The qPCR was used to detect the expression of common lncRNAs to investigate their differential expression in IH and normal tissues. The results showed that among a variety of lncRNAs, only LncRNA MCM3AP-AS1 was up-regulated in IH compared to normal tissue (Figure 1A). We then compared the expression of these lncRNAs in hemangioma cell lines and normal cell lines, and our experimental results demonstrated that the expression of LncRNA MCM3AP-AS1 was significantly up-regulated in HemECs compared to the HUVECs that was taken as controls (Figure 1B). Since LncRNA MCM3AP-AS1 was up-regulated in both IH tissues and HemECs, the data from the SEER database were used to analyze whether LncRNA MCM3AP-AS1 affects the prognosis of hemangioma patients. The result indicated that patients with low expression of LncRNA MCM3AP-AS1 had significantly better overall survival (Figure 1C). These results suggest that the overexpression of lncRNA MCM3AP-AS1 aggravates the progression of IH.

Previous research has reported the tumor-promoting effect of LncRNAs on several tumors. We explored whether LncRNA MCM3AP-AS1 has the similar effect on IH cells. CCK-8 analysis demonstrated that the cell viability of HemECs was significantly reduced by knock-down of LncRNA MCM3AP-AS1 (Figure 2A). We further analyzed whether LncRNA MCM3AP-AS1 induces apoptosis in HemECs. FACS analysis was employed to estimate the effect of silence of LncRNA MCM3AP-AS1 on the apoptosis in HemECs. The results demonstrated that the apoptotic ratio of HemECs was significantly increased after knock-down of LncRNA MCM3AP-AS1 (Figure 2B). To examine whether LncRNA MCM3AP-AS1 was linked to cell cycle arrest in HemECs, we analyzed the effects of knock-down of LncRNA MCM3AP-AS1 on cell cycle of HemECs, and found that the percentage of cells in the G1 phase was significantly increased (Figure 2C). The above results demonstrated that knockdown of LncRNA MCM3AP-AS1 inhibited proliferation, induced apoptosis of HemECs, and arrested hemangioma cells in the G1phase of cell cycle.

To explore which miRNAs functioned as the sponge of LncRNA MCM3AP-AS1 to co-regulate the expression of downstream genes, we searched the starBase v3.0 (http://starbase.sysu.edu.cn/index.php) and found that LncRNA MCM3AP-AS1 harbored a putative binding site for miR-138-5p (Figure 3A). To further evaluate this conjecture, we construct the LncRNA MCM3AP-AS1-WT or LncRNA MCM3AP-AS1-Mut luciferase reporter and transfected these reporters into HemECs together with miR-138-5p mimic or its NC mimic. Dual-luciferase reporter assays showed that miR-138-5p mimic significantly reduced the luciferase activity of LncRNA MCM3AP-AS1-WT reporter, whereas exhibited modest effects on the activity of LncRNA MCM3AP-AS1-MUT reporter (Figure 3B). qPCR results showed that miR-138-5p mimics significantly reduced the expression of LncRNA MCM3AP-AS1 (Figure 3C). Then we detected the expression of miR-138-5p in HemECs and IH clinical tissues. As expected, the expression of miR-138-5p was significantly decreased in both IH cells and clinical samples compared with control groups (Figures 3D,E). These results demonstrated that LncRNA MCM3AP-AS1 directly targeted miR-138-5p.

We then predicted the downstream target genes of miR-138-5p using the three databases and Venn analysis showed two overlapping genes (Figure 4A). Through further binding site analysis, we found that the downstream regulatory gene of miR-138-5p was the hypoxia-inducible factor-1alpha (HIF-1α) (Figure 4B). HIF-1α-WT and HIF-1α--Mut luciferase reporters were constructed and transfected into HemECs together with miR-138-5p mimic or its NC mimic. Dual-luciferase reporter assays showed that miR-138-5p mimic significantly reduced the luciferase activity of HIF-1α-WT reporter, whereas exhibited no significant effects on the activity of HIF-1α-MUT reporter (Figure 4C). qRT-PCR results showed that miR-138-5p mimics significantly reduced the expression of HIF-1α (Figure 4D). Further analysis of clinical patient information from the database demonstrated that patients with low expression of HIF-1α had significantly better overall survival (Figure 4E).

Previous studies have shown that the abnormally elevated glycolysis level of tumor cells was reversed by inhibiting the expression of HIF-1α, which forced tumor cells to return to the metabolic mode of glucose oxidative phosphorylation, increased the energy consumption of tumor cells, and induced the death of tumor cells. Therefore, we examined the effect of HIF-1α on glycolysis in IH. Our results showed that LncRNA MCM3AP-AS1 significantly increased glucose consumption of HemECs, but this effect was partially reversed by adding HIF-1α inhibitor (DPT) (Figure 5A). Since the abnormally increased glycolysis of tumor cells was mainly manifested as increased pyruvate production and lactic acid production, we also tested the effect of HIF-1α on lactic acid production in IH. Results revealed that LncRNA MCM3AP-AS1 significantly increased lactic acid production of HemECs, but this effect was partially reversed by adding HIF-1α inhibitor (DPT) (Figure 5B). We also examined the expression of several key genes involved in glycolysis. The qPCR results showed that LncRNA MCM3AP-AS1 largely increased the expression of GLUT1, LDH and HK2, while HIF-1α inhibitor (DPT) partially reverse the enhancement of these genes expression induced by LncRNA MCM3AP-AS1 on IH (Figure 5C).

We further evaluated whether LncRNA MCM3AP-AS1 exerted regulatory roles via miR-138-5p/HIF-1α pathway in HemECs. CCK-8 analysis demonstrated that LncRNA MCM3AP-AS1 knockdown alone decreased the number of HemECs, while miR‐138-5p inhibitor alone or siHIF-1α significantly attenuated the proliferation-inhibiting effects of siLncRNA MCM3AP-AS1 on HemECs (Figure 6A). Similarly, the FACS analysis results demonstrated that the apoptotic ratio of HemECs was significantly increased after knock-down of LncRNA MCM3AP-AS1. However, miR‐138-5p inhibitor alone or siHIF-1α significantly reversed the apoptosis-inducing effects of siLncRNA MCM3AP-AS1 on HemECs (Figure 6B). The cell cycle analysis revealed that most HemECs were arrested at G0/G1phase of the cell cycle in cells with LncRNA MCM3AP-AS1 knock-down. Interestingly, miR‐138-5p inhibitor alone or siHIF-1α partially reversed the effect induced by siLncRNA MCM3AP-AS1 on HemECs (Figure 6C). Further analysis showed that knock-down of LncRNA MCM3AP-AS1 significantly decreased the glucose consumption and lactate production while miR‐138-5p inhibitor alone or siHIF-1α significantly reversed these effects of siLncRNA MCM3AP-AS1 on HemECs (Figures 6D,E).

Previous results have demonstrated that LncRNA MCM3AP-AS1 and miR-138-5p played an opposite role in the progression of IH disease, and miR-138-5p negatively regulates HIF-1α expression. Therefore, we examined the expression of these three genes in clinical tissues. IHC was employed to evaluate the expression of LncRNA MCM3AP-AS1 on the same ISH which was used for the evaluation of miR-138-5p expression. We found that both of LncRNA MCM3AP-AS1 and miR-138-5p were expressed in three clinical tissues. The same analysis strategy was used to examine the expression of miR-138-5p and HIF-1α (Figures 7A-C). The above analysis demonstrated that the expression of LncRNA MCM3AP-AS1 is negatively correlated with miR‐138-5p, and miR‐138-5p negatively correlated with HIF-1α in IH tissues.

Twenty-two infantile hemangioma specimens including normal subcutaneous tissues and infantile hemangioma tissues in the involuting stage (four males and eight females; median age, 7 months) and proliferating stage (two males and eight females; median age, 6 months), were collected in the plastic surgery department of our hospital from October 2019 to October 2020. All the samples were immediately frozen at −80°C for further analysis. The informed consent was obtained from the parents of patients. The experiments were approved by the Ethics Committee for Experiment of Nantong University.

HemECs were isolated from IH tissues in the proliferating phase as previously described (Khan et al., 2006). HemECs were cultured in human endothelial-serum free medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) with 5% CO₂ at 37°C.

All the specimens were detached quickly and immersed in lysis solution (4305895, Thermo Scientific) at the ratio of 1:6, then the samples were homogenized using an ultrasound homogenizer. Total RNA extraction was performed as the supplier’s protocol (AM 1912, Thermo Scientific). NanoDrop ND-1000 spectrophotometer (Thermo Scientific) was used to evaluate the quantity and purity of RNA. Complementary DNAs were obtained by the reverse transcription reaction with a reverse transcription kit (RR037A, Takara) and applied as templates to determine the expression of target genes by PCR. All the primers were designed by Primer Premier 5.0 software, and synthesized by Sangon Biotech (Shanghai, China). The primers were designed and synthesized as follows: hsa-miR-138-5p, 5′-gtc​gta​tcc​agt​gca​ggg​tcc​gag​gta​ttc​gca​ctg​gat​acg​acc​ggc​ct-3′; LncRNA-MCM3AP-AS1, forward: 5′-GCT​GCT​AAT​GGC​AAC​ACT​GA-3′ and reverse: 5′-AGGTGCTGTCTG GTGGAGAT-3′. GAPDH, forward: 5′-CAG GAG​GCA​TTG​CTG​ATG​AT-3′ and reverse: 5′-GAA​GGC​TGG​GGC​TCA​TTT-3′. LncRNA H19 forward: 5′-ACC​ACT​GCA​CTA​CCT​GAC​TC-3′, and reverse: 5′-CCG​CAG​GGG​GTG​GCC​ATG​AA-3′; LncRNA AC092718.4 forward: 5′-GCA​ACT​CCT​AGA​TTC​GAT​AC-3′, and reverse: 5′-CAC​GCG​TAT​GGT​AAC​ATG​CT-3′; LncRNA NEAT1 forward: 5′-TTCCGTGCT TCCTCTTCTGT-3′ and reverse: 5′-CAG​GGT​GTC​CTC​CAC​CTT​TA-3′; LncRNA PARD6G-AS1, forward: 5′-ATG​CTG​CAA​CTT​GTA​ACT-3′ and reverse: 5′-ACT​TGC​GAC​TTG​ACA​CTT​AGA​TT-3′;

CCK-8 assay was used to assess the cell viability of HemECs and EoMA cells. Briefly, HemECs and EoMA cells (about 10,000 per well) were seeded into 96-well plates. After 24, 48, and 72 h, CCK-8 solution (10 μL) was added into each well and incubated with HemECs and EoMA cells at 37 °C for 3 h. Then the light absorbance was measured at the wavelength of 450 nm to assess the cell viability of HemECs and EoMA cells.

The cells were seeded into 6-well plates. For cell cycle analysis, the cells were transfected with the different plasmids for 48 h. Then, the cells were washed by PBS (4°C), and collected with cold 70% ethanol (4°C) followed by washing with PBS. After the above operations, the cells were incubated with 1 ml of 20 mg/ml propidium iodide (PI) which contained RNase (1 mg/ml) in PBS for 30 min followed by fluorescence-activated cell sorting (FACS) assay. For the apoptosis assay, the cells were collected after treatment with the different plasmids for 48 h. The cells were washed with cold (4°C) PBS, followed by incubation with PI and Annexin V-EGFP according to the procedures specified in the kit (KeyGen Biotech Co. Ltd., Nanjing, China). Then, the processed cells were inspected using FACS assay.

For the luciferase reporter assay, a dual luciferase reporter assay system (Promega corporation) was used to detect the binding between miR-138-5p and LncRNA MCM3AP-AS1 or 3′-UTR of HIF-1α according to the manufacturer’s protocols. HemECs were seeded onto a 6-well plate and cultured for 24 h, cells were co-transfected with pGL3-MCM3AP-AS1 WT, or pGL3-MCM3AP-AS1 Mut (pGL3-HIF-1α 3′UTR WT or pGL3-HIF-1α 3′UTR Mut) and miR-138-5p mimic/NC mimic. After 48 h, luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, United States).

The paraffin-embedded tissues were cut into 4 μm thick sections and sections heated at 56°C for 5 min. After that, xylene and ethanol were used for deparaffinization. The sections were sealed with 3% H₂O₂ for 10 min, hybridized with 20 μl pre hybridizing solution in 42°C incubators for 3.5 h, and hybridized with 20 μl probe (The corresponding probe for detecting genes) solution (synthesized by Sangon Biotech Co., Ltd., Shanghai, China) in 40°C incubators for 20 h. The hybridization was coated with 3% BSA for 2 h in 37°C, washed and sealed. Biotinylated secondary anti-digoxin, streptavidin-biotin peroxidase complex ABC and DAB were added in turn according to the instructions of the ISH kit. Finally, the samples were observed under a light microscope.

As for immunohistochemical analysis, paraffin-embedded tissue sections were deparaffinized by xylene and hydrated with gradient ethanol. Then, the sections were treated with citrate buffer (pH = 6) for 20 min and immersed in 3% H₂O₂ for 10 min in a humidified chamber. After washing with PBS 5 min 3 times and blocked for 30 min with 10% goat serum, sections were incubated with rabbit HIF-1α antibody (1:400 Abcam, Cambridge, MA, United States) in a humidified chamber at 4°C overnight with a two-step protocol. Five randomly selected fields were captured under high-power magnification (×200) using a bright-field microscope (Olympus, Tokyo, Japan) and analyzed using Image-Pro Plus v6.2 software (Media Cybernetics, Silver Spring, MD).

The ENCORI database (http://starbase.sysu.edu.cn/), miRWalk database (http://mirwalk.umm.uni-heidelberg.de/) and miRDB database (http://www.mirdb.org) are powerful databases to study non-coding RNAs, such as LncRNAs, miRNAs and circRNAs. These databases were used to predict the binding sites between LncRNA MCM3AP-AS1 and miR-138-5p or miR-138-5p and the 3′-UTR of HIF-1α.

All the experiments were repeated at least three times independently. Data were analyzed using SPSS (SPSS 12.0, SPSS (IBM) Inc., Illinois, United States). The categorical variants were assigned a numerical value. Data were presented as mean ± standard deviation. Unpaired Student’s t tests or one-way ANOVA was used for data comparison (SPSS 12.0, SPSS (IBM) Inc., Illinois, United States). Statistical significance was determined at defined p < 0.05.