MicroRNA-520a discourages lung cancer pathogenesis and progression involving the downregulation of RRM2 and Wnt signaling deficits

Background Increasingly evidence has noted the critical functions of microRNAs (miRNAs) in disease control including cancer progression. This paper aimed to explore the functions of miR-520a in lung cancer (LC) and the downstream molecules implicated. Aberrantly expressed miRNAs in LC tissues were screened out by miRNA microarrays. miR-520a expression in LC tissues and cell lines was determined, and the correlation between miR-520a level and survival rate of patients was analyzed. Altered expression of miR-520a was introduced to evaluate its function in LC cell malignant behaviors. The target mRNA and the potential signaling pathway mediated by miR-520a were figured out. Xenograft tumors were induced in mice to test the role of miR-520a in tumorigenesis in vivo . Poor expression of miR-520a was found in LC tissues and cell lines. A higher miR-520a level indicated a better survival rate in LC patients. Overexpression of miR-520a led to declines in cell viability, proliferation, migration, invasion and resistance to apoptosis. The target mRNAs of miR-520a were enriched on the Wnt signaling. miR-520a inactivated the Wnt pathway. miR-520a could bind to RRM2 and downregulate RRM2 expression in LC cells. Overexpression of RRM2 promoted the malignant behaviors of cancer cells, but this promotion was inhibited by miR-520a. Overexpression of miR-520a also inhibited the tumor growth and metastasis in nude mice. The present study provided evidence that miR-520a could inhibit LC progression through RRM2 downregulation and Wnt signaling deficit. This paper may offer novel ideas concerning LC treatment. the dark for 30 minutes, washed in methanol and PBS for two times, and then loaded with 4', 6-diamidino-2-phenylindole (DAPI) and incubated for 20 minutes. The labeling was observed under a fluorescent microscope (XSP-BM13C, Shanghai CSOIF. Co., Ltd., Shanghai, China) at a 400 × magnification with 3 random fields included. The EdU-positive cells were stained in red and all cells were stained in blue by DAPI. The cell proliferation rate = number of proliferation cells/total cells × 100%. Three independent experiments were performed. behaviors of cells and tumor growth in LC by directly binding to RRM2 and the subsequent Wnt signaling defect. These findings can provide novel insights into the gene-based therapy for LC treatment. We also hope more researches in this field will be conducted to develop more understandings to improve the therapeutic


Introduction
Cancer remains the greatest healthy concern across the globe, and lung cancer (LC) is the most prevailing cancer type which occupies nearly a quarter of cancer-related mortality according to the Cancer Statistics in 2020 [1]. According to the pathological type, LC is categorized into two main subtypes including non-small cell lung cancer (NSCLC, taking up to nearly 85% of all cases) and small cell lung cancer (15% of all cases) [2]. In China, LC has the greatest mortality rate among all cancer types [3]. Despite the improvements in traditional therapeutic regimens including surgery, radiotherapy and chemotherapy in the past decades, the treating outcome of LC patients remains unfavorable [4]. A major reason is that a considerable number of LC patients are initially found at advanced stages with metastatic properties, and the overall 5-year survival rate of these patients are extremely low at about 5% [1]. Identifying new interventions targeting LC growth and metastasis is of great importance to offer better opportunities and hopes to LC patients.
Gene-based therapy has been a promising target for disease treatment and aroused wide concerns.
Approximately 97% of all human genomes are transcribed to non-coding (nc) RNAs and can regulate the molecular processes at DNA-RNA-protein levels including in tumorigenesis, though without protein-coding capacities [5]. microRNAs (miRNAs) are a largely studied short ncRNAs which are renowned for their capacity in post-transcriptionally controlling gene expression by primarily interacting with the 3'-UTR of target mRNAs [6]. Owing to the potent gene-modifying functions, miRNAs can regulate diverse fundamental cellular processes including apoptosis, proliferation, maintenance of cell differentiation, and tumorigenesis in several cancers including LC [7]. The microarray analysis in this study identified miR-520a as an aberrantly lowly expressed miRNA in LC tissues. miR-520a has been found as a tumor inhibitor in many human malignancies. It showed inhibitory functions on proliferation, invasion and migration in gastric cancer cells by targeting spindle and kinetochore associated 2 [8]. The miR-520a-3p has been documented to be implicated in several competing endogenous RNA (ceRNA) networks (miRNAs working as sponges for other ncRNAs), whose downregulation by other RNAs may facilitate NSCLC malignancy [9,10]. But the independent roles of miR-520a in LC and the downstream molecules involved remains largely unknown. Here, the integrated bio-information analyses suggested that ribonucleotide reductase subunit 2 (RRM2) is a potential mRNA target of miR-520a. Overexpression of RRM2 has been confirmed with oncogenic roles in many malignancies including gastric cancer [11] and pancreatic cancer [12]. Herein, we hypothesized that miRNA-520a could inhibit LC progression in tumor growth and metastasis regards, with both cell and animal experiments performed to validate this hypothesis.

Clinical sample collection
During the period from January 2015 to January 2016, 24 patients pathologically diagnosed as LC and admitted into Shandong Provincial Chest Hospital were recruited into the research. Patients with other chronic diseases, or with chemo/radio therapy history, or with a family history of malignancy were excluded. The tumor tissues and the adjacent normal tissues (over 5 cm away from tumor) were resected during surgery and instantly frozen in liquid nitrogen and then in a -80℃ ice box. A threeyear follow-up study was performed on each patient. This study was ratified and supervised by the Ethics Committee of Shandong Provincial Chest Hospital. Signed informed consent was collected from each eligible patient. The characteristic information of all participants is presented in Table 1.

Hematoxylin and eosin (HE) staining
The collected lung tissues from patients and liver and lung tissues from mice (see below) were embedded in paraffin and cut into slices, which were successively baked at 60℃ for one hour, soaked in xylene I, II and III (10 minutes for each), soaked in different concentrations of alcohol for 5 min, washed in water for 5 minutes. Afterwards, the slices were stained in hematoxylin solution for 8 minutes, differentiated in 0.5% hydrochloric acid-ethanol mixture for 10 seconds, soaked in ammonia for 40 seconds, and then stained with 0.5% eosin for 5 minutes. The slices were washed in distilled water for 1 minute after each staining. Next, the slices were soaked in xylene and alcohol again, sealed with neutral balsam (Solarbio, Science & Technology Co., Ltd., Beijing, China), and then observed under a microscope (BX53, Olympus Optical Co., Ltd, Tokyo, Japan) with 5 random fields selected. The nuclei were stained in red, while the cytoplasm in pink. Three independent experiments were performed.

Microarray analysis
Total RNA from 15 mg either LC tissues or normal lung tissues was extracted using a miRNeasy Kit (Qiagen GmbH, Hilden, Germany) and a mirVana miRNA Isolation Kit (Thermo Fisher Scientific, Carlsbad, CA, USA), respectively, and the RNA purity was examined by a Nano Drop (Thermo Fisher Scientific). Subsequently, 100 ng RNA was collected and labeled using a miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Inc. Wakefield, MA, USA). Then, the samples were hybridized with the Human V2 mi RNA Microarrays (Agilent) at 200 rpm at 60ºC for a total of 20 hours. The images were scanned and analyzed using a DNA Microarray Scanner (Agilent), while the data were obtained and analyzed by GeneSpring GX (Agilent).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA from tissues and cells was separated by the TRIzol Reagent (Thermo Fisher). Then, the RNA was transcribed to cDNA by a high-capacity cDNA Reverse Transcription Kit (Thermo Fisher).
Subsequently, real-time qPCR was performed using TaqMan microRNA assay kits (Thermo Fisher) on a 7900HT fast Real-time PCR system (Applied Biosystems, Foster City, CA). The fold change of the acquired Data was measured using the 2 -ΔΔCt method with U6 as the internal control for miRNA while GAPDH for RRM2. The primer sequences are exhibited in Table 2.
(Guangzhou, Guangdong, China). Cell transfection was performed on a Lipofectamine 2000 DNA 6 Transfection Reagent (Thermo Fisher). In brief, when the cell confluence reached 80%, the Lipofectamine Reagent was diluted in Opti-MEM, while another Opti-MEM was used to dilute DNA. The diluted DNA was added in the diluted Lipofectamine 2000 at a ratio of 1:1 and incubated for 5 minutes. After that, the cells were added with DNA-lipid compound and cultured at 37℃ for 2 days.

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
Well growing cells were resuspended to 5 × 10 4 /mL and sorted into 96-well plates at 100 μL per well till the cells reaching a density of 5 × 10 3 cells/well (the marginal pores were filled with sterile phosphate buffer saline (PBS) to eliminate the potential edge effect). Three duplicated groups were set for each group. The plates were incubated in a 37℃ incubator with 5% CO 2 . One plate was taken out for measurement every 24 hours. In brief, each well was loaded with 20 μL MTT solution (Solarbio) and then incubated for four more hours. Then, the culture medium in wells was discarded, followed by loading of 150 μL dimethyl sulphoxide to fully dissolve the crystal violet. Then the optical density at 570 nm of each well was determined using a microplate reader (Varioskan LUX, Thermo Fisher). The obtained data were analyzed to produce an MTT-proliferation curve.

5-ethynyl-2'-deoxyuridine (EdU) labeling assay
Exponentially growing cells were sorted into 6-well plates. When the confluence got to 80%, 10 μM EdU solution was loaded into each well (Genecopoeia, USA). Then the cells were incubated for 2 hours, washed in PBS, fixed in 4% paraformaldehyde for 30 minutes, incubated in glycine solution, and rinsed with PBS containing 0.5% TritonX-100. Subsequently, the cells were added with Andy

Flow cytometry
Apoptosis of cells was determined by an Annexin V-FITC Apoptosis Detection Kit (Nacalai, Tesque, Japan). The treated cells were cultivated at 37℃ with 5% CO 2 for 48 hours, centrifuged at 1000 rpm for 3 minutes, washed in PBS for twice, and then centrifuged again. Then, 20 μL Annexin V Binding Buffer (10 ×) was added into 200 μL PBS. Cells were resuspended in PBS, and then filled with 10 μL Annexin V-FITC conjugate and 5 μL PI solution, and then cultivated at room temperature in the dark for 15 minutes. Then the cells were added with 300 μL binding buffer, and the excitation wavelength at 488 nm was determined using a flow cytometer (Beckman Coulter, USA).

Xenograft tumors in nude mice
A total of 12 female nude mice (strain: BALB/c; 3-4 weeks old, 14 ± 2 g) were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were fed at constant 25℃ and 45% humidity with free access to food and water. The mice were randomly allocated into 4 groups (miR-520a control, miR-520a mimic × A549/H460 cells), 3 in each. As for cell implantation, the exponentially growing A549/H460 cells with stable miR-520a mimic and mimic control transfection were adjusted to 1 × 10 7 cells/mL. Then, each mouse was transplanted with 20 μL cell suspension 9 through axillary injection, and then the growth of tumor in vivo was photographed and recorded. The tumor volume was recorded every 7 days, which was calculated as a × b 2 /2 (where 'a' refers to the long diameter while 'b' refers to the short one), and a growth curve was produced. Twenty-eight days later, the mice were euthanized by overdose of 1% pentobarbital sodium (150 mg/kg), and the tumor was collected and weighed. All procedures were performed in line with the guidelines of the Animal Ethics Committee of Shandong Provincial Chest Hospital. Great attempts were made to reduce the usage and pain of animals.

Tumor metastasis in vivo
Another 12 nude mice were numbered by weight and allocated into 4 groups for tumor metastasis measurement. The cells with stable transfection were injected into mice through the caudal veins, and the mice were euthanized by overdose of 1% pentobarbital sodium on the 45 th day post injection.
Thereafter, the lung and liver tissues of mice were collected, embedded in paraffin, cut into slices, and subjected to HE staining to observe the formation of metastases. The staining was observed under the microscope with 5 non-overlapping fields included.

Statistical analysis
SPSS 22.0 (IBM Corp. Armonk, NY, USA) was used for data analysis. Measurement data were exhibited as mean ± standard derivation (SD). Data were analyzed using the t test (two groups) and one-way or two-way analysis of variance (ANOVA) followed with Tukey's multiple comparison test (multiple groups). The p value was obtained from two-tailed tests, and p < 0.05 was regarded to have statistical significance.

miR-520a is poorly existed in LC tissues and cells
Tissue samples from 3 LC patients were collected and observed. It was found that the cells in the LC tissues presented destructed cell structure and shrinking nuclei, indicating sever lung injury (Fig. 1A).
Then, the RNA in tissues was extracted for microRNA analysis, which suggested that miR-520a is notably down-regulated in tumor tissues (Fig. 1B). A similar result was found by RT-qPCR, which identified lower expression of miR-520a in LC tissues than that in the paired adjacent tissues (Fig. 1C).
The following study suggested that patients with higher levels of miR-520a have better prognosis and longer lifetime (Fig. 1D). In addition, miR-520a expression in LC cell lines H273, H23, A549 and H460 and in normal pulmonary epithelial cell line 16HBE was determined as well. The RT-qPCR results suggested that all LC cell lines showed decreased miR-520a expression as relative to the 16 HBE cells (Fig. 1E). Among them, the A549 and H460 cells, with lowest expression of miR-520a, were transfected with miR-520 mimic or the mimic control for the subsequent experiments (Fig. 1F).

Artificial up-regulation of miR-520a inhibits LC progression
Following the above findings and administration, it was found that over-expression of miR-520a by miRNA mimic led to a notable decline in cell number of EdU-positive cells in both A549 and H460 cells with reduced cell viability ( Fig. 2A). The MTT assay results suggested that both the number of proliferated cells and the proliferation rate of cells were reduced following miR-520a overexpression ( Fig. 2B). In terms of cell migration and invasion, the Transwell assay results suggested notable declines in cell migration rate and number of migrated cells, as well as declines in number of invaded cells in both A549 and H460 cell lines following miR-520a mimic administration (Fig. 2C-D). In addition, according to the flow cytometry, it was found that miR-520 overexpression led to increased number PI-Annexin V-double-positive cells, indicating an increase in cell apoptosis rate (Fig. 2E). From the molecular perspective, it was found that miR-520a mimic led to a declined level of Bcl-2 but increased levels of Bax and cleaved caspase-3 (Fig. 2F). Collectively, these results suggested that miR-520a could inhibit the progression of LC.

miR-520a directly binds to RRM2 to mediate the Wnt signaling pathway
To further identify the potential downstream molecules implicated in the effect of miR-520a, we first predict the target mRNAs of miR-520a on StarBase, TargetScan, miRDB and miRBase, with 219 mRNAs found intersected (Fig. 3A). Then, a GO enrichment analysis was performed, which identified that the Wnt signaling was highly enriched by these genes (Fig. 3B) and a total of 18 genes were enriched in this signaling pathway. Then, the levels of Wnt signaling pathway-related factors Cyclin D1 and β-catenin in A549 and H460 cell lines were measured. The western blot analysis results identified that the Cyclin D1 and β-catenin levels in both cell lines were declined following miR-520a 11 overexpression (Fig. 3C), indicating that miR-520a inactivated the Wnt signaling. Subsequently, we tested the expression of mRNAs enriching in the Wnt signaling in tissues using RT-qPCR. The results suggested that the expression of SDC1, YWHAZ and RRM2 was notably increased in tumor tissues as compared to the paired normal tissues (Fig. 3D). Then the luciferase assays validated that only RRM2 presented a targeting relationship with miR-520a (Fig. 3E). The correlation analysis showed an inverse relationship between miR-520a and RRM2 in LC tumor tissues (Fig. 3F). Accordingly, the RRM2 expression was found higher in LC cell lines than that in 16HBE cells (Fig. 3G). Also, miR-520a mimic was found to decrease the RRM2 expression in A549 and H460 cells (Fig. 3H). In addition, overexpression of RRM2 in cells resulted in increased levels of Cyclin D1 and β-catenin in A549 and H460 cells (Fig. 3I), indicating that miR-520a might inactivate the Wnt signaling pathway through down-regulating RRM2 expression.

miR-520a inhibits the oncogenic effects of RRM2 on LC progression
To confirm the involvement of RRM2 in the miR-520a-mediated events in LC, A549 and H460 cells introduced with RRM2 OE vector were further transfected with miR-520a mimic (Fig. 4A). It was found that the increased cell viability by RRM2 OE vector transfection alone was reversed by further miR-520a transfection (Fig. 4B), In addition, co-transfection of miR-520a and RRM2 OE vector resulted in a decreased number in migrated cells and invaded cells, as well as increased number of apoptotic cells compared to the RRM2 OE vector transfection alone (Fig. 4C-E). These results, collectively, suggested that miR-520a directly binds to RRM2 to inhibit the malignant behaviors of LC cells.

miR-520a inhibits tumor growth and metastasis in vivo
A549 and H460 cell lines with stable miR-520a mimic/control transfection were implanted into nude mice. Then the tumor volume in mice was measured every 7 days, and it was found that overexpression of miR-520a led to a significant decline in tumor growth rate in mice (Fig. 5A). Twentyeight days later when the mice were euthanized, it was found that miR-520a mimic also led to reduced tumor weight in nude mice (Fig. 5B). In addition, the HE staining results suggested that the number of metastatic nodules in lung (Fig. 5C) and liver (Fig. 5D) tissues was decreased by miR-520a mimic. 12 Discussion LC has been remaining as the most prevailing cancer and consequently the biggest cause of cancermorbidity worldwide with the survival rate of patients at late stages particularly low, leaving understanding the biology of LC and identifying novel molecules involved in pathogenesis of great urgency [13]. miRNA-targeted therapies, including miRNA replacement and miRNA reduction where oligonucleotides, virus-based constructs or small molecule compounds are administrated to restore expression of suppressive miRNAs or inhibit expression of oncogenic miRNAs [14]. The present study evidenced that miR-520a played potent tumor suppressing roles in LC development in both cell models and animal models, during which the target down-regulation of RRM2 and Wnt signaling defect were involved.
A dysregulated miRNA profiling is often linked to the onset and development of diseases including cancers. Here, a miRNA microarray analysis suggested that miR-520a was down-regulated in LC tumor samples. This was validated by RT-qPCR, which presented that miR-520a was expressed at poor levels in LC tissues and cell lines as relative to the normal ones. A higher level of miR-520a was associated with a better prognosis in LC patients, and artificial upregulation of miR-520a inhibited proliferation, migration, invasion, and resistance to death of LC cell lines. Emerging study have emphasized the core functions of miRNAs in LC pathogenesis. For instance, miR-218 was found to act as a strong tumor suppressor in LC, inhibiting the malignant behaviors of LC cells and tumor growth by targeting interleukin-6 receptor and JAK3 [15]. On the other hand, miR-421 was found as an oncogene, which promoted proliferation and invasion potentials in NSCLC cells by targeting PCDC4 [16]. In terms of miR-520a, it was reported with tumor-inhibiting roles in many neoplastic diseases.
Upregulation of miR-520a-3p by lidocaine, for example, was found to block proliferation and trigger apoptosis of colorectal cancer cells [17]. The similar trends were found in osteosarcoma cells [18] and breast cancer [19]. As for in LC, recent studies have noted the involvement of miR-520a in several ceRNA networks with tumor-suppressing functions as well [9,10]. Here, our study validated the independent anti-cancer roles of miR-520a. In addition, the increased cleaved caspase-3 level and Bax/Bcl-2 ratio identified the pro-apoptotic role of miR-520a in a cytokine perspective. Furthermore, the growth and metastases of xenograft tumors in nude mice were also inhibited by overexpression of miR-520a.
The direct targets of miR-520a were little concerned in the above-mentioned researches concerning its role in LC. In the present study, an integrated bioinformation analysis based on the data from 4 well-known RNA prediction systems was performed, with 219 mRNAs were found intersected, namely, the common outcomes. Then a GO enrichment analysis found that the Wnt signaling pathway was enriched by these mRNAs. The Wnt signaling is important for the development and homepstasis by regulating their endogenous stem cells, whose aberrant activation has been recognized as a critical factor in the onset, maintenance and development of many cancers [20]. There is no exception for LC, in which Wnt inactivation has been reviewed as a promising therapeutic option for LC treatment [21,22]. In addition, it was found that miR-520a led to declines in Cyclin D1 and β-catenin levels in cells, indicating the implication of Wnt defect in the miR-520a-mediated events. Furthermore, our study found that SDC1, YWHAZ and RRM2 were enriched in this signaling pathway, while only RRM2 was confirmed to have a binding relationship with miR-520a according to the luciferase assays. Then we found RRM2 was highly expressed in LC tissues and cell lines. RRM2 is a cell cycle dependent factor that was suggested to play oncogenic roles in many cancers such as adrenocortical cancer [23], glioma [24] and neuroblastoma [25]. Interestingly, RRM2 has also been involved in several ceRNA networks and its upregulation has been found to trigger cell proliferation, drug-resistance and tumor growth in LC [26,27]. Importantly, artificial up-regulation of RRM2 in cells promoted cell viability, migration, invasion and resistance to apoptosis. Accordingly, further experiments found that overexpression of miR-520a blocked the above events, indicating that RRM2 is a downstream target of miR-520a during its regulation in LC progression.

Conclusion
To sum up, the current study evidenced that miR-520a could inhibit malignant behaviors of cells and tumor growth in LC by directly binding to RRM2 and the subsequent Wnt signaling defect. These findings can provide novel insights into the gene-based therapy for LC treatment. We also hope more researches in this field will be conducted to develop more understandings to improve the therapeutic 14 efficacy in LC treatment.   F, miR-520a expression in A549 and H460 cells after miR-520a mimic/control transfection determined by RT-qPCR (* p < 0.05, two-way ANOVA).