- Open Access
Expression of microRNA-454 in TGF-β1-stimulated hepatic stellate cells and in mouse livers infected with Schistosoma japonicum
- Dandan Zhu†1,
- Xue He†1,
- Yinong Duan1Email author,
- Jinling Chen1,
- Jianxin Wang1, 2,
- Xiaolei Sun1,
- Hongyan Qian3,
- Jinrong Feng1,
- Wei Sun1,
- Feifan Xu1 and
- Lingbo Zhang1
© Zhu et al.; licensee BioMed Central Ltd. 2014
Received: 24 December 2013
Accepted: 17 March 2014
Published: 31 March 2014
In the process of hepatic fibrosis, hepatic stellate cells (HSCs) can be activated by many inflammatory cytokines. The transforming growth factor-β1 (TGF-β1) is one of the main profibrogenic mediators. Recently, some studies have also shown that microRNAs (miRNAs) play essential roles in the progress of liver fibrosis by being involved in the differentiation, fat metabolism and ECM production of HSCs.
The expression of miR-454 in LX-2 cells treated with TGF-β1 and in the fibrotic livers with Schistosoma japonicum infection was detected by qRT-PCR. The role of miR-454 on LX-2 cells was then analyzed by Western blot, flow cytometry and luciferase assay.
The results showed that the expression of miR-454 was down-regulated in the TGF-β1-treated LX-2 cells and miR-454 could inhibit the activation of HSCs by directly targeting Smad4. However, we found that miR-454 had no effect on cell cycle and cell proliferation in TGF-β1-treated LX-2. Besides these, miR-454 was found to be regulated in the process of Schistosoma japonicum infection.
All the results suggested that miR-454 could provide a novel therapeutic approach for treating liver fibrosis, especially the liver fibrosis induced by Schistosoma japonicum.
Hepatic fibrosis, characterized by the excessive deposition of extracellular matrix (ECM), occurs in many types of liver diseases and reflects a balance between liver repair and scar formation . Inflammation plays a key role in the development of liver fibrosis. During the progression of liver fibrosis, macrophages, lymphocytes, fibroblasts and other inflammatory cells can be stimulated by etiological factors . Hepatic stellate cells (HSCs), which have been considered to be the major effector cells, can be activated by many inflammatory cytokines and undergo myofibroblastic transdifferentiation in the progress of liver fibrosis [1, 3]. During this progress, the transforming growth factor-β1 (TGF-β1) is recognized as one of the main profibrogenic mediators [4, 5] and plays a key role in the development of inflammation and subsequent liver fibrosis.
MicroRNAs (miRNAs) are endogenous, small and noncoding RNAs, which have the ability to regulate gene expression in a mature form by binding to the 3′-untranslated region (3′-UTR) of target mRNAs and repressing translation or inducing mRNA cleavage [6, 7]. Previous studies have revealed that miRNAs play indispensable roles in the progress of liver fibrosis by being involved in the differentiation, fat metabolism and ECM production of HSCs [8, 9] and the proliferation and apoptosis of HSCs [10–12]. Over-expression of miR-146a can suppress TGF-β1-induced HSC proliferation and induce HSC apoptosis . miR-15b and miR-16 are essential for HSC apoptosis by targeting Bcl-2 through the caspase signaling pathway . miR-335 can also inhibit HSC migration by decreasing the tenascin-C (TNC) expression . Recently, the miR-454 family has been reported to be up-regulated in human colorectal cancer tissues and cell lines by targeting Smad4 . However, there are no prior studies in which the effect of miR-454 on TGF-β1-induced HSC activation is considered. Therefore, in the present study, we attempted to observe the expression of miR-454 in activated HSCs and in the fibrotic livers, and then to study the role of miR-454 on HSC activation.
Animals and Schistosoma japonicum (S. japonicum) -infected liver fibrosis models
Healthy 4-6-wk-old male ICR mice were obtained from the Laboratory Animal Center of Nantong University. S. japonicum cercariae released from infected intermediate host snail Oncomelania hupensis were provided by the Jiangsu Institute of Parasitic Diseases (Wuxi, China). To construct the models infected with S. japonicum, mice were percutaneously infected with 20 ± 2 cercariae of S. japonicum and sacrificed on the 8th week after infection. HE staining and sirius-red staining were performed to confirm that the liver fibrosis models were constructed successfully. Animal care and experimental procedures were approved by the Animal Ethics Committee of Nantong University.
Cell culture and treatment
An immortalized human HSCs line, LX-2 cell line, was obtained from Xiang Ya Central Experiment Laboratory. LX-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA) in a humidified incubator at 37°C with 5% CO2. LX-2 cells were plated in a 6-well plate and cultured for 24 h before transfection. Then mimics, inhibitors of miR-454 or the nonspecific (NS)-miRNA were transfected into the cells at a final concentration of 100 nmol/l using lipofectamine 2000 (Invitrogen, USA) according to the manufacturers instructions. The culture medium was discarded after transfection for 4-6 h and replaced with the fresh medium or the medium with TGF-β1 (Sigma, USA) at the concentration of 5 ng/ml for 48 h. The sequences of the miR-454 mimics, inhibitors and the NS-miRNA were all designed and synthesized by Genepharma Company in Shanghai, China.
Construction and luciferase assay of 3′-UTR of Smad4
The wild-type and mutant sequences of the 3′-UTR of human Smad4 were amplified from LX-2 cells and cloned into the psi-CHECK-2 luciferase vector. For dual-luciferase reporter assays, the wild-type Luc-Smad4 or mutant Luc-Smad4 plasmids and miRNAs were co-transfected into LX-2 cells using lipofectamine 2000. After transfection for 48 h, the cells were collected and luciferase activity was analyzed by the dual-luciferase assay kit (Promega, USA).
RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was isolated using the Trizol reagent (Invitrogen, USA) according to the manufacturer’s instruction and then reverse transcribed into cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). QRT-PCR was performed according to the protocol of SYBR Premix Ex Taq RT-PCR Kit (Takara, Japan) in the Eco Real-time PCR system (Illumina, USA). The miRNAs were extracted using RNAiso for Small RNA (Takara, Japan) and reverse transcribed for qRT-PCR using SYBR PrimeScript miRNA RT-PCR integrative kit (Takara, Japan) according to the manufacturer’s protocol. The sense primers for miRNA qRT-PCR were synthesized by Invitrogen (China) [10, 13], and the universal anti-sense primer was obtained from Takara.
Proteins from LX-2 were extracted using RIPA lysis buffer (Beyotime, China) and quantified by the Bradford method (Sangon, China). Then the proteins were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 10% nonfat dry milk and then probed with primary antibodies against α-smooth muscle actin (α-SMA, Santa Cruz Biotechnology, USA), Smad4 (Santa Cruz Biotechnology, USA), PCNA (Abcam, USA) and glyceraldehyde phosphate dehydrogenase (GAPDH, Goodhere, China) at 4°C overnight. The membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Then the membranes were visualized with ECL-chemiluminescent kit (Merck, Germany).
The cell proliferation of LX-2 was determined using MTT assay. Firstly, the cells were plated at a density of 5 × 103 cells/well in a 96-well culture plate for 24 h. After transfection with miR-454 mimics or NS-miRNA for 4-6 h, the culture medium was replaced with the fresh medium or the medium with TGF-β1. After LX-2 cells were pulsed with MTT (Sigma, USA) for 4 h, 100 μl of dimethylsulphoxide (DMSO) was added in the medium to dissolve the formazan products. The optical density (OD) was determined on an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek) at 490 nm wavelength. All experiments were performed in triplicate and repeated at least three times.
The cell cycle phase was determined by fluorescence activating cell sorter (FACS) analysis with propidium iodide (PI, Sigma, USA) staining. LX-2 cells were transfected with miR-454 mimics or NS-miRNA and treated with or without TGF-β1. After fixation, cells were treated with RNase A to digest RNA in the cells and stained with PI at 37°C for 30 min. The cells were then assessed by flow cytometry and the data were analyzed. All experiments were repeated at least three times.
All experiments were performed in triplicate and the results were presented as mean ± SEM. The data were analyzed by One-Way ANOVA (LSD) using the SPSS 15.0 software to determine their significant differences. A value of P<0.05 was considered statistically significant.
α-SMA is up-regulated in TGF-β1-treated LX-2 cells
Expression of miR-454 is down-regulated in TGF-β1-treated LX-2 cells
Over-expression of miR-454 cannot regulate the proliferation and cell cycle distribution of LX-2 cells
Over-expression of miR-454 inhibits the expression of α-SMA in TGF-β1-treated LX-2 cells
Smad4 is a direct target of miR-454 in HSCs
Furthermore, in order to investigate whether Smad4 was directly targeted by miR-454 expression, we constructed the luciferase reporter plasmids containing wild-type or mutant 3′-UTR sequences of Smad4. The data showed that the luciferase reporter activity of the reporter plasmids containing the wild-type 3′-UTR of Smad4, but not the mutant 3′-UTR of Smad4, could be reduced by miR-454 mimics and increased by miR-454 inhibitor (Figure 5C). Therefore, miR-454 could inhibit TGF-β1-induced LX-2 activation by the suppression of Smad4 expression.
Expression of miR-454 is down-regulated in S. japonicum-induced liver fibrosis models
Liver fibrosis, which is characterized by an accumulation of ECM, is a common response to many liver injuries. During the progression of liver fibrosis, quiescent HSCs, which can be provoked by hepatitis B and C viruses, nonalcoholic steatohepatitis, alcoholism and schistosome infection, can activate into a myofibroblast-like phenotype [2, 4]. Recently, some studies have focused on the functions of miRNAs on liver fibrosis [8, 9, 15]. Previous studies have shown that miR-181b expression could be increased in HSC-T6 cells treated with TGF-β1 and miR-181b mimics could significantly promote the proliferation of HSC-T6 cells by directly targeting p27 . The expression of miR-150 and miR-194 were both down-regulated in the fibrotic livers of rats induced by bile duct-ligation (BDL), while over-expression of miR-150 and miR-194 could inhibit the proliferation of LX-2 cells . In addition, miR-21 significantly activated hepatic stellate cells through the PTEN/Akt pathway . Consistent with the results obtained by Ji et al. that the expression of miR-454 was depressed in activated rat HSCs, we found that the expression of miR-454 was down-regulated in LX-2 cells treated with TGF-β1.
TGF-β1/Smads signaling pathway is a key mediator to induce HSC activation and liver fibrosis . TGF-β1 can promote the formation of ECM through stimulation of the synthesis and secretion of Type-1 or Type-2 collagen and α-SMA [18, 19]. In addition, previous studies have indicated that anti-sense Smad4 gene could block TGF-β1 signal transduction by reducing the expression of Smad4 to inhibit the production of ECM and ameliorate hepatic fibrosis [20–22]. In this research, we also found that TGF-β1 could induce the activation of HSCs and reduce the expression of miR-454. Besides these, over-expression of miR-454 could inhibit the expression of α-SMA induced by TGF-β1 stimulation. However, miR-454 did not affect the proliferation and cell cycle in TGF-β1-treated LX-2 cells. Since previous studies have shown that the expression of miR-454 was significantly up-regulated in colon cancer and in HCT116 cells, and miR-454 contributed to colon tumorigenesis by directly repressing the expression of Smad4 , we further investigated whether Smad4 was also a direct target of miR-454 to regulate the expression of α-SMA in TGF-β1-treated LX-2 cells. Our experiments demonstrated that miR-454 could influence the level of Smad4 expression in TGF-β1-treated LX-2 cells, and over-expression of miR-454 could inhibit the luciferase activity of the wild-type Luc-Smad4 plasmid, but not the mutant-type Luc-Smad4 plasmid. Meanwhile, miR-454 inhibitor could increase luciferase activity of the wild-type 3′-UTR of Smad4 in LX-2 cells. These data indicated that miR-454 may down-regulate the expression of α-SMA and therefore inhibit the activation of TGF-β1-treated HSCs by directly targeting Smad4. However, since previous researches have found that over-expression of miR-146a could also inhibit TGF-β1-induced HSC proliferation by targeting Smad4 , we cannot rule out the possibility that other miRNAs could participate in the modulation of HSC activation and proliferation. We also cannot rule out the possibility that miR-454 could inhibit HSC activation through other signaling pathways, since multiple mRNAs are predicted by some softwares to be targeted by the same miRNA. Hence, further studies are needed to reveal the complex relationship among miRNAs (miR-146a and others), targets and hepatic fibrosis.
Schistosomiasis, which is caused by schistosomes, is one of the most common causes of hepatic fibrosis. Granulomatous inflammation is an initial characteristic manifestation of liver fibrosis induced by schistosomes. Recently, some studies have reported that the expression of some miRNAs can be regulated in the mouse liver infected with S. japonicum, and it suggests that miRNAs may be involved in the process of inflammatory granuloma formation and the subsequent hepatic fibrosis [23, 24]. Meanwhile, studies have also focused on the expression of miRNA profiles in the host liver infected with schistosomes to understand the molecular mechanisms of schistosomal hepatopathy, which differs from other chronic hepatopathy . In this study, we also observed the expression of miR-454 in the mouse liver during S. japonicum infection. The results showed that the level of the miR-454 was down-regulated in the fibrotic livers infected with S. japonicum for 8 weeks. On the contrary, the levels of α-SMA and Smad4 expression were all up-regulated. Since the tendency of the expression of Smad4 was opposite to that of miR-454 in the process of hepatic fibrosis induced by S. japonicum infection, we hypothesized that miR-454 may be involved in the progression of liver fibrosis induced by S. japonicum infection through the TGF-β1/Smad4 pathway.
In summary, our results indicated that miR-454 could inhibit the activation of HSCs by directly targeting Smad4. Moreover, miR-454 was regulated in the process of hepatic fibrosis induced by S. japonicum infection. Thus, miR-454 may provide a novel therapeutic approach for treating liver fibrosis, especially the liver fibrosis induced by S. japonicum.
This work was supported by a grant from the National Natural Science Foundation of China (No. 81171589) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
- Lee UE, Friedman SL: Mechanisms of hepatic fibrogenesis. Best Pract Res Clin Gastroenterol. 2011, 25 (2): 195-206. 10.1016/j.bpg.2011.02.005.PubMed CentralView ArticlePubMedGoogle Scholar
- Wynn TA, Barron L: Macrophages: master regulators of inflammation and fibrosis. Semin Liver Dis. 2010, 30 (3): 245-257. 10.1055/s-0030-1255354.PubMed CentralView ArticlePubMedGoogle Scholar
- Popov Y, Schuppan D: Targeting liver fibrosis: strategies for development and validation of antifibrotic therapies. Hepatology. 2009, 50 (4): 1294-1306. 10.1002/hep.23123.View ArticlePubMedGoogle Scholar
- Bataller R, Brenner DA: Liver fibrosis. J Clin Invest. 2005, 115 (2): 209-218. 10.1172/JCI24282.PubMed CentralView ArticlePubMedGoogle Scholar
- El-Lakkany NM, Hammam OA, El-Maadawy WH, Badawy AA, Ain-Shoka AA, Ebeid FA: Anti-inflammatory/anti-fibrotic effects of the hepatoprotective silymarin and the schistosomicide praziquantel against Schistosoma mansoni-induced liver fibrosis. Parasit Vectors. 2012, 5: 9-22. 10.1186/1756-3305-5-9.PubMed CentralView ArticlePubMedGoogle Scholar
- Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004, 116 (2): 281-297. 10.1016/S0092-8674(04)00045-5.View ArticlePubMedGoogle Scholar
- Chitwood DH, Timmermans MC: Target mimics modulate miRNAs. Nat Genet. 2007, 39 (8): 935-936. 10.1038/ng0807-935.View ArticlePubMedGoogle Scholar
- Wang B, Li W, Guo K, Xiao Y, Wang Y, Fan J: miR-181b promotes hepatic stellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosis patients. Biochem Biophys Res Commun. 2012, 421 (1): 4-8. 10.1016/j.bbrc.2012.03.025.View ArticlePubMedGoogle Scholar
- 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 (12): 1714-1725. 10.1016/j.yexcr.2011.05.001.View ArticlePubMedGoogle 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 (4): 759-766. 10.1016/j.febslet.2009.01.034.View ArticlePubMedGoogle Scholar
- He Y, Huang C, Sun X, Long XR, Lv XW, Li J: MicroRNA-146a modulates TGF-beta1-induced hepatic stellate cell proliferation by targeting SMAD4. Cell Signal. 2012, 24 (10): 1923-1930. 10.1016/j.cellsig.2012.06.003.View ArticlePubMedGoogle Scholar
- Guo CJ, Pan Q, Li DG, Sun H, Liu BW: miR-15b and miR-16 are implicated in activation of the rat hepatic stellate cell: An essential role for apoptosis. J Hepatol. 2009, 50 (4): 766-778. 10.1016/j.jhep.2008.11.025.View ArticlePubMedGoogle Scholar
- Liu L, Nie J, Chen L, Dong G, Du X, Wu X, Tang Y, Han W: The oncogenic role of microRNA-130a/301a/454 in human colorectal cancer via targeting Smad4 expression. PLoS One. 2013, 8 (2): e55532-10.1371/journal.pone.0055532.PubMed CentralView ArticlePubMedGoogle Scholar
- Inagaki Y, Okazaki I: Emerging insights into Transforming growth factor beta Smad signal in hepatic fibrogenesis. Gut. 2007, 56 (2): 284-292. 10.1136/gut.2005.088690.PubMed CentralView ArticlePubMedGoogle Scholar
- Venugopal SK, Jiang J, Kim TH, Li Y, Wang SS, Torok NJ, Wu J, Zern MA: Liver fibrosis causes downregulation of miRNA-150 and miRNA-194 in hepatic stellate cells, and their overexpression causes decreased stellate cell activation. Am J Physiol Gastrointest Liver Physiol. 2010, 298 (1): G101-G106. 10.1152/ajpgi.00220.2009.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei J, Feng L, Li Z, Xu G, Fan X: MicroRNA-21 activates hepatic stellate cells via PTEN/Akt signaling. Biomed Pharmacother. 2013, 67 (5): 387-392. 10.1016/j.biopha.2013.03.014.View ArticlePubMedGoogle Scholar
- Cheng K, Yang N, Mahato RI: TGF-β1 gene silencing for treating liver fibrosis. Mol Pharm. 2009, 6 (3): 772-779. 10.1021/mp9000469.PubMed CentralView ArticlePubMedGoogle Scholar
- Baarsma HA, Menzen MH, Halayko AJ, Meurs H, Kerstjens HA, Gosens R: Beta-Catenin signaling is required for TGF-beta1-induced extracellular matrix production by airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2011, 301 (6): L956-L965. 10.1152/ajplung.00123.2011.View ArticlePubMedGoogle Scholar
- Chen BL, Peng J, Li QF, Yang M, Wang Y, Chen W: Exogenous bone morphogenetic protein-7 reduces hepatic fibrosis in Schistosoma japonicum-infected mice via transforming growth factor-beta/Smad signaling. World J Gastroenterol. 2013, 19 (9): 1405-1415. 10.3748/wjg.v19.i9.1405.PubMed CentralView ArticlePubMedGoogle Scholar
- Dong MX, Jia Y, Zhang YB, Li CC, Geng YT, Zhou L, Li XY, Liu JC, Niu YC: Emodin protects rat liver from CCl(4)-induced fibrogenesis via inhibition of hepatic stellate cells activation. World J Gastroenterol. 2009, 15 (38): 4753-4762. 10.3748/wjg.15.4753.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang KL, Chang WT, Hung KC, Li EI, Chuang CC: Inhibition of transforming growth factor-beta-induced liver fibrosis by a retinoic acid derivative via the suppression of Col 1A2 promoter activity. Biochem Biophys Res Commun. 2008, 373 (2): 219-223. 10.1016/j.bbrc.2008.05.192.View ArticlePubMedGoogle Scholar
- Xu XB, He ZP, Leng XS, Liang ZQ, Peng JR, Zhang HY, Xiao M, Zhang H, Liu CL, Zhang XD: Effects of Smad4 on liver fibrosis and hepatocarcinogenesis in mice treated with CCl4/ethanol. Zhonghua Gan Zang Bing Za Zhi. 2010, 18 (2): 119-123.PubMedGoogle Scholar
- Cai P, Piao X, Liu S, Hou N, Wang H, Chen Q: MicroRNA-gene expression network in murine liver during Schistosoma japonicum infection. PLoS One. 2013, 8 (6): e67037-10.1371/journal.pone.0067037.PubMed CentralView ArticlePubMedGoogle Scholar
- He X, Sai X, Chen C, Zhang Y, Xu X, Zhang D, Pan W: Host serum miR-223 is a potential new biomarker for Schistosoma japonicum infection and the response to chemotherapy. Parasit Vectors. 2013, 6: 272-279. 10.1186/1756-3305-6-272.PubMed CentralView ArticlePubMedGoogle Scholar
- Han H, Peng J, Hong Y, Zhang M, Han Y, Liu D, Fu Z, Shi Y, Xu J, Tao J, Lin J: MicroRNA expression profile in different tissues of BALB/c mice in the early phase of Schistosoma japonicum infection. Mol Biochem Parasitol. 2013, 188 (1): 1-9. 10.1016/j.molbiopara.2013.02.001.View ArticlePubMedGoogle Scholar
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