miRNA-338-3p/CDK4 signaling pathway suppressed hepatic stellate cell activation and proliferation
- Bensong Duan†1,
- Jiangfeng Hu†1Email author,
- Tongyangzi Zhang2,
- Xu Luo3,
- Yi Zhou3,
- Shun Liu4,
- Liang Zhu3,
- Cheng Wu5,
- Wenxiang Liu6,
- Chao Chen6Email author and
- Hengjun Gao7, 8Email author
© The Author(s). 2017
Received: 7 October 2016
Accepted: 7 January 2017
Published: 17 January 2017
Activated hepatic stellate cell (HSC) is the main fibrogenic cell type in the injured liver. miRNA plays an important role in activation and proliferation of HSC.
Our previous study examined the expression profiles of microRNAs in quiescent and activated HSC. Real-time PCR and western blot were used to detect the expression of Collagen type I (Col 1) and Alpha-Smooth Muscle Actin (α-SMA). CCK-8 and Edu assay was used to measure the proliferation rate of HSC. Luciferase reporter gene assay was used to tested the binding between miR-338-3p and Cyclin-dependent kinase 4 (CDK4).
We found overexpression of miR-338-3p could inhibit Col 1 and α-SMA, two major HSC activation markers, whereas miR-338-3p inhibitor could promote them. Besides, miR-338-3p overexpression could suppress the growth rate of HSC. Further, we found that CDK4, a pleiotropic signaling protein, was a direct target gene of miR-338-3p. Moreover, we found that overexpression of CDK4 could block the effects of miR-338-3p.
We found miR-338-3p is an anti-fibrotic miRNA which inhibits cell activation and proliferation. Our findings suggest that miR-338-3p/CDK4 signaling pathway participates in the regulation of HSC activation and growth and may act as a novel target for further anti-fibrotic therapy.
KeywordsLiver fibrosis miR-338 CDK4
Liver fibrosis is a common consequence of most chronic liver diseases . Liver fibrosis received more attention until the hepatic stellate cell (HSC) was identified as the main ECM-producing cells in the injured liver . Under the normal physiologic condition, HSCs reside in the space of disse and store a large amount of vitamin A. After suffering from liver injury, HSCs will be activated, and then proliferate, eventually transdifferentiate into myofibroblast-like cells .
microRNAs (miRNAs), short (~22 nt) conceding RNA molecules, can directly regulate gene expression by binding to the 3’UTR region of target mRNA to participate in lots of regulation of physiological process and diseases [4–8]. Recently, researchers focused on the role of miRNA in liver fibrosis pathophysiology to determine their regulatory effects on proliferation, differentiation of HSC [9–11]. Several abnormally expressed miRNAs were found and identified between quiescent and activated HSCs by using miRNA array or RT-PCR [12–19]. Previous studies have reported that miRNAs were critically involved in the activation of HSCs. Among them, miR-29b precursor allowed activated HSCs to switch to a more quiescent state . Similarly, overexpression of miR-27a/b could lead HSCs to a quiescent phenotype . microRNA-338 (miR-338), a newly identified miRNA, played a crucial role in a variety of carcinomas. Aberrant expression of miR-338 was closely related to cell proliferation, invasion, early detection and clinic pathologic variables in liver cancer, colorectal cancer, gastric cancer and neuroblastoma [21–25]. In previous research, our miRNA microarray data have found altered expression of miR-338-3p during culture activation of HSC . However, little is known about the role of miR-338-3p in liver fibrosis.
Cyclin-dependent kinase 4 (CDK4) is found to be involved in cell cycle regulation. Activation of cyclin D—CDK4 promotes the cell cycle progression through G1/S transition . Inhibition of CDK4 shows promising efficacy on advanced breast cancer . In liver tissue and hepatoma cells, CDK4/6 inhibition is a potent mediator of cytostasis . However, whether the CDK4 participates in the fibrogenic process and regulates HSC activation and proliferation remains largely unknown. In this study, RT-PCR data suggested that miR-338-3p expression in fully activated HSCs were significantly decreased compared with that in quiescent HSCs. Transforming growth factor (TGF-β) is deemed to be the most potent fibrogenic cytokine. The results showed that there was a negative relationship between TGF-β and miR-338-3p. Therefore, we speculated that miR-338-3p was closely associated with HSCs function. Then, we found overexpression of miR-338-3p could suppress HSCs activation and proliferation while inhibition of miR-338-3p could promote HSCs activation and proliferation. Based on the Bioinformatics prediction, we found that CDK4 was a potential target gene of miR-338-3p. Further luciferase reporter assay and RT-PCR confirmed their complementary binding. Moreover, our results indicated that overexpression of CDK4 could partially block miR-338-3p-inhibited cell activation and proliferation.
Primary rat HSCs, cell lines and culture
The isolation method of primary rat HSCs was according to the previous literature . Primary Rat HSCs, HSC-T6 and HEK293T (human embryonic kidney cell line) were kindly gifted from Dr. Gao (Tongji University, Shanghai, China). The primary cells and cell lines were cultured in DMEM (Dulbecco’s modified Eagle’s medium, Thermo, Waltham, MA, USA) containing 10% FBS (Fetal bovine serum, FBS, Gibco, Grand Island, NY, USA) at 37 °C in a humidified atmosphere of 5% CO2.
Wild-type 3’UTR containing predicted miR-338-3p binding sites were amplified from HSC-T6 genomic DNA and inserted into the PGL3 luciferase reporter vector. Mutant 3’UTR was generated using the Quick Change Lighting Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The CDK4 expression vector was obtained by cloning the CDK4-coding sequence into the pcDNA.
Total RNA was extracted from cultured HSCs using Trizol Reagent (Takara, Dalian, China). The primer sequences used for mRNA detection in this study were listed as follows: GAPDH (PF: CAGTGCCAGCCTCGTCTCAT, PR: AGGGGCCATCCACAGTCTTC); ColI (PF: ATCCTGCCGATGTCGCTAT, PR: CCACAAGCGTGCTGTAGGT); α-SMA (PF: CCGAGATCTCACCGACTACC, PR: TCCAGAGCGACATAGCACAG); CDK4 (PF: GAAGAAGAAGCGGAGGAAGAGG, PR: TTAGGTTAGTGCGGGAATGAAT).
CCK-8 assay and Edu assay
Cell proliferation was performed using CCK-8 assay (Dojindo, Japan) and Edu (Ribibio, Guangzhou, China) assay. For CCK-8, HSC-T6 was transfected with the miR-338 precursor, miR-338 inhibitor (Ribibio, Guangzhou, China) or pcDNA-CDK4 in 96 well culture plates. Proliferation rates were tested at 24, 48 and 72 h after transfection. The EdU assay was conducted according to the protocol of Ribibio Edu Kit.
Luciferase assay was performed with the Dual Luciferase Reporter Assay System (Promega, Madison, WI). Transfection was carried out in 48 well plates using Fugen (Roche). There were two groups. One was co-transfected with 200 ng wild-type-CDK4-3’UTR, 20 nm miR-338 precursor, and 20 ng Renilla. Another was co-transfected with 200mutant CDK4 3’UTR without binding site of miR-338-3p, 20 nm miR-338 precursor, and 20 ng Renilla. 48 h later, Firefly and Renilla luciferase activities were tested.
Cells were lysed in SDS sample buffer. Antibodies against GAPDH (Biogot, 1:5000 dilution, Nanjing, China), Col1 (Abcam, 1:1500 dilution, Cambridge, MA, USA), α-sma (Sigma, 1:1000 dilution, Shanghai, China) and CDK4 (Biogot, 1:3000 dilution, Nanjing, China) were used in this study. Signals were visualized with ImageQuant LAS 4000 (GE Healthcare Life Sciences).
The statistical analysis in our study was performed by using SPSS 22.0. Data were given as mean ± SEM. Two tailed t-test was used to determine between two groups. Statistical significance level was set at p < 0.05.
miR-338-3p was downregulated in fully activated HSCs
Overexpression of miR-338-3p suppressed HSC activation while inhibition of it promoted HSC activation
Overexpression of miR-338 suppressed HSC-T6 proliferation
miR-338 repressed CDK4 expression by directly binding to its 3’UTR region
CDK4 rescued miR-338-inhibition of activation and proliferation of HSCs
We conduct CCK-8 assay to determine the proliferation of HSC-T6 transfected with CDK4 plasmid or control empty vector. The results showed that cell transfected with CDK4 plasmid displayed a higher proliferative capacity compared with the control group (Fig. 4a). Besides, we found cells transfected with CDK4 could promote cell activation with upregulation of Col1 and α-Sma (Fig. 4b, c). We co-transfected HSC-T6 cells with miR-338 precursor and CDK4 plasmid to investigate whether CDK4 would rescue the inhibition effect of miR-338 on the cell activation and proliferation or not. The results suggested that overexpression of CDK4 partially block the repression effect of miR-338 on the activation and proliferation of HSC-T6. The growth curves of three groups (Control, Pre-miR-338/pcDNA, Pre-miR-338/CDK4) were shown in Fig. 4d. The expression of activation associated markers, Col1 andα-Sma, was showed in Fig. 4e-f.
Liver fibrosis is a scarring response to liver damage. It’s a common pathological process for most of the liver disorder. A small number of patients go on to progress cirrhosis and/or hepatocellular carcinoma. Fortunately, liver fibrosis can be reversed if the inflammation was controlled .
Aberrant expression of miRNAs have been involved in liver fibrosis and regarded as a potential treatment strategy. Intervening miRNAs expression could assist activated HSCs to return to a quiescent phenotype. miRNA microarray and RT-PCR was carried out to determine abnormally expressed miRNAs during HSCs activation. Our results suggested that miR-338-3p was significantly downregulated in this process. miR-338 is located on chromosome 17q25.3 with a length of 22 nt and produces two mature forms, miR-338-3p and miR-338-5p. miR-338 was first reported in neurodegeneration and gradually studied in various disease . In hepatocellular carcinoma, miR-338 downregulation was associated with tumor size, TNM stage, vascular invasion and in trahepatic metastasis [21, 22]. In colorectal carcinoma, miR-338 expression was significantly increased in both blood and tissue samples. It might appear to be a potential biomarker for early detection in colorectal carcinoma . In gastric carcinoma, miR-338 was epigenetically silenced and its reduction was related to pathological variables. Overexpression of miR-338 could suppress cell proliferation, migration, invasion and tumorigenicity . Moreover, combined with other six miRNAs, miR-338 could be used to predict gastric cancer prognosis . Despite in cancer, miR-338 was also involved in idiopathic pulmonary fibrosis .
This is the first study to identify the biological function of miR-338-3p in liver fibrosis. Our results demonstrated that miR-338 precursor transfection suppressed the activation and proliferation of HSC-T6, whereas inhibition of miR-338-3p promoted cell activation and proliferation.
To understand the underlying mechanism of miR-338-mediated inhibition of proliferation, we identified CDK4, a member of the cyclin-dependent kinase family, as a candidate target gene. CDK4 usually work with Cyclin D to regulate the cell cycle in G1/S stage. Aberrant activation of CDK4 was closely associated with various kinds of carcinomas. CDK4 expression is significantly upregulated in lung cancer tissues and function as an important element for cell proliferation [35, 36]. In breast cancer, inhibition of CDK4 can induce G1 arrest . These observations suggest that inhibition of CDK4 might be beneficial for cancer treatment. An increasing body of clinical trials targeting CDK4 has been launched. However, the role of CDK4 in liver fibrosis remains largely unknown. In this study, Luciferase reporter assay showed that there was a combination of miR-338 and CDK4. Hence, we deduced that miR-338-3p inhibited HSCs’ activation and proliferation likely through silencing CDK4. Our data indicated that restoring CDK4 expression could partially rescue miR-338-inhibited cell activation and proliferation.
In conclusion, our study identified a new anti-fibrosis miRNA that may play an important role in the development of liver fibrosis.
Cyclin-dependent kinase 4
- Col 1:
Collagen type I
Hepatic stellate cell
Alpha-Smooth Muscle Actin
We thank Tongji University School of Medicine for technical help.
This work was supported by grants from National Natural Science Foundation of China [grants 81402756, 81101644, 11472300, 11272342]; Science and Technology Research Project from Guangxi Education Department (YB2014067). The funding body approved the study design and the methods that were used for data acquisition and analysis, but had no involvement with the interpretation of data or with the composition of the manuscript.
Availability of data and materials
All the data supporting the findings of the study is contained within the manuscript.
Conceived and designed the experiments: BD, JH, HG, CC Performed the experiments: JH, BD, TZ, XL. Analyzed the data: JH, SL, CW, YZ. Wrote the paper: JH, BD, LZ, WL. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study was carried out in strict accordance with the International Council for Laboratory Animal Science. All animal experiments were conducted in accordance with protocols approved by the Experimental Animal Ethical Committee of Tongji University.
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- Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115:209–18.View ArticlePubMedPubMed CentralGoogle Scholar
- Gabele E, Brenner DA, Rippe RA. Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell. Front Biosci. 2003;8:d69–77.View ArticlePubMedGoogle Scholar
- Marra F. Hepatic stellate cells and the regulation of liver inflammation. J Hepatol. 1999;31:1120–30.View ArticlePubMedGoogle Scholar
- Liu Q, Wang G, Chen Y, Li G, Yang D, Kang J. A miR-590/Acvr2a/Rad51b axis regulates DNA damage repair during mESC proliferation. Stem Cell Rep. 2014;3:1103–17.View ArticleGoogle Scholar
- Yan IK, Wang X, Asmann YW, Haga H, Patel T. Circulating extracellular RNA markers of liver regeneration. PLoS One. 2016;11:e0155888.View ArticlePubMedPubMed CentralGoogle Scholar
- Seeliger C, Balmayor ER, van Griensven M. miRNAs related to skeletal diseases. Stem Cells Dev. 2016;25(17):1261-1281.Google Scholar
- Cheng CJ, Bahal R, Babar IA, Pincus Z, Barrera F, Liu C, Svoronos A, Braddock DT, Glazer PM, Engelman DM, et al. MicroRNA silencing for cancer therapy targeted to the tumour microenvironment. Nature. 2015;518:107–10.View ArticlePubMedGoogle Scholar
- Schickel R, Boyerinas B, Park SM, Peter ME. MicroRNAs: key players in the immune system, differentiation, tumorigenesis and cell death. Oncogene. 2008;27:5959–74.View ArticlePubMedGoogle Scholar
- Yu F, Guo Y, Chen B, Dong P, Zheng J. MicroRNA-17-5p activates hepatic stellate cells through targeting of Smad7. Lab Investig. 2015;95:781–9.View ArticlePubMedGoogle Scholar
- Sekiya Y, Ogawa T, Yoshizato K, Ikeda K, Kawada N. Suppression of hepatic stellate cell activation by microRNA-29b. Biochem Biophys Res Commun. 2011;412:74–9.View ArticlePubMedGoogle Scholar
- Zhao J, Tang N, Wu K, Dai W, Ye C, Shi J, Zhang J, Ning B, Zeng X, Lin Y. MiR-21 simultaneously regulates ERK1 signaling in HSC activation and hepatocyte EMT in hepatic fibrosis. PLoS One. 2014;9:e108005.View ArticlePubMedPubMed CentralGoogle Scholar
- Roderburg C, Urban GW, Bettermann K, Vucur M, Zimmermann H, Schmidt S, Janssen J, Koppe C, Knolle P, Castoldi M, et al. Micro-RNA profiling reveals a role for miR-29 in human and murine liver fibrosis. Hepatology. 2011;53:209–18.View ArticlePubMedGoogle Scholar
- Kitano M, Bloomston P.M. Hepatic Stellate Cells and microRNAs in Pathogenesis of Liver Fibrosis. J Clin Med. 2016;5(3):38. doi:https://doi.org/10.3390/jcm5030038.
- Chen C, Wu CQ, Zhang ZQ, Yao DK, Zhu L. Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation. Exp Cell Res. 2011;317:1714–25.View ArticlePubMedGoogle Scholar
- Cushing L, Kuang PP, Qian J, Shao F, Wu J, Little F, Thannickal VJ, Cardoso WV, Lu J. miR-29 is a major regulator of genes associated with pulmonary fibrosis. Am J Respir Cell Mol Biol. 2011;45:287–94.View ArticlePubMedGoogle Scholar
- Guo CJ, Pan Q, Cheng T, Jiang B, Chen GY, Li DG. Changes in microRNAs associated with hepatic stellate cell activation status identify signaling pathways. FEBS J. 2009;276:5163–76.View ArticlePubMedGoogle Scholar
- Li WQ, Chen C, Xu MD, Guo J, Li YM, Xia QM, Liu HM, He J, Yu HY, Zhu L. The rno-miR-34 family is upregulated and targets ACSL1 in dimethylnitrosamine-induced hepatic fibrosis in rats. FEBS J. 2011;278:1522–32.View ArticlePubMedGoogle Scholar
- Hu J, Chen C, Liu Q, Liu B, Song C, Zhu S, Wu C, Liu S, Yu H, Yao D, et al. The role of the miR-31/FIH1 pathway in TGF-beta-induced liver fibrosis. Clin Sci (Lond). 2015;129:305–17.View ArticleGoogle Scholar
- Wu K, Ye C, Lin L, Chu Y, Ji M, Dai W, Zeng X, Lin Y. Inhibiting miR-21 attenuates experimental hepatic fibrosis by suppressing both ERK1 pathway in HSC and hepatocyte EMT. Clin Sci. 2016;130(16):1469-1480.Google Scholar
- Ji J, Zhang J, Huang G, Qian J, Wang X, Mei S. Over-expressed microRNA-27a and 27b influence fat accumulation and cell proliferation during rat hepatic stellate cell activation. FEBS Lett. 2009;583:759–66.View ArticlePubMedGoogle Scholar
- Huang XH, Chen JS, Wang Q, Chen XL, Wen L, Chen LZ, Bi J, Zhang LJ, Su Q, Zeng WT. miR-338-3p suppresses invasion of liver cancer cell by targeting smoothened. J Pathol. 2011;225:463–72.View ArticlePubMedGoogle Scholar
- Huang XH, Wang Q, Chen JS, Fu XH, Chen XL, Chen LZ, Li W, Bi J, Zhang LJ, Fu Q, et al. Bead-based microarray analysis of microRNA expression in hepatocellular carcinoma: miR-338 is downregulated. Hepatol Res. 2009;39:786–94.View ArticlePubMedGoogle Scholar
- Yong FL, Law CW, Wang CW. Potentiality of a triple microRNA classifier: miR-193a-3p, miR-23a and miR-338-5p for early detection of colorectal cancer. BMC Cancer. 2013;13:280.View ArticlePubMedPubMed CentralGoogle Scholar
- Li P, Chen X, Su L, Li C, Zhi Q, Yu B, Sheng H, Wang J, Feng R, Cai Q, et al. Epigenetic silencing of miR-338-3p contributes to tumorigenicity in gastric cancer by targeting SSX2IP. PLoS One. 2013;8:e66782.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen X, Pan M, Han L, Lu H, Hao X, Dong Q. miR-338-3p suppresses neuroblastoma proliferation, invasion and migration through targeting PREX2a. FEBS Lett. 2013;587:3729–37.View ArticlePubMedGoogle Scholar
- Jia X, Liu B, Shi X, Ye M, Zhang F, Liu H. Roles of the ERK, JNK/AP-1/cyclin D1-CDK4 pathway in silica-induced cell cycle changes in human embryo lung fibroblast cells. Cell Biol Int. 2011;35:697–704.View ArticlePubMedGoogle Scholar
- O’Sullivan CC. CDK4/6 inhibitors for the treatment of advanced hormone receptor positive breast cancer and beyond: 2016 update. Expert Opin Pharmacother. 2016;17:1657-1667.Google Scholar
- Rivadeneira DB, Mayhew CN, Thangavel C, Sotillo E, Reed CA, Grana X, Knudsen ES. Proliferative suppression by CDK4/6 inhibition: complex function of the retinoblastoma pathway in liver tissue and hepatoma cells. Gastroenterology. 2010;138:1920–30.View ArticlePubMedPubMed CentralGoogle Scholar
- Riccalton-Banks L, Bhandari R, Fry J, Shakesheff KM. A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue. Mol Cell Biochem. 2003;248:97–102.View ArticlePubMedGoogle Scholar
- Sun K, Deng HJ, Lei ST, Dong JQ, Li GX. miRNA-338-3p suppresses cell growth of human colorectal carcinoma by targeting smoothened. World J Gastroenterol. 2013;19:2197–207.View ArticlePubMedPubMed CentralGoogle Scholar
- Perez-Tamayo R. Cirrhosis of the liver: a reversible disease? Pathol Annu. 1979;14(Pt 2):183–213.PubMedGoogle Scholar
- Saba R, Goodman CD, Huzarewich RL, Robertson C, Booth SA. A miRNA signature of prion induced neurodegeneration. PLoS One. 2008;3:e3652.View ArticlePubMedPubMed CentralGoogle Scholar
- Li X, Zhang Y, Zhang Y, Ding J, Wu K, Fan D. Survival prediction of gastric cancer by a seven-microRNA signature. Gut. 2010;59:579–85.View ArticlePubMedGoogle Scholar
- Zhang H, Liu X, Chen S, Wu J, Ye X, Xu L, Chen H, Zhang D, Tan R, Wang Y. Tectorigenin inhibits the in vitro proliferation and enhances miR-338* expression of pulmonary fibroblasts in rats with idiopathic pulmonary fibrosis. J Ethnopharmacol. 2010;131:165–73.View ArticlePubMedGoogle Scholar
- Lingfei K, Pingzhang Y, Zhengguo L, Jianhua G, Yaowu Z. A study on p16, pRb, cdk4 and cyclinD1 expression in non-small cell lung cancers. Cancer Lett. 1998;130:93–101.View ArticlePubMedGoogle Scholar
- Wu A, Wu B, Guo J, Luo W, Wu D, Yang H, Zhen Y, Yu X, Wang H, Zhou Y, et al. Elevated expression of CDK4 in lung cancer. J Transl Med. 2011;9:38.View ArticlePubMedPubMed CentralGoogle Scholar
- Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. Cancer Res. 1996;56:2973–8.PubMedGoogle Scholar