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MiR-125b-1-3p Exerts Antitumor Functions in Lung Carcinoma Cells by Targeting S1PR1

Background: MicroRNAs (miRNAs) have been extensively studied over the decades and have been identified as potential molecular targets for cancer therapy. To date, many miRNAs have been found participating in the tumorigenesis of non-small cell lung cancer (NSCLC). The present study was designed to evaluate the functions of miR-125b-1-3p in NSCLC cells. Methods: MiR-125b-1-3p expression was detected in tissue samples from 21 NSCLC patients and in NSCLC cell lines using the real-time polymerase chain reaction. A549 cell lines were transfected with a miR-125b-1-3p mimic or miR-125b-1-3p antisense. Cell counting kit-8, wound healing, Matrigel invasion assays, and flow cytometry were used to assess the effects of these transfections on cell growth, migration, invasion, and apoptosis, respectively. Western blotting was used to detect apoptosis-related proteins, expression of S1PR1, and the phosphorylation status of STAT3. Significant differences between groups were estimated using Student's t-test or a one-way analysis of variance. Results: MiR-125b-1-3p was downregulated in NSCLC samples and cell lines. Overexpression of miR-125b-1-3p inhibited NSCLC cell proliferation (37.8 ± 9.1%, t = 3.191, P = 0.013), migration (42.3 ± 6.7%, t = 6.321, P = 0.003), and invasion (57.6 ± 11.3%, t = 4.112, P = 0.001) and simultaneously induced more NSCLC cell apoptosis (2.76 ± 0.78 folds, t = 3.772, P = 0.001). MiR-125b-1-3p antisense resulted in completely opposite results. S1PR1 was found as the target gene of miR-125b-1-3p. Overexpression of miR-125b-1-3p inhibited S1PR1 protein expression (27.4 ± 6.1% of control, t = 4.083, P = 0.007). In addition, S1PR1 siRNA decreased STAT3 phosphorylation (16.4 ± 0.14% of control, t = 3.023, P = 0.015), as in cells overexpressing miR-125b-1-3p (16.7 ± 0.17% of control, t = 4.162, P = 0.026). Conclusion: Our results suggest that miR-125b-1-3p exerts antitumor functions in NSCLC cells by targeting S1PR1.


Ethical approval
This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. Informed consents were obtained from all participants.

Patient samples and cell culture
From January 2015 to October 2017, 21 pairs of NSCLC biopsies and matched adjacent nontumor tissues were collected from patients at the First Affiliated Hospital of Wenzhou Medical University. All of the NSCLC patients were pathologically confirmed and tumor node metastasis (TNM) staging classifications were determined by the Union for International Cancer Control/American Joint Committee on Cancer 7 th edition for the lung. None of the patients received antitumor treatment before. Clinical characteristics of the patients are summarized in Table 1. All the samples were divided into a miRNA high expression group (n = 10) and low expression group (n = 11) using the median miR-125b-1-3p as the cutoff point. The NSCLC cell lines such as A549, H450, H1299, and 16-HBE normal lung bronchus epithelial cells were purchased from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Rockford, IL, USA), 100 U/L penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 .

RNA extraction, complementary DNA synthesis, and the quantitative real-time polymerase chain reaction
Total RNA was extracted using TRIZOL reagent (Invitrogen, Waltham, MA, USA) followed by reverse transcription. Complementary DNAs were then used as templates for quantitative real-time polymerase chain reaction with iQ™ SYBR ® Green Supermix (Bio-Rad, Hercules, CA, USA). U6 was used as the control for normalization. Relative mRNA expression levels were calculated using the 2 −ΔΔCT method. All experiments were repeated at least three times.

Clonogenic assay
For the colony formation assay, 500 cells were plated in six-well plates and cultured in medium containing 10% fetal bovine serum. The medium was refreshed every 3 days. After incubation for 14 days, cells were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). Visible colonies were counted manually. Triplicate wells were measured for each group.

Cell migration and invasion assays
For migration assay, cells were seeded in a serum-free medium in the upper Transwell chambers (Corning, New York, USA). Twenty-four hours later, cells were fixed with methanol and stained by 0.1% crystal violet. Migrated cells were counted under microscope and statistically analyzed. For invasion assay, Transwell chambers were coated with Matrigel (BD Bioscience, San Jose, CA, USA) to growth the cells for 24 h. Residual cells on the upper Transwell chamber were counted and statistically analyzed.

Cell apoptosis assays
An Annexin V-FITC/propidium iodide (PI) Double Staining Kit (BD Biosciences, San Jose, CA, USA) was used to detect cellular apoptosis according to the manufacturer's instructions. Cells were collected, washed with cold phosphate-buffered saline, and resuspended at 1 × 10 6 cells/ml in 1 ml binding buffer. Next, 5 µl Annexin V-FITC and 5 µl PI were added and incubated for 15 min at room temperature in the dark. Cells were detected by flow cytometry. All experiments were repeated three times.

Luciferase assays
The sequence in the 3'-untranslated region (UTR) of the human S1PR1 gene targeted by miR-125b-1-3p was predicted using microRNA.org (http://www.microrna.org/). The 3'-UTR of S1PR1 and a sequence with mutations of two nucleotides in the miR-125b-1-3p target site were cloned into a pGL3 promoter vector to generate the recombinant constructs: wild-type and mutant 3'-UTRs, respectively. For the luciferase assay, A549 cells were co-transfected with wild-type and mutant 3'-UTRs of S1PR1 and the miR-125b-1-3p mimic or scrambled controls (NC). Luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System according to the manufacturer's instructions (Promega, Madison, WI, USA) at 48 h posttransfection.

Statistical analyses
All statistical analyses were performed using SPSS 18.0 software (IBM, Chicago, IL, USA). Data from at least three independent experiments, each performed in triplicate, were presented as means ± standard deviation (SD). Significant differences between groups were estimated using a one-way analysis of variance (ANOVA). A P < 0.05 was considered as statistically significant.

dIscussIon
The prevalence of NSCLC is increasing worldwide, and the prognosis of NSCLC patients remains poor. Therefore, improved therapeutic strategies for NSCLC patients are vital for the management of NSCLC. In recent years, miRNAs have emerged as potential antitumor agents due to its ability to affect the development of various types of cancers including NSCLC. [9] Indeed, a better clarification of the functions of miRNAs in the pathogenesis of NSCLC may help in the search for more effective NSCLC therapies.
MiR-125b-1-3p has been found to play an important role in cancer and immunosuppression. [10] MiR-125b-1-3p was reportedly downregulated significantly in Kaposi's sarcoma. [11] In contrast, miR-125b-1-3p was found elevated in DICER1-mutated pleuropulmonary blastoma. [12] This study found that miR-125b-1-3p was downregulated in NSCLC cells. Those discrepancies may be due to different cancer types, since mounting evidences indicate that miRNAs are highly tissue specific and can act as either oncogenes or tumor suppressors. [13,14] For instance, miR-205 can play a dual role, depending on the specific tumor type and target genes. [15] Therefore, further investigations of miR-125b-1-3p in more cancer types are necessary. Noteworthy, we also revealed that miR-125b-1-3p exerts antitumor effects at least partly through induction of apoptosis. Dysregulated apoptosis is a common cause of cancer, and the treatment of NSCLC is usually impeded by the intrinsic or acquired resistance to apoptosis induced by anticancer agents. [16] Many studies revealed that miRNAs could overcome the resistance to chemotherapy in NSCLC. For example, miR-101 could enhance paclitaxel (PTX)-induced apoptosis in NSCLC. [17] MiR-216b was able to sensitize NSCLC cells to cisplatin-induced apoptosis. [18] Moreover, miR-129-5p could also overcome resistance to cisplatin in NSCLC cells. [19] Hence, it would be interesting to investigate whether miR-125-1-3p also possesses the ability to overcome chemoresistance in NSCLC.
To understand the functional mechanism of miRNAs, it is critical to identify targets involved in their regulation. SIPR1 was further identified as a direct functional target of miR-125-1-3p in NSCLC cells. S1PR1 is a G-protein-coupled receptor of the bioactive lipid, sphingosine-1-phosphate (S1P). [20,21] Tumor cells secrete S1P, promoting growth, survival, motility, and metastasis through binding to S1PRs in a autocrine/paracrine manner. [22] A very recent study showed that upregulation of S1PR1 could lead to the proliferation and invasion of NSCLC cells. [23] Considering S1PR1 is the predominant one compared with other S1PRs in NSCLC cells, the ability of miR-125-1-3p to repress S1PR1 might be applied as a promising strategy against NSCLC.
Aberrant STAT3 signaling is often observed in various cancers including the NSCLC. Inhibition of STAT3 signals would reduce cell proliferation and increase apoptosis of cancer cells. Many studies have been conducted to investigate the correlation between miRNAs and STAT3. For instance, miR-206 could inhibit STAT3 signaling and thereby attenuates the growth and angiogenesis of NSCLC cells. [24] MiR-124 inhibits the proliferation of NSCLC cells and functions as a tumor suppressor through targeting STAT3. [25] MiR-9600 regulates tumor progression and promotes PTX sensitivity in NSCLC through altering expression of STAT3. [26] These studies suggest that STAT3 is subjected to the regulation of various miRNAs. In this study, we found that miR-125-1-3p was also involved in the STAT3 signaling pathway. Although the mechanisms of how miR-125-1-3p affects the STAT3 signaling are not fully revealed in this study. Given that S1PR1 is crucial for the activation of STAT3 in cancer cell. [27] MiR-125-1-3p may affect the STAT3 signaling through the regulation of S1PR1, and further investigations are needed to test this hypothesis.
There are limitations of this study. First, the number of samples in this analysis is relatively small. Second, there is a lack of survival analysis due to unable to collect the survival data of patients. Third, the investigation was performed based on cell lines and future in vivo analysis is needed. In summary, we revealed that miR-125b-1-3p was downregulated and function as a tumor suppressor in NSCLC. S1PR1 was identified as a direct target of miR-125b-1-3p in NSCLC cells. Moreover, STAT3 signaling pathway was also affected by the miR-125b-1-3p. Our findings may help the development of novel therapeutic targets for the treatment of NSCLC patients.

Financial support and sponsorship
This work was supported by the grants from the Natural Science Foundation of Zhejiang Province (No. LY14H160042) and Wenzhou Municipal Foundation of Science and Technology (No. Y20160049).