A novel lncRNA MCM3AP-AS1 promotes the growth of hepatocellular carcinoma by targeting miR-194-5p/FOXA1 axis

A total of 80 pairs of HCC and tumor-adjacent tissues were collected from patients who underwent hepatectomy at the First Affiliated Hospital of Xi’an Jiaotong University (Xi’an, China). None of HCC patients received any pre-operative treatments, such as radiofrequency ablation (RFA), transcatheter arterial chemoembolization (TACE), immunotherapy and targeted therapy. The tissue samples were confirmed by two histopathologists. All samples were immediately snap-frozen in liquid nitrogen and subsequently stored at − 80 °C until RNA extraction and protein isolation. The demographic and clinicopathological features for HCC patients were described in Table 1.

The human immortalized normal hepatocyte cell line LO2 and HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721 were maintained in our lab [26]. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin, Sigma, St-Louis, MO, USA) in a humidified incubator containing 5% CO₂ at 37 °C.

Two lentivector-mediated short-hairpin MCM3AP-AS1 (sh-MCM3AP-AS1–1 and sh-MCM3AP-AS1–2) and non-targeting plasmids (sh-control) were designed and synthesized by Geneseed Biotech (Guangzhou, China). Lentivirus infection of HCC cells was performed in the presence of Polybrene (8 ng/ml). The cDNA encoding MCM3AP-AS1 was PCR-amplified by the Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix (Thermo-Fisher Scientific, Waltham, MA, USA) and subcloned into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA). The empty plasmid pcDNA3.1 was used as negative control (EV). Hsa-miR-194-5p mimics and negative control mimics were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The plasmid expressing FOXA1 was previously described [27]. A small interfering RNA (siRNA) targeting AGO2 and scrambled siRNA were obtained from Geneseed Biotech. Plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol.

TRIzol reagent (Invitrogen) was used for total RNA isolation from HCC tissues and cultured cells. Total RNA was reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo-Fisher Scientific). qRT-PCR analyses were performed using SYBR® Premix Ex Taq™ II (Takara, Dalian, China) and Taqman UniversalMaster Mix II (Life Technologies Corporation, Carlsbad, CA, United States) on an ABI PRISM 7300 Sequence Detection system (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturers’ instructions. The 2^-ΔΔCt method was used to calculate the relative gene expression normalized by GAPDH and U6. The sequences of the primers were listed in Table 2.

HCC tissues and cells were lysed with RIPA buffer (Beyotime, Shanghai, China) and protein concentrations were quantified with a BCA protein assay kit II (BIO-RAD, Hercules, CA, USA). Protein samples were separated by 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Invitrogen). The membranes were incubated with rabbit-anti-human FOXA1 (ab170933; Abcam, Cambridge, MA, USA), rabbit-anti-human PARP1 (ab191217; Abcam), rabbit-anti-human caspase-3 (ab32351; Abcam), rabbit-anti-human caspase-7 (ab32522; Abcam), rabbit-anti-human Cyclin D1 (ab134175; Abcam), rabbit-anti-human p21 (ab109520; Abcam), mouse-anti-human β-actin (ab8226; Abcam) and mouse-anti-human GAPDH primary antibody (sc-47,724; Santa Cruz Biotechnology, Santa Cruz, Dallas, TX, USA) overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (NXA931–1ML and NA934-1ML, GE Healthcare Life Sciences, Beijing, China) were used to incubate the membranes for 1 h at room temperature. The blot signals on the membrane were visualized with ECL reagents (Millipore, Plano, TX, USA) and detected using Amersham™ Imager 680 from GE Healthcare Life Sciences.

HCC cells suspensions were added to a 96-well plate at a density of 1 × 10⁴/mL. A total of 10 μL of CCK-8 solution (Dojindo, Tokyo, Honshu, Japan) was added to each well at the same time every day for 3 days. Finally, the absorbance at 450 nm was measured using a microplate reader (Thermo-Fisher Scientific) after a 2 h incubation.

Forty-eight hours after transfection, HCC cells (1 × 10³ per well) were seeded in a 6-well plate and cultured with complete medium for 2 weeks. Cell colonies were fixed with 4% paraformaldehyde for 30 min and stained with 0.5% crystal violet for 30 min at room temperature.

EdU incorporation assay was performed with the EdU kit (Roche, Indianapolis, IN, USA) in accordance with the manufacturer’s instruction. Results were acquired using the Zeiss fluorescence photomicroscope (Carl Zeiss, Oberkochen, Germany) and quantified via counting at least five random fields.

For cell cycle analysis, cells were collected and fixed using 70% ethanol. After washing with PBS and subsequently washing with stain buffer, 1 × 10⁶ cells were resuspended in 0.5 mL of PI/RNase Staining Buffer (BD biosciences, San Jose, CA, USA), and cells were incubated for 15 min at room temperature (RT), protected from light. A FACSCanto II flow cytometer (BD biosciences) was used to analyze cell cycle distribution. For apoptosis assay, the PE Annexin V Apoptosis Detection Kit I (BD biosciences) was used following the manufacturer’s protocols. Cells were harvested and washed with pre-cold PBS buffer twice. Then, 5 μl of PE Annexin V and 5 μl of 7-AAD solution were added to each sample, and cells were incubated for 15 min at RT. FACSCanto II flow cytometer was used to measure the cell apoptosis.

BALB/c nude mice (male, 4–5-week-old, 18-20 g) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and randomly divided into two groups (n = 6 per group). Hep3B cells (1 × 10⁶ per injection) that were transfected with sh-MCM3AP-AS1 and sh-control, respectively, were implanted into the right flank of the mice via subcutaneous injection. Tumor volumes were measured every 3 days after being apparently observed and calculated with the following formula: Volume = (length × width²)/2. After 3 weeks, all mice were sacrificed under anesthesia. Tumor tissues were harvested and subjected to immunohistochemistry for Ki-67 staining [28]. The animal experiments were approved by the Animal Care and Use Committee of Xi’an Jiaotong University.

HepG2 cells were transfected with biotinylated wild type (wt) miR-194-5p, mutant (mt) miR-194-5p and negative control (NC) (Guangzhou RiboBio Co., Ltd). Cell lysates were harvested 48 h after transfection and incubated with Dynabeads M-280 Streptavidin (Invitrogen, CA, USA) for 3 h at 4 °C according to the manufacturer’s protocol. Then, the beads were washed three times with ice-cold lysis buffer and once with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0 and 500 mM NaCl) [29]. The bound RNAs were purified using TRIzol for the qRT-PCR analysis.

RIP assay was performed with the EZ-Magna RIP Kit (Millipore, Bedford, MA, USA) and a AGO2 antibody (Millipore) as previously described [26]. qRT-PCR was carried out to detect co-precipitated RNAs.

The sequence of 3′-UTR of FOXA1 or MCM3AP-AS1 was amplified from human genomic DNA. Then these sequences were respectively subcloned into pGL3 luciferase reporter vector (Promega, Madison, WI, USA). The potential miR-194-5p binding sites were mutated by the Quick-change site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). The wt (mt) 3′-UTR of FOXA1 vector or wt (mt) MCM3AP-AS1 vector and control mimics or miR-194-5p mimics were co-transfected into HepG2 and SMMC-7721 cells. The luciferase activity was measured and normalized as previously described [26].

Cy3-labeled MCM3AP-AS1 probe were obtained from RiboBio (Guangzhou, China). Subcellular localization of MCM3AP-AS1 was detected by the FISH Kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions [30].

The exact copy numbers of MCM3AP-AS1 and miR-194-5p transcripts per Hep3B and HepG2 cell were quantified by using quantitative real-time RT-PCR assay. In this assay, serially diluted RT-PCR products of MCM3AP-AS1 and miR-194-5p were used as templates to formulate standard curves, and then, the exact copies of MCM3AP-AS1 and miR-194-5p per cell were calculated accordingly.

To further disclose differentially expressed lncRNAs in HCC, we analyzed a microarray data comparing the expression of lncRNAs in 50 HCC tissues and 5 adjacent non-tumor tissues from the GEO database with the accession number GSE65485. Thirty-seven lncRNAs were differentially expressed in HCC tissues compared to adjacent non-tumor tissues (Table 3). A novel lncRNA MCM3AP-AS1 (also known as MCM3APAS), which was 2.84-fold higher in HCC tissues than that in adjacent non-tumor tissues, caught our attention. Next, we search for the expression pattern of MCM3AP-AS1 in HCC based on TCGA data from starBase V3.0 [31]. The results showed that MCM3AP-AS1 in HCC tissues was significantly higher than that in normal liver tissues (P < 0.0001, Additional file 1: Figure S1). Furthermore, qRT-PCR analysis of MCM3AP-AS1 in 80 pairs of HCC and matched tumor-adjacent tissues revealed that MCM3AP-AS1 was significantly overexpressed in HCC tissues compared to tumor-adjacent tissues (P < 0.0001, Fig. 1a). Elevated expression of MCM3AP-AS1 also observed in HCC cell lines (HepG2, Hep3B, SMMC-7721 and Huh7) compared to LO2 cells (P < 0.05, Fig. 1b). Moreover, based on two other GEO datasets (GSE45436 and GSE54236) from R2: Genomics Analysis and Visualization Platform (http://​r2.​amc.​nl), we found that MCM3AP-AS1 expression was prominently higher in HCC tissues compared to normal liver tissues (P < 0.0001, Fig. 1c and d). Thus, these results indicated that MCM3AP-AS1 upregulation was a frequent event in HCC.

Next, we aimed to reveal the clinical significance of MCM3AP-AS1 in HCC. TCGA data from R2: Genomics Analysis and Visualization Platform (http://​r2.​amc.​nl) revealed that MCM3AP-AS1 was more highly expressed in HCC with high tumor grades (G3 + G4) than that in HCC with low tumor grades (G1 + G2) (P = 0.0032, Fig. 2a). Furthermore, MCM3AP-AS1 was also more highly expressed in HCC with advanced tumor stages (III-IV) than that in HCC with early tumor stages (I-II) (P = 0.0013, Fig. 2b). We divided HCC patients into tow subgroups (low/high MCM3AP-AS1 level) by using the median of the cohort as a cut-off value. As shown in Table 1, the correlation analysis between MCM3AP-AS1 expression and clinicopathologic characteristics of these 80 HCC patients indicated that high expression of MCM3AP-AS1 was positively correlated with large tumor size (P = 0.006), high tumor grade (P = 0.039), and advanced TNM stages (P = 0.004). Kaplan-Meier survival analysis showed that HCC patients with high MCM3AP-AS1 expression had a significant poorer overall survival than those with low MCM3AP-AS1 expression (P = 0.0054, Fig. 2c). Furthermore, TCGA data from OncoLnc (http://​www.​oncolnc.​org/​) further demonstrated that high MCM3AP-AS1 expression also indicated poor survival of HCC patients (P = 0.0112, Fig. 2d). Collectively, our data showed that high MCM3AP-AS1 expression was associated with poor clinical outcomes of HCC patients.

Since MCM3AP-AS1 expression was associated with tumor size, we next disclosed the biological roles of MCM3AP-AS1 in HCC cell growth. MCM3AP-AS1 were stably depleted in HepG2 and Hep3B cells with different specific shRNAs (P < 0.05, Fig. 3a). CCK-8 assays indicated that MCM3AP-AS1 knockdown significantly inhibited the proliferation of HepG2 and Hep3B cells (P < 0.05, Fig. 3b). Colony formation and EdU incorporation assays also indicated that MCM3AP-AS1 silencing prominently suppressed the growth of HepG2 and Hep3B cells (P < 0.05. Figure 3c and d). Moreover, flow cytometry assays revealed that the percentage of apoptotic HCC cells were obviously increased by MCM3AP-AS1 knockdown (P < 0.05. Figure 3e). Depletion of MCM3AP-AS1 led to cell cycle arrest at G1 phase in HepG2 and Hep3B cells (P < 0.05, Fig. 3f). Furthermore, MCM3AP-AS1 knockdown led to increased levels of cleaved PARP1, cleaved caspase-3, cleaved caspase-7 and p21, and decreased Cyclin D1 expression in HCC cells (P < 0.05, Additional file 2: Figure S2). Notably, MCM3AP-AS1 knockdown did not significantly affected the growth of LO2 cells, which had low MCM3AP-AS1 expression (Additional file 3: Figure S3). Thus, these results showed that knockdown of MCM3AP-AS1 repressed the proliferation, cell cycle progression and induced apoptosis of HCC cells in vitro.

To further elucidate the biological roles of MCM3AP-AS1 in HCC tumorigenesis in vivo, Hep3B cells with MCM3AP-AS1 knockdown were implanted into nude mice via subcutaneous injection. The results of tumor growth curves and tumor weight indicated that MCM3AP-AS1 knockdown obviously reduced tumor growth in mice (P < 0.05, Fig. 4a and b). Tumor tissues were harvested for qRT-PCR analysis of MCM3AP-AS1. We confirmed that lower expression of MCM3AP-AS1 was detected in tumor tissues arising from MCM3AP-AS1 knockdown group compared to control group (P < 0.05, Fig. 4c). Ki-67 immunostaining indicated that the subcutaneous tumors formed by MCM3AP-AS1 knockdown Hep3B cells showed less Ki-67 positive cells compared to those formed by control Hep3B cells (P < 0.05, Fig. 4d). Altogether, these results suggested that MCM3AP-AS1 knockdown suppressed HCC tumorigenesis in vivo.

To investigate the mechanisms underlying the role of MCM3AP-AS1 in HCC, we predicted 31 miRNAs containing binding site of MCM3AP-AS1 by using starBase V3.0. Among these miRNAs, we found that only low expression of miR-194-5p and miR-23c associated with poor prognosis of HCC patients based on TCGA data from OncoLnc (P < 0.05, Additional file 4: Figure S4). Thus, we detected expression of miR-194-5p and miR-23c in HepG2 cells after MCM3AP-AS1 knockdown. Our data revealed that MCM3AP-AS1 knockdown significantly increased miR-194-5p expression rather than miR-23c (P < 0.05, Fig. 5a). Consistent results were confirmed in Hep3B cells (P < 0.05, Fig. 5a). The expression of miR-194-5p in tumor tissues from MCM3AP-AS1 knockdown injected mice was significantly higher than that in control mice (P < 0.05, Additional file 5: Figure S5A). Reduced expression of miR-194-5p was confirmed in HCC tissues compared to paracancerous tissues (P < 0.0001, Additional file 6: Figure S6A). Analysis of TCGA data and our HCC cases indicated a significant inverse correlation between MCM3AP-AS1 and miR-194-5p expression (P < 0.05, Additional file 6: Figure S6B and S6C). Furthermore, luciferase reporter assay revealed that miR-194-5p overexpression reduced the luciferase activity of vectors containing wt MCM3AP-AS1, but it did not influence the luciferase activity of vectors containing mt MCM3AP-AS1 in both SMMC-7721 and HepG2 cells (P < 0.05, Fig. 5b and Additional file 7: Figure S7A). Notably, we found that MCM3AP-AS1 level was reduced after overexpression of miR-194-5p in HCC cells (P < 0.05, Additional file 7: Figure S7B). Moreover, MCM3AP-AS1 was pulled down by biotin-labeled miR-194-5p, while mutagenesis of the binding sites for MCMC3AP-AS1 in miR-194-5p disrupted the interaction between MCM3AP-AS1 and miR-194-5p (P < 0.05, Fig. 5c). MCM3AP-AS1 mainly located in the cytoplasm (Additional file 8: Figure S8A). qRT-PCR found that the expression level of MCM3AP-AS1 was approximately 100 copies per cell, and mature miR-194-5p levels were approximately 200 copies per cell (Additional file 8: Figure S8B), suggesting that the abundance of MCM3AP-AS1 and miR-194-5p was comparable. Thus, our data demonstrated that MCM3AP-AS1 was associated with miR-194-5p and acted as a competing endogenous RNA (ceRNA) in HCC cells. Since AGO2 is an critical component of the RNA-induced silencing complex (RISC) and acts as a key regulator of miRNA functions [32]. We conducted anti-AGO2 RIP in HepG2 cells transiently overexpressing miR-194-5p. Endogenous MCM3AP-AS1 pull-down by AGO2 was significantly enriched in miR-194-5p-transfected cells (P < 0.05, Fig. 5d). In addition, AGO2 silencing impaired AGO2 dependent degradation of MCM3AP-AS1 and resulted in increased expression of MCM3AP-AS1, whereas miR-194-5p stability was reduced by AGO2 knockdown in HepG2 cells (P < 0.05, Fig. 5e). These data demonstrated that miR-194-5p bound to MCM3AP-AS1 and induced the degradation of MCM3AP-AS1 in HCC cells.

By using starBase V3.0, we found 48 candidate targets containing the complementary site for the seed region of miR-194-5p in four different prediction databases (targetScan, picTar, PITA and miRanada). Among these genes, only thyroid hormone receptor interactor 12 (TRIP12) [33], cullin 4B (CUL4B) [34], FOXA1 [27, 35, 36], BTB domain containing 7 (BTBD7) [37] and heparin binding EGF like growth factor (HBEGF) [38] were reported to be oncogenes in HCC. Then, we screened the mRNA levels of these genes after miR-194-5p overexpression in HepG2 cells. We found that miR-194-5p overexpression only reduced the level of FOXA1 mRNA (P < 0.05, Fig. 6a). Furthermore, FOXA1 protein expression was significantly down-regulated by miR-194-5p in both HepG2 and Hep3B cells (P < 0.05, Fig. 6a and b). The expression of FOXA1 protein in tumor tissues from MCM3AP-AS1 knockdown injected mice was significantly lower than that in control mice (P < 0.05, Additional file 5: Figure S5B). Furthermore, HCC tissues with high MCM3AP-AS1 or low miR-194-5p level showed an obvious higher level of FOXA1 protein compared to cases with low MCM3AP-AS1 or high miR-194-5p level (P < 0.05, Additional file 6: Figure S6D and S6E). Luciferase reporter assay indicated that miR-194-5p overexpression markedly decreased the luciferase activity of vectors containing wt 3’UTR of FOXA1 rather than mt 3’UTR of FOXA1 (P < 0.05, Fig. 6c). Moreover, MCM3AP-AS1 abolished the inhibitory effect of miR-194-5p on the luciferase activity of vectors containing wt 3’UTR of FOXA1 (P < 0.05, Fig. 6d). Meanwhile, MCM3AP-AS1 overexpression prominently increased FOXA1 expression, which was reversed by miR-194-5p restoration in SMMC-7721 cells (P < 0.05, Fig. 6e). Overexpression of miR-194-5p obviously reduced FOXA1 level in HepG2 cells, while FOXA1 downregulation was abrogated by MCM3AP-AS1 overexpression (P < 0.05, Fig. 6f). Taken together, MCM3AP-AS1 functioned as a ceRNA to promote FOXA1 expression by sponging miR-194-5p in HCC.

To explore whether the FOXA1 was critical for cell proliferation restriction, G1 arrest and apoptosis upon MCM3AP-AS1 knockdown, HepG2 cells with MCM3AP-AS1 knockdown were transfected with FOXA1 expression vectors. CCK8 assays indicated that restoration of FOXA1 attenuated the proliferation suppressive role of MCM3AP-AS1 knockdown in HepG2 cells (P < 0.05, Fig. 7a). Flow cytometry assays revealed that FOXA1 restoration reversed apoptosis and G1 arrest in HepG2 cells with MCM3AP-AS1 knockdown (P < 0.05, Fig. 7b and c). Moreover, colony formation and EdU incorporation assays also indicated that overexpression of FOXA1 attenuated the growth arrest of HepG2 cells induced by MCM3AP-AS1 knockdown (P < 0.05, Fig. 7d and e). Thus, these results strongly suggested that MCM3PA-AS1 knockdown induced cell proliferation restriction, cell cycle arrest and apoptosis was at least partially mediated by FOXA1 inhibition in HCC.