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Long non-coding RNA MCM3AP-AS1 facilitates colorectal cancer progression by regulating the microRNA-599/ARPP19 axis

Colorectal cancer (CRC) is one of the most aggressive malignancies worldwide. Increasing evidence has indicated that microRNA (miR)-599 is involved in the occurrence and development of different types of tumors, such as breast cancer and glioma. However, the role of miR-599 in CRC remains unclear. Thus, the present study aimed to identify the regulatory mechanism of miR-599 in CRC progression. Reverse transcription-quantitative PCR was used to analyze the expression levels of MCM3AP-AS1, miR-599 and ARPP19, and Cell Counting Kit-8 and Transwell assays were used to determine the cell proliferation and migration of CRC cells. In addition, a Dual-luciferase reporter assay was used to analyze the direct interaction between miR-599 and MCM3AP-AS1 or ARPP19. Reverse transcription-quantitative PCR analysis demonstrated that miR-599 expression decreased in patients with CRC and in CRC cell lines, while miR-599 overexpression inhibited cell proliferation and migration abilities in vitro. MCM3AP-AS1 was identified as a molecular sponge of miR-599, and further investigation indicated that MCM3AP-AS1 silencing inhibited cell proliferation and migration of the CRC cell lines. In addition, ARPP19 was identified as a target gene of miR-599, and MCM3AP-AS1-knockdown decreased ARPP19 mRNA expression and increased miR-599 expression. Furthermore, silencing ARPP19 inhibited the proliferation and migration of the CRC cell lines. The results also demonstrated that MCM3AP-AS1 promoted CRC cell progression by regulating the miR-599/ARPP19 axis. Taken together, the results of the present study suggest that MCM3AP-AS1 may be a novel therapeutic target for patients with CRC.


Introduction
Colorectal cancer (CRC) is one of the most common malignancies with 1.4 million new cases and >690,000 associated mortalities in 2012 (1). Recent advancements in prognosis and therapeutic strategies in CRC have been made (2); however, due to metastasis and the fast-growing nature, the 5-year survival rate of patients with CRC remains poor, at only ~14% (3)(4)(5). Thus, it remains critical to develop novel reliable molecular markers for CRC treatment.
MicroRNAs (miRNAs/miRs) are small RNA molecules, 21-25 nucleotides in length, which have the ability to induce mRNA degradation or repress translation by binding to the 3'-untranslated region of RNA transcripts (6). It has been reported that dysregulated miRNAs are frequently involved in different types of human cancer, as both oncogenes and tumor suppressors. For example, overexpression of miR-187 inhibits cell proliferation and metastasis of glioma by downregulating SMAD1 expression (7). miR-599 has been reported to act as a tumor suppressor in several types of cancer. For example, miR-599 suppresses glioma progression by targeting RAB27B (8). However, the regulatory mechanism of miR-599 in CRC remains unclear.
Long non-coding (lnc)RNAs are RNA molecules, >200 nucleotides in length, which are involved in different types of cellular processes, such as proliferation, migration and differentiation (9)(10)(11)(12)(13). Previous studies have demonstrated that lncRNAs are important regulatory factors that participate in cell proliferation, invasion and metastasis (14)(15)(16). For example, lncRNA RBM5-AS1 promotes the aggressive behaviors of oral squamous cell carcinoma by regulating the miR-1285-3p/YAP1 axis (17). A previous study also demonstrated that lncRNAs can regulate the progression of different types of cancer by sponging miRNAs. For example, lncRNA RBMS3-AS3 acts as a miR-4534 sponge to inhibit prostate cancer progression by upregulating VASH1 expression (18). lncRNA SNHG17 acts as a ceRNA of miR-324-3p to contribute the progression of osteosarcoma (19). MCM3AP-AS1 has been reported to be involved in several types of cancer. For example, Yang et al (20) demonstrated that the MCM3AP-AS1/miR-211/KLF5/AGGF1 axis regulates glioblastoma angiogenesis, while Li et al (21) reported that overexpression of lncRNA MCM3AP-AS1 promotes lung cancer progression via the miR-340-5p/KPNA4 axis. In addition, Yang et al (22) revealed that MCM3AP-AS1 sponges miR-138 by modulating FOXK1 expression to promote pancreatic cancer cell proliferation and migration. However, the molecular mechanism of MCM3AP-AS1 in CRC remains largely unknown.
The present study investigated the effects and mechanisms of lncRNA MCM3AP-AS1 on the proliferation and migration of CRC cells.

Materials and methods
Patients and tissue samples. CRC tissues and adjacent normal tissues were collected from 30 patients (18 males and 12 females) with a median age of 56 years (range, 38-67 years) between January 2018 and January 2019 at The Bishan Hospital of Chongqing (Chongqing, China). Adjacent normal tissues of the patients were at least 1-cm away from the tumor tissues. The specimens were immediately preserved at -80˚C following surgical resection. The present study was approved by the Ethics Committee of The Bishan Hospital of Chongqing and written informed consent was provided by all patients prior to the study start. Reverse transcription-quantitative (RT-q)PCR. Total RNA was extracted from CRC tissues, adjacent normal tissues, HCT-116, SW620, SW480, LoVo and NCM460 cells using TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and reverse transcribed into cDNA using the RT kit (cat. no. RR047A; Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. qPCR was subsequently performed using the SYBR-Green PCR Master Mix kit (cat. no. DRR041A; Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. The following thermocycling conditions were used for qPCR: Pre-denaturation at 95˚C for 15 sec, denaturation at 94˚C for 30 sec, annealing at 60˚C for 20 sec and extension at 72˚C for 40 sec for 40 cycles. The primers were designed as follows: MCM3AP-AS1 forward, 5'-CGA TTT GGC GTT AAC TTA TTG A-3'; and reverse, 5'-ATC ATT ACA GAA CAT AAT CAA CAG GTA-3'; ARPP19 forward, 5'-ACT CAT GCA GCG GAG CTC TAG-3'; and reverse, 5'-GTT CAG AGA TGA AGG TCC GAA CA-3'; miR-599 forward, 5'-TCG GCA GGU UCA AGC CAG GGG-3'; and reverse, 5'-CAC TCA ACT GGT GTC GTG GA-3'; GAPDH forward, 5'-CGG AGT CAA CGG ATT TGG TCG TAT-3'; and reverse, 5'-AGC CTT CTC CAT GGT GGT GAA GAC-3'; U6 forward, 5'-GCT TCG GCA GCA CAT ATA CTA AAA T-3'; and reverse, 5'-CGC TTC ACG AAT TTG CGT GTC AT-3'. GAPDH was used as an internal control for MCM3AP-AS1 and ARPP19, while U6 was used as an internal control for miR-599. Relative mRNA expression levels were quantified using the 2 -ΔΔCq method (23).
Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was assessed via the CCK-8 assay (Dojindo Molecular Technologies, Inc.). Briefly, HCT-116 and SW480 cells were seeded into 96-well plates at a density of 2x10 4 cells/well. Following transfection for 48 h, CCK-8 reagent was added to the wells at 37˚C for 2 h, according to the manufacturer's instructions and cell proliferation was subsequently analyzed at a wavelength of 450 nm, using a microplate reader (Thermo Fisher Scientific, Inc.).
Bioinformatic prediction and Dual-luciferase reporter assay. StarBase 2.0 (http://starbase.sysu.edu.cn) was used to predict the potential downstream targets of MCM3AP-AS1 and miR-599. To assess the binding ability between different RNAs, wild type or mutant fragments (MCM3AP-AS1 and ARPP19) were amplified and cloned into the pGL3 luciferase vector (Promega Corporation). The wild-type or mutant fragments were subsequently transfected into 293T cells (American Type Culture Collection), which had been co-transfected with miR-599 mimics or NC mimics. Transfection was performed using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 48 h at 37˚C, a Dual-luciferase reporter system (Promega Corporation) was used to measure the luciferase gene activity. Firefly luciferase activity was normalized to that of Renilla luciferase.
Migration assay. The migration efficiency of the CRC cell lines was assessed via the Transwell assay. The transfected HCT-116 and SW480 cells were plated in the upper chamber at the density of 2x10 5 cells per well in serum-free DMEM. DMEM supplemented with 10% FBS was plated in the lower chambers. Following incubation for 48 h at 37˚C, the migratory cells were stained with 0.1% crystal violet for 20 min at room temperature.and counted under a light microscope (magnification, x200; Olympus Corporation).
Statistical analysis. Statistical analysis was performed using SPSS v20.0 software (IBM Corp.). All experiments were performed in triplicate and data are presented as the mean ± standard deviation. Comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey's post hoc test. Comparisons between CRC tissues and adjacent normal tissues were performed using a paired Student's t-test, while comparisons between the experimental and control groups was performed using an unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Results
Decreased miR-599 expression affects the migratory ability of CRC cell lines. The results demonstrated that miR-599 expression was significantly decreased in the CRC tissues and cell lines (HCT-116, SW620, SW480 and LoVo) compared with that in adjacent normal tissues and the NCM460 cell line (Fig. 1A and B). Especially, miR-599 expression was significantly higher in HCT-116 and SW480 cells, compared with NCM460 cells. Thus, HCT-116 and SW480 cells were selected to perform subsequent experiments. RT-qPCR analysis demonstrated that transfection with miR-599 mimics significantly enhanced miR-599 expression in HCT-116 and SW480 cells (Fig. 1C). Furthermore, the results from the CCK-8 assay indicated that overexpression of miR-599 significantly inhibited cell proliferation (Fig. 1D), while the results from the Transwell assay demonstrated that miR-599 overexpression significantly decreased the migratory ability of the CRC cell lines (Fig. 1E). Collectively, these results suggest that miR-599 has an antitumor effect on CRC cell lines.
MCM3AP-AS1 sponges miR-599 to accelerate CRC progression. lncRNAs exert their functions by interacting with downstream target miRNAs (24). In the present study, a potential target site between miR-599 and MCM3AP-AS1 was predicted using StarBase software ( Fig. 2A). A Dual-luciferase reporter assay was performed for validation, and the results demonstrated that transfection with miR-599 mimics significantly suppressed the luciferase activity of wild-type MCM3AP-AS1; however, no effect was observed on mutant MCM3AP-AS1 (Fig. 2B). In addition, MCM3AP-AS1 mRNA expression was significantly elevated in the CRC cell lines compared with that in the NCM460 cell line (Fig. 2C).

MCM3AP-AS1 accelerates CRC progression via the miR-599/ARPP19 axis.
To determine whether MCM3AP-AS1 promoted CRC progression via the miR-599/ARPP19 axis, a series of rescue experiments were performed. The results demonstrated that ARPP19 mRNA expression significantly decreased following transfection with sh-ARPP19 in SW480 cells (Fig. 4A). In addition, SW480 cell proliferation and migration were attenuated following ARPP19-knockdown, respectively ( Fig. 4B and C). SW480 cells were transfected with pcDNA3.1, pcDNA3.1/MCM3AP-AS1 and pcDNA.1/MCM3AP-AS1+miR-599, and the results demonstrated that MCM3AP-AS1 and ARPP19 expression levels increased following overexpression of MCM3AP-AS1, the effects of which were partially reversed following transfection with the miR-599 mimic ( Fig. 4D and E). In addition, miR-599 expression decreased following overexpression of MCM3AP-AS1, the effect of which was partially reversed following transfection with the miR-599 mimic (Fig. 4F). The results from the CCK-8 and Transwell assays demonstrated that overexpression of MCM3AP-AS1 promoted SW480 cell proliferation and migration, respectively, the effects of which were partially reversed following overexpression of miR-599 ( Fig. 4G and H). Taken together, these results suggest that MCM3AP-AS1 sponges miR-599 and increases ARPP19 mRNA expression to accelerate CRC progression.

Discussion
Increasing evidence has indicated that miRNAs may have an antitumor role in different types of tumor. For example, miR-192 has been demonstrated to inhibit the progression of lung cancer bone metastasis by regulating TRIM44 (28). miR-506-3p inhibits papillary thyroid cancer cells tumorigenesis via targeting YAP1 (29). miR-296-5p suppresses the tumorigenesis of esophageal squamous cell carcinoma cells via inhibiting STAT3 signaling (30). The present study demonstrated that miR-599 was involved in CRC, whereby its expression was decreased in the examined CRC cell lines. In addition, overexpression of miR-599 inhibited cell proliferation and migration in CRC.
lncRNAs have been reported to act as competing endogenous (ce)RNAs that sponge miRNAs. For example, lncRNA FAL1 acts as a ceRNA of miR-1236 to promote cell proliferation and migration in hepatocellular carcinoma (HCC) (31), while lncRNA-UCA1 acts as a ceRNA of Sox4 to promote cell proliferation in esophageal cancer (32). In the present study, MCM3AP-AS1 was demonstrated to negatively regulate miR-599 via direct interaction. Notably, MCM3AP-AS1 has been reported to participate in the progression of different types of cancer. For example, Liang et al (33) demonstrated that MCM3AP-AS1 promotes proliferation and invasion in papillary thyroid cancer by regulating the miR-211-5p/SPARC axis. In addition, Wang et al (34) reported that MCM3AP-AS1 promotes HCC progression by regulating the miR-194-5p/FOXA1 axis. However, the molecular mechanism of MCM3AP-AS1 in CRC remains unclear. The results from the present study demonstrated that MCM3AP-AS1 sponged miR-599 to accelerate CRC progression. In addition, MCM3AP-AS1 mRNA expression was increased in the CRC cell lines. In vitro functional assays were performed and the results demonstrated that MCM3AP-AS1-knockdown significantly decreased the proliferation and migration of CRC cells. Collectively, the results of the present study demonstrated that MCM3AP-AS1 accelerated CRC progression by binding to miR-599.
Previous studies have reported that miRNAs are involved in tumor progression by targeting mRNAs. For example, miR-26a inhibits thyroid cancer cell proliferation by targeting ARPP19 (35). In addition, miR-9-5p targets GOT1 to inhibit pancreatic cancer cell proliferation, invasion and metabolism (36). In the present study, bioinformatics analysis and the Dual-luciferase reporter assay confirmed that miR-599 directly binds to ARPP19. Furthermore, MCM3AP-AS1-knockdown decreased ARPP19 mRNA expression by promoting miR-599 expression. The results also demonstrated that ARPP19-knockdown attenuated the proliferation and migration of CRC cells. In addition, overexpression of MCM3AP-AS1 increased ARPP19 mRNA expression, the effect of which was reversed following transfection with miR-599 mimics. Notably, MCM3AP-AS1 promoted CRC progression via the miR-599/ARPP19 axis.
In conclusion, the results of the present study suggest that lncRNA MCM3AP-AS1 exerts its biological functions by modulating the miR-599/ARPP19 axis, thus MCM3AP-AS1 may be an effective therapeutic target for CRC. However, the current study is limited by the small sample size. Besides, in vivo interactions among MCM3AP-AS1, miR-599 and ARPP19 have not been explored. Future research is required to include in vivo experiments and enroll more patients to further verify these conclusions.