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miR-629–5p promotes growth and metastasis of hepatocellular carcinoma by activating β-catenin
Experimental Cell Research 380 (2019) 124–130
Contents lists available at ScienceDirect
Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr
Research article
miR-629–5p promotes growth and metastasis of hepatocellular carcinoma by activating β-catenin
T
Xin Taoa,1, Xiaoxia Yangb,1, Kexin Wub, Liang Yangb, Yufei Huangb, Qian Jinb, Suling Chena,∗ a b
Department of Infectious Disease, Heping Hospital Attached to Changzhi Medical College, Changzhi, 046000, China Changzhi Medical College, Changzhi, 046000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: miR-629–5p Hepatocellular carcinoma Proliferation Invasion β-catenin
Aberrant miR-629–5p expression in several cancer types has been reported. Nonetheless, its potential effect and mechanism of action on tumor growth and metastasis in hepatocellular carcinoma (HCC) have rarely been analyzed. In this study, we found that miR-629–5p was upregulated in HCC tissue samples as compared to matched adjacent-tissue samples. Overexpression of miR-629–5p promoted the proliferation, migration, and invasiveness of human HCC cells in vitro, whereas miR-629–5p knockdown reduced these parameters. Consistently, miR-629–5p overexpression accelerated tumor growth and metastasis in a nude mouse model. Mechanistically, miR-629–5p directly targeted the 3′ untranslated region (3′UTR) of the secreted frizzled-related protein 2 (SFRP2) mRNA and suppressed its expression, resulting in the activation of β-catenin. Inhibition of βcatenin abrogated miR-629-5p–induced growth and invasiveness. Collectively, these results suggest that miR629–5p activates β-catenin signaling by downregulating SFRP2 and thus promotes the growth and metastasis of HCC. These data open up new prospects for HCC treatment.
1. Introduction Hepatocellular carcinoma (HCC) is a common malignant human tumor worldwide [1]. Due to its high malignancy potential, HCC ranks as the fifth leading cause of cancer-related deaths in the United States, resulting in more than 30,000 deaths in 2018 [1]. Although the treatments were improved dramatically in recent years, the prognosis of HCC patients remains unsatisfactory. Thus, it is important to further explore novel biomarkers and potential therapeutic targets to improve the therapeutic responsiveness and prognosis of HCC. MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that regulate gene expression by binding to the 3′ untranslated region (3′UTR) of target mRNA, thereby inducing mRNA degradation or protein synthesis repression [2]. Accumulating evidence indicates that miRNAs can function in human carcinogenesis as novel types of oncogenes or tumor suppressors [3,4]. To date, a group of miRNAs has been reported to regulate the growth and metastasis of HCC and is believed to constitute promising markers for the diagnosis and prognosis of this disease, e.g., miR-139, miR-26a, miR-532–3p, and miR-665 [5–8]. It has been shown that miR-629–5p is dysregulated in several types of cancer and plays an oncogenic role via the regulation of diverse cellular
functions such as cell proliferation, apoptosis, migration, and invasion [9–11]. Nevertheless, the biological effect of miR-629–5p in HCC remains unknown. In this study, we found that the expression of miR-629–5p was upregulated in HCC cell lines and tissue samples. Furthermore, in vitro and in vivo assays showed that miR-629–5p promoted the growth and metastasis of HCC by repressing SFRP2 and activating β-catenin. These observations provide a clearer understanding of the mechanism by which miR-629–5p promotes HCC. 2. Materials and methods 2.1. Tissue samples and cell lines Twenty human HCC tissue samples and matched normal adjacenttissue samples were obtained from Heping Hospital Attached to Changzhi Medical College. Each tumor tissue sample was snap-frozen in lipid nitrogen and then stored at −80 °C. Informed consent was obtained from each patient, and the use of human tissues was approved by the Medical Ethics and Human Clinical Trial Committee of Heping Hospital Attached to Changzhi Medical College. Human HCC cell lines
∗
Corresponding author. Department of Infectious Disease, Heping Hospital Attached to Changzhi Medical College, No. 110 South Yanan Road, Changzhi, 046000, China. E-mail address: [email protected] (S. Chen). 1 Equal contributors. https://doi.org/10.1016/j.yexcr.2019.03.042 Received 14 January 2019; Received in revised form 29 March 2019; Accepted 30 March 2019 Available online 04 April 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.
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2.6. Wound-healing assay and transwell invasion assay
MHCC97L, MHCC97H, and HCCLM3 and nontransformed hepatic cell line L02 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% of fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified incubator with 5% CO2.
Wound-healing assay was performed to assess the cell migration ability. Transfected cells were seeded in 6-well plates and cultured to 80–90% confluence. The cell monolayer was scratched with a sterile 200 μl pipette tip, and the detached cells were removed with a PBS wash. After cultivation in the complete DMEM medium for 48 h, the cells were photographed. The distance traveled by the cells between the two boundaries of the wound was calculated. For the Transwell invasion assay, transfected cells were seeded onto the membrane of the upper chamber with Matrigel in a serum-free medium. The lower chamber was filled with the complete DMEM medium (containing 10% of fetal bovine serum as a chemoattractant). After incubation at 37 °C for 48 h, the invading cells that got attached to the lower surface of the membrane were stained with crystal violet and counted under a microscope.
2.2. Quantitative reverse-transcription polymerase chain reaction (qRTPCR) Total-RNA samples including miRNAs were isolated with the miRNeasy Mini Kit (Qiagen, Shanghai, China). Expression levels of mRNA and miRNA were examined in triplicate by qRT-PCR. For mRNA analysis, complementary DNA (cDNA) synthesis from total RNA was performed with random hexamer primers. For miR-629–5p analysis, specific RT primers were used. cDNA was then amplified by means of the SYBR® Green PCR Master Mix. Relative expression was normalized to U6 or β-actin expression and was calculated by the 2−ΔΔCt method.
2.7. Luciferase reporter assay
2.3. Western blotting
L02 and MHCC97L cells were seeded in 96-well plates and transiently transfected with an appropriate reporter plasmid and miRNA using Lipofectamine 2000; 48 h later, the cells were harvested and lysed, and luciferase activity was measured via the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Luciferase reporter assays were performed in duplicate and repeated in three independent experiments.
Western blotting assay was performed as described previously [12]. Cells or tissues were lysed in RIPA buffer supplemented with a Protease Inhibitor Cocktail (Pierce, Appleton, WI, USA). The lysates were analyzed using the BCA Protein Assay Kit (Pierce). Proteins were separated via SDS-PAGE in a 10% gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were subsequently blocked with 5% nonfat milk and incubated with antibodies against SFRP2, β-catenin, cyclin D1, and β-actin (Proteintech, Wuhan, China) (primary antibodies) overnight at 4 °C. After a wash with Tris-buffered saline supplemented with 0.1% of Tween 20 (TBST), membranes were incubated with a secondary antibody for 1 h at room temperature. Signals were detected by means of an Enhanced Chemiluminescence system (Pierce).
2.8. In vivo tumor growth and metastasis assays The HCC model in mice was set up as described previously [13]. For the in vivo tumor growth assay, 2 × 106 MHCC97L cells infected with the miR-629-5p–expressing lentivirus or control lentivirus were subcutaneously injected into the flank of each nude mouse (BALB/c-nu/nu; five in each group) (SLRC, Shanghai, China). Four weeks later, the subcutaneous tumors were excised, weighed, and implanted into the liver of a nude mouse (five in each group). The mice were euthanized after 6 weeks, and then the lungs were collected, fixed with phosphatebuffered neutral formalin, sectioned serially, and stained with hematoxylin and eosin (H&E) for standard histological examination. All the procedures were approved by the Institutional Research Ethics Committee at Changzhi Medical College.
2.4. Oligonucleotide transfection and plasmid construction MiR-629–5p mimics, a miR-629–5p inhibitor, β-catenin small interfering RNA (siRNA), and the corresponding negative control were purchased from Ribobio (Guangzhou, China). Oligonucleotide transfection was performed using Lipofectamine 2000 (Invitrogen). The wild-type (WT) or mutant (MUT) 3′UTR of secreted frizzled-related protein 2 (SFRP2) mRNA containing a putative binding site for miR629–5p was cloned into the psiCHECK-2 luciferase reporter vector. SFRP2 without the 3′UTR region was cloned into the multiple cloning site of pcDNA3.1 to generate an SFRP2 overexpression plasmid. The constructs were verified by DNA sequencing.
2.9. Immunohistochemical staining This assay was carried out similarly to previously described methods [14]. In breif, tissues were fixed in phosphate-buffered neutral formalin, embedded in paraffin, and cut into 5-μm-thick sections. The latter were deparaffinized, rehydrated, and microwave-heated in sodium citrate buffer for antigen retrieval. The tissue sections were incubated with a primary antibody (against Ki67) at a 1:100 dilution overnight at 4 °C. Detection of the primary antibody was performed for 1 h at room temperature using a goat anti-rabbit IgG antibody conjugated with horseradish peroxidase, and the results were visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB) as a substrate.
2.5. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and colony formation assay The number of viable cells was evaluated by the MTT assay. Briefly, transfected cells (5 × 103 per well) seeded in 96-well plates were cultured in the growth medium. After cultivation for 24, 48, 72, and 96 h, 100 μl of the sterile MTT dye (0.5 mg/ml, Sigma) was added into each well with incubation for 4 h at 37 °C, followed by removal of the culture medium and addition of 150 μl of dimethyl sulphoxide (Sigma, St. Louis, MO, USA). Absorbance was measured at 490 nm. All the experiments were conducted in triplicate. For the colony formation assay, transfected cells were seeded in 6-well plates at a density of 300 cells per well and cultured at 37 °C for 2 weeks. The medium was changed every 3 days. At the end of the incubation, the cells were fixed with 100% methanol and stained with 0.1% crystal violet. Megascopic cell colonies were counted in Image-Pro Plus 5.0 (Media Cybernetics, Bethesda, MD).
2.10. Statistical analysis GraphPad Prism 5.0 software was used for statistical calculations. All data are presented as means ± standard deviation (SD). The twotailed Student t-test was performed to analyze differences between two groups. Data with P values less than 0.05 were considered statistically significant. 125
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Fig. 1. The levels of miR-629–5p expression in HCC. (A) MiR-629–5p expression levels were assessed by qRT-PCR in three HCC cell lines and L02 cells. (B) qRT-PCR analysis of miR-629–5p expression levels in 20 HCC tissue samples and matched normal tissue samples. (C) Kaplan–Meier analysis of survival of HCC patients with a distinct expression level of miR-629–5p in the tumor (data from database TCGA). *P < 0.05, **P < 0.01.
Fig. 2. MiR-629–5p promotes HCC cell proliferation, migration, and invasion in vitro. (A) Relative miR-629–5p levels in MHCC97L cells transfected with miR-629–5p or the negative control (miR-ctrl) were measured by qRT-PCR. (B) Cell viability was determined by the MTT assay of MHCC97L cells transfected with miR-629–5p or miR-ctrl. (C) The colony formation assay was performed on the transfected cells. (D) The wound-healing rates of the cells. (E) Transwell invasion assay. (F) qRT-PCR analysis of miR-629–5p expression in MHCC97H cells transfected with the miR-629–5p inhibitor (in) or the negative control RNA (NC). The knockdown of miR629–5p suppressed cell viability (G), the colony formation ability (H), migration (I), and invasion (J). *P < 0.05, **P < 0.01.
3. Results
tissue samples compared with matched normal tissue samples (Fig. 1B). Furthermore, statistical analysis of data from The Cancer Gene Atlas (TCGA) database indicated that patients with high miR-629–5p levels in the tumor had shorter overall survival than did patients with low miR629–5p expression in the tumor (Fig. 1C). These results suggested that upregulation of miR-629–5p may affect HCC initiation and progression.
3.1. miR-629–5p is overexpressed in HCC In this study, the expression levels of miR-629–5p were first measured by qRT-PCR in several HCC cell lines and in nontransformed hepatic cell line L02. The miR-629–5p level was significantly higher in HCC cell lines relative to L02 cells (Fig. 1A). In agreement with this result, miR-629–5p turned out to be markedly upregulated in HCC
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Fig. 3. MiR-629–5p promoted tumor proliferation and metastasis in a mouse model of HCC. (A) MHCC97L cells infected with a miR-629-5p–expressing lentivirus or a negative control lentivirus were used to set up the mouse model. Photographs of tumors after 4 weeks are shown. (B) Diagrams showing the calculated tumor volume and weight in each group. (C) Representative photographs of immunohistochemical staining of the Ki67 antigen in tumors from nude mice. The percentage of Ki67positive cells is given on the right. (D) Representative H&E-stained sections of the lung tissues; quantitative analysis of tumor foci is shown on the right. *P < 0.05, **P < 0.01.
uncovered higher percentages of Ki67-positive cells in the miR-629–5p group compared to the control group (Fig. 3C). We next examined the mice for lung metastasis from the tumor derived from MHCC97L cells. As shown in Fig. 3D, the lung metastatic nodules were significantly more numerous in the miR-629–5p group than in the control group (Fig. 3D). Taken together, these results meant that miR-629–5p suppressed tumor growth and metastasis in HCC in vivo.
3.2. miR-629–5p promotes HCC cell proliferation, invasion, and migration in vitro To evaluate the possible involvement of miR-629–5p in HCC, miR629–5p mimics were transfected into MHCC97L cells, and the efficiency of transfection was validated by miRNA qRT-PCR (Fig. 2A). Cell viability was then assessed by the MTT assay, and the results revealed that overexpression of miR-629–5p obviously increased the viability of MHCC97L cells as compared to the corresponding control (Fig. 2B). This phenomenon was next confirmed by the colony formation assay, which showed that forced expression of miR-629–5p notably enhanced the colony formation ability of these cells (Fig. 2C). After that, woundhealing and Transwell invasion assays were carried out to further explore the activities of miR-629–5p in HCC cells. As presented in Fig. 2D and 48 h after administration of a scratch, the gap between cells was narrower in the MHCC97L cell group overexpressing miR-629–5p than in the negative control group, indicating that overexpression of miR629–5p may promote cell migration. In addition, the results of the Transwell invasion assay revealed that miR-629–5p overexpression enhanced the invasion ability of HCC cells (Fig. 2E). In contrast, miR629–5p knockdown in MHCC97H cells via the miR-629–5p inhibitor significantly suppressed cell proliferation, migration, and invasion as compared to the negative control (Fig. 2F–J). These results suggested that miR-629–5p acts as an oncogenic miRNA in HCC cell lines in vitro.
3.4. SFRP2 is a direct target of mir-629–5p Using TargetScan database, we identified the secreted frizzled-related protein 2 (SFRP2) as a likely target gene of miR-629–5p because its mRNA contains a putative miR-629–5p target site in its 3′UTR (Fig. 4A). Western blot and qRT-PCR analyses suggested that both the protein and mRNA levels of SFRP2 were significantly downregulated in miR-629-5p–overexpressing cells and upregulated in miR-629–5p knockdown cells (Fig. 4B and C). Furthermore, Western blot analysis of the tumor tissue samples confirmed decreased SFRP2 protein levels in miR-629-5p–overexpressing tumors (Fig. 4C). We then performed the luciferase assays, and the results indicated that miR-629–5p markedly decreased the luciferase activity of the wild-type SFRP2 3′UTR in both L02 and MHCC97L cells, whereas this suppressive effect was abrogated after the miR-629-5p–binding site was mutated in SFRP2 3′UTR (Fig. 4D), suggesting that miR-629–5p directly inhibited SFRP2 expression by binding to the 3′UTR of its mRNA. Subsequently, we performed restoration assays on MHCC97L cells to determine whether SFRP2 mediated the oncogenic influence of miR-629–5p. As illustrated in Fig. 4E, SFRP2 overexpression abrogated miR-629-5p–induced cell growth and invasion. Altogether, these results indicated that miR629–5p promoted HCC cell proliferation and invasion by targeting SFRP2 mRNA.
3.3. miR-629–5p promotes tumor growth and metastasis in vivo To validate the above observations in vitro, we examined the in vivo relevance of miR-629-5p–mediated regulation of HCC growth and metastasis in a mouse model of HCC. As depicted in Fig. 3A and B, miR629–5p promoted tumor growth in vivo, and the tumors derived from miR-629-5p–overexpressing MHCC97L cells had larger volume and weight than those of the control tumors. Accordingly, immunohistochemical staining for the cell proliferation marker Ki67 127
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Fig. 4. SFRP2 was proved to be a potential target gene of miR-629–5p. (A) miR-629–5p and its putative binding sequences in the 3′UTR of SFRP2 mRNA. (B) The expression level of SFRP2 mRNA in HCC cells after alteration of miR-629–5p expression according to qRT-PCR analysis. (C) The expression level of the SFRP2 protein in HCC cells or subcutaneous tumors after alteration of miR-629–5p expression, according to a Western blot. (D) The luciferase reporter assay was conducted to determine direct targeting of the SFRP2 mRNA 3′UTR by miR-629–5p. (E) SFRP2 plasmids were transfected into miR-629-5p–overexpressing MHCC97L cells, and then Western blot, MTT, and Transwell invasion assays were performed. **P < 0.01 as compared with the miR-ctrl group, &P < 0.05 and &&P < 0.01 as compared with the miR-629–5p group.
3.5. miR-629–5p promotes β-catenin activity in HCC cells
629–5p expression was significantly higher in HCC cell lines and tissue samples; (2) miR-629–5p significantly promoted HCC cell proliferation and invasion both in vitro and in vivo; (3) SFRP2 mRNA is a direct target of miR-629–5p; (4) through binding to the 3′UTR of SFRP2 mRNA, miR629–5p activated β-catenin signaling and promoted HCC cell proliferation and invasion. The miR-629–5p gene was mapped to the chromosomal 15q23 locus, which has been reported to be dysregulated and play an oncogenic part in several cancers [9–11]. For example, miR-629 expression is significantly higher in human clear cell renal cell carcinoma and enhances motility and invasiveness of these cells via TGF-β/Smad signaling [9]. Furthermore, upregulated miR-629 promotes human pancreatic cancer progression by downregulating FOXO3 [10]. In addition, miR-629–5p functions as an oncogene by improving proliferation and migration and by repressing apoptosis and fluorouracil (5-FU) sensitivity during colorectal cancer progression by directly downregulating CXXC finger protein 4 [11]. In agreement with these data, we found here that miR-629–5p expression is significantly higher in HCC cell lines and tissue samples. MiR-629–5p overexpression was able to promote HCC cell proliferation, migration, and invasion in vitro, whereas miR-629–5p knockdown suppressed cell proliferation and invasiveness. Moreover, miR-629–5p overexpression accelerated tumor growth and metastasis in our nude-mouse model, thus pointing to the oncogenic activity of miR-629–5p in human HCC. Next, we attempted to determine the mechanism by which miR629–5p promotes HCC growth and metastasis. In database TargetScan, we identified SFRP2 as a likely target gene of miR-629–5p because
Given that SFRP2 functions as a negative regulator of the Wnt–βcatenin signaling pathway [15,16], we measured the levels of β-catenin and its downstream effector, cyclin D1, in HCC cells after alteration of miR-629–5p expression. As shown in Fig. 5A, overexpression of miR629–5p enhanced the expression of total β-catenin and cyclin D1 and resulted in substantial nuclear accumulation of β-catenin. In contrast, inhibition of miR-629–5p expression obviously downregulated β-catenin and cyclin D1. Likewise, miR-629–5p overexpression markedly increased the transactivating activity of β-catenin in MHCC97L cells, as determined by a β-catenin reporter assay (Fig. 5B), suggesting that miR629–5p overexpression can enhance β-catenin nuclear translocation. To confirm the role of β-catenin activation in miR-629-5p–induced cell proliferation and invasion, we analyzed the impact of knocking β-catenin down with a specific siRNA (Fig. 5C). Functional assays showed that the β-catenin knockdown abrogated miR-629-5p–induced growth and invasiveness. These results suggested that β-catenin signaling is a functional mediator of miR-374a–induced proliferation and invasiveness of HCC cells. 4. Discussion More and more miRNAs have been shown to participate in various tumor processes, including tumor initiation, progression, and metastasis [17]. In this study, we investigated the biological role of miR629–5p in HCC growth and metastasis and demonstrated that (1) miR128
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Fig. 5. Wnt/β-catenin signaling mediates the effects of miR-629–5p. (A) Western blot analysis of the expression levels of total and nuclear β-catenin and cyclin D1 in HCC cells after alteration of miR-629–5p expression. (B) The indicated cells transfected with TOPflash or FOPflash and Renilla pRL-TK plasmids were subjected to dual-luciferase assays 48 h after transfection. Reporter activity was examined and normalized to Renilla luciferase activity. (C) β-catenin siRNA (siβ-catenin) was transfected into miR-629-5p–overexpressing MHCC97L cells, and then Western blot, MTT, and Transwell invasion assays were carried out. **P < 0.01 as compared with the miR-ctrl group, &P < 0.05 and &&P < 0.01 as compared with the miR-629–5p group.
Acknowledgements
SFRP2 mRNA contains a putative miR-629–5p target site in its 3′UTR. Further experiments revealed that the protein and mRNA levels of SFRP2 are significantly lower in miR-629-5p–overexpressing cells and higher in miR-629–5p knockdown cells. Additionally, luciferase reporter assays confirmed that miR-629–5p can directly target the 3′UTR of SFRP2 mRNA. Secreted frizzled-related proteins (SFRPs) are extracellular signaling molecules that antagonize the Wnt/β-catenin signaling pathway [15]. As a member of the SFRP family, SFRP2 is usually inactivated by promoter methylation in different human cancers including HCC and serves as an epigenetic tumor biomarker [15,18–20]. As a result, SFRP2 appears to act as a tumor suppressor in several cancers, including melanoma, pituitary corticotroph adenoma, and triple-negative breast cancer [20–22]. On the other hand, SFRP2 has been reported to have oncogenic properties during progression of p53 mutation–associated osteosarcoma [23]. In the present study, we demonstrated that SFRP2 overexpression abrogates miR-629-5p–induced HCC cell growth and invasion. Furthermore, miR-629-5p–mediated downregulation of SFRP2 leads to β-catenin activation in HCC cells. Knockdown of β-catenin prevented miR-629-5p–induced HCC cell growth and invasion. The data were similar to the findings in pancreatic cancer, in which βcatenin was enhanced in miR-629-overexpressed pancreatic cancer cells [10]. To our knowledge, this study is the first to explore the underlying mechanism by which miR-629–5p promotes β-catenin activity in HCC cells. In conclusion, this study provides novel evidence that miR-629–5p promotes HCC growth and metastasis by downregulating SFRP2 and thereby enhancing β-catenin signaling. Our results suggest that these signaling molecules may be potential targets for future prevention or treatment of human HCC.
This work was supported by the Scientific Research Project of Shanxi Health and Family Planning Commission in 2016 (201602030). References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2018, CA A Cancer J. Clin. 68 (2018) 7–30. [2] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell 136 (2009) 215–233. [3] X. Zhao, W. Dou, L. He, S. Liang, J. Tie, C. Liu, T. Li, Y. Lu, P. Mo, Y. Shi, K. Wu, Y. Nie, D. Fan, MicroRNA-7 functions as an anti-metastatic microRNA in gastric cancer by targeting insulin-like growth factor-1 receptor, Oncogene 32 (2013) 1363–1372. [4] J. Cai, H. Guan, L. Fang, Y. Yang, X. Zhu, J. Yuan, J. Wu, M. Li, MicroRNA-374a activates Wnt/beta-catenin signaling to promote breast cancer metastasis, J. Clin. Investig. 123 (2013) 566–579. [5] C.C. Wong, C.M. Wong, E.K. Tung, S.L. Au, J.M. Lee, R.T. Poon, K. Man, I.O. Ng, The microRNA miR-139 suppresses metastasis and progression of hepatocellular carcinoma by down-regulating Rho-kinase 2, Gastroenterology 140 (2011) 322–331. [6] X. Yang, X.F. Zhang, X. Lu, H.L. Jia, L. Liang, Q.Z. Dong, Q.H. Ye, L.X. Qin, MicroRNA-26a suppresses angiogenesis in human hepatocellular carcinoma by targeting hepatocyte growth factor-cMet pathway, Hepatology 59 (2014) 1874–1885. [7] J. Han, F. Wang, Y. Lan, J. Wang, C. Nie, Y. Liang, R. Song, T. Zheng, S. Pan, T. Pei, C. Xie, G. Yang, X. Liu, M. Zhu, Y. Wang, Y. Liu, F. Meng, Y. Cui, B. Zhang, X. Meng, J. Zhang, L. Liu, KIFC1 regulated by miR-532-3p promotes epithelial-to-mesenchymal transition and metastasis of hepatocellular carcinoma via gankyrin/AKT signaling, Oncogene 38 (2018) 406–420. [8] Y. Hu, C. Yang, S. Yang, F. Cheng, J. Rao, X. Wang, miR-665 promotes hepatocellular carcinoma cell migration, invasion, and proliferation by decreasing Hippo signaling through targeting PTPRB, Cell Death Dis. 9 (2018) 954. [9] K. Jingushi, Y. Ueda, K. Kitae, H. Hase, H. Egawa, I. Ohshio, R. Kawakami, Y. Kashiwagi, Y. Tsukada, T. Kobayashi, W. Nakata, K. Fujita, M. Uemura, N. Nonomura, K. Tsujikawa, miR-629 targets TRIM33 to promote tgfbeta/smad signaling and metastatic phenotypes in ccRCC, Mol. Canc. Res. 13 (2015) 565–574. [10] H. Yan, Q. Li, J. Wu, W. Hu, J. Jiang, L. Shi, X. Yang, D. Zhu, M. Ji, C. Wu, MiR-629 promotes human pancreatic cancer progression by targeting FOXO3, Cell Death Dis. 8 (2017) e3154. [11] J. Lu, S. Lu, J. Li, Q. Yu, L. Liu, Q. Li, MiR-629-5p promotes colorectal cancer progression through targetting CXXC finger protein 4, Biosci. Rep. 38 (2018). [12] S. Chen, F. Li, H. Chai, X. Tao, H. Wang, A. Ji, miR-502 inhibits cell proliferation and tumor growth in hepatocellular carcinoma through suppressing phosphoinositide 3-kinase catalytic subunit gamma, Biochem. Biophys. Res. Commun. 464
Competing interests None.
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X. Tao, et al.
[19] H. Wang, X.L. Duan, X.L. Qi, L. Meng, Y.S. Xu, T. Wu, P.G. Dai, Concurrent hypermethylation of SFRP2 and DKK2 activates the Wnt/beta-catenin pathway and is associated with poor prognosis in patients with gastric cancer, Mol Cells 40 (2017) 45–53. [20] S. Liu, Z. Wang, Z. Liu, S. Shi, Z. Zhang, J. Zhang, H. Lin, miR-221/222 activate the Wnt/beta-catenin signaling to promote triple-negative breast cancer, J. Mol. Cell Biol. 10 (2018) 302–315. [21] X. Luo, B. Wei, A. Chen, H. Zhao, K. Huang, J. Chen, Methylation-mediated loss of SFRP2 enhances melanoma cell invasion via Wnt signaling, Am J Transl Res 8 (2016) 1502–1509. [22] J. Ren, F. Jian, H. Jiang, Y. Sun, S. Pan, C. Gu, X. Chen, W. Wang, G. Ning, L. Bian, Q. Sun, Decreased expression of SFRP2 promotes development of the pituitary corticotroph adenoma by upregulating Wnt signaling, Int. J. Oncol. 52 (2018) 1934–1946. [23] H. Kim, S. Yoo, R. Zhou, A. Xu, J.M. Bernitz, Y. Yuan, A.M. Gomes, M.G. Daniel, J. Su, E.G. Demicco, J. Zhu, K.A. Moore, D.F. Lee, I.R. Lemischka, C. Schaniel, Oncogenic role of SFRP2 in p53-mutant osteosarcoma development via autocrine and paracrine mechanism, Proc. Natl. Acad. Sci. U. S. A. 115 (2018) E11128–E11137.
(2015) 500–505. [13] F. Fang, R.M. Chang, L. Yu, X. Lei, S. Xiao, H. Yang, L.Y. Yang, MicroRNA-188-5p suppresses tumor cell proliferation and metastasis by directly targeting FGF5 in hepatocellular carcinoma, J. Hepatol. 63 (2015) 874–885. [14] Y. Yan, Y.C. Luo, H.Y. Wan, J. Wang, P.P. Zhang, M. Liu, X. Li, S. Li, H. Tang, MicroRNA-10a is involved in the metastatic process by regulating Eph tyrosine kinase receptor A4-mediated epithelial-mesenchymal transition and adhesion in hepatoma cells, Hepatology 57 (2013) 667–677. [15] Y.L. Shih, C.B. Hsieh, H.C. Lai, M.D. Yan, T.Y. Hsieh, Y.C. Chao, Y.W. Lin, SFRP1 suppressed hepatoma cells growth through Wnt canonical signaling pathway, Int. J. Cancer 121 (2007) 1028–1035. [16] Y. Guo, L. Chen, C. Sun, C. Yu, MicroRNA-500a promotes migration and invasion in hepatocellular carcinoma by activating the Wnt/beta-catenin signaling pathway, Biomed. Pharmacother. 91 (2017) 13–20. [17] K. Nakao, H. Miyaaki, T. Ichikawa, Antitumor function of microRNA-122 against hepatocellular carcinoma, J. Gastroenterol. 49 (2014) 589–593. [18] H. Takagi, S. Sasaki, H. Suzuki, M. Toyota, R. Maruyama, M. Nojima, H. Yamamoto, M. Omata, T. Tokino, K. Imai, Y. Shinomura, Frequent epigenetic inactivation of SFRP genes in hepatocellular carcinoma, J. Gastroenterol. 43 (2008) 378–389.
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Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr
Research article
miR-629–5p promotes growth and metastasis of hepatocellular carcinoma by activating β-catenin
T
Xin Taoa,1, Xiaoxia Yangb,1, Kexin Wub, Liang Yangb, Yufei Huangb, Qian Jinb, Suling Chena,∗ a b
Department of Infectious Disease, Heping Hospital Attached to Changzhi Medical College, Changzhi, 046000, China Changzhi Medical College, Changzhi, 046000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: miR-629–5p Hepatocellular carcinoma Proliferation Invasion β-catenin
Aberrant miR-629–5p expression in several cancer types has been reported. Nonetheless, its potential effect and mechanism of action on tumor growth and metastasis in hepatocellular carcinoma (HCC) have rarely been analyzed. In this study, we found that miR-629–5p was upregulated in HCC tissue samples as compared to matched adjacent-tissue samples. Overexpression of miR-629–5p promoted the proliferation, migration, and invasiveness of human HCC cells in vitro, whereas miR-629–5p knockdown reduced these parameters. Consistently, miR-629–5p overexpression accelerated tumor growth and metastasis in a nude mouse model. Mechanistically, miR-629–5p directly targeted the 3′ untranslated region (3′UTR) of the secreted frizzled-related protein 2 (SFRP2) mRNA and suppressed its expression, resulting in the activation of β-catenin. Inhibition of βcatenin abrogated miR-629-5p–induced growth and invasiveness. Collectively, these results suggest that miR629–5p activates β-catenin signaling by downregulating SFRP2 and thus promotes the growth and metastasis of HCC. These data open up new prospects for HCC treatment.
1. Introduction Hepatocellular carcinoma (HCC) is a common malignant human tumor worldwide [1]. Due to its high malignancy potential, HCC ranks as the fifth leading cause of cancer-related deaths in the United States, resulting in more than 30,000 deaths in 2018 [1]. Although the treatments were improved dramatically in recent years, the prognosis of HCC patients remains unsatisfactory. Thus, it is important to further explore novel biomarkers and potential therapeutic targets to improve the therapeutic responsiveness and prognosis of HCC. MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that regulate gene expression by binding to the 3′ untranslated region (3′UTR) of target mRNA, thereby inducing mRNA degradation or protein synthesis repression [2]. Accumulating evidence indicates that miRNAs can function in human carcinogenesis as novel types of oncogenes or tumor suppressors [3,4]. To date, a group of miRNAs has been reported to regulate the growth and metastasis of HCC and is believed to constitute promising markers for the diagnosis and prognosis of this disease, e.g., miR-139, miR-26a, miR-532–3p, and miR-665 [5–8]. It has been shown that miR-629–5p is dysregulated in several types of cancer and plays an oncogenic role via the regulation of diverse cellular
functions such as cell proliferation, apoptosis, migration, and invasion [9–11]. Nevertheless, the biological effect of miR-629–5p in HCC remains unknown. In this study, we found that the expression of miR-629–5p was upregulated in HCC cell lines and tissue samples. Furthermore, in vitro and in vivo assays showed that miR-629–5p promoted the growth and metastasis of HCC by repressing SFRP2 and activating β-catenin. These observations provide a clearer understanding of the mechanism by which miR-629–5p promotes HCC. 2. Materials and methods 2.1. Tissue samples and cell lines Twenty human HCC tissue samples and matched normal adjacenttissue samples were obtained from Heping Hospital Attached to Changzhi Medical College. Each tumor tissue sample was snap-frozen in lipid nitrogen and then stored at −80 °C. Informed consent was obtained from each patient, and the use of human tissues was approved by the Medical Ethics and Human Clinical Trial Committee of Heping Hospital Attached to Changzhi Medical College. Human HCC cell lines
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Corresponding author. Department of Infectious Disease, Heping Hospital Attached to Changzhi Medical College, No. 110 South Yanan Road, Changzhi, 046000, China. E-mail address: [email protected] (S. Chen). 1 Equal contributors. https://doi.org/10.1016/j.yexcr.2019.03.042 Received 14 January 2019; Received in revised form 29 March 2019; Accepted 30 March 2019 Available online 04 April 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.
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2.6. Wound-healing assay and transwell invasion assay
MHCC97L, MHCC97H, and HCCLM3 and nontransformed hepatic cell line L02 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% of fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified incubator with 5% CO2.
Wound-healing assay was performed to assess the cell migration ability. Transfected cells were seeded in 6-well plates and cultured to 80–90% confluence. The cell monolayer was scratched with a sterile 200 μl pipette tip, and the detached cells were removed with a PBS wash. After cultivation in the complete DMEM medium for 48 h, the cells were photographed. The distance traveled by the cells between the two boundaries of the wound was calculated. For the Transwell invasion assay, transfected cells were seeded onto the membrane of the upper chamber with Matrigel in a serum-free medium. The lower chamber was filled with the complete DMEM medium (containing 10% of fetal bovine serum as a chemoattractant). After incubation at 37 °C for 48 h, the invading cells that got attached to the lower surface of the membrane were stained with crystal violet and counted under a microscope.
2.2. Quantitative reverse-transcription polymerase chain reaction (qRTPCR) Total-RNA samples including miRNAs were isolated with the miRNeasy Mini Kit (Qiagen, Shanghai, China). Expression levels of mRNA and miRNA were examined in triplicate by qRT-PCR. For mRNA analysis, complementary DNA (cDNA) synthesis from total RNA was performed with random hexamer primers. For miR-629–5p analysis, specific RT primers were used. cDNA was then amplified by means of the SYBR® Green PCR Master Mix. Relative expression was normalized to U6 or β-actin expression and was calculated by the 2−ΔΔCt method.
2.7. Luciferase reporter assay
2.3. Western blotting
L02 and MHCC97L cells were seeded in 96-well plates and transiently transfected with an appropriate reporter plasmid and miRNA using Lipofectamine 2000; 48 h later, the cells were harvested and lysed, and luciferase activity was measured via the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Luciferase reporter assays were performed in duplicate and repeated in three independent experiments.
Western blotting assay was performed as described previously [12]. Cells or tissues were lysed in RIPA buffer supplemented with a Protease Inhibitor Cocktail (Pierce, Appleton, WI, USA). The lysates were analyzed using the BCA Protein Assay Kit (Pierce). Proteins were separated via SDS-PAGE in a 10% gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were subsequently blocked with 5% nonfat milk and incubated with antibodies against SFRP2, β-catenin, cyclin D1, and β-actin (Proteintech, Wuhan, China) (primary antibodies) overnight at 4 °C. After a wash with Tris-buffered saline supplemented with 0.1% of Tween 20 (TBST), membranes were incubated with a secondary antibody for 1 h at room temperature. Signals were detected by means of an Enhanced Chemiluminescence system (Pierce).
2.8. In vivo tumor growth and metastasis assays The HCC model in mice was set up as described previously [13]. For the in vivo tumor growth assay, 2 × 106 MHCC97L cells infected with the miR-629-5p–expressing lentivirus or control lentivirus were subcutaneously injected into the flank of each nude mouse (BALB/c-nu/nu; five in each group) (SLRC, Shanghai, China). Four weeks later, the subcutaneous tumors were excised, weighed, and implanted into the liver of a nude mouse (five in each group). The mice were euthanized after 6 weeks, and then the lungs were collected, fixed with phosphatebuffered neutral formalin, sectioned serially, and stained with hematoxylin and eosin (H&E) for standard histological examination. All the procedures were approved by the Institutional Research Ethics Committee at Changzhi Medical College.
2.4. Oligonucleotide transfection and plasmid construction MiR-629–5p mimics, a miR-629–5p inhibitor, β-catenin small interfering RNA (siRNA), and the corresponding negative control were purchased from Ribobio (Guangzhou, China). Oligonucleotide transfection was performed using Lipofectamine 2000 (Invitrogen). The wild-type (WT) or mutant (MUT) 3′UTR of secreted frizzled-related protein 2 (SFRP2) mRNA containing a putative binding site for miR629–5p was cloned into the psiCHECK-2 luciferase reporter vector. SFRP2 without the 3′UTR region was cloned into the multiple cloning site of pcDNA3.1 to generate an SFRP2 overexpression plasmid. The constructs were verified by DNA sequencing.
2.9. Immunohistochemical staining This assay was carried out similarly to previously described methods [14]. In breif, tissues were fixed in phosphate-buffered neutral formalin, embedded in paraffin, and cut into 5-μm-thick sections. The latter were deparaffinized, rehydrated, and microwave-heated in sodium citrate buffer for antigen retrieval. The tissue sections were incubated with a primary antibody (against Ki67) at a 1:100 dilution overnight at 4 °C. Detection of the primary antibody was performed for 1 h at room temperature using a goat anti-rabbit IgG antibody conjugated with horseradish peroxidase, and the results were visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB) as a substrate.
2.5. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and colony formation assay The number of viable cells was evaluated by the MTT assay. Briefly, transfected cells (5 × 103 per well) seeded in 96-well plates were cultured in the growth medium. After cultivation for 24, 48, 72, and 96 h, 100 μl of the sterile MTT dye (0.5 mg/ml, Sigma) was added into each well with incubation for 4 h at 37 °C, followed by removal of the culture medium and addition of 150 μl of dimethyl sulphoxide (Sigma, St. Louis, MO, USA). Absorbance was measured at 490 nm. All the experiments were conducted in triplicate. For the colony formation assay, transfected cells were seeded in 6-well plates at a density of 300 cells per well and cultured at 37 °C for 2 weeks. The medium was changed every 3 days. At the end of the incubation, the cells were fixed with 100% methanol and stained with 0.1% crystal violet. Megascopic cell colonies were counted in Image-Pro Plus 5.0 (Media Cybernetics, Bethesda, MD).
2.10. Statistical analysis GraphPad Prism 5.0 software was used for statistical calculations. All data are presented as means ± standard deviation (SD). The twotailed Student t-test was performed to analyze differences between two groups. Data with P values less than 0.05 were considered statistically significant. 125
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Fig. 1. The levels of miR-629–5p expression in HCC. (A) MiR-629–5p expression levels were assessed by qRT-PCR in three HCC cell lines and L02 cells. (B) qRT-PCR analysis of miR-629–5p expression levels in 20 HCC tissue samples and matched normal tissue samples. (C) Kaplan–Meier analysis of survival of HCC patients with a distinct expression level of miR-629–5p in the tumor (data from database TCGA). *P < 0.05, **P < 0.01.
Fig. 2. MiR-629–5p promotes HCC cell proliferation, migration, and invasion in vitro. (A) Relative miR-629–5p levels in MHCC97L cells transfected with miR-629–5p or the negative control (miR-ctrl) were measured by qRT-PCR. (B) Cell viability was determined by the MTT assay of MHCC97L cells transfected with miR-629–5p or miR-ctrl. (C) The colony formation assay was performed on the transfected cells. (D) The wound-healing rates of the cells. (E) Transwell invasion assay. (F) qRT-PCR analysis of miR-629–5p expression in MHCC97H cells transfected with the miR-629–5p inhibitor (in) or the negative control RNA (NC). The knockdown of miR629–5p suppressed cell viability (G), the colony formation ability (H), migration (I), and invasion (J). *P < 0.05, **P < 0.01.
3. Results
tissue samples compared with matched normal tissue samples (Fig. 1B). Furthermore, statistical analysis of data from The Cancer Gene Atlas (TCGA) database indicated that patients with high miR-629–5p levels in the tumor had shorter overall survival than did patients with low miR629–5p expression in the tumor (Fig. 1C). These results suggested that upregulation of miR-629–5p may affect HCC initiation and progression.
3.1. miR-629–5p is overexpressed in HCC In this study, the expression levels of miR-629–5p were first measured by qRT-PCR in several HCC cell lines and in nontransformed hepatic cell line L02. The miR-629–5p level was significantly higher in HCC cell lines relative to L02 cells (Fig. 1A). In agreement with this result, miR-629–5p turned out to be markedly upregulated in HCC
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Fig. 3. MiR-629–5p promoted tumor proliferation and metastasis in a mouse model of HCC. (A) MHCC97L cells infected with a miR-629-5p–expressing lentivirus or a negative control lentivirus were used to set up the mouse model. Photographs of tumors after 4 weeks are shown. (B) Diagrams showing the calculated tumor volume and weight in each group. (C) Representative photographs of immunohistochemical staining of the Ki67 antigen in tumors from nude mice. The percentage of Ki67positive cells is given on the right. (D) Representative H&E-stained sections of the lung tissues; quantitative analysis of tumor foci is shown on the right. *P < 0.05, **P < 0.01.
uncovered higher percentages of Ki67-positive cells in the miR-629–5p group compared to the control group (Fig. 3C). We next examined the mice for lung metastasis from the tumor derived from MHCC97L cells. As shown in Fig. 3D, the lung metastatic nodules were significantly more numerous in the miR-629–5p group than in the control group (Fig. 3D). Taken together, these results meant that miR-629–5p suppressed tumor growth and metastasis in HCC in vivo.
3.2. miR-629–5p promotes HCC cell proliferation, invasion, and migration in vitro To evaluate the possible involvement of miR-629–5p in HCC, miR629–5p mimics were transfected into MHCC97L cells, and the efficiency of transfection was validated by miRNA qRT-PCR (Fig. 2A). Cell viability was then assessed by the MTT assay, and the results revealed that overexpression of miR-629–5p obviously increased the viability of MHCC97L cells as compared to the corresponding control (Fig. 2B). This phenomenon was next confirmed by the colony formation assay, which showed that forced expression of miR-629–5p notably enhanced the colony formation ability of these cells (Fig. 2C). After that, woundhealing and Transwell invasion assays were carried out to further explore the activities of miR-629–5p in HCC cells. As presented in Fig. 2D and 48 h after administration of a scratch, the gap between cells was narrower in the MHCC97L cell group overexpressing miR-629–5p than in the negative control group, indicating that overexpression of miR629–5p may promote cell migration. In addition, the results of the Transwell invasion assay revealed that miR-629–5p overexpression enhanced the invasion ability of HCC cells (Fig. 2E). In contrast, miR629–5p knockdown in MHCC97H cells via the miR-629–5p inhibitor significantly suppressed cell proliferation, migration, and invasion as compared to the negative control (Fig. 2F–J). These results suggested that miR-629–5p acts as an oncogenic miRNA in HCC cell lines in vitro.
3.4. SFRP2 is a direct target of mir-629–5p Using TargetScan database, we identified the secreted frizzled-related protein 2 (SFRP2) as a likely target gene of miR-629–5p because its mRNA contains a putative miR-629–5p target site in its 3′UTR (Fig. 4A). Western blot and qRT-PCR analyses suggested that both the protein and mRNA levels of SFRP2 were significantly downregulated in miR-629-5p–overexpressing cells and upregulated in miR-629–5p knockdown cells (Fig. 4B and C). Furthermore, Western blot analysis of the tumor tissue samples confirmed decreased SFRP2 protein levels in miR-629-5p–overexpressing tumors (Fig. 4C). We then performed the luciferase assays, and the results indicated that miR-629–5p markedly decreased the luciferase activity of the wild-type SFRP2 3′UTR in both L02 and MHCC97L cells, whereas this suppressive effect was abrogated after the miR-629-5p–binding site was mutated in SFRP2 3′UTR (Fig. 4D), suggesting that miR-629–5p directly inhibited SFRP2 expression by binding to the 3′UTR of its mRNA. Subsequently, we performed restoration assays on MHCC97L cells to determine whether SFRP2 mediated the oncogenic influence of miR-629–5p. As illustrated in Fig. 4E, SFRP2 overexpression abrogated miR-629-5p–induced cell growth and invasion. Altogether, these results indicated that miR629–5p promoted HCC cell proliferation and invasion by targeting SFRP2 mRNA.
3.3. miR-629–5p promotes tumor growth and metastasis in vivo To validate the above observations in vitro, we examined the in vivo relevance of miR-629-5p–mediated regulation of HCC growth and metastasis in a mouse model of HCC. As depicted in Fig. 3A and B, miR629–5p promoted tumor growth in vivo, and the tumors derived from miR-629-5p–overexpressing MHCC97L cells had larger volume and weight than those of the control tumors. Accordingly, immunohistochemical staining for the cell proliferation marker Ki67 127
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Fig. 4. SFRP2 was proved to be a potential target gene of miR-629–5p. (A) miR-629–5p and its putative binding sequences in the 3′UTR of SFRP2 mRNA. (B) The expression level of SFRP2 mRNA in HCC cells after alteration of miR-629–5p expression according to qRT-PCR analysis. (C) The expression level of the SFRP2 protein in HCC cells or subcutaneous tumors after alteration of miR-629–5p expression, according to a Western blot. (D) The luciferase reporter assay was conducted to determine direct targeting of the SFRP2 mRNA 3′UTR by miR-629–5p. (E) SFRP2 plasmids were transfected into miR-629-5p–overexpressing MHCC97L cells, and then Western blot, MTT, and Transwell invasion assays were performed. **P < 0.01 as compared with the miR-ctrl group, &P < 0.05 and &&P < 0.01 as compared with the miR-629–5p group.
3.5. miR-629–5p promotes β-catenin activity in HCC cells
629–5p expression was significantly higher in HCC cell lines and tissue samples; (2) miR-629–5p significantly promoted HCC cell proliferation and invasion both in vitro and in vivo; (3) SFRP2 mRNA is a direct target of miR-629–5p; (4) through binding to the 3′UTR of SFRP2 mRNA, miR629–5p activated β-catenin signaling and promoted HCC cell proliferation and invasion. The miR-629–5p gene was mapped to the chromosomal 15q23 locus, which has been reported to be dysregulated and play an oncogenic part in several cancers [9–11]. For example, miR-629 expression is significantly higher in human clear cell renal cell carcinoma and enhances motility and invasiveness of these cells via TGF-β/Smad signaling [9]. Furthermore, upregulated miR-629 promotes human pancreatic cancer progression by downregulating FOXO3 [10]. In addition, miR-629–5p functions as an oncogene by improving proliferation and migration and by repressing apoptosis and fluorouracil (5-FU) sensitivity during colorectal cancer progression by directly downregulating CXXC finger protein 4 [11]. In agreement with these data, we found here that miR-629–5p expression is significantly higher in HCC cell lines and tissue samples. MiR-629–5p overexpression was able to promote HCC cell proliferation, migration, and invasion in vitro, whereas miR-629–5p knockdown suppressed cell proliferation and invasiveness. Moreover, miR-629–5p overexpression accelerated tumor growth and metastasis in our nude-mouse model, thus pointing to the oncogenic activity of miR-629–5p in human HCC. Next, we attempted to determine the mechanism by which miR629–5p promotes HCC growth and metastasis. In database TargetScan, we identified SFRP2 as a likely target gene of miR-629–5p because
Given that SFRP2 functions as a negative regulator of the Wnt–βcatenin signaling pathway [15,16], we measured the levels of β-catenin and its downstream effector, cyclin D1, in HCC cells after alteration of miR-629–5p expression. As shown in Fig. 5A, overexpression of miR629–5p enhanced the expression of total β-catenin and cyclin D1 and resulted in substantial nuclear accumulation of β-catenin. In contrast, inhibition of miR-629–5p expression obviously downregulated β-catenin and cyclin D1. Likewise, miR-629–5p overexpression markedly increased the transactivating activity of β-catenin in MHCC97L cells, as determined by a β-catenin reporter assay (Fig. 5B), suggesting that miR629–5p overexpression can enhance β-catenin nuclear translocation. To confirm the role of β-catenin activation in miR-629-5p–induced cell proliferation and invasion, we analyzed the impact of knocking β-catenin down with a specific siRNA (Fig. 5C). Functional assays showed that the β-catenin knockdown abrogated miR-629-5p–induced growth and invasiveness. These results suggested that β-catenin signaling is a functional mediator of miR-374a–induced proliferation and invasiveness of HCC cells. 4. Discussion More and more miRNAs have been shown to participate in various tumor processes, including tumor initiation, progression, and metastasis [17]. In this study, we investigated the biological role of miR629–5p in HCC growth and metastasis and demonstrated that (1) miR128
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Fig. 5. Wnt/β-catenin signaling mediates the effects of miR-629–5p. (A) Western blot analysis of the expression levels of total and nuclear β-catenin and cyclin D1 in HCC cells after alteration of miR-629–5p expression. (B) The indicated cells transfected with TOPflash or FOPflash and Renilla pRL-TK plasmids were subjected to dual-luciferase assays 48 h after transfection. Reporter activity was examined and normalized to Renilla luciferase activity. (C) β-catenin siRNA (siβ-catenin) was transfected into miR-629-5p–overexpressing MHCC97L cells, and then Western blot, MTT, and Transwell invasion assays were carried out. **P < 0.01 as compared with the miR-ctrl group, &P < 0.05 and &&P < 0.01 as compared with the miR-629–5p group.
Acknowledgements
SFRP2 mRNA contains a putative miR-629–5p target site in its 3′UTR. Further experiments revealed that the protein and mRNA levels of SFRP2 are significantly lower in miR-629-5p–overexpressing cells and higher in miR-629–5p knockdown cells. Additionally, luciferase reporter assays confirmed that miR-629–5p can directly target the 3′UTR of SFRP2 mRNA. Secreted frizzled-related proteins (SFRPs) are extracellular signaling molecules that antagonize the Wnt/β-catenin signaling pathway [15]. As a member of the SFRP family, SFRP2 is usually inactivated by promoter methylation in different human cancers including HCC and serves as an epigenetic tumor biomarker [15,18–20]. As a result, SFRP2 appears to act as a tumor suppressor in several cancers, including melanoma, pituitary corticotroph adenoma, and triple-negative breast cancer [20–22]. On the other hand, SFRP2 has been reported to have oncogenic properties during progression of p53 mutation–associated osteosarcoma [23]. In the present study, we demonstrated that SFRP2 overexpression abrogates miR-629-5p–induced HCC cell growth and invasion. Furthermore, miR-629-5p–mediated downregulation of SFRP2 leads to β-catenin activation in HCC cells. Knockdown of β-catenin prevented miR-629-5p–induced HCC cell growth and invasion. The data were similar to the findings in pancreatic cancer, in which βcatenin was enhanced in miR-629-overexpressed pancreatic cancer cells [10]. To our knowledge, this study is the first to explore the underlying mechanism by which miR-629–5p promotes β-catenin activity in HCC cells. In conclusion, this study provides novel evidence that miR-629–5p promotes HCC growth and metastasis by downregulating SFRP2 and thereby enhancing β-catenin signaling. Our results suggest that these signaling molecules may be potential targets for future prevention or treatment of human HCC.
This work was supported by the Scientific Research Project of Shanxi Health and Family Planning Commission in 2016 (201602030). References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2018, CA A Cancer J. Clin. 68 (2018) 7–30. [2] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell 136 (2009) 215–233. [3] X. Zhao, W. Dou, L. He, S. Liang, J. Tie, C. Liu, T. Li, Y. Lu, P. Mo, Y. Shi, K. Wu, Y. Nie, D. Fan, MicroRNA-7 functions as an anti-metastatic microRNA in gastric cancer by targeting insulin-like growth factor-1 receptor, Oncogene 32 (2013) 1363–1372. [4] J. Cai, H. Guan, L. Fang, Y. Yang, X. Zhu, J. Yuan, J. Wu, M. Li, MicroRNA-374a activates Wnt/beta-catenin signaling to promote breast cancer metastasis, J. Clin. Investig. 123 (2013) 566–579. [5] C.C. Wong, C.M. Wong, E.K. Tung, S.L. Au, J.M. Lee, R.T. Poon, K. Man, I.O. Ng, The microRNA miR-139 suppresses metastasis and progression of hepatocellular carcinoma by down-regulating Rho-kinase 2, Gastroenterology 140 (2011) 322–331. [6] X. Yang, X.F. Zhang, X. Lu, H.L. Jia, L. Liang, Q.Z. Dong, Q.H. Ye, L.X. Qin, MicroRNA-26a suppresses angiogenesis in human hepatocellular carcinoma by targeting hepatocyte growth factor-cMet pathway, Hepatology 59 (2014) 1874–1885. [7] J. Han, F. Wang, Y. Lan, J. Wang, C. Nie, Y. Liang, R. Song, T. Zheng, S. Pan, T. Pei, C. Xie, G. Yang, X. Liu, M. Zhu, Y. Wang, Y. Liu, F. Meng, Y. Cui, B. Zhang, X. Meng, J. Zhang, L. Liu, KIFC1 regulated by miR-532-3p promotes epithelial-to-mesenchymal transition and metastasis of hepatocellular carcinoma via gankyrin/AKT signaling, Oncogene 38 (2018) 406–420. [8] Y. Hu, C. Yang, S. Yang, F. Cheng, J. Rao, X. Wang, miR-665 promotes hepatocellular carcinoma cell migration, invasion, and proliferation by decreasing Hippo signaling through targeting PTPRB, Cell Death Dis. 9 (2018) 954. [9] K. Jingushi, Y. Ueda, K. Kitae, H. Hase, H. Egawa, I. Ohshio, R. Kawakami, Y. Kashiwagi, Y. Tsukada, T. Kobayashi, W. Nakata, K. Fujita, M. Uemura, N. Nonomura, K. Tsujikawa, miR-629 targets TRIM33 to promote tgfbeta/smad signaling and metastatic phenotypes in ccRCC, Mol. Canc. Res. 13 (2015) 565–574. [10] H. Yan, Q. Li, J. Wu, W. Hu, J. Jiang, L. Shi, X. Yang, D. Zhu, M. Ji, C. Wu, MiR-629 promotes human pancreatic cancer progression by targeting FOXO3, Cell Death Dis. 8 (2017) e3154. [11] J. Lu, S. Lu, J. Li, Q. Yu, L. Liu, Q. Li, MiR-629-5p promotes colorectal cancer progression through targetting CXXC finger protein 4, Biosci. Rep. 38 (2018). [12] S. Chen, F. Li, H. Chai, X. Tao, H. Wang, A. Ji, miR-502 inhibits cell proliferation and tumor growth in hepatocellular carcinoma through suppressing phosphoinositide 3-kinase catalytic subunit gamma, Biochem. Biophys. Res. Commun. 464
Competing interests None.
129
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X. Tao, et al.
[19] H. Wang, X.L. Duan, X.L. Qi, L. Meng, Y.S. Xu, T. Wu, P.G. Dai, Concurrent hypermethylation of SFRP2 and DKK2 activates the Wnt/beta-catenin pathway and is associated with poor prognosis in patients with gastric cancer, Mol Cells 40 (2017) 45–53. [20] S. Liu, Z. Wang, Z. Liu, S. Shi, Z. Zhang, J. Zhang, H. Lin, miR-221/222 activate the Wnt/beta-catenin signaling to promote triple-negative breast cancer, J. Mol. Cell Biol. 10 (2018) 302–315. [21] X. Luo, B. Wei, A. Chen, H. Zhao, K. Huang, J. Chen, Methylation-mediated loss of SFRP2 enhances melanoma cell invasion via Wnt signaling, Am J Transl Res 8 (2016) 1502–1509. [22] J. Ren, F. Jian, H. Jiang, Y. Sun, S. Pan, C. Gu, X. Chen, W. Wang, G. Ning, L. Bian, Q. Sun, Decreased expression of SFRP2 promotes development of the pituitary corticotroph adenoma by upregulating Wnt signaling, Int. J. Oncol. 52 (2018) 1934–1946. [23] H. Kim, S. Yoo, R. Zhou, A. Xu, J.M. Bernitz, Y. Yuan, A.M. Gomes, M.G. Daniel, J. Su, E.G. Demicco, J. Zhu, K.A. Moore, D.F. Lee, I.R. Lemischka, C. Schaniel, Oncogenic role of SFRP2 in p53-mutant osteosarcoma development via autocrine and paracrine mechanism, Proc. Natl. Acad. Sci. U. S. A. 115 (2018) E11128–E11137.
(2015) 500–505. [13] F. Fang, R.M. Chang, L. Yu, X. Lei, S. Xiao, H. Yang, L.Y. Yang, MicroRNA-188-5p suppresses tumor cell proliferation and metastasis by directly targeting FGF5 in hepatocellular carcinoma, J. Hepatol. 63 (2015) 874–885. [14] Y. Yan, Y.C. Luo, H.Y. Wan, J. Wang, P.P. Zhang, M. Liu, X. Li, S. Li, H. Tang, MicroRNA-10a is involved in the metastatic process by regulating Eph tyrosine kinase receptor A4-mediated epithelial-mesenchymal transition and adhesion in hepatoma cells, Hepatology 57 (2013) 667–677. [15] Y.L. Shih, C.B. Hsieh, H.C. Lai, M.D. Yan, T.Y. Hsieh, Y.C. Chao, Y.W. Lin, SFRP1 suppressed hepatoma cells growth through Wnt canonical signaling pathway, Int. J. Cancer 121 (2007) 1028–1035. [16] Y. Guo, L. Chen, C. Sun, C. Yu, MicroRNA-500a promotes migration and invasion in hepatocellular carcinoma by activating the Wnt/beta-catenin signaling pathway, Biomed. Pharmacother. 91 (2017) 13–20. [17] K. Nakao, H. Miyaaki, T. Ichikawa, Antitumor function of microRNA-122 against hepatocellular carcinoma, J. Gastroenterol. 49 (2014) 589–593. [18] H. Takagi, S. Sasaki, H. Suzuki, M. Toyota, R. Maruyama, M. Nojima, H. Yamamoto, M. Omata, T. Tokino, K. Imai, Y. Shinomura, Frequent epigenetic inactivation of SFRP genes in hepatocellular carcinoma, J. Gastroenterol. 43 (2008) 378–389.
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