GeneCards (genecards.org/) database was used to explore the upstream regulatory mechanism of DKK3 in tumor cells. Prediction of lncRNA and miRNA, and miRNA and target gene binding sites was performed using TargetScan (targetscan.org/vert_72/) and miRanda (microrna.org/microrna/home.do) databases. The Atlas of non-coding RNAs in Cancer (TANRIC) database (ibl.mdanderson.org/tanric/design/basic/download.html), which is an interactive data analysis and visualization platform (19), was used to analyze the expression of lnc-MICAL2-1 in BC tissues and calculate the correlation coefficient between lnc-MICAL2-1 and DKK3 expression, which was subsequently displayed as a scatter diagram. In addition, TANRIC was used to study the relationship between lnc-MICAL2-1 expression, clinical characteristics and overall survival in BC. The corresponding clinical data of the patients in TANRIC were obtained from The Cancer Genome Atlas (TCGA) data portal (portal.gdc.cancer.gov/; normal, n=105; tumor, n=837).

MDA-MB-231, MCF-7 and 293T cells cell lines were purchased from the American Type Culture Collection and cultured as described previously (20). MDA-MB-231 and MCF-7 cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). 293T cells were cultured in DMEM containing 10% FBS, 2 mM L-glutamine and 50 µg/ml gentamicin. All cells were cultured at 37°C in a humidified incubator with 5% CO₂.

miR-25 mimics (5′-AGGCGGAGACUUGGGCAAUUG-3′), miR-25 inhibitor (5′-CAAUUGCCCAAGUCUCCGCCU-3′), miR-25 mimics control (5′-UUGUACUACACAAAAGUACUG-3′) and miR-25 inhibitor control (5′-CAGUACU-UUUGUGUAGUACAA-3′) were purchased from Sangon Biotech Co., Ltd. Cells were seeded (4×10⁵ cells/well) into 6-well cell culture plates containing 2 ml complete medium. Ay 50–80% confluence, MCF-7 cells were transfected with miR-25 mimics (50 nM), miR-25 mimics control (50 nM), miR-25 inhibitor (100 nM) or miR-25 inhibitor control (100 nM) using riboFECT CP Transfection kit (Guangzhou RiboBio Co., Ltd.) at 37°C for 48 h. pcDNA3.1/V5-His-TOPO vector (Invitrogen; Thermo Fisher Scientific, Inc.) was used to construct the lnc-MICAL2-1 overexpression and knockdown vectors. pcDNA3.1/V5-His-TOPO empty vector was used as the overexpression negative control. The sequences of the shRNAs were as follows: sh-lnc-MICAL2-1 (2 µg; 5′-AATGGCAAACACTGAGCTGCT-3′), sh-NC (2 µg; 5′-TTCTCCGAACGTGTCACGT-3′). Vectors (2 µg) were transfected into cells using FuGENE^® HD transfection reagent (Roche Diagnostics GmbH) at 37°C for 48 h, according to the manufacturer's protocol. After 48 h of transfection, transfection efficiencies were determined using reverse transcription-quantitative PCR (RT-qPCR).

A CCK-8 assay (Dojindo Molecular Technologies, Inc.) was used to study cell viability. A total of 2×10³ cells/well were plated in 96-well plates and cultured for 24, 48, 72 and 96 h at 37°C. Subsequently, 10 µl CCK-8 solution was added to each well and incubated with the cells at 37°C with 5% CO₂ for 4 h. The absorbance was measured at a wavelength of 450 nm.

Cells were cultured for 24 h until the cell confluence reached ~90%. A scratch was then made in the cell monolayer using a pipette tip to create an artificial wound. The cells were washed twice with PBS and incubated with serum-free DMEM at 37°C. The cells were visualized in the same position after 0, 24 and 48 h to determine the migratory distance. Images were obtained under a light microscope. Wound healing was calculated according to the following formula: Relative wound healing (%)=(0 h scratch distance-24/48 h scratch distance)/0 h scratch distance ×100.

The Transwell assays were performed as described by Li et al (21). Briefly, the cells were cultured in serum-free DMEM, then trypsinized. The cell density was adjusted to 1×10⁶ cells/ml, and cells were seeded into the upper chamber of a 24-well Transwell culture plate. DMEM supplemented with 10% FBS was added to the lower chamber of the Transwell plate. The cells were then incubated for 24 h for evaluation of migration. After 24 h, the cells on the surface of the lower chamber membrane were fixed with 5% glutaraldehyde for 15 min at room temperature and then stained with 0.1% crystal violet for 20 min at room temperature. Stained cells were visualized using a light microscope in three randomly selected fields of view. To evaluate the invasive ability of cells, 200 µl cells (1×10⁶ cells/ml) were seeded into the upper chambers of Transwell plates with 50 mg/l Matrigel (8 µm; cat. no. CLS3374; Corning, Inc.) and serum-free medium after Matrigel precoating at 37°C for 1 h. 500 µl DMEM supplemented with 10% FBS was added to the lower chamber. Following incubation at 37°C for 24 h, the cells remaining in the upper chamber were removed, whereas cells in the lower chamber were fixed with 5% glutaraldehyde for 15 min at 4°C, washed with PBS and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) for 20 min at room temperature. Stained cells were visualized using a light microscope in three randomly selected fields of view.

After 24 h of transfection, cells were digested with 0.25% trypsin to prepare a single cell suspension. The cells were then seeded into a 6-well culture plate at a density of 500 cells/well (three repeats/experimental group) and cultured at 37°C in a 5% CO₂ incubator. The culture medium was changed every 3 days during the culture period. After 15 days, the culture was terminated upon the appearance of visible colonies in the culture dish. The medium was subsequently discarded, the cells were washed with pre-cooled (4°C) PBS 2–3 times, and then fixed with 5 ml pure methanol for 15 min at room temperature. The cells were washed another three times with PBS and subsequently stained with Giemsa staining solution for 15 min at room temperature. Stained cells were visualized using a light microscope (magnification, ×5) to count the number of colonies with >10 cells.

Biotinylated miR-25-5p probe (5′-AGGCGGAGACUUGGGCAAUUG-Biotin-3′) and control probe (5′-GTTAACGGGTTCAGAGGCGGA-Biotin-3′) were synthesized by Sangon Biotech Co., Ltd. First, cells (1×10⁷) were lysed with 500 µl RIP lysis buffer centrifuged at 4°C and 12,000 × g for 15 min, and the cell lysate in the supernatant was collected. The Pierce Magnetic RNA-Protein Pull-Down kit (Thermo Fisher Scientific, Inc.) was used for RNA pull-down experiments. Biotinylated RNA probes (miR-25-5p probe and control probe) and 400 µl streptavidin magnetic beads (Thermo Fisher Scientific, Inc.) were incubated with cell lysates overnight at 4°C. After proteinase K digestion at 55°C for 20 min, the RNA attached to the magnetic beads was separated twice with washing buffer, and then centrifuged at 5,000 × g for 30 sec at 4°C to collect the Bio-miR/mRNA mixture, after which RT-qPCR was performed.

TargetScan (targetscan.org/vert_72/) and miRanda (microrna.org/microrna/home.do) databases were used to predict the target genes of miR-25. The reporter vector pmirGLO-DKK3-wild-type (wt), pmirGLO-DKK3-mutant (mut), pmirGLO-MICAL2-1-wt and pmirGLO-MICAL2-1-mut plasmids, or synthetic miR-NC mimics (forward, 5′-UUGUACUACACAAAAGUACUG-3′ and reverse, 5′-GUACUUUUGUGUAGUACAAUU-3′) or miR-25 mimics (forward, 5′-AGGCGGAGACUUGGGCAAUUG-3′ and reverse, 5′-AUUGCCCAAGUCUCCGCCUUU-3′) were obtained from Sangon Biotech Co., Ltd. Upon cell confluence reaching 70–80%, each reporter plasmid was co-transfected into 293T cells alongside miR-NC mimics or miR-25 mimics using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 24 h after transfection, the relative firefly luciferase activity was detected using a Dual-Glo Luciferase Reporter assay system, according to the manufacturer's protocol (Promega Corporation). The relative luciferase activity was normalized to Renilla luciferase activity.

Cells transfected with the lnc-MICAL2-1 overexpression or control NC vector for 48 h, or transfected with lnc-MICAL2-1 knockdown or control NC vector were washed with PBS once and fixed with methanol for 15 min at room temperature. Subsequently, cells were incubated with an anti-β-catenin primary antibody (1:500; cat. no. ab32572; Abcam) overnight at 4°C. After primary antibody incubation, the cells were incubated with a TRITC-conjugated goat anti-rabbit IgG antibody (cat. no. TA130015 1:200; OriGene Technologies, Inc.) for 1 h at room temperature. The nuclei were counterstained with DAPI for 5 min at room temperature. Stained cells were visualized using a fluorescence microscope.

After 72 h of transfection, cells were trypsinized and 2×10⁵ cells/ml were plated in a 6-well culture plate. The cells were fixed with 75% ice-cold ethanol at 4°C for at least 30 min, then washed with PBS to remove the fixative solution. Following the addition of 100 µl RNase, the cells were incubated in a water bath at 37°C for 30 min. Cells were subsequently incubated with 400 µl propidium iodide (PI; Sigma-Aldrich; Merck KGaA) at 4°C in the dark for 30 min. A ACEA NovoCyte flow cytometer (ACEA Bioscience, Inc.) with NovoExpress Software (version 1.0.2; ACEA Biosciences, Inc.) was used for cell cycle distribution analysis.

Cells in the logarithmic growth phase were collected and seeded into a 6-well plate at a density of 1×10⁵ cells/well, then incubated with 5% CO₂ at 37°C for 18 h. Cells were then transfected with lnc-MICAL2-1 overexpression, knockdown or NC vectors. After 48 h, 1–5×10⁵ cells were harvested and resuspended in 1X PBS. A total of 5 µl Annexin V-FITC and 5 µl PI was added and incubated at room temperature for 5 min in the dark. Apoptotic cells were analyzed using a flow cytometer (FACSCalibur; BD Biosciences) with BD Cell Quest Pro Software (version 3.3; Becton, Dickinson and Company).

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The purity of total RNA was detected and quantified using an UV spectrophotometer (260 nm to calculate RNA concentration and 260/280 nm to evaluate RNA purity). Then, total RNA (1 µg) was reverse transcribed into cDNA using a reverse transcription system (cat. no. A3500; Promega Corporation), according to the manufacturer's protocol. Primer3web software (version 4.1.0; http://primer3.ut.ee/) was used to design the following primers for qPCR: GAPDH (NM_001256799.3) forward, 5′-GAAAGCCTGCCGGTGACTAA-3′ and reverse, 5′-TTCCCGTTCTCAGCCTTGAC-3′; DKK3 (NM_001018057.2) forward, 5′-CCTGGCAAACTTACCTCCCA-3′ and reverse, 5′-CATTTTGGTGCAGTGACCCC-3′; lnc-MICAL2-1 forward, 5′-TCAGCCTGCCTGCCAATATG-3′ and reverse, 5′-AAGCAGTGCTGCAAGGAAGA-3′; miR-25-5p forward, 5′-AGGCGGAGACTTGGGCAATTG-3′ and reverse, 5′-GTGCGTGTCGTGGAGTCG-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACACAATTG-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. qPCR was performed on an ABI 7500 Real-Time PCR Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min; 40 cycles of denaturation at 95°C for 10 sec, annealing and extension at 60°C for 1 min. mRNA and miRNA expression levels were determined using the 2^−ΔΔCq method (22). GAPDH was used as the mRNA housekeeping gene and U6 was used as the miRNA housekeeping gene.

Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA protein assay kit (Bio-Rad Laboratories, Inc.), loaded on a 12% SDS gel and resolved using 10% SDS-PAGE (NuPAGE; Invitrogen; Thermo Fisher Scientific, Inc.). The separated proteins were subsequently transferred to PVDF membranes (Whatman plc; Cytiva) and blocked with 5% non-fat dry milk powder (Merck KGaA) at room temperature for 2 h. The PVDF membrane was then incubated with DKK3 (1:1,000; cat. no. ab187532; Abcam) primary antibodies at 4°C overnight. After the primary antibody incubation, the membranes were washed with TBS-Tween 20 (0.05%) three times and incubated with a goat anti-rabbit secondary antibody (cat. no. bs-0295G-HRP; 1:3,000; BIOSS) for 2 h at room temperature. Protein bands were visualized using chemiluminescence reagent on a gel imaging system (Thermo Fisher Scientific, Inc.). The band densities were analyzed using ImageJ software (version 4.6; National Institutes of Health). The gray value of the target protein was normalized to the gray value of the internal reference protein, GAPDH, and the data are presented as the relative content of the target protein in a sample.

A total of 63 male BALB/c-nude mice (5-week-old; weight 18–20 g; Chengdu Dashuo Laboratory Animal Co., Ltd.) were maintained in aseptic conditions at 25°C and 40–60% humidity, with 12-h light/dark cycles, and free access to food and water. Mice were subcutaneously injected with 1×10⁷ MCF-7 cells stably transfected with lnc-MICAL2-1 overexpression, knockdown or NC. After inoculation, the tumor volume was measured once a week. After 6 weeks, the mice (n=6 per group) were euthanized via cervical dislocation, the tumor tissue was harvested, and the weight and volume were measured. The volume of the tumor was calculated using the following equation: Volume=length × width² ×0.5, where the length represented the longest diameter and the width represented the shortest diameter of the tumor. The maximum tumor diameter observed in the present study was 1.84 cm and the maximum tumor volume was 1,935.3 mm³. To evaluate the survival of mice, 15 mice from each group were observed until the 10th week. If ulceration, infection or necrosis occurred, or mice were on the verge of death, the experiment was terminated and the mice were euthanized. The survival rate of tumor-bearing mice after inoculation with tumor cells was analyzed using the Kaplan-Meier method. The log-rank (Mantel-Cox) test was used to determine statistical differences in the survival curves. All animal procedures and experimental methods were approved by the Committee on the Ethics of Animal Experiments of Southern Medical University (approval no. SYXK 2020–0012). This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (23), and in accordance with the Animal Research: Reporting In Vivo Experiments guidelines (24).

Tumor tissues were fixed with 4% neutral formaldehyde for 24 h at room temperature, routinely embedded in paraffin and cut into 4-µm slices. After conventional dewaxing, the sections were stained with hematoxylin at room temperature for 5 min and eosin for 3 min at room temperature. Then, graded alcohol dehydration, xylene clearing and neutral gum sealing were performed. The morphological changes of tumor cells were observed under a light microscope.

Tumor tissues were embedded in paraffin, sectioned into 5-µm slices, heated for 2 h at 60°C, Subsequently, the tissues were deparaffinized with xylene I and II (each for 10 min), and dehydrated in a gradient alcohol series. Heat-mediated antigen retrieval was performed with citrate buffer (pH 6), then the sections were immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase, followed by blocking with 5% bovine serum albumin (Wuhan Servicebio Technology Co., Ltd.) for 30 min at room temperature. The sections were subsequently incubated with 50 µl anti-DKK3 rabbit monoclonal antibody (1:100; cat. no. ab187532; Abcam), MMP-25 (1:100; cat. no. ab56309; Abcam) overnight at 4°C. Following the primary antibody incubation, the sections were washed with PBS for 2 min and incubated with 50 µl biotinylated goat anti-rabbit IgG (1:100; cat. no. bs-0295G-HRP; BIOSS) at 37°C for 30 min. Sections were then washed with PBS three times (15 min each), and then incubated with high sensitivity streptavidin-HRP conjugate (Biosynthesis Biotechnology Co., Ltd.) for 30 min at 37°C. Sections were stained with DAB (Biosynthesis Biotechnology Co., Ltd.) for 8 min at room temperature and hematoxylin for 5 min at room temperature, then dehydrated in gradient ethanol for 1 min, made transparent with xylene and sealed with neutral gum. The immunostaining images were captured using a light microscope (magnification, ×400). The integrated optical density in the selected area was calculated in five non-overlapping fields of view of each section as the statistical area. Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) was used for the analysis.

Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software, Inc.). Statistical differences between two groups were compared using the non-parametric Mann Whitney-U test, whereas a Kruskal-Wallis test and Dunn's post hoc test was used for the comparisons between more than two groups. The differential expression of lncRNAs was analyzed using an unpaired Student's t-test between tumor and adjacent normal tissues. Spearman's rank correlation analysis was used to determine the correlation between lncRNA and mRNA expression. The nonparametric Kaplan-Meier method was used for survival analysis. The log-rank (Mantel-Cox) test was used to compare the difference between survival curves. P<0.05 was considered to indicate a statistically significant difference.

To determine the underlying mechanism of downregulated DKK3 expression in BC, the GeneCards bioinformatics tool was used. The results revealed that lnc-MICAL2-1 may regulate DKK3 expression via modulation of miR-25. The expression levels of lnc-MICAL2-1 in the tumor tissues of patients with breast invasive carcinoma (BRCA) obtained from TCGA database were analyzed. The results demonstrated that the expression levels of lnc-MICAL2-1 were downregulated in human BRCA tissues compared with those in normal tissues The results also found that the expression levels of lnc-MICAL2-1 were significantly downregulated in tumor tissues (n=837) compared with in adjacent normal tissues (n=105) (Fig. 1A). Breast cancer is divided according to the expression matrix by molecular profiling (GEP). For example, PAM50-based GEP can divide breast cancer into different subtypes, including normal-like, basal, luminal A, luminal B and Her2. Analysis of lnc-MICAL2-1 expression in the PAM50 subtype of BRCA samples identified significant differences between the basal and luminal A subtypes (Fig. 1B). Correlation analysis in basal, luminal B and human epidermal growth factor receptor 2 (Her2) subtypes of BC identified a positive correlation between lnc-MICAL2-1 and DKK3 mRNA expression in the basal (correlation, 0.3569581; P<0.05; n=139), luminal B (correlation, 0.4869612; P<0.05; n=191) and Her2 (correlation, 0.4401873; P<0.05; n=67) subtypes (Fig. 1C-E). According to the median expression of lnc-MICAL2-1 in all patients, the patients were divided into high expression and low expression groups. Survival analysis showed that high expression levels of lnc-MICAL2-1 decreased the overall survival rate of BC and was thus indicative of a poorer prognosis (Fig. 1F). The association between the expression of lnc-MICAL2-1 and the estrogen receptor, progesterone receptor and therapy of patients with BC was statistically significant (P<0.05), but there was no association with staging and HER2 expression (P>0.05) (Fig. 1G-K).

To further understand the effects of lnc-MICAL2-1 on BC cells, lnc-MICAL2-1 overexpression and knockdown vectors were constructed and transfected into the BC cell lines, MCF-7 and MBA-MD-231 (Fig. 2A and B). The results revealed that the optical density value was decreased following the overexpression of lnc-MICAL2-1 in BC cells, suggesting that cell proliferation was inhibited. Conversely, the proliferative ability of cells with lnc-MICAL2-1 knockdown was significantly increased (Fig. 2C and D). In addition, a wound healing assay was performed using transfected MCF-7 cells to determine the effects on cell migration (Fig. 2E). The results demonstrated that cell migration was inhibited following the overexpression of lnc-MICAL2-1, whereas the knockdown of lnc-MICAL2-1 promoted cell migration (Fig. 2F). The results of the Transwell assays indicated that the overexpression of lnc-MICAL2-1 inhibited the invasive and migratory ability of MCF-7 cells (Fig. 2G-J). The data from the colony formation assay experiments further validated that the overexpression of lnc-MICAL2-1 inhibited cell proliferation (Fig. 2K and L).

A positive correlation was identified between lnc-MICAL2-1 and DKK3 mRNA expression in BC tissues (correlation, 0.632; n=47; P<0.05; Fig. 3A). Subsequently, lnc-MICAL2-1 was overexpressed or knocked down in MCF-7 and MDA-MB-231 cells. The results revealed that in MCF-7 cells overexpressing lnc-MICAL2-1, the mRNA and protein expression levels of DKK3 were significantly upregulated. Conversely, following the knockdown of lnc-MICAL2-1 expression in MCF-7 cells, the mRNA and protein expression levels of DKK3 were significantly downregulated (Fig. 3B-D). Therefore, it was hypothesized that lnc-MICAL2-1 may exert a positive regulatory effect on DKK3 expression. Bioinformatics analysis identified 13 complementary base pairings between miR-25 and lnc-MICAL2-1 (Fig. 3E). Similarly, complementary base pairings were identified between miR-25 and DKK3 (Fig. 3F). A previous study reported that miR-25 targeted DKK3 and regulated the Wnt/β-catenin signaling pathway (12). Therefore, dual-luciferase reporter and RNA pull-down assays were performed to validate the binding. The results of dual-luciferase reporter showed that compared with miRNA NC, the luciferase activity in wt-lnc-MICAL2-1 cells transfected with miRNA-25 mimic was significantly decreased (P<0.01), whereas the luciferase activity in mut-lnc-MICAL2-1 cells was not affected (P>0.05) (Fig. 3G). The results revealed that lnc-MICAL2-1 was able to sponge miR-25. This relationship was further determined using the RNA pull-down assay. The results indicated that lnc-MICAL2-1 could be pulled down by miRNA-25 (Fig. 3H). The results of the dual-luciferase reporter assay showed that compared with the miRNA NC group, the luciferase activity in wt-DKK3 cells transfected with miRNA-25 mimic was significantly decreased (P<0.01), but the luciferase activity in mut-DKK3 cells was not affected (P>0.05) (Fig. 3I). This relationship was further determined using the RNA pull-down assay, whereby DKK3 was pulled down by miRNA-25 (Fig. 3J). These findings suggested that lnc-MICAL2-1 may have reduced the degradation of DKK3 mRNA by sponging miR-25, thereby exerting an indirect positive regulatory effect on DKK3.

To explore the regulatory effect of lnc-MICAL2-1 on the DKK3-mediated Wnt signaling pathway, a lnc-MICAL2-1 overexpression plasmid and miR-25 mimic were co-transfected into MCF-7 cells, and shRNA lnc-MICAL2-1 and miR-25 inhibitor were co-transfected into MCF-7 cells. Transfection efficiency of miR-25 mimic and inhibitor were determined by RT-qPCR in MCF-7 cells, and the results showed that the transfection was successful (Fig. 4A). Furthermore, the results revealed that the mRNA and protein expression levels of DKK3 in cells co-transfected with the lnc-MICAL2-1 overexpression vector and miR-25 mimic were downregulated compared with in cells only transfected with the lnc-MICAL2-1 overexpression vector (Fig. 4B-D). Conversely, the mRNA and protein expression levels of DKK3 in cells co-transfected with shRNA lnc-MICAL2-1 and miR-25 inhibitor were upregulated compared with in cells transfected with shRNA lnc-MICAL2-1 only (Fig. 4E-G). In addition, the overexpression of lnc-MICAL2-1 decreased β-catenin nuclear localization, whereas shRNA lnc-MICAL2-1 increased the translocation of β-catenin into the nucleus (Fig. 4H). Next, the effect of lnc-MICAL2-1 on the β-catenin signaling pathway-mediated apoptosis of MCF-7 cells was explored. Activation of the Wnt/β-catenin pathway affects cell apoptosis and cell cycle progression (25). The data revealed that in the lnc-MICAL2-1 knockdown cells, the number of cells in the S phase of the cell cycle was significantly increased compared with that in the control cells (Fig. 4I and J), whereas the levels of apoptosis were significantly increased following the overexpression of lnc-MICAL2-1 in MCF-7 cells compared with in the control cells (Fig. 4K and L).

To further explore the inhibitory function of lnc-MICAL2-1 on BC cells, a BC xenograft model was established. The overexpression of lnc-MICAL2-1 significantly reduced tumor weight (Fig. 5A) and volume (Fig. 5B) compared with in the control and lnc-MICAL2-1 knockdown groups. The survival curve also revealed that the overexpression of lnc-MICAL2-1 significantly prolonged the survival time of mice compared with in the control group (Fig. 5C).

H&E staining showed that NC group tumors grew rapidly with a different morphology. Inflammatory infiltration was observed around the edges of the tumor tissues, as well as a small number of necrotizing cells. The tumors in the lnc-MICAL2-1 group displayed a reduced number of tumor cells, increased cell degeneration and necrosis, and an enhanced tissue response (Fig. 5D). The results of immunohistochemistry showed that the knockdown of lnc-MICAL2-1 downregulated the expression levels of DKK3, whereas the expression levels of β-catenin and MMP-25 were upregulated (Fig. 5D and E). These findings suggested that lnc-MICAL2-1 expression may be positively associated with DKK3 expression, whereas DKK3 expression may be negatively associated with the expression of β-catenin and MMP-25 (Fig. 5D and E).