Microrna‑574‑3P Inhibits the Malignant Behavior of Liver Cancer Cells by Targeting ADAM28

ONCOLOGY LETTERS 20: 3015-3023, 2020

MicroRNA‑574‑3p inhibits the malignant behavior of liver cancer cells by targeting ADAM28

ZHONGMING ZHA, FUXIN JIA, PINGAN HU, ERHUI MAI and TING LEI

Department of Hepatobiliary Surgery, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang, Henan 471000, P.R. China

Received September 27, 2019; Accepted April 29, 2020

DOI: 10.3892/ol.2020.11852

Abstract. Liver cancer is one of the most common and worldwide (1,2). Currently, surgical resection combined with aggressive tumors, and usually leads to a poor clinical outcome. chemotherapy, radiotherapy, liver implant and intervention Increasing evidence has demonstrated the important functions remains the major therapeutic strategy for patients with liver of microRNAs (miRs) in tumor progression. miR‑574‑3p has cancer (3). Although notable progress has been made in been reported as a tumor suppressor and potential therapeutic the diagnosis and treatment of this disease, the outcome of target in various types of cancer. However, the underlying patients with liver cancer remains poor due to metastasis and mechanism of the effects of miR‑574‑3p in liver cancer remains recurrence (4,5). Therefore, the identification of novel factors unknown. In the present study, reverse transcription‑quantitative involved in the development of liver cancer is required to PCR was performed to detect miR‑574‑3p expression in liver improve the prognosis of patients with this disease. cancer tissues, and the influence of miR‑574‑3p on cell growth MicroRNAs (miRNAs/miRs) are a class of non‑coding was evaluated using the Cell Counting Kit‑8 assay, and cell single‑stranded RNAs consisting of 19‑25 nucleotides migration and flow cytometry analyses. The present study that negatively regulate gene expression (6‑8). miRNAs revealed that miR‑574‑3p expression was downregulated in bind the 3'‑untranslated region (UTR) of target mRNAs, liver cancer tissues and cell lines. miR‑574‑3p overexpression, leading to translation inhibition or mRNA degradation (9). achieved by transfecting miR‑574‑3p mimics into liver cancer Bioinformatics analysis has indicated that the expression of cells, reduced cell proliferation and migration, and promoted up to 30% of human genes is regulated by miRNAs, which cell apoptosis. Mechanistically, ADAM metallopeptidase is consistent with the increasing involvement of miRNAs in a domain 28 (ADAM28) was identified as a miR‑574‑3p target wide range of biological processes, such as cell proliferation, via binding to the 3'‑untranslated region of the ADAM28 differentiation and apoptosis (10). Notably, accumulating mRNA. Gain‑of‑function of miR‑574‑3p downregulated evidence has demonstrated the key roles of miRNAs in the the expression levels of ADAM28 in liver cancer cells. progression of liver cancer by acting as tumor suppressors Additionally, overexpression of ADAM28 significantly or oncogenes (11). For example, miR‑548b suppresses the attenuated the suppressive effect of miR‑574‑3p on the growth proliferation and invasion of liver cancer cells by targeting of liver cancer cells. The present results provide novel insights specificity protein 1 (12). Previous studies have revealed that into the function of the miR‑574‑3p/ADAM28 signaling miR‑574‑3p acts as a tumor suppressor in numerous types of pathway in liver cancer. cancer (13‑17), such as esophageal squamous cell carcinoma (ESCC), in which miR‑574‑3p is downregulated and may Introduction be a predictor of the postoperative outcome of patients with ESCC (13). Next generation sequencing data has indicated Liver cancer is one of the most common malignancies and that miR‑574‑3p is a promising prognostic marker for patients has been a leading cause of cancer‑associated mortality with breast cancer (17). Additionally, miR‑574‑3p expression is downregulated in prostate cancer tissues and inhibits tumorigenesis (14). Previous studies have revealed that the dysregulation of serum miR‑574‑3p in patients with liver cancer serves as an important biomarker for the diagnosis of Correspondence to: Dr Ting Lei, Department of Hepatobiliary Surgery, Luoyang Central Hospital Affiliated to Zhengzhou liver cancer (18,19). However, to the best of our knowledge, University, 288 Zhongzhouzhong Road, Luoyang, Henan 471000, the function of miR‑574‑3p in liver cancer has not been fully P. R. Ch i na explored. E‑mail: [email protected] The ADAM metallopeptidases (ADAMs) are characterized by sequence similarity to the reprolysin family of snake venom Key words: microRNA‑574‑3p, ADAM metallopeptidase endopeptidases, which share the metalloproteinase domain domain 28, liver cancer with matrix metalloproteinases (20). Increasing evidence has demonstrated that ADAM proteins serve important roles in multiple biological processes, such as cell proliferation, 3016 ZHA et al: FUNCTION OF miR-574-3p IN LIVER CANCER migration and adhesion (21). The critical function of ADAMs cDNA synthesis kit (Thermo Fisher Scientific, Inc.) with a in human diseases, particularly cancer, has been illustrated temperature protocol of 25̊C for 5 min, followed by 42̊C for by a previous study (22). ADAM domain 28 (ADAM28) 60 min and 70̊C for 5 min. RT‑qPCR was performed with the is a member of the ADAMs family that is overexpressed All‑in‑One™ miRNA qRT‑PCR detection kit (GeneCopoeia, in numerous types of cancer, including breast cancer, lung Inc.) to detect the levels of miR‑574‑3p. The expression levels cancer and acute myeloid leukemia (23‑25). Mechanistically, of ADAM28 were quantified using the SYBR Premix Ex ADAM28 promotes tumorigenesis by cleaving molecules Taq II (Takara Biotechnology Co., Ltd.). The thermocycling involved in cancer metastasis, such as connective tissue conditions were: 95̊C for 1 min, followed by 40 cycles at 95̊C growth factor, insulin‑like growth factor binding protein‑3 for 10 sec, 58̊C for 30 sec and 72̊C for 30 sec. miR‑574‑3p and von Willebrand's factor (26). However, the involvement of expression was normalized to U6 RNA, while ADAM28 ADAM28 in liver cancer has not been fully established. expression was normalized to GAPDH. The primers used in In the present study, miR‑574‑3p expression in liver the present study were: miR‑574‑3p forward, 5'‑ATCGGAAGT cancer tissues and cell lines was analyzed to reveal the role TGAGTGAGCCGCGTC‑3' and reverse, 5'‑GCCGTGAGT of miR‑574‑3p in the progression of liver cancer. Additionally, CAGGAGTGGT‑3'; U6 forward, 5'‑GCTTCGGCAGCACAT the effects of miR‑574‑3p on the growth of liver cancer cells ATACTAAAAT‑3' and reverse, 5'‑CGCTTCAGAATTTGC and its potential downstream targets were explored. GTGTCAT‑3'; ADAM28 forward, 5'‑GTAAAAGAGAGA CCCAAGAGCCAG‑3' and reverse, 5'‑GTAGTCCTTGAC Materials and methods AGGTGCTGATG‑3'; GAPDH forward, 5'‑CACCTGCGC TGTGTGGACT‑3' and reverse, 5'‑GGATGGCTGATGTGT Clinical samples. Liver cancer tissues and paired adjacent CGGGTGG‑3'. The relative expression levels of miR‑574‑3p normal tissues (~5 cm from the margin of the tumor tissues) were were determined using the 2‑ΔΔCq method (27). The ratio collected by surgical resection from 50 patients diagnosed at (R=RQCancer/RQNormal; RQ, relative quantification) between Luoyang Central Hospital Affiliated to Zhengzhou University the relative expression levels of miR‑574‑3p and ADAM28 (Luoyang, China) between May 2013 and October 2014. in liver cancer and adjacent normal tissues was calculated. Patients who received radiotherapy or chemotherapy before Expression was considered to be downregulated when R<0.7 surgery were excluded. Tissues were frozen in liquid nitrogen and upregulated when R>1.3 (28). prior to the experiments. The pathological characteristics of patients with liver cancer enrolled in the present study are Luciferase reporter assay. The wild‑type (WT) or mutant provided in Table SI. The present study was approved by the (Mut) 3'‑UTR sequences of ADAM28 containing the potential Ethics Committee of Luoyang Central Hospital Affiliated seeding region of miR‑574‑3p were inserted into the pmirGLO to Zhengzhou University and performed according to the vector (Promega Corporation). Cells (5,000 cells/well) Declaration of Helsinki. Written informed consent was were seeded into a 96‑well plate and co‑transfected with obtained from all participants enrolled in the present study. pmirGLO‑UTR‑WT or pmirGLO‑UTR‑Mut with miR‑574‑3p mimics or control miRNA using Lipofectamine® 2000 Cell culture and transfection. Liver cancer cell lines, including (Invitrogen; Thermo Fisher Scientific, Inc.). Following 48 h of HepG2, Huh7 and Hep3B were obtained from The Cell Bank of transfection, cells were harvested and the luciferase activity Type Culture Collection of the Chinese Academy of Sciences. was detected using a Dual‑luciferase Reporter assay kit The immortalized normal liver THLE‑3 epithelial cell line was (Promega Corporation) with an iMark Fluorometer Microplate purchased from the American Type Culture Collection. Cells Reader (Bio‑Rad Laboratories, Inc.). Renilla luciferase activity were cultured in DMEM supplemented with 10% FBS (both was detected for normalization. The assay was performed with Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin three independent repeats. and 100 U/ml penicillin (Sigma‑Aldrich; Merck KGaA). Cells 3 were cultured at 37̊C with 5% CO2. Cell Counting Kit‑8 (CCK‑8) assay. A total of 2x10 cells/well The miR‑574‑3p mimics (5'‑CACGCUCAUGCACAC expressing miR‑574‑3p mimics or control miRNA were seeded ACCCACA‑3') and negative control miRNA (5'‑GUUCGU into a 96‑well plate. Cell proliferation was evaluated after 1, 2, ACGUACACUGUUCA‑3') were purchased from Guangzhou 3, 4 and 5 days using the CCK‑8 assay (Dojindo Molecular RiboBio Co., Ltd. The ADAM28 expression plasmid was Technologies, Inc.) according to the manufacturer's protocol. constructed by inserting the full cDNA sequence of ADAM28 Briefly, 10 µl CCK‑8 solution was added to the medium and into the backbone of a pcDNA 3.1+ vector (Addgene, Inc.). incubated at 37̊C for 3 h. The absorbance of each well was When the cell confluence reached 70‑80%, miRNAs (50 nM) measured at 450 nm using the EnSpire 2300 Multilabel Reader or pcDNA‑ADAM28 (0.5 µg) were transfected into cells using (PerkinElmer, Inc.). Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were Target prediction. The potential targets of miR‑574‑3p were harvested after 48 h of transfection for further analysis. predicted using miRDB (http://mirdb.org/; version 6.0) and TargetScan (http://www.targetscan.org/vert_72/; release RNA extraction and reverse transcription‑quantitative PCR no. 7.2) by inputting the name of the miRNA as ‘miR‑574‑3p’. (RT‑qPCR). Total RNA was extracted from tissues or cells using the TRIzol® reagent at room temperature (Invitrogen; Western blotting. Proteins were extracted from cells using Thermo Fisher Scientific, Inc.) according to the manufacturer's RIPA buffer containing protease inhibitor (Beyotime Institute protocol. RT was performed using the Revert Aid First Strand of Biotechnology). The protein concentration was determined ONCOLOGY LETTERS 20: 3015-3023, 2020 3017

Figure 1. miR‑574‑3p expression is downregulated in liver cancer. (A) miR‑574‑3p expression in paired liver cancer and adjacent normal tissues detected by RT‑qPCR. (B) miR‑574‑3p expression in liver cancer cell lines, including HepG2, Huh7 and Hep3B, and in the immortalized normal liver THLE‑3 cell line measured by RT‑qPCR. **P<0.01; ***P<0.001 vs. THLE‑3 cells. miR, microRNA; OD450, optical density at 450 nm; RT‑qPCR, reverse transcription‑quantitative PCR; RQ, relative quantification.

using a bicinchoninic acid protein assay kit (Beyotime Institute binding buffer and stained with Annexin V‑FITC for of Biotechnology). An equal amount of protein (20 µg/lane) 15 min at room temperature in the dark. Subsequently, cells was separated by 15% SDS‑PAGE and transferred onto PVDF were incubated with propidium iodide for 5 min at room membranes (EMD Millipore). The membranes were blocked temperature. Apoptosis was analyzed using a FACScan flow with 5% skimmed milk in TBS buffer at room temperature for cytometer (BD Biosciences) and the data (both early and late 1 h, followed by incubation with ADAM28 (cat. no. sc‑393877; apoptosis) were analyzed using the Cellquest software v10.6.4 Santa Cruz Biotechnology, Inc.) and GAPDH (cat. no. ab9485; (BD Biosciences). Abcam) primary antibodies (both 1:1,000 dilution) over- night at 4̊C. After three washes with TBS‑Tween-20 (0.1%), Statistical analysis. Data are presented as the mean ± SD the membranes were incubated with goat anti‑mouse from three independent experiments. Statistical analysis was (cat. no. 170‑6516) or goat anti‑rabbit (cat. no. 170‑6515) performed using SPSS v16.0 (SPSS, Inc.). Paired Student's t‑test IgG (H+L)‑horseradish peroxidase‑conjugated secondary was used to determine the significance between adjacent and antibodies (both 1:5,000 dilution; Bio‑Rad Laboratories, Inc.) cancer tissues. Unpaired Student's t‑test was used for analyzing for 2 h at room temperature. The bands were visualized using differences between miRNAs and miR mimics, and between the Enhanced Chemiluminescence Detection reagent (Pierce; pcDNA‑vector and pcDNA‑ADAM28 groups. One‑way Thermo Fisher Scientific, Inc.). ANOVA followed by Tukey's post hoc test was used for the statistical analysis of differences among multiple groups. The Cell migration. The migration of liver cancer cells was correlation between the expression levels of miR‑574‑3p and determined using Transwell chambers (8‑µm pore size; ADAM28 was analyzed using Spearman's correlation test. Corning Inc.) following the manufacturer's protocol. A P<0.05 was considered to indicate a statistically significant total of 5x104 cells transfected with miR‑574‑3p mimics or difference. miR‑NC were seeded into the upper chamber containing 200 µl medium without serum. Medium supplemented with Results 10% FBS was added into the lower chamber and served as the chemoattractant. After incubation for 24 h at 37̊C, cells in the miR‑574‑3p expression is downregulated in liver cancer. To upper chamber were removed using a cell scraper and those investigate miR‑574‑3p expression in liver cancer, the expres- attached to the lower surface of the membrane were fixed sion levels of miR‑574‑3p in 50 paired cancer and adjacent with 4% paraformaldehyde at room temperature for 10 min. normal tissues were detected via RT‑qPCR. The data indicated Subsequently cells were stained with 0.1% crystal violet that miR‑574‑3p expression was significantly downregulated in (Beyotime Institute of Biotechnology) for 15 min at room cancer tissues compared with in non‑tumor tissues (Fig. 1A). temperature and counted using an inverted light microscope Accordingly, miR‑574‑3p expression was downregulated in (magnification, x100). the liver cancer HepG2, Huh7 and Hep3B cell lines compared with in the immortalized normal liver THLE‑3 cell line Cell apoptosis. Apoptosis of liver cancer cells was determined (Fig. 1B). The present data indicate that miR‑574‑3p expression using the FITC Annexin V Apoptosis Detection kit (Beijing 4A is downregulated in liver cancer. Biotech Co., Ltd.) according to the manufacturer's protocol. Briefly, 1x10 6 cells/well were transfected with miR‑574‑3p Overexpression of miR‑574‑3p inhibits the proliferation of mimics or control miRNA, collected after 48 h of transfection liver cancer cells. To characterize the effect of miR‑574‑3p and washed twice with PBS. Cells were resuspended with in liver cancer, miR‑574‑3p was overexpressed by transfecting 3018 ZHA et al: FUNCTION OF miR-574-3p IN LIVER CANCER

Figure 2. Overexpression of miR‑574‑3p inhibits the growth of liver cancer cells. (A) miR‑574‑3p expression measured by reverse transcription‑quantitative PCR after liver cancer cells were transfected with miR‑574‑3p mimics or corresponding controls. Cell Counting Kit‑8 assay indicating the proliferation of (B) HepG2 and (C) Huh7 cells transfected with miR‑574‑3p mimics or control miRNA. (D) Overexpression of miR‑574‑3p inhibited the migration of liver cancer cells. (E) Transfection of miR‑574‑3p mimics increased the apoptosis of Huh7 and HepG2 cells. *P<0.05; **P<0.01; ***P<0.001 vs. control miRNA. OD, optical density; miRNA/miR, microRNA; OD450, optical density at 450 nm; PI, propidium iodide; FITC, fluorescein isothiocyanate.

miR‑574‑3p mimics into HepG2 and Huh7 cells. The a suppressive role of miR‑574‑3p in the tumorigenesis of liver transfection efficiency was analyzed using an RT‑qPCR assay cancer. (Fig. 2A). The effects of miR‑574‑3p on the proliferation of cancer cells were determined using the CCK‑8 assay. ADAM28 is a target of miR‑574‑3p in liver cancer. To further As indicated in Fig. 2B and C, miR‑574‑3p overexpression understand the functional mechanism of miR‑574‑3p in significantly suppressed the proliferation of both HepG2 and liver cancer, potential targets of miR‑574‑3p were predicted. Huh7 cells after 4 and 5 days. Additionally, the suppressive The bioinformatics analysis suggested that ADAM28 was effect of miR‑574‑3p in liver cancer was investigated by a putative target of miR‑574‑3p. The potential binding site evaluating the impact of miR‑574‑3p on cell migration. The of miR‑574‑3p at the 3'‑UTR of ADAM28 is presented in present results revealed that transfection of miR‑574‑3p Fig. 3A. To validate the binding between miR‑574‑3p and inhibited the migratory capacity of HepG2 and Huh7 cells the 3'‑UTR of ADAM28, a luciferase reporter assay was (Fig. 2D). Additionally, the flow cytometry analysis indicated performed by co‑transfecting luciferase vectors expressing a that transfection with miR‑574‑3p mimics significantly WT or Mut 3'‑UTR of ADAM28, and a miR‑574‑3p mimic. increased apoptosis of both HepG2 and Huh7 cells (Fig. 2E). miR‑574‑3p overexpression significantly decreased the lucif- The present results suggest that miR‑574‑3p overexpression erase activity of cells carrying the WT but not the Mut 3'‑UTR inhibits the malignant abilities of liver cancer cells, indicating of ADAM28 (Fig. 3B and C). To illustrate whether the binding ONCOLOGY LETTERS 20: 3015-3023, 2020 3019

Figure 3. ADAM28 is a target of miR‑574‑3p in liver cancer. (A) Putative miR‑574‑3p binding sequence in the 3'‑UTR of ADAM28 predicted using the miRDB database. Relative luciferase activity of (B) HepG2 and (C) Huh7 cells co‑transfected with miR‑574‑3p mimics with wild‑type or mutant 3'‑UTR of ADAM28. ADAM28 expression measured by (D) reverse transcription‑quantitative PCR and (E) western blot analysis after transfection with miR‑574‑3p mimics. ***P<0.001 vs. control miRNA. UTR, untranslated region; ADAM28, ADAM metallopeptidase domain 28; miRNA/miR, microRNA. of miR‑574‑3p affected the stability of ADAM28 mRNA, with in matched adjacent normal tissues. The correlation RT‑qPCR was performed in liver cancer cells expressing the between miR‑574‑3p and ADAM28 expression was analyzed miR‑574‑3p mimic or the control miRNA. The results revealed by Spearman's correlation test. The data revealed that the that miR‑574‑3p overexpression significantly decreased expression levels of miR‑574‑3p were inversely correlated with the mRNA expression levels of ADAM28 in both HepG2 those of ADAM28 in cancer tissues (Fig. 4B). Additionally, and Huh7 cells (Fig. 3D). Similarly, the protein expression the mRNA expression levels of ADAM28 in liver cancer cell levels of ADAM28 were decreased by miR‑574‑3p transfec- lines were detected by RT‑qPCR. The results revealed that tion (Fig. 3E). The present results suggest that ADAM28 is ADAM28 expression was significantly upregulated in liver a target of miR‑574‑3p and that it is negatively regulated by cancer cells compared with in the immortalized normal liver miR‑574‑3p in liver cancer. THLE‑3 cell line (Fig. 4C). The present findings suggest that ADAM28 expression is upregulated in liver cancer. miR‑574‑3p expression is inversely correlated with ADAM28 expression in liver cancer. Since miR‑574‑3p negatively Recovered ADAM28 expression reverses the inhibitory effect regulated ADAM28 expression in liver cancer cells, the of miR‑574‑3p in liver cancer cells. To confirm the involvement abundance of ADAM28 in liver cancer tissues was evaluated of ADAM28 in the anticancer activity of miR‑574‑3p in liver by RT‑qPCR. As indicated in Fig. 4A, ADAM28 expression cancer cells, HepG2 and Huh7 cells were transfected with was significantly upregulated in cancer tissues compared the pcDNA‑ADAM28 plasmid. ADAM28 overexpression 3020 ZHA et al: FUNCTION OF miR-574-3p IN LIVER CANCER

Figure 4. ADAM28 expression is inversely correlated with that of miR‑574‑3p in liver cancer. (A) ADAM28 expression in paired liver cancer and adjacent normal tissues detected by RT‑qPCR. (B) Correlation between the expression levels of miR‑574‑3p and ADAM28 analyzed by Spearman's correlation test. (C) ADAM28 expression in liver cancer cell lines and the normal THLE‑3 cell line investigated by RT‑qPCR. ***P<0.001 vs. THLE‑3 cells. RT‑qPCR, reverse transcription‑quantitative PCR; ADAM28, ADAM metallopeptidase domain 28; miR, microRNA; RQ, relative quantification.

was analyzed by RT‑qPCR and western blot analysis, biomarker for the prognosis and therapy monitoring of patients respectively (Fig. 5A and B). Functional analysis revealed with head and neck squamous cell cancer (34). miR‑574‑3p that overexpression of ADAM28 significantly attenuated the overexpression induces cell cycle arrest in response to X‑ray inhibitory effect of miR‑574‑3p on the proliferation of liver irradiation by targeting the production of the enhancer of cancer cells after 5 days (Fig. 5C and D). The present results rudimentary homolog protein (35). In the present study, suggest that the tumor suppressive role of miR‑574‑3p in liver miR‑574‑3p was markedly downregulated in liver cancer cancer is partially mediated by ADAM28 downregulation. tissues and cell lines. Further studies are required to explore the underlying mechanisms that contribute to decreased Discussion miR‑574‑3p expression in liver cancer. Additionally, to strengthen the potential clinical significance of miR‑574‑3p in Increasing evidence has demonstrated that miRNAs are liver cancer, the association between miR‑574‑3p expression abnormally expressed in liver cancer and regulate cancer and the prognosis of patients with liver cancer should progression (29,30). Therefore, understanding the mechanisms be investigated using a larger sample size. In the present of miRNAs in liver cancer may provide novel targets for the study, miR‑574‑3p transfection inhibited cell proliferation treatment of this disease, and may improve the outcome of and migration, and induced apoptosis of liver cancer cells, patients with liver cancer. In the present study, the expression suggesting a tumor suppressive role of miR‑574‑3p in liver levels and the functional mechanism of miR‑574‑3p in liver cancer. However, experiments with loss‑of‑function of cancer were investigated. miR‑574‑3p downregulation was miR‑574‑3p and in vivo assays in mice are required to fully observed in liver cancer tissues and cell lines, highlighting the characterize the inhibitory effects of miR‑574‑3p on the potential clinical significance of miR‑574‑3p in liver cancer. development of liver cancer. Interestingly, some miRNAs, The tumor suppressive function of miR‑574‑3p has been such as miR‑23b‑3p, can promote or inhibit tumor progression reported in several types of cancer, such as ovarian and in different types of cancer depending on the alternation of gastric cancer, and osteosarcoma (13,15,18,31‑33). A recent downstream targets (36). miR‑574‑3p exerts tumor‑promoting study revealed that miR‑574‑3p inhibits the metastasis and effects in osteosarcoma (OS), and miR‑574‑3p overexpression chemoresistance of epithelial ovarian cancer (32). Circulating promotes the growth of OS cells by targeting SMAD family miR‑574‑3p in the plasma has been proposed as a promising member 4 (31). A recent study revealed that serum miR‑574‑3p ONCOLOGY LETTERS 20: 3015-3023, 2020 3021

Figure 5. Overexpression of ADAM28 reverses the suppressive effect of miR‑574‑3p on the growth of liver cancer cells. HepG2 and Huh7 cells were transfected with pcDNA‑vector or pcDNA‑ADAM28, and the overexpression of ADAM28 was confirmed by (A) reverse transcription‑quantitative PCR and (B) western blot analysis. Proliferation of (C) HepG2 and (D) Huh7 cells detected by a Cell Counting Kit‑8 assay following transfection of miR‑574‑3p and ADAM28. **P<0.01; ***P<0.001 vs. pcDNA‑vector. OD450, optical density at 450 nm; ADAM28, ADAM metallopeptidase domain 28; miR, microRNA.

expression in patients with liver cancer is higher than that in with 3'‑UTR‑mutated ADAM28 to investigate its effects on patients with cirrhosis or in healthy controls (18), suggesting miR‑574‑3p‑mediated proliferation of liver cancer cells. an oncogenic function of miR‑574‑3p in cancer. It is possible In conclusion, the present study revealed that miR‑574‑3p that miR‑574‑3p serves promoting or inhibitory roles in expression was downregulated in liver cancer tissues. liver cancer depending on the targeted proteins. It would be Overexpression of miR‑574‑3p inhibited the malignant useful to investigate the expression levels and functions of phenotype of liver cancer cells by targeting ADAM28. The miR‑574‑3p in other types of cancer to confirm the suppressive present results shed light on the critical involvement of the or oncogenic role of miR‑574‑3p in cancer. miR‑574‑3p/ADAM28 signaling pathway in liver cancer. Based on the involvement of ADAMs in cancer, ADAMs Further studies should explore the therapeutic potential of can be regulated by miRNAs to modulate the progression miR‑574‑3p and identify other genome‑wide targets in addition of tumorigenesis (37‑45). A previous study revealed that to ADAM28. miR‑552 targets ADAM28 in colorectal cancer and enhances the metastasis of cancer cells (26). Additionally, ADAM28 Acknowledgements is negatively regulated by miR‑198 in colorectal cancer and inhibits cancer cell proliferation (42). In the present study, Not applicable. miR‑574‑3p bound the 3'‑UTR of ADAM28 and reduced ADAM28 expression in liver cancer cells, suggesting that Funding miR‑574‑3p targets ADAM28 and negatively regulates its expression. Consistently, the expression levels of ADAM28 No funding was received. were inversely correlated with those of miR‑574‑3p in liver cancer tissues. Overexpression of ADAM28 attenuated the Availability of data and materials inhibitory effect of miR‑574‑3p on the proliferation of liver cancer cells. To further validate that miR‑574‑3p suppresses The datasets used and/or analyzed during the current study the growth of liver cancer cells by targeting ADAM28, it are available from the corresponding author on reasonable would be useful to perform rescue assays by transfecting request. 3022 ZHA et al: FUNCTION OF miR-574-3p IN LIVER CANCER

Authors' contributions 16. Ujihira T, Ikeda K, Suzuki T, Yamaga R, Sato W, Horie‑Inoue K, Shigekawa T, Osaki A, Saeki T, Okamoto K, et al: MicroRNA‑574‑3p, identified by microRNA library‑based func- ZZ and TL designed the study. ZZ performed most of the tional screening, modulates tamoxifen response in breast cancer. experiments and analyzed the data. FJ, PH and EM contributed Sci Rep 5: 7641, 2015. 17. Krishnan P, Ghosh S, Wang B, Li D, Narasimhan A, Berendt R, to performing the experiments, and analyzed and interpreted Graham K, Mackey JR, Kovalchuk O and Damaraju S: Next the data. ZZ and TL wrote the manuscript. All authors read generation sequencing profiling identifies miR‑574‑3p and and approved the final manuscript. miR‑660‑5p as potential novel prognostic markers for breast cancer. BMC Genomics 16: 735, 2015. 18. Shen X, Xue Y, Cong H, Wang X and Ju S: Dysregulation of serum Ethics approval and consent to participate microRNA‑574‑3p and its clinical significance in hepatocellular carcinoma. Ann Clin Biochem 55: 478‑484, 2018. 19. Gui J, Tian Y, Wen X, Zhang W, Zhang P, Gao J, Run W, Tian L, The present study was approved by the Ethics Committee of Jia X and Gao Y: Serum microRNA characterization identifies Luoyang Central Hospital Affiliated to Zhengzhou University miR‑885‑5p as a potential marker for detecting liver pathologies. (Luoyang, China). Written informed consent was provided by Clin Sci (Lond) 120: 183‑193, 2011. 20. Tortorella MD, Malfait F, Barve RA, Shieh HS and Malfait AM: all patients. A review of the ADAMTS family, pharmaceutical targets of the future. Curr Pharm Des 15: 2359‑2374, 2009. Patient consent for publication 21. Reiss K and Saftig P: The ‘a disintegrin and metalloprotease’ (ADAM) family of sheddases: Physiological and cellular functions. Semin Cell Dev Biol 20: 126‑137, 2009. Not applicable. 22. Duffy MJ, McKiernan E, O'Donovan N and McGowan PM: Role of ADAMs in cancer formation and progression. Clin Cancer Res 15: 1140‑1144, 2009. Competing interests 23. Mitsui Y, Mochizuki S, Kodama T, Shimoda M, Ohtsuka T, Shiomi T, Chijiiwa M, Ikeda T, Kitajima M and Okada Y: The authors declare that they have no competing interests. ADAM28 is overexpressed in human breast carcinomas: Implications for carcinoma cell proliferation through cleavage of insulin‑like growth factor binding protein‑3. Cancer Res 66: References 9913‑9920, 2006. 24. Mochizuki S, Shimoda M, Abe H, Miyamae Y, Kuramoto J, 1. Di Sandro S, Danieli M, Ferla F, Lauterio A, Carlis RD, Aramaki‑Hattori N, Ishii K, Ueno H, Miyakoshi A, Kojoh K Benuzzi L, Buscemi V, Pezzoli I and Carlis LD: The current role and Okada Y: Selective inhibition of ADAM28 suppresses lung of laparoscopic resection for HCC: A systematic review of past carcinoma cell growth and metastasis. Mol Cancer Ther 17: ten years. Transl Gastroenterol Hepatol 3: 68, 2018. 2427‑2438, 2018. 2. Aghemo A: Update on HCC management and review of the new 25. Zhang JM, Wang CC, Zhang GC, Jiang Q, Yang SM, Fu HX, EASL guidelines. Gastroenterol Hepatol (N Y) 14: 384‑386, 2018. Wang QM, Zhu XL, Zhu HH, Jiang H, et al: ADAM28 3. Bolondi L, Piscaglia F, Camaggi V, Grazi GL and Cavallari A: promotes tumor growth and dissemination of acute myeloid Review article: Liver transplantation for HCC. Treatment options leukemia through IGFBP‑3 degradation and IGF‑I‑induced cell on the waiting list. Aliment Pharmacol Ther 17 (Suppl 2): proliferation. Cancer Lett 442: 193‑201, 2019. S145‑S150, 2003. 26. Wang J, Li H, Wang Y, Wang L, Yan X, Zhang D, Ma X, 4. Liu CY, Chen KF and Chen PJ: Treatment of liver cancer. Cold Du Y, Liu X and Yang Y: MicroRNA‑552 enhances metastatic Spring Harb Perspect Med 5: a021535, 2015. capacity of colorectal cancer cells by targeting a disintegrin and 5. Marengo A, Rosso C and Bugianesi E: Liver cancer: Connections metalloprotease 28. Oncotarget 7: 70194‑70210, 2016. with obesity, fatty liver, and cirrhosis. Annu Rev Med 67: 103‑117, 27. Livak KJ and Schmittgen TD: Analysis of relative gene expres- 2016. sion data using real‑time quantitative PCR and the 2(‑Delta Delta 6. Ambros V: The functions of animal microRNAs. Nature 431: C(T)) method. Methods 25: 402‑408, 2001. 350‑355, 2004. 28. Grossi I, Arici B, Portolani N, De Petro G and Salvi A: 7. Bartel DP: MicroRNAs: Genomics, biogenesis, mechanism, and Clinical and biological significance of miR‑23b and miR‑193a function. Cell 116: 281‑297, 2004. in human hepatocellular carcinoma. Oncotarget 8: 6955‑6969, 8. Mohr AM and Mott JL: Overview of microRNA biology. Semin 2017. Liver Dis 35: 3‑11, 2015. 29. Huang X and Jia Z: Construction of HCC‑targeting artificial 9. Fabian MR, Sonenberg N and Filipowicz W: Regulation of miRNAs using natural miRNA precursors. Exp Ther Med 6: mRNA translation and stability by microRNAs. Annu Rev 209‑215, 2013. Biochem 79: 351‑379, 2010. 30. Lou W, Liu J, Ding B, Chen D, Xu L, Ding J, Jiang D, Zhou L, 10. Gentilin E, Degli Uberti E and Zatelli MC: Strategies to Zheng S and Fan W: Identification of potential miRNA‑mRNA use microRNAs as therapeutic targets. Best Pract Res Clin regulatory network contributing to pathogenesis of HBV‑related Endocrinol Metab 30: 629‑639, 2016. HCC. J Transl Med 17: 7, 2019. 11. Mizuguchi Y, Takizawa T, Yoshida H and Uchida E: Dysregulated 31. Xu H, Liu X, Zhou J, Chen X and Zhao J: miR‑574‑3p acts as a miRNA in progression of hepatocellular carcinoma: A systematic tumor promoter in osteosarcoma by targeting SMAD4 signaling review. Hepatol Res 46: 391‑406, 2016. pathway. Oncol Lett 12: 5247‑5253, 2016. 12. Qiu H, Zhang G, Song B and Jia J: MicroRNA‑548b inhibits 32. Zheng J, Zhou Y, Li XJ and Hu JM: miR‑574‑3p exerts as a tumor proliferation and invasion of hepatocellular carcinoma cells suppressor in ovarian cancer through inhibiting MMP3 expres- by directly targeting specificity protein 1. Exp Ther Med 18: sion. Eur Rev Med Pharmacol Sci 23: 6839‑6848, 2019. 2332‑2340, 2019. 33. Zhang P, Zhu J, Zheng Y, Zhang H, Sun H and Gao S: 13. Okumura T, Kojima H, Miwa T, Sekine S, Hashimoto I, Hojo S, miRNA‑574‑3p inhibits metastasis and chemoresistance of Nagata T and Shimada Y: The expression of microRNA 574‑3p epithelial ovarian cancer (EOC) by negatively regulating as a predictor of postoperative outcome in patients with esopha- epidermal growth factor receptor (EGFR). Am J Transl Res 11: geal squamous cell carcinoma. World J Surg Oncol 14: 228, 4151‑4165, 2019. 2016. 34. Summerer I, Unger K, Braselmann H, Schuettrumpf L, 14. Chiyomaru T, Yamamura S, Fukuhara S, Hidaka H, Majid S, Maihoefer C, Baumeister P, Kirchner T, Niyazi M, Sage E, Saini S, Arora S, Deng G, Shahryari V, Chang I, et al: Genistein Specht HM, et al: Circulating microRNAs as prognostic therapy up‑regulates tumor suppressor microRNA‑574‑3p in prostate biomarkers in head and neck cancer patients. Br J Cancer 113: cancer. PLoS One 8: e58929, 2013. 76‑82, 2015. 15. Wang M, Zhang R, Zhang S, Xu R and Yang Q: MicroRNA‑574‑3p 35. Ishikawa K, Ishikawa A, Shoji Y and Imai T: A genotoxic regulates epithelial mesenchymal transition and cisplatin resis- stress‑responsive miRNA, miR‑574‑3p, delays cell growth by tance via targeting ZEB1 in human gastric carcinoma cells. suppressing the enhancer of rudimentary homolog gene in vitro. Gene 700: 110‑119, 2019. Int J Mol Sci 15: 2971‑2990, 2014. ONCOLOGY LETTERS 20: 3015-3023, 2020 3023

36. Grossi I, Salvi A, Baiocchi G, Portolani N and De Petro G: 42. Li LX, Lam IH, Liang FF, Yi SP, Ye LF, Wang JT, Guo WW and Functional role of microRNA‑23b‑3p in cancer biology. Xu M: miR‑198 affects the proliferation and apoptosis of colorectal Microrna 7: 156‑166, 2018. cancer through regulation of ADAM28/JAK‑STAT signaling 37. Li H, Guo X, Li Q, Ran P, Xiang X, Yuan Y, Dong T, Zhu B, pathway. Eur Rev Med Pharmacol Sci 23: 1487‑1493, 2019. Wang L, Li F, et al: Long non‑coding RNA 1308 promotes 43. Ge X, Cui H, Zhou Y, Yin D, Feng Y, Xin Q, Xu X, Liu W, Liu S cell invasion by regulating the miR‑124/ADAM 15 axis in and Zhang Q: miR‑320a modulates cell growth and chemosen- non‑small‑cell lung cancer cells. Cancer Manag Res 10: sitivity via regulating ADAM10 in gastric cancer. Mol Med 6599‑6609, 2018. Rep 16: 9664‑9670, 2017. 38. Yang X, Cui Y, Yang F, Sun C and Gao X: MicroRNA302a 44. Li J, Lu M, Jin J, Lu X, Xu T and Jin S: miR‑449a suppresses suppresses cell proliferation, migration and invasion in osteosar- tamoxifen resistance in human breast cancer cells by targeting coma by targeting ADAM9. Mol Med Rep 16: 3565‑3572, 2017. ADAM22. Cell Physiol Biochem 50: 136‑149, 2018. 39. Ji T, Zhang X and Li W: MicroRNA543 inhibits proliferation, 45. van Kampen JGM, van Hooij O, Jansen CF, Smit FP, van Noort PI, invasion and induces apoptosis of glioblastoma cells by directly Schultz I, Schaapveld RQJ, Schalken JA and Verhaegh GW: targeting ADAM9. Mol Med Rep 16: 6419‑6427, 2017. miRNA‑520f reverses epithelial‑to‑mesenchymal transition by 40. Hua Y, Liang C, Miao C, Wang S, Su S, Shao P, Liu B, Bao M, targeting ADAM9 and TGFBR2. Cancer Res 77: 2008‑2017, 2017. Zhu J, Xu A, et al: MicroRNA‑126 inhibits proliferation and metastasis in prostate cancer via regulation of ADAM9. Oncol Lett 15: 9051‑9060, 2018. 41. Liu Q, Jiang J, Fu Y, Liu T, Yu Y and Zhang X: miR‑129‑5p functions as a tumor suppressor in gastric cancer progression through targeting ADAM9. Biomed Pharmacother 105: 420‑427, 2018.