MiR‐372‐3p promotes cell growth and metastasis by targeting FGF9 in lung squamous cell carcinoma

Abstract The aim of this study was to study the role of miR‐372‐3p in lung squamous cell carcinoma (LSCC) cell proliferation and invasion by suppressing FGF9. RT‐PCR was used to determine miR‐372‐3p and FGF9 mRNA expression in tissues and cells. Western blot was used to determine FGF9 expression in tissues and NCI‐H520 cell line. Dual luciferase reporter gene assay was conducted to confirm that FGF9 can be directly targeted by miR‐372‐3p. MTT, colony formation assays were conducted to investigate the effects of ectopic miR‐372‐3p and FGF9 expression on NCI‐H520 cell growth. Flow cytometry was used to analyze the influence of miR‐372‐3p and FGF9 expression on cell cycle distribution and apoptosis. Transwell assay was also conducted to see the effects of miR‐372‐3p and FGF9 expression on NCI‐H520 cell invasiveness. MiR‐372‐3p was found significantly overexpressed in both LSCC tissues and cell lines, whereas FGF9 mRNA was found underexpressed in LSCC tissues. MiR‐372‐3p directly bound to wild‐type FGF9 mRNA 3′UTR, therefore led to the reduction in FGF9 expression. The upregulation of FGF9 or the downregulation of miR‐372‐3p substantially retarded LSCC cell growth, mitosis, and invasion. MiR‐372‐3p enhanced LSCC cell proliferation and invasion through inhibiting FGF9.


Introduction
Lung cancer is the principal cause of deaths related to cancer worldwide [1]. Non-small-cell lung cancers (NSCLC) account for 80% of lung cancer, and are classified into four subtypes according to their histological and pathological features: lung adenocarcinoma, lung squamous cell carcinoma (LSCC), lung large cell carcinoma, and lung neuroendocrine cancer [2,3]. Approximately 40% of patients with NSCLC that are at advanced stage hardly survive more than 5 years despite of traditional chemotherapy, molecular-targeted therapy or chemoradiotherapy [2][3][4]. Therefore, novel effective treatments are demanded to tackle the short survival time of NSCLC.
The abnormal activation of fibroblast growth factor (FGF) signaling pathways is required for tissue homeostasis, repair, angiogenesis, organogenesis, and is involved in carcinogenesis [5][6][7][8][9][10], indicating its potential role of a target for therapeutic intervention [5,10,11]. By interacting with FGF receptors (FGFRs) and FGF ligands, FGFs modulate cell proliferation, differentiation and mobility [9]. FGF9, belonging to the FGF family, its aberrant expression has been identified in diverse tumors, such as breast, prostate, endometrioid, and lung cancers, indicating its tumor biomarker role in various cancers [12][13][14][15]. Furthermore, Wang et al. indicated that FGF9 was upregulated in patients with lung adenocarcinoma, and that aberrant FGF9 expression might inhibit the progression of lung adenocarcinoma [16]. However, little is known about the role of FGF9 during LSCC.
MiRNAs, a class of small single strand RNA molecules, modulate the expression of approximately a third of human genes, and affect diverse cellular activities such as cell proliferation, cycle, apoptosis, and differentiation [17]. Circulating miRNAs have great potential to serve as diagnostic/prognostic biomarkers for diverse cancers, such as gastric, bladder, breast, prostate, renal, and lung cancers [18][19][20][21][22][23]. MiRNA-371-373 (miR-371-373) cluster, a human homolog of the mouse miR-290-295 cluster is initially reported to be merely expressed in human embryonic stem cells, implicating its role in stem cell multipotency [24]. Recent studies have also confirmed that the miR-371-373 cluster is usually deregulated in human tumors such as hepatoblastoma, colorectal cancer, and testicular germ cell tumors [25][26][27]. MiRNAs function as tumor suppressors or tumor facilitators by directly binding to target 3′UTRs. MiR-373, for instance, has been found to facilitate tumor invasion and metastasis by inhibiting CD44 [28]. We herein conducted this study to explore the role of miR-372-3p in LSCC.
In this study, we identified FGF9 as a target gene of miR-372-3p. We also confirmed a converse correlation between miR-372-3p and FGF9 in LSCC tissues and cells. The direct suppression of FGF9 by miR-372-3p and the potential effects of miR-372-3p as a tumor facilitator in LSCC have been validated at both experimental and clinical levels.

MaterialsandMethods Clinicalsamples
Twenty LSCC and corresponding adjacent normal tissues were obtained from those underwent pneumonectomy from April 2015 to December 2016 in The Fourth Affiliated Hospital of China Medical University. The pathological type of each tumor sample was confirmed by experienced pathologists. Fresh samples were frozen in the liquid nitrogen prior to RNA extraction. None of the patients had ever received any adjuvant chemotherapy or radiotherapy before surgery. Informed consent was signed by every patient. The study was approved by the Ethical Committee of The Fourth Affiliated Hospital of China Medical University.

RNAisolationandRT-PCRanalysis
Total RNA was extracted from frozen tissues using Trizol (Invitrogen) following the manufacturer's protocols. Both miR-372-3p and FGF9 mRNA were reversely transcribed to cDNAs using the Reverse Transcription System Kit (Invitrogen). RT-PCR was carried out following the instruction of SYBR ® Premix EX Taq kit (TaKaRa, Japan). cDNAs were then amplified (primer sequences are listed in Table 1). FGF9 mRNA expression was normalized to GAPDH expression and miR-372-3p to U6 snRNA, respectively. The relative expression of miR-372-3p and FGF9 mRNA were calculated by 2 −∆∆C τ method.

Dualluciferasereportergeneassay
A wild-type or mutated FGF9 3′-UTR segment was inserted between the XhoI and PmeI restriction sites of the pMiRGLO vector (Sangon Co., LTD, ShangHai, China). The reconstructed vectors were then cotransfected with miR-mimics or miR-NCs into NCI-H520 cells and incubated for 48 h. Ultimately, the activities of Firefly and Renilla luciferases were detected by Dual-Luciferase Reporter Assay System (Promega, Madison, USA). The luciferase activities of firefly were normalized against Renilla to represent the relative luciferase activity of cells with different transfection.
20 μL MTT solution (5 mg/mL) was added into each well once a day for days. After 4 h's incubation, the original culture solution was discarded and 200 μL DMSO was added to dissolve the excessive crystals. Optical absorbance of cells was measured using a multifunctional microplate reader at 490 nm.

Colonyformationassay
Approximately 200 cells were seeded in a 24-well plate and cultured in RPMI-1640 supplemented with 10% FBS for 2 weeks. Then the media were removed and the colonies were washed twice with PBS. Ultimately, cells were fixed by 4% methanol, stained with Giemsa for 15 min. The number of naked-eye-can-see colonies was counted under an optical microscope. Transwellassay 1×10 5 cells were seeded into the Transwell inserts with 8 μm-pore membranes coated with Matrigel (Corning Incorporated, NY). Serum free media were added to the inserts, whereas 20% FBS media were added to the bottom chambers. Cells were then cultured for 24 h at 37°C. Cells on the upper surface of the membrane were removed and the rest of the cells were fixed with 4% methanol for 15 min, washed twice by phosphate-buffered saline (PBS) and stained by crystal violet for 15 min. Invasive cells were observed under an optical microscope and the number of invaded cells in ten randomly selected 20× fields was counted.

Westernblot
Proteins were lysed using radioimmunoprecipitation assay (RIPA, Sigma-Aldrich) lysate buffer and the protein concentrations were determined using the bicinchoninic acid assay (BCA) reagent kit (Beyotime, Jiangsu, China). Proteins in samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (50 μg/lane). The separated proteins were electro-transferred onto polyvinylidene fluoride (PVDF) membranes, and subsequently incubated with nonfat milk for 2 h at the room temperature. The PVDF membranes were incubated with the primary antibodies (anti-FGF9, 1:1,000, anti-GAPDH, 1:1,000) at 4°C overnight. The intensity of protein bands was quantified using ImageJ software (NIH, USA).

Statisticalanalysis
Statistical analyses were performed by SPSS19.0 (IBM, USA). The data were shown as mean ± standard deviation (x±s). The Student's t-test was used to analyze the significance of the difference between any two groups, whereas one-way ANOVA was used among more than two groups. Kaplan-Meier analysis was performed to analyze the survival rates of patients with LSCC. Differences were regarded statistically significant if P values were less than 0.05.

MiR-372-3pandFGF9expressioninLSCC tissuesandcelllines
We performed RT-PCR to quantify the expression of miR-372-3p in LSCC tissues and adjacent tissues. As shown in Figure 1A-B, the results indicated that miR-372-3p was significantly upregulated in LSCC tissues and cell lines compared with adjacent tissues and normal cells, respectively (P < 0.05). We chose NCI-H520 cell line for the subsequent experiments in vitro due to its highest miR-372-3p level.
On the other hand, FGF9 mRNA expression was also determined by RT-PCR. The relative expression level of FGF9 mRNA was obviously lower in LSCC tissues compared with that in normal tissues ( Fig. 1C; P < 0.05).

MiR-372-3ppromotedLSCCcellviabilityand growth
To explore the effects of miR-372-3p on LSCC cell growth, MTT and colony formation assays were performed on NCI-H520 cells. The viability of cells transfected with miR-372-3p inhibitors were dramatically weaker than those transfected with NCs (Fig. 3A, P < 0.05). Meanwhile, the viability of those transfected with FGF9 cDNAs was obviously suppressed compared with those transfected with NCs (P < 0.05).
Similarly, the colony formation results showed that the transfection of either FGF9 cDNAs or miR-372-3p inhibitors led to the decreased cell proliferation (Fig. 3B).
To further detect the influence of miR-372-3p and FGF9 ectopic expression on LSCC cell invasiveness, we performed Transwell assay. As shown in Figure 3E, the repression of miR-372-3p led to the reduced LSCC cell invasion, suggesting that miR-372-3p could promote LSCC cell invasiveness (P < 0.05). Meanwhile, exogenous FGF9 also substantially inhibited the LSCC cell mobility (P < 0.05).

Discussion
Nowadays, with the deterioration of the environment, aggravation of the air pollution and prevalence of smoking, lung cancer gradually become a common cancer which severely threatens people's health and life. According to an investigation conducted by the GLOBOCAN project (http://globocan.iarc.fr/), the mortality and incidence rate of lung cancer were 16.7% and 23.2% respectively in 2012 and it has become the third most common cancer. Therefore, it is necessary and meaningful to explore the mechanism of tumorigenesis of the lung cancer.
MicroRNAs (miRs) have been widely accepted as a regulator of human cancers and it can be either cancer promoters or cancer suppressors. A plethora of studies have demonstrated that miR-372 has a strong influence on some human cancers like gastric cancer, hepatocellular cancer, and colorectal cancer by regulating specific genes [29][30][31]. MiR-372-3p, on the other hand, has not been reported to influence carcinogenesis too much, however, was discovered to suppress insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) therefore led to the decrease in proliferation and invasiveness in renal cell carcinoma [32]. In this study, we explored the influence of miR-372-3p on lung squamous cell carcinoma (LSCC), and to explain the mechanism behind the effects, the targeting relationship between miR-372-3p and gene FGF9 was also revealed.
Although miR-372-3p has been found to regulate cancer development by suppressing various genes in various tumors [32][33][34], it was worth mentioning that the FGF9 was chosen as the candidate target gene using bioinformatics analysis. In this study, we found that miR-372-3p depressed the expression of FGF9 directly. On the basis of the discovery that miR-372-3p was overexpressed and FGF9 was underexpressed compared with adjacent tissues in three LSCC cell lines, we hypothesize that FGF9 seemed FGF9 belongs to the fibroblast growth factor (FGF) family that participates in a spectrum of biological processes including tumor growth and invasion. FGF9 is specifically important in glial cell growth and development. It has been reported to suppress renal tumor metastases [35]. However, it has also been discovered to facilitate gastric cancer invasion and growth [36]. Besides, FGF9 was believed to indicate a poor prognosis of nonsmall-cell lung cancer, colon cancer, and prostate cancer [15,37,38]. However, we found that FGF9 was underexpressed in LSCC tissues, which suggested its tumor suppressor role rather than a tumor facilitator role. The different roles of FGF9 may due to the different context of different tumors, and further experiments are warranted to confirm its role in LSCC. FGF9 was proved to be targeted by miRNAs such as miR-155-5p and miR-140-5p in lung dysplasia and hepatic cell carcinoma [39,40]. In our study, we also confirmed that FGF9 could be directly suppressed by miR-372-3p, which contributed to the comprehensive understanding of LSCC carcinogenesis.
Taken together, exogenous miR-372-3p significantly suppressed FGF9 expression, and simultaneously, LSCC cells FGF9 can be directly repressed by miR-372-3p. This interaction between miR-372-3p and FGF9 in LSCC led to the increase in tumor growth and invasiveness. We thus conclude that miR-372-3p could promote LSCC development by suppressing FGF9 expression.
Our research provided novel insight into the treatment of LSCC. Traditional treatments for LSCC are radiotherapy, chemotherapy, and lobe resection, which always do harm to patients and have a high risk of side effects and recurrence rates. Our study provides therapists a basis for further treatments of LSCC by targeting miR-372-3p or FGF9. However, some limitations remain in our study that FGF9 is not the only gene that influencing the progression of LSCC, moreover, other miRNAs may regulate the expression of FGF9 except for miR-372-3p in LSCC. Therefore, our future work will be dedicated to overcome the limitations, specifically, by investigating the relationship between other genes and the progression of LSCC.
In conclusion, the expression of miR-372-3p and FGF9 in LSCC tissues and cells were determined and the effects of miR-372-3p and FGF9 on LSCC cell activities have been studied. Besides, we confirmed the binding relationship between miR-372-3p and FGF9. So we come to that conclusion that miR-372-3p could probably promote LSCC growth and invasion by suppressing FGF9 expression.