibitor. (D) The relationship of miR-875-5p and EGFR was analyzed by correlation analysis. * Compared with miR-NC mimic, ** p < 0.01; # compared with miR-NC inhibitor, ## p < 0.01

MicroRNA-875-5p inhibited cell proliferation and metastasis by targeting EGFR
This study further investigated the co-regulatory effects of miR-875-5p and EGFR on cervical cancer cells. The expression of EGFR ( Figure 5 A) was clearly decreased in cells co-transfected with miR-875-5p mimic + vector, whereas EGFR expression was increased by co-transfection with miR-NC + EGFR (p < 0.01). Furthermore, these effects on EGFR expression level were also additive in cells co-transfected with miR-875-5p mimic + EGFR, meaning both the negative effect of miR-875-5p and the positive effect of EGFR were evident (p < 0.01), suggesting that miR-875-5p mimic and EGFR vector were both successfully transfected into cells. Overexpression of EGFR decreased cell viability (Figure 5 B), while it also inhibited the increase in cell viability produced by overexpression of miR-875-5p (p < 0.001).
Flow cytometry results ( Figure 5 C and D) showed that EGFR overexpression decreased the number of G0/G1-phase cells and increased the number of S-phase cells (p < 0.01). At the same time, the increase in G0/G1-phase cell number caused by miR-875-5p overexpression was mitigated by transfection with EGFR vector (p < 0.01). In the wound healing experiment shown in Figure 5 E, the relative wound width in cells co-transfected with miR-NC + EGFR was significantly reduced compared to miR-NC + vector (p < 0.01). Moreover, the increase in relative wound width caused by miR-875-5p overexpression was decreased by cotransfection of EGFR vector (p < 0.01). In addition, in cell invasion assays (Figure 5 F), EGFR overexpression increased the number of invading cells, while it inhibited the reduction in invasive cell number induced by miR-875-5p overexpression (p < 0.01). Migration of cells co-transfected with miR-875-5p mimic + EGFR was examined using wound healing assays. (F) Cell invasion was measured using a Transwell assay. * denotes the results compared with miR-NC mimic + vector, ** p < 0.01, *** p < 0.001; # denotes the results compared with miR-875-5p mimic + EGFR, ## p < 0.01

DISCUSSION
The etiology of cervical cancer is closely related to chromosomal mutations, human papilloma virus infection, and single nucleotide polymorphisms, especially abnormal expression of miRNA [5][6][7]. MicroRNA-875-5p acts as a tumor suppressor gene to inhibit progression of a variety of tumors, by targeting different target genes. MicroRNA-875-5p is rarely reported in cervical cancer. A study of pancreatic cancer found that miR-875-5p was significantly lower in tissues adjacent to cancer [12]. Like the previous study, the present study found that miR-875-5p was expressed at low levels in cervical cancer tissues, suggesting that miR-875-5p might play an oncogenic role in cervical cancer. In addition, this study also showed that the expression of miR-875-5p in cervical cancer tissues was correlated with several clinicopathological parameters, suggesting that under-expression of miR-875-5p might promote the development of cervical cancer.
Further results from this study showed that miR-875-5p suppressed cell viability and metastasis and promoted cell cycle block, confirming that miR-875-5p is a tumor suppressor gene for cervical cancer. The mechanism by which miR-875-5p inhibits tumors may be that it suppresses the translation or degradation of target genes, and then binds the target gene mRNAs to form an interconnected regulatory network, thereby regulating mRNA expression [13].
Epidermal growth factor receptor is activated by epidermal growth factor, which affects cell growth and differentiation. Studies have shown that EGFR is overexpressed or aberrantly expressed in various tumors, including cervical cancer [11]. Epidermal growth factor receptor and tumor cell proliferation, tumor invasion, and infiltration may have bidirectional regulatory effects [14]. Immunohistochemical detection of EGFR expression in cervical cancer tissues revealed that EGFR protein expression gradually increased with clinical stage and with increasing incidence of positivity [15]. Some miRNAs, such as miR-125a-5p and miR-2861, can also regulate the proliferative activity and apoptosis of cervical cancer cells by regulating the expression of EGFR [16].
In order to better understand the potential mechanism of miR-875-5p, this study used the Targetscan website to predict the direct binding of miR-875-5p to the EGFR 3'-UTR, and validated that EGFR was a direct target of miR-875-5p. In addition, the correlation analysis found that miR-875-5p in cervical cancer was negatively correlated with EGFR expression, indicating that miR-875-5p expression negatively regulates EGFR expression. This finding presumably indicated the potential involvement of EGFR in miR-875-5p-mediated development of cervical cancer. The results of this study showed that the suppression of cell viability, migration, and invasion were alleviated by EGFR overexpression, suggesting that EGFR is indeed involved in the mechanism of cervical cancer mediation by miR-875-5p. To be precise, miR-875-5p affected cervical cancer cell proliferation and metastasis by binding to the 3' UTR of EGFR, reducing the protein expression of EGFR.

CONCLUSION
MicroRNA-875-5p is expressed at low levels and is negatively correlated with EGFR expression. It inhibits cell growth and metastasis in cervical cancer by suppressing EGFR expression. These findings not only expand the molecular mechanisms of cervical cancer development but also provide new targets for cervical cancer treatment. Future studies should examine the expression of signaling molecules downstream of miR-875-5p/EGFR.