miR-206 inhibits estrogen-induced proliferation and invasion of ER-α36 positive gastric cancer cells

Objective To explore the biological role of miR-206 in inhibiting the proliferation and invasion of estrogen-induced ER-α36 positive gastric cancer cells and its related mechanisms. Methods MTT and tranwell assays were used to detect the proliferation and invasion of BGC-823 cells under different concentrations and time of ER stimulation. RT-PCR was used to detect the effect of ER stimulation on the content of miR-206 in gastric cancer cells. miR-206 mimics were transfected into BGC-823 cells to detect cell proliferation and invasion; luciferase reporter assay was used to determine whether miR-206 targets the 3’-untranslated region of the CDK14-encoding gene (3’ -UTR). Results We confirmed that estrogen promoted the proliferation and invasion of BGC-823 cells; As the concentration of estrogen increases, the level of miR206 in the cell gradually rises.Overexpression of miR-206 significantly decreased the estrogen-induced BGC-823 cells. Proliferative and invasive ability; miR-206 inhibits the expression of CDK14 by directly binding to the 3’-UTR of CDK14 mRNA, thereby inhibiting the proliferation and invasion of gastric cancer cells. Conclusions Our results suggest that miR-206 inhibits estrogen-induced proliferation and invasion of ER-α36-positive gastric cancer cells by inhibiting CDK14 and may be a promising therapeutic option for anticancer therapy.

Furthermore, cyclin-dependent kinases (CDKs) are essentially serine-threonine kinases that play an important role in the cell cycle [6]. Due to their regulatory effects on the cell cycle, CDKs have long been thought to be promising targets of anticancer therapy [7]. According to reports, CDK overexpression is present in human cancers such as GC, ovarian cancer, breast cancer, lung cancer and colorectal cancer and is associated with the prognosis of cancer patients [8]. In breast, oesophageal, and GC cells, CDK14, also known as PFTK1 (PFTAIRE protein kinase 1), is moderately to highly expressed [9][10][11]. Because miRNA-mediated gene regulation is involved in a variety of stem cell mechanisms, miRNA dysregulation is linked to cancer development and metastasis [12,13]. Therefore, it is thought that the presence of regulatory miRNAs is required for the pathogenesis of GC during the differentiation process, where cells change to specialised types. The aforementioned process affects and downregulates the expression of miR-206, which has been shown to have significant effects on cellular differentiation and functions in human rhabdomyosarcoma (RMS) cells. As a result, miR-206 re-expression promotes myogenic differentiation and inhibits tumour growth [14]. Therefore, miR-206 has been identified as a potential target of anticancer therapy [15]. According to breast cancer studies, miR-206 is downregulated in ERα-positive breast cancer cells and directly inhibits ERα expression in MCF-7 cells [16]. Moreover, lower miR-206 expression levels have been linked to late TNM stage, lymph node metastasis, a relatively low total survival rate and increased metastatic potential [17]. However, the underlying molecular mechanisms are not yet fully understood. In this study, it was hypothesised that miR-206 inhibited the proliferation and invasion of GC cells. On this basis, the potential mechanism was discussed, with the goal of providing a reference for clinical treatment of GC.
Materials and Methods

Cell culture and transfection
Cell culture and transfection were carried out using a self-owned ER-α36-positive human BGC-823 cell line. BGC-823 cells were cultured in RPMI-1640 containing 10% foetal bovine serum (FBS) plus 100 μg/mL streptomycin at 37°C in a humid incubator with 5% CO 2 . To counteract the effect of oestrogen contained in the medium, RPMI-1640 was replaced with phenol red-free RPMI 1640 supplemented with 5% dextran/charcoal-stripped FBS 24 hours before transfection.
Transfection of the cells with miR-206 mimic was accomplished by mixing the transfection reagent Lipofectamine 2000 (Invitrogen, USA) with 100 pmol siRNA oligomer in a reduced serum medium.

E2 stimulation
For 3 hours, BGC-823 cells were treated with 0.1, 1 and 10 mM E2. Following that, cell viability, migration and invasion rates were all evaluated.

Cell viability assay
An MTT assay was performed to analyse cell viability in each group. In a 96-well culture plate, a total of 5,000 cells were inoculated. After incubating at 37°C, 5% CO 2 for 12, 24, 48 and 72 hours, 20 µL of the MTT solution was added to each well (5 mg/ml, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Then, the cells were incubated at 37°C for another 4 hours before adding 150 μl dimethyl sulfoxide.
After reaction at room temperature for 10 min, the optical density at 570 nm was measured using the Multiskan FC microplate photometer to assess cell proliferation rate.

Statistical analysis
The data were expressed as mean ± SD of three independent experiments. The statistical software SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) was used for one-way analysis of variance (ANOVA) to analyse the differences between groups. P < 0.05 was considered statistically significant.

Stimulation of proliferation and invasion of ER-α36-positive GC cells by oestrogen-signalling
Firstly, the expression levels of ER-α in ER-α36-positive GC cells were detected by cell immunofluorescence assay, the results of which showed that ER-α was highly expressed in ER-α36-positive GC cells (Fig. 1A). Afterwards, the dose-

Exertion of biological effects of miR-206 through specific inhibition of CDK14
Since the proliferation and invasion of GC cells were inhibited by miR-206, the potential mechanism of this effect was studied herein. For this purpose, the online algorithm TargetScan 6.2 was used for bioinformatics analysis in order to predict the potential miRNA targets of miR-206. Among the miRNA targets found to be involved in cell proliferation and invasion, CDK14 was the most favourable. The results of Western blot showed that, under oestrogen stimulation, the expression of CDK14 strongly increased; and with the increase of oestrogen dose, the expression of CDK14 gradually increased (Fig. 4A). TargetScan predicted that the binding sequence in the 3'-UTR of CDK14 is a significant match for miR-206 (Fig. 4B). Moreover, luciferase assay showed that the overexpression of miR-206 inhibited the luciferase activity in cells transfected with WT 3'-UTR plasmid carrying CDK14 gene. In addition, the luciferase activity in MUT CDK14 3'-UTR vectors was not affected by miR-206 transfection (Figs. 4B and C). Therefore, the above data indicated that CDK14 transcripts were the real targets of miR-206. It has been confirmed that continuous exposure to E2 increases the risk of breast cancer. Moreover, the use of inhibition of E2-mediated signalling for the treatment of oestrogen-dependent GC has been suggested [18,19]. This study revealed that E2 stimulation could significantly increase the viability and invasion of ER-α-positive GC cells. These findings show that few miRs are involved in the development of GC.
Further, few miRs have been shown to act as oncogenes or tumour suppressors in GC [20][21][22][23]. For instance, it was previously found that miR-939 was significantly downregulated in GC, which was relevant to poor disease-free survival and was considered to be an independent prognostic factor for overall survival [24]. Therefore, expanding the understanding of the miR in GC may contribute to the development of effective strategies for GC treatment.
The progression of tumour metastasis is a continuous process that involves the acquisition of several features through which malignant cells can spread from the primary tumour and colonise at secondary sites. CDK14, a serine/threonine-protein kinase associated with cell division cycle 2, interacts with cyclin D3 and acts as an essential regulator of CDK-cyclins (CCNs) and cell cycle progression [8]. miR-206 may target cyclins; this combination has dramatic effects that further affect cell proliferation and invasion. This suggests that miR-206 is a powerful regulator of proliferation and invasion. The results of this study show that the mechanism of the effect of miR-206 on cell proliferation is relevant to CDK14. Although miR-206 has been reported to activate the apoptosis in HeLa cells [25], apoptosis induced by miR-206 in GC cells was not noted in this study. These data indicate the different tumour suppressive effects of miR-206, including induction of G1 cell cycle arrest and inhibition of cell migration. Therefore, this study showed that miR-206 inhibits the proliferation and invasion in GC cells. Additionally, it was proved that CDK14 is subject to the post-transcriptional regulation by miR-206 and that proliferation and invasion are inhibited by this mechanism. The effects of ER-miR-206-CDK14 were introduced in detail in this study, providing the mechanism for the proliferation and invasion of GC cells and may be used in new therapeutic or diagnostic methods.

Compliance with ethics guidelines
The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of Minhang Hospital, Fudan University.

Conflict-of-interest statement
All authors declare no financial or commercial conflicts of interest. Data sharing statement: All data generated or analyzed during this study are included in this published article.

Funding
This work was supported by the Project of Shanghai Minhang District Science and Technology Committee (2020MHZ076).

Authors' contributions
Chunyan Yuan and Yuanyuan Yang contributed equally to this work.
Chunyan Yuan wrote the manuscript.
Yuanyuan Yang performed literature review and followed-up.
Xia Sheng revised the manuscript.
Xuming Wang revised the manuscript.
All the authors read and approved the final manuscript.