Long non-coding RNA MCM3AP antisense RNA 1 promotes non-small cell lung cancer progression through targeting microRNA-195-5p

ABSTRACT Lung cancer (LC) ranks first among all causes of cancer-related death, with non-small cell lung cancer (NSCLC) taking up 85% of lung cancer cases. Although lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) has been reported to be an oncogenic factor in NSCLC, its detailed mechanism in NSCLC is unknown. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine MCM3AP-AS1, microRNA (miR)-195-5p and E2F transcription factor 3 (E2F3) mRNA expressions in NSCLC tissues and cells. Western blot was utilized to determine the expression levels of E2F3, BCL2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), E-cadherin and N-cadherin. CCK-8 and Transwell assays were conducted to examine cell proliferation, migration and invasion, respectively. Dual-luciferase reporter assay and RNA immunoprecipitation experiments were used to determine the regulatory relationships between MCM3AP-AS1 and miR-195-5p, and miR-195-5p and E2F3. We demonstrated that MCM3AP-AS1 was overexpressed in NSCLC tissues and cells, and MCM3AP-AS1 overexpression accelerated the proliferation, migration and invasion of NSCLC cells. In addition, MCM3AP-AS1 overexpression markedly up-modulated Bcl-2 expression and repressed Bax expression; MCM3AP-AS1 overexpression also significantly up-regulated N-cadherin expression and suppressed E-cadherin expression in NSCLC cells. What is more, in NSCLC cells, miR-195-5p was a target of MCM3AP-AS1, and the latter worked as a molecular sponge for miR-195-5p to regulate E2F3 expression. Collectively, MCM3AP-AS1, serving as a competitive endogenous RNA (ceRNA) to regulate miR-195-5p/E2F3 axis, promotes NSCLC progression, which is a promising therapeutic target for NSCLC.


Introduction
Lung cancer (LC) is the commonest malignancy in the world with the highest mortality [1]. Nonsmall cell lung cancer (NSCLC), as the main pathological subtype of LC, accounts for more than 80% of cases of LC [2]. The treatment strategies of NSCLC mainly include surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy [3]. Although a lot of efforts have been made to improve the therapy of NSCLC, the five-year survival rate of the patients is only about 18% [4,5]. Therefore, it is necessary to have a better understanding of the molecular mechanisms underlying NSCLC development to further improve the prognosis of the patients.
In recent years, accumulating studies find that long non-coding RNAs (lncRNAs) are aberrantly expressed in diverse tumors, which is involved in tumorigenesis and disease development [6,7]. For instance, lncRNA TPTEP1, SPRY4-IT1 and ZEB2-AS1 are reported to be aberrantly expressed in NSCLC and modulate biological processes such as proliferation and metastasis of cancer cells [8][9][10]. Previous researches show that lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) is associated with the progression of some tumors, such as prostate carcinoma, gastric carcinoma and cervical carcinoma [11][12][13]. Reportedly, in NSCLC, MCM3AP-AS1 expression is also upregulated, and the proliferation, migration and angiogenesis of NSCLC cells are impeded by MCM3AP-AS1 knockdown [14]. However, the detailed mechanism of its effects in NSCLC is vague.

Sample collection
From June 2017 to August 2018, sixty-three pairs of matched NSCLC tissues and tissues of resection margin were taken from the tissue bank of the Cancer Hospital of the University of Chinese Academy of Sciences. All patients were histologically confirmed as NSCLC by pathologists at the Cancer Hospital of the University of Chinese Academy of Sciences and were not suffering from any other malignancies, and no neoadjuvant therapy was given prior to surgery. Among the patients, there are 38 males and 25 females, aged from 41-74 years old (average: 52.4). Patients receiving neoadjuvant therapy, patients with mixed NSCLC/small cell histology or CNS metastases were excluded. The surgically resected NSCLC tissues and paracancerous tissues (at least 5 cm away from the tumor margin) were collected and stored in liquid nitrogen immediately after resection during surgery. All specimens were confirmed as NSCLC or normal lung tissue by postoperative pathological examination. All patients who provided tissues signed informed consent before the surgery, and all of them agreed to the use of their samples in scientific research. This research was endorsed by the Research Ethics Committee of Cancer Hospital of the University of Chinese Academy of Sciences (Approval Number: 2017-05).

qRT-PCR
Total RNA from tissues and cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was reversely transcribed into cDNA using the reverse transcription kit (Applied Biosystems, Foster City, CA, USA). cDNA was used as a template, and qRT-PCR was conducted using SYBR® Premix-Ex-Taq™ (Takara, Tokyo, Japan) on ABI7300 PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Additionally, the relative expression of miR-195-5p was normalized to U6, and the relative expressions of MCM3AP-AS1 and E2F3 mRNA were normalized to GAPDH, with 2 −ΔΔCt method [23]. Primer sequences were shown in Table 1.

Cell viability
A549 and H226 cells were inoculated into 96-well plates at 1.0 × 10 3 /well before 10 μL of cell counting kit (CCK-8) reagent (MedChem Express, Monmouth Junction, New Jersey, USA) was added into the wells at the 1st, 2nd, and 3rd d, respectively, and OD 450 values were measured on a microplate reader after 2 h of incubation.

EdU assay
A549 and H226 cells were inoculated into 24-well plates, respectively, and cultured for 24 h. After that, the cells were incubated with 200 μL of 50 μmol/L EdU medium (RiboBio Co., LTD, Guangzhou, China) for 2 h, rinsed with PBS and then fixed with paraformaldehyde for 10 min. Following that, the cells were incubated for 5 min with 200 μL of 2 mg/ml of glycine and rinsed with PBS for 5 min. One hundred microliters of PBS with 0.5% Triton X-100 was added to each well, and the cells were placed on a shaker for 10 min and then washed with PBS for 5 min. Subsequently, the cells were stained with Apollo® solution in the dark for 30 min, and nuclear DNA was counterstained with DAPI. Then, the cells were observed, and the images were collected using a fluorescent microscope in the dark.

Transwell assay
Migration assays were performed using Transwell chambers (8 µM pore size; Corning, Beijing, China). After dispersing A549 and H226 cells with 0.25% trypsin, the cells were centrifuged and resuspended with serum-free medium. 5 × 10 4 cells were inoculated into each chamber before the complete medium was added in the 24-well plate. After 24 h incubation at 37°C, the unmigrated cells were removed. The cells on the Transwell membranes were fixed with 4% paraformaldehyde for 10 min and stained with 0.5% crystal violet solution. After being rinsed with tap water, the migrated cells were counted under an inverted microscope. Invasion experiments were performed with Transwell chambers coated with Matrigel, and the remaining procedures were the same as the migration assay.

Western blot
Cellular protein was extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and protein concentration was determined using a BCA kit (Beyotime Biotechnology, Shanghai, China). After SDS-PAGE, the protein samples were transferred to PVDF membranes and blocked with 5% skimmed milk for 1 h at room temperature. The corresponding primary antibodies were then added to interact with the proteins at 4°C overnight, and then the membrane was rinsed with tris buffered saline with Tween 20 (TBST). Next, the secondary antibodies were added to incubate the membrane for 2 h at room temperature before the membrane was rinsed with TBST again, and then ECL chemiluminescent kit (Millipore, Billerica, MA, USA) was added onto the membrane to develop the bands. The antibodies used in this study, including anti-E2F3 (ab50917, 1:500), anti-Bax (ab32503, 1:500), anti-Bcl-2 (ab185002, 1:500), anti-E-cadherin (ab11512, 1:500), anti-N-cadherin (ab18203, 1:500) and antiβ-actin (ab179467, 1:2000), were all bought from Abcam (Shanghai, China).

RIP experiment
A549 and H226 cells were transfected with pcDNA3.1-MCM3AP-AS1 vector or pcDNA3.1 vector. Forty-eight hours later, the cells were subjected to RIP assays using anti-Ago2 antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer's instruction. qRT-PCR was conducted to detect MCM3AP-AS1 and miR-195-5p expressions in the immunoprecipitate.

Lung metastasis model in vivo
The animal experiments were approved by the Animal Research Ethics Committee of Cancer Hospital of the University of Chinese Academy of Sciences (Approval Number: 2020-06). Ten male BALB/c nude mice (6-8 weeks old, average weight: 24.5 g ± 2.20) were purchased from Zhejiang Province Experimental Animal Center (Hangzhou, China). Mice were maintained under standard housing conditions (23°C, 40% humidity, 12 h light cycles, and free access to food and water). The mice were randomly divided into two groups (NC group and MCM3AP-AS1 overexpression group, n = 5 in per group). A549 cells (2 × 10 7 cells/per mouse) were injected into each mouse via tail vein. Three weeks later, the mice were euthanized with 100% oxygen/5% isoflurane, and the bilateral thoracotomy was used to confirm the death. Lung tissues were removed after lavage, fixed in 10% neutral formalin for 36 hours, and embedded in paraffin. Then, hematoxylin/eosin staining was performed for pathological examination of lung metastatic nodules of the mice. The number of pulmonary metastatic nodules in each section was counted in 5 randomly selected visual fields under the microscope (Olympus, Tokyo, China).

Statistical analysis
All the experiments were conducted in triplicate. The data were processed by GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA), plotted and represented as mean ± standard deviation. To make the comparison between two groups, One-Sample Kolmogorov-Smirnov test was used to examine whether the data are normally distributed. For the data which were normally distributed, independent sample t test was used. For the data which were skewed distributed, paired sample Wilcoxon signed rank test was used. One-way ANOVA test was performed to make the comparison among three or more groups. If there was a significant difference, Newman-Keuls analysis was performed to make the comparison between two groups. Pearson correlation analysis was used to determine the correlation. P < 0.05 signified statistical significance.

Results
In this study, we performed a series of experiments to investigate the biological function of MCM3AP-AS1 in NSCLC progression, and explore the regulatory mechanism of MCM3AP-AS1/miR-195-5p/E2F3 axis, which were aimed to help clarify the mechanism of NSCLC progression.

MCM3AP-AS1 and E2F3 expressions were up-regulated, and miR-195-5p expression was down-regulated in NSCLC tissue specimens
First of all, gene expression analysis and survival analysis were conducted using the GEPIA database. As shown, MCM3AP-AS1 expression was upregulated in both lung adenocarcinoma tissues (LUAD) and lung squamous carcinoma (LUSC) tissues; additionally, MCM3AP-AS1 overexpression was significantly linked to a shorter overall survival time of NSCLC patients (Figure 1a-b). Then, the expression patterns of MCM3AP-AS1, miR-195-5p and E2F3 in paired specimens collected from 63 patients with NSCLC were examined by qRT-PCR, respectively. We found significant up-regulation of both MCM3AP-AS1 and E2F3 expressions and downregulation of miR-195-5p expression in NSCLC tissue specimens, in comparison with these in paired adjacent normal tissues (Figure 1c-e). Pearson's correlation analysis further indicated that MCM3AP-AS1 and E2F3 were negatively correlated with miR-195-5p; conversely, MCM3AP-AS1 expression was positively correlated with E2F3 expression in NSCLC samples (Figure 1f-h). StarBase database also suggested that MCM3AP-AS1 expression and E2F3 expression were negatively correlated with miR-195-5p expression in NSCLC samples; conversely, MCM3AP-AS1 expression was positively correlated with E2F3 expression (Supplementary Figure 1a-c). Moreover, statistically, high MCM3AP-AS1 expression was closely associated with the larger tumor size, low differentiation and higher TNM stage of the NSCLC patients ( Table  2). These results suggested that MCM3AP-AS1 was an oncogenic factor for NSCLC and might have regulatory functions on miR-195-5p and E2F3.

The effect of MCM3AP-AS1/miR-195-5p axis on the proliferation of NSCLC cells
To clarify the function of MCM3AP-AS1 in NSCLC progression, A549 cells were cotransfected with MCM3AP-AS1 overexpression plasmid and miR-195-5p mimics; H226 cells were co-transfected with sh-MCM3AP-AS1#1 and miR-195-5p inhibitors, and qRT-PCR showed that the transfection was successful (Figure 3a). The proliferation of these two cell lines was assessed by CCK-8 and EdU assays, and the data presented that the proliferation of NSCLC cells was markedly increased after the transfection with MCM3AP-AS1 overexpression plasmid, while miR-195-5p mimics abolished this effect; the proliferation of NSCLC cells was remarkably decreased after the transfection with sh-MCM3AP-AS1#1, while cotransfection of miR-195-5p inhibitors reversed this effect (Figure 3b-d). Western blot assay suggested that after MCM3AP-AS1 overexpression, Bcl-2 protein level was increased and BAX protein level was decreased, while miR-195-5p mimics counteracted such effect; after knockdown MCM3AP-AS1, Bcl-2 protein level was declined and Bax protein level was elevated, while miR-195-5p inhibitors reversed this effect (Figure 3ef). These results suggested that MCM3AP-AS1 promoted the growth and repressed the apoptosis of NSCLC cells, which was mediated by its inhibitory function on miR-195-5p.

The effect of MCM3AP-AS1/miR-195-5p axis on the metastasis of NSCLC cells
The migration and invasion of these two cell lines were assessed using Transwell assay, and the results showed that the number of migrated and invaded cells was significantly raised after NSCLC cells were transfected with MCM3AP-AS1 overexpression plasmid, while miR-195-5p mimics abrogated such effect (Figure 4a-b); the number of migrated and invaded cells was markedly reduced after the transfection with sh-MCM3AP-AS1#1 while miR-195-5p inhibitors reversed such effect (Figure 4c-d). After MCM3AP-AS1 overexpression, E-cadherin protein level was decreased and N-cadherin protein level was increased, while miR-195-5p mimics reversed this effect; after knockdown MCM3AP-AS1, E-cadherin protein level was elevated and N-cadherin protein level was declined, while miR-195-5p inhibitors reversed such effect (Figure 4e-f). Lung metastasis experiments in nude mice indicated that overexpression of MCM3AP-AS1 promoted lung metastasis in vivo (Supplementary Figure 1d). These results manifested that MCM3AP-AS1 facilitated the metastasis of NSCLC, which was mediated by its inhibitory function on miR-195-5p.

Discussion
LncRNA can regulate the expressions of downstream genes at the different levels, including chromatin modification, transcription or posttranscription, thus participating in various biological processes, including cell proliferation, migration and apoptosis [24,25]. The role of lncRNA in cancer biology is reported in a lot of previous research. For example, in NSCLC, lncRNA KCNQ1OT1 and AWPPH expressions are significantly up-modulated in tumor tissues, and their high expressions were correlated to adverse prognosis; KCNQ1OT1 and AWPPH can enhance the proliferation, migration and invasion of NSCLC cells, respectively [26,27]. Reportedly, MCM3AP, which serves as an essential modulator in DNA replication by acetylating micro-chromosome maintenance protein 3 (MCM3), impedes cell cycle progression and modulates gene expression in tumors [28]. MCM3AP-AS1 is the lncRNA antisense of MCM3AP gene. MCM3AP-AS1 is overexpressed in hepatocellular carcinoma tissues and MCM3AP-AS1 knockdown inhibits the malignant phenotypes of hepatocellular carcinoma cells; mechanistically, MCM3AP-AS1 promotes epidermal growth factor receptor (EGFR) expression by the adsorption of miR-455, which in turn enhances the metastasis of hepatocellular carcinoma [29]. Another study reveals that MCM3AP-AS1 regulates the progression of hepatocellular carcinoma via miR-194-5p/ FOXA1 axis [30]. In NSCLC, MCM3AP-AS1 knockdown impedes cell proliferation and migration, and YY1 transcription factor (YY1) mediates the transcription of MCM3AP-AS1 in NSCLC [14]. In this study, it was verified that MCM3AP-AS1 expression was up-modulated in NSCLC, and its overexpression was associated with unfavorable prognosis of patients and MCM3AP-AS1 facilitated the proliferation and metastasis of NSCLC cells. Our data prove that MCM3AP-AS1 is a promising biomarker and therapy target for NSCLC, which is consistent with the previous reports [14].
E2F3, which is an important cell cycle regulator and figures prominently in regulating cell proliferation, apoptosis and differentiation, belongs to the E2F transcriptional regulatory family and is closely related to tumorigenesis [40][41][42]. Reportedly, E2F3 expression is upregulated in tumors, such as osteosarcoma and breast cancer, and enhances the proliferation and colony formation of tumor cells [42,43]. In NSCLC, E2F3 improves the malignancy of cancer cells by increasing the expressions of cyclinD1, cyclinD2, and CDK4 while inhibiting p21 and p57 expressions [44,45]. The present work verified the binding sites between miR-195-5p and E2F3 3ʹUTR and proved that MCM3AP-AS1 could up-regulate E2F3 expression by adsorbing miR-195-5p, which explained the mechanism of E2F3 dysfunction in NSCLC. Importantly, our data also suggest that MCM3AP-AS1, miR-195-5p and E2F3 form a novel ceRNA network to take part in regulating NSCLC progression.
MCM3AP-AS1 as a biomarker to predict the prognosis of NSCLC patients' needs to be further verified by a larger cohort of patients from different medical centers. Second, whether MCM3AP-AS1 can promote other malignant phenotypes of NSCLC cells, such as drug resistance and radioresistance, needs further investigation. Furthermore, there are other potential downstream miRNAs of MCM3AP-AS1 remained to be screened and validated in the future.