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MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling Ai Shen a, b, Daofeng Zheng a, Yunhai Luo a, Tong Mou a, Qingsong Chen a, Zuotian Huang a, Zhongjun Wu a, * a b
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China Hepatobiliary Pancreatic Tumor Center, Chongqing University Cancer Hospital, Chongqing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 October 2019 Accepted 27 October 2019 Available online xxx
Ischemia and reperfusion (I/R) injury is a common cause of hepatocyte injury and liver dysfunction during liver transplantation, but its mechanism is needed further explored. We aimed to investigate whether STING pathway activation is involved in the liver I/R and further determine the role of the microRNA(miR)-24-3p in liver I/R injury in mice. Our data showed that STING mRNA level was negatively related with miR-24-3p in livers of I/R-treated mice. Next, we identified that STING could be bound by miR-24-3p by bioinformatic and luciferase report assay. Moreover, downregulation of STING alleviated the protein expression of p-IRF3 and the serum level of inflammatory factor and aminotransferase in I/R mice model. Furthermore, transfection of I/R treated mice with exogenous miR-24-3p significantly inhibited the protein expression of STING and p-IRF3 in liver, and attenuated serum inflammatory cytokines release, as well as the dysfunction and apoptosis of liver in I/R model in vivo. This study suggests that miR-24-3p may ameliorate inflammatory response and cellular apoptosis in hepatic I/R process by targeting STING, which might be a potential therapeutic target for preventing liver I/R development and progression. © 2019 Elsevier Inc. All rights reserved.
Keywords: MicroRNA-24-3p Stimulator of interferon genes Interferon regulatory factor 3 Inflammation Liver ischemia and reperfusion injury
1. Introduction Liver ischemia and reperfusion (I/R) injury has been recognized as the major cause of organ dysfunction during transplantation, which increases the risk of mortality [1e4]. The pathology of I/R injury involves innate immune system activation and apoptosis. I/R triggers several danger-associated molecular patterns (DAMPs) such as self-DNA, releasing from the injured cells, which can be recognized by the pattern recognition receptor (PRR) or signal sensor, eventually leading to a local inflammatory response and the occurrence of cell apoptosis [5e7]. However, the exact mechanism of I/R injury was uncertainty. Stimulator of interferon gene (STING), known as transmembrane protein 173 (TMEM173), can be activated by the cytoplasmic DNA sensor under stress condition, which plays a critical role in regulating the innate immune response. In the biological condition, STING is located in endoplasmic reticulum (ER) [8]. Upon
* Corresponding author. Friendship Road 1, Yuzhong District, 400016, Chongqing, China. E-mail address: [email protected] (Z. Wu).
DNA sensor stimulating, STING can be secreted from ER and become activation, facilitating the ER stress, inflammation and apoptosis [9,10]. As the key downstream element of STING, interferon regulatory factor 3 (IRF3) mediates the transcription of type I interferon (IFN-a and IFN-b) and inflammatory cytokines such as IL1b, IL-6, and TNF-a [9,11]. Recently, it has been reported that STINGIRF3 pathway activation induced the inflammatory response and cellular apoptosis in liver disorders such as alcoholic liver disease (ALD) [12] and nonalcoholic fatty liver disease (NAFLD) [11]. Chung KW and its colleagues reported that ablation of STING can limit renal inflammation and fibrosis [13]. However, it remains to be elucidated whether STING pathway activation is involved in hepatic I/R injury process. MicroRNA (miRNA, miR), 20e24 nucleotide in length, is a class of endogenous noncoding RNAs. miRNA downregulates targeted gene expression by directly binding to its 30 -untranslated regions (30 -UTRs) [14,15], which participates in various biological and pathological processes. miR-24-3p has emerged as a crucial miRNA that can negatively regulate the inflammatory reaction, apoptosis and tumor growth, which is predicted to bind the 30 -UTR of the STING mRNA through the TargetScan 7.2. miR-24-3p can prevent
https://doi.org/10.1016/j.bbrc.2019.10.182 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
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cardiomyocytes from I/R in mouse [16]. However, whether and how miR-24-3p regulates hepatic I/R injury remains to be elucidated. In this study, we found that STING pathway activation is involved in progression of the liver I/R injury in mice. miR-24-3p exerts anti-inflammation and anti-apoptosis effects in the mice liver I/R model via modulating the STING pathway, which may provide a new approach for therapy and prophylaxis of hepatic I/R injury. 2. Materials and methods 2.1. Establishment of liver I/R model Seventy percent liver ischemia and reperfusion mice model were built referring to the previous described [17]. Mice were purchased from Animal Center of Chongqing Medical University. Before operation, mice were fed in specific pathogen free with a 12h light-dark cycle with food and water ad libitum in a temperaturecontrolled environment (22 ± 2 C and 55 ± 10% relative humidity) for 7 days. All the experiment protocols were approved by Ethics Committee on Animal Research of Chongqing Medical University. For the experiment setting, mice were ischemic for 1 h, and followed by reperfusion for 3, 6, 9, 12 h as needed. Mice for sham group were laparotomized only. 2.2. qRT-PCR Total RNA was extracted from liver tissue of mice by TRIzol (Invitrogen, USA), then, reverse transcription and qRT-PCR were performed following the manufacturer’s protocol. Primers for qRTPCR as follow: U6 forward, 50 -GCTCGCTTCGGCAGCACA-30 , reverse, 50 -GAGGTATTCGCACCAGAG GA-30 . miR-24-3p forward, 50 -CGCA GTGGCTCAGTTCAGCA-30 , reverse 50 -AGTGCGTGTCGTGG AGTCG-30 . STING forward, 50 -GGTCACCGCTCCAAATATGTAG-30 , reverse, 50 CAGTAGTC CAAGTTCGTGCGA-3’; GAPDH forward, 50 -AGCGA GACCCCACTAACA-30 , reverse, 50 -GGGGC TAAGCAGTTGGTG-30 . 2.3. Western-blot Liver tissues were lysed by RIPA (beyotime, China) and total proteins were collected, then boiled with loading buffer (beyotime, China) for 10 min. Western-blot was performed according to manufacturer’s introduction. Primary antibodies were used as follow: STING (1:1000, Abcam, USA), p-IRF3 (1:1000, Cell Signaling Technology, USA), b-actin (1:1000, Boster, China). Secondary antibodies were needed as follow: The goat anti-rabbit (1:3000, ZSGBBio, China) and anti-mouse (1:3000, ZSGB-Bio, China). 2.4. Aminotransferase Serum AST and ALT were measured by microplate assay (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s manuals. 2.5. Transfection miR-24-3p mimic and its control (Genepharma, China) were separately transfected into mice with Lipofectamine 6000 (beyontime, China) at 24 h before I/R model induction. The final concentration was 100 nM. Mice were separately transfected with si-STINGs and its control (Genepharma, China) by Lipofectamine 6000 at 24 h prior to the I/R model establishment, of which the final concentration was 50 nM.
2.6. Luciferase activity Dual luciferase reporter assay was conducted following the direction. Briefly, 293T cells were co-transfected with pmirGLOSTING 30 UTR WT or pmirGLO-STING 30 UTR-Mut with miR-24-3p control or miR-24-3p mimic and pRL-TK (Promega, USA) using Lipofectamine 6000 (Beyotime, China). The final concentration was 10nM. After culture for 48 h, the relative luciferase activity of each well was determined. 2.7. Enzyme-linked immuno sorbent assay Serum of mice were collected and diluted, then, enzyme-linked immuno sorbent assay (ELISA) (Thermo Fisher Scientific, USA) were conducted according to the manufacturer’s introduction. 2.8. HE staining Liver tissues were harvested and fixed with paraffin, then 4 mm per section was cut and stained with hematoxylin and eosin (HE). The liver tissue injury was observed at 200 magnification under microscope and evaluated by SUZUKI’s criteria [18]. 2.9. TUNEL staining Apoptosis of liver tissue was assessed by In Situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol. The number of TUNEL-positive cells per field at 200 magnification was counted to estimate the level of liver tissue apoptosis. 2.10. Statistical analysis All data were analyzed by SPSS 21.0 and expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparisons tests were conducted for multiple-group comparisons, while the Student’s ttest for two groups. Pearson was performed to confirm the correlation between two groups. In all analyses, differences at P < 0.05 were considered statistically significant. 3. Results 3.1. The STING mRNA level was negatively correlated with miR-243p level in I/R treated liver tissue To determine the level of STING mRNA and miR-24-3p in liver tissue of I/R treated mice, qRT-PCR assay was performed. Our data revealed that after ischemia 1 h, STING mRNA increased in liver with extending the reperfusion time, and peaked at 9 h as compared to the sham group(p < 0.001) (Fig. 1A). While level of miR-24-3p decreased as early as at 6 h after reperfusion in liver of I/ R group as compared to the sham group, and reached to the bottom after reperfusion for 9 h, respectively (p < 0.05 and p < 0.01) (Fig. 1B). Moreover, the Pearson analysis showed that the STING mRNA level has negative correlation with the level of miR-24-3p in the I/R liver of mice (p < 0.0001, r ¼ 0.8706) (Fig. 1C). Meanwhile, as shown in Fig. 1D and E, the serum aminotransferase levels in mice with 1 h ischemia and 3, 6, 9, 12 h reperfusion were demonstrated. After I/R, the serum level of AST and ALT were increased and peaked at I1R9 (both are p < 0.001). Therefore, I1R9 was chosen as the standard time of liver I/R in the following experiment. 3.2. STING was targeted by miR-24-3p directly According to the TargetScan 7.2, we identified the 30 -UTR of
Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
A. Shen et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Fig. 1. miR-24-3p negatively correlated with STING during hepatic I/R in mice. A. Relative mRNA level of STING in liver of mice; B. Relative mRNA level of miR-24-3p in liver of mice; C. Pearson correlative analysis between miR-24-3p and STING mRNA; D and E. Serum level of AST and ALT, respectively. All experiments were repetitively conducted at least three times. (n ¼ 6). *P < 0.05, * *P < 0.01, * * *P < 0.001, n.s. P > 0.05 vs. Sham. Sham, sham operation; I1R3, ischemia for 1 h and reperfusion for 3 h; I1R6, ischemia for 1 h and reperfusion for 6 h; I1R9, ischemia for 1 h and reperfusion for 9 h; I1R12, ischemia for 1 h and reperfusion for 12 h.
STING was the potential binding site of miR-24-3p (Fig. 2A). Meanwhile, luciferase reporter gene assay was also conducted to confirm miR-24-3p binding to the 30 -UTR of STING. The wild-type and mutant gene sequence of STING were shown in Fig. 2B. When co-transfection of 293T cells with miR-24-3p mimic and wildtype STING, the luciferase activity was significantly downregulated as compared to the miR-24-3p control cells (p < 0.01)
(Fig. 2C). However, the luciferase activity nearly the same between 293T cells after co-transfection of miR-24-3p mimic or miR-24-3p control and mutant STING (p > 0.05) (Fig. 2D). 3.3. STING was a key regulator in liver I/R injury To explore the role of STING in the I/R-induced livers of mice,
Fig. 2. STING was the target gene of miR-24-3p. A. TargetScan 7.2 predicted that STING was the target gene of miR-24-3p; B. The base-pairing interaction between the miR-24-3p and 30 UTR of STING mRNA; C and D. Dual-luciferase reporter assay demonstrated that miR-24-3p repressed luciferase activity of STING 30 UTR-WT in 293T cells. All experiments were repetitively conducted at least three times. * *P < 0.01, n.s. P > 0.05. WT, wild type; MUT, mutant.
Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
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mice separately received caudal vein injection with siSTINGs and its control before subjecting to liver I/R. The STING mRNA level was obviously decreased in livers by the siSTING1 and siSTING2 precondition as compared to the I/R group (both are p < 0.001) (Fig. 3A). Due to the same inhibiting effect between siSTING1 and siSTING2 injection, we selected one of them to perform the following test. We observed that STING and p-IRF3 protein level increased in the I/R group as compared to the sham group (both are p < 0.001), which was reversed by the siSTING pretreatment (both are p < 0.01) (Fig. 3BeD). Furthermore, the level of proinflammation cytokines IL-6 and TNF-a decreased in the I/ R þ siSTING group as compared to the I/R group (both are p < 0.001) (Fig. 3E and F). So as to the aminotransferase level in serum of mice (both are p < 0.001) (Fig. 3G and H). 3.4. miR-24-3p protected against apoptosis and inflammation in liver I/R mice by modulating STING To investigate the role of miR-24-3p in the liver I/R injury, mice received injection with Ago-miR-24-3p mimic and Ago-miR-24-3p control before subjecting to I/R, respectively. By qRT-PCR, the AgomiR-24-3p mimic significantly increased miR-24-3p level in the liver tissue as compared to the I/R group (p < 0.001) (Fig. 4A). Moreover, liver function was evaluated by measurement of serum ALT and AST levels in each group. The results showed that the level of ALT and AST decreased in the I/R þ Ago-miR-24-3p mimic group as compared to the I/R group (both are p < 0.001) (Fig. 4B and C). Furthermore, the serum level of IL-6 and TNF-a was suppressed by the Ago-miR-24-3p mimic pretreatment as compared to the I/R group (both are p < 0.001) (Fig. 4D and E). To determine the antiapoptotic effect of miR-24-3p on the I/R mice, TUNEL staining was conducted. We observed that the number of TUNEL positive cells reduced in the I/R þ Ago-miR-24-3p group as compared to the I/R group (p < 0.001) (Fig. 4F and G). The HE staining data showed that exogenous Ago-miR-24-3p alleviated cell necrosis and inflammatory cell infiltration in liver tissue, and decreased the SUZUKI’s score as compared to the I/R group (p < 0.001) (Fig. 4H and I). In addition, exogenous Ago-miR-24 mimic administration
can reverse the upregulation of STING and p-IRF3 protein expression induced by liver I/R in mice (both are p < 0.01) (Fig. 4J-L). 4. Discussion In this current study, we identified miR-24-3p as a critical modulator of inflammation and apoptosis in liver I/R process. Our work demonstrates that miR-24 may exert its protective effects in hepatic I/R model in vivo by modulating the STING pathway. The STING pathway is emerging as an important regulator of innate immune cell behavior [19]. Activation of STING pathway has been reported to correlate with better clinical outcomes in human cancer patients [20e23]. Growing evidence has shown that STINGIRF3 pathway activation mediated inflammation and cellular apoptosis, which participates in several disease progression [24e26]. It has been reported that STING-IRF3 pathway activation promotes ER stress and hepatocyte apoptosis in early alcoholic liver disease [12]. Furthermore, overexpression of STING and p-IRF3 in the livers of an HFD-induced obese mouse model can lead to cell metabolic disorders, apoptosis and inflammation [11]. Moreover, the STING signaling pathway probably arises to detect aberrant cytosolic DNA species from an infectious agent or leakage of selfDNA from the nucleus of damaged cells [27]. Considering the importance of STING-IRF3 in innate immune response, we investigated whether STING activation occurs in the liver I/R process. We measured the STING and p-IRF3 protein expression in vivo and found that their protein expression significantly elevated in the I/R liver tissue, paralleling by upregulated serum level of proinflammatory cytokines and aminotransferase. Furthermore, to determine whether STING activation contributes to the pathology of liver I/R, we transfected mice with STING siRNA to downregulate STING expression in liver of mice. Under I/R condition, STING knocking down can significantly decrease IL-6 and TNF-a releasing and attenuate the aminotransferase alterations, which is similar to the previous reports [11]. These results indicate that STING activation participates in the I/R induced liver dysfunction and inflammation response. In recent decades, several miRNAs have been indicated as bio-
Fig. 3. STING play a crucial role in liver I/R injury regulation in mice. A. Selection and verification of silence RNA of STING; B-D. Relative protein expression of STING and p-IRF3 in liver of mice by western blot assay; E and F. Serum level of TNF-a and IL-6 by ELISA, respectively; G and H. Serum level of AST and ALT, respectively. All experiments were repetitively conducted at least three times. (n ¼ 6). * *P < 0.01, * * *P < 0.001, n.s. P > 0.05.
Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
Fig. 4. miR-24-3p may alleviate hepatic I/R injury via STING pathway. A. mRNA expression of miR-24-3p in liver of mice; B and C. Serum level of AST and ALT, respectively; D and E. Serum level of TNF-a and IL-6 by ELISA, respectively; F and G. TUNEL staining and evaluation of liver tissue apoptosis during hepatic I/R in mice (200 ); H and I. HE staining (200 ) and SUZUKI’s grades of liver tissue injury during hepatic I/R in mice; J-L. Protein expression of STING and p-IRF3 of liver in mice by western blot assay. All experiments were repetitively conducted at least three times. (n ¼ 6). * *P < 0.01, * * *P < 0.001, n.s. P > 0.05.
Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
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marker or innovative therapeutic target in the I/R elicited organ injury [28]. However, it remains unknown whether STING activation in liver I/R process results from some miRNA molecule modulation. According to TargetScan 7.2 prediction, miR-24-3p is identified to bind the 3’ UTR site of STING, thereby downregulating STING mRNA. Also, our luciferase results confirmed this. It has been widely documented that miR-24-3p plays a key role in inflammation, apoptosis and tumor growth [29e32]. In this current study, we firstly determined the miR-24-3p alteration during the liver I/R process. By qRT-PCR assay, miR-24-3p level downregulated in the I/ R liver tissue, which was negatively related to the STING mRNA. To further investigate the role of miR-24 in the pathological alteration and liver dysfunction during I/R, exogenous miR-24 was utilized in I/R mice model. We found that miR-24-3p can interact with STING thereby reducing the inflammatory factor releasing, downregulating serum AST and ALT, and alleviating hepatic tissue injury and apoptosis. Similar results were showed in the previous research [16,31,33,34]. In conclusion, we indicate that STING-IRF3 activation is involved in the pathological progression of hepatic I/R injury, which can be attenuated by exogenous miR-24-3p. Our findings suggest that modulation of the STING pathway in the liver by administering miR-24-3p might be a potential therapeutic strategy for the liver I/R injury. Funding This study was funded by grants from the National Natural Science Foundation of China (No. 81873592) and the graduate tutor team construction project of Chongqing Municipal Education Commission Foundation, China (No. dstd201801). Acknowledgements We thank the Laboratory Research Center in the First Affiliated Hospital of Chongqing Medical University for their technical support and guidance. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.182. References [1] H.K. Eltzschig, T. Eckle, Ischemia and reperfusion–from mechanism to translation, Nat. Med. 17 (2011) 1391e1401. [2] S.P. Monga, Lipid metabolic reprogramming in hepatic ischemia-reperfusion injury, Nat. Med. 24 (2018) 6e7. [3] D. Ni, H. Wei, W. Chen, Q. Bao, Z.T. Rosenkrans, T.E. Barnhart, C.A. Ferreira, Y. Wang, H. Yao, T. Sun, D. Jiang, S. Li, T. Cao, Z. Liu, J.W. Engle, P. Hu, X. Lan, W. Cai, Ceria nanoparticles meet hepatic ischemia-reperfusion injury: the perfect imperfection, Adv. Mater. 31 (2019), e1902956. [4] C. Peralta, M.B. Jimenez-Castro, J. Gracia-Sancho, Hepatic ischemia and reperfusion injury: effects on the liver sinusoidal milieu, J. Hepatol. 59 (2013) 1094e1106. [5] J. Evankovich, S.W. Cho, R. Zhang, J. Cardinal, R. Dhupar, L. Zhang, J.R. Klune, J. Zlotnicki, T. Billiar, A. Tsung, High mobility group box 1 release from hepatocytes during ischemia and reperfusion injury is mediated by decreased histone deacetylase activity, J. Biol. Chem. 285 (2010) 39888e39897. [6] K.M. Quesnelle, P.V. Bystrom, L.H. Toledo-Pereyra, Molecular responses to ischemia and reperfusion in the liver, Arch. Toxicol. 89 (2015) 651e657. [7] Y. Zhai, R.W. Busuttil, J.W. Kupiec-Weglinski, Liver ischemia and reperfusion injury: new insights into mechanisms of innate-adaptive immune-mediated tissue inflammation, Am. J. Transplant. 11 (2011) 1563e1569. [8] H. Ishikawa, G.N. Barber, STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling, Nature 455 (2008) 674e678. [9] H. Ishikawa, Z. Ma, G.N. Barber, STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity, Nature 461 (2009) 788e792.
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Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
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MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling Ai Shen a, b, Daofeng Zheng a, Yunhai Luo a, Tong Mou a, Qingsong Chen a, Zuotian Huang a, Zhongjun Wu a, * a b
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China Hepatobiliary Pancreatic Tumor Center, Chongqing University Cancer Hospital, Chongqing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 October 2019 Accepted 27 October 2019 Available online xxx
Ischemia and reperfusion (I/R) injury is a common cause of hepatocyte injury and liver dysfunction during liver transplantation, but its mechanism is needed further explored. We aimed to investigate whether STING pathway activation is involved in the liver I/R and further determine the role of the microRNA(miR)-24-3p in liver I/R injury in mice. Our data showed that STING mRNA level was negatively related with miR-24-3p in livers of I/R-treated mice. Next, we identified that STING could be bound by miR-24-3p by bioinformatic and luciferase report assay. Moreover, downregulation of STING alleviated the protein expression of p-IRF3 and the serum level of inflammatory factor and aminotransferase in I/R mice model. Furthermore, transfection of I/R treated mice with exogenous miR-24-3p significantly inhibited the protein expression of STING and p-IRF3 in liver, and attenuated serum inflammatory cytokines release, as well as the dysfunction and apoptosis of liver in I/R model in vivo. This study suggests that miR-24-3p may ameliorate inflammatory response and cellular apoptosis in hepatic I/R process by targeting STING, which might be a potential therapeutic target for preventing liver I/R development and progression. © 2019 Elsevier Inc. All rights reserved.
Keywords: MicroRNA-24-3p Stimulator of interferon genes Interferon regulatory factor 3 Inflammation Liver ischemia and reperfusion injury
1. Introduction Liver ischemia and reperfusion (I/R) injury has been recognized as the major cause of organ dysfunction during transplantation, which increases the risk of mortality [1e4]. The pathology of I/R injury involves innate immune system activation and apoptosis. I/R triggers several danger-associated molecular patterns (DAMPs) such as self-DNA, releasing from the injured cells, which can be recognized by the pattern recognition receptor (PRR) or signal sensor, eventually leading to a local inflammatory response and the occurrence of cell apoptosis [5e7]. However, the exact mechanism of I/R injury was uncertainty. Stimulator of interferon gene (STING), known as transmembrane protein 173 (TMEM173), can be activated by the cytoplasmic DNA sensor under stress condition, which plays a critical role in regulating the innate immune response. In the biological condition, STING is located in endoplasmic reticulum (ER) [8]. Upon
* Corresponding author. Friendship Road 1, Yuzhong District, 400016, Chongqing, China. E-mail address: [email protected] (Z. Wu).
DNA sensor stimulating, STING can be secreted from ER and become activation, facilitating the ER stress, inflammation and apoptosis [9,10]. As the key downstream element of STING, interferon regulatory factor 3 (IRF3) mediates the transcription of type I interferon (IFN-a and IFN-b) and inflammatory cytokines such as IL1b, IL-6, and TNF-a [9,11]. Recently, it has been reported that STINGIRF3 pathway activation induced the inflammatory response and cellular apoptosis in liver disorders such as alcoholic liver disease (ALD) [12] and nonalcoholic fatty liver disease (NAFLD) [11]. Chung KW and its colleagues reported that ablation of STING can limit renal inflammation and fibrosis [13]. However, it remains to be elucidated whether STING pathway activation is involved in hepatic I/R injury process. MicroRNA (miRNA, miR), 20e24 nucleotide in length, is a class of endogenous noncoding RNAs. miRNA downregulates targeted gene expression by directly binding to its 30 -untranslated regions (30 -UTRs) [14,15], which participates in various biological and pathological processes. miR-24-3p has emerged as a crucial miRNA that can negatively regulate the inflammatory reaction, apoptosis and tumor growth, which is predicted to bind the 30 -UTR of the STING mRNA through the TargetScan 7.2. miR-24-3p can prevent
https://doi.org/10.1016/j.bbrc.2019.10.182 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
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cardiomyocytes from I/R in mouse [16]. However, whether and how miR-24-3p regulates hepatic I/R injury remains to be elucidated. In this study, we found that STING pathway activation is involved in progression of the liver I/R injury in mice. miR-24-3p exerts anti-inflammation and anti-apoptosis effects in the mice liver I/R model via modulating the STING pathway, which may provide a new approach for therapy and prophylaxis of hepatic I/R injury. 2. Materials and methods 2.1. Establishment of liver I/R model Seventy percent liver ischemia and reperfusion mice model were built referring to the previous described [17]. Mice were purchased from Animal Center of Chongqing Medical University. Before operation, mice were fed in specific pathogen free with a 12h light-dark cycle with food and water ad libitum in a temperaturecontrolled environment (22 ± 2 C and 55 ± 10% relative humidity) for 7 days. All the experiment protocols were approved by Ethics Committee on Animal Research of Chongqing Medical University. For the experiment setting, mice were ischemic for 1 h, and followed by reperfusion for 3, 6, 9, 12 h as needed. Mice for sham group were laparotomized only. 2.2. qRT-PCR Total RNA was extracted from liver tissue of mice by TRIzol (Invitrogen, USA), then, reverse transcription and qRT-PCR were performed following the manufacturer’s protocol. Primers for qRTPCR as follow: U6 forward, 50 -GCTCGCTTCGGCAGCACA-30 , reverse, 50 -GAGGTATTCGCACCAGAG GA-30 . miR-24-3p forward, 50 -CGCA GTGGCTCAGTTCAGCA-30 , reverse 50 -AGTGCGTGTCGTGG AGTCG-30 . STING forward, 50 -GGTCACCGCTCCAAATATGTAG-30 , reverse, 50 CAGTAGTC CAAGTTCGTGCGA-3’; GAPDH forward, 50 -AGCGA GACCCCACTAACA-30 , reverse, 50 -GGGGC TAAGCAGTTGGTG-30 . 2.3. Western-blot Liver tissues were lysed by RIPA (beyotime, China) and total proteins were collected, then boiled with loading buffer (beyotime, China) for 10 min. Western-blot was performed according to manufacturer’s introduction. Primary antibodies were used as follow: STING (1:1000, Abcam, USA), p-IRF3 (1:1000, Cell Signaling Technology, USA), b-actin (1:1000, Boster, China). Secondary antibodies were needed as follow: The goat anti-rabbit (1:3000, ZSGBBio, China) and anti-mouse (1:3000, ZSGB-Bio, China). 2.4. Aminotransferase Serum AST and ALT were measured by microplate assay (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s manuals. 2.5. Transfection miR-24-3p mimic and its control (Genepharma, China) were separately transfected into mice with Lipofectamine 6000 (beyontime, China) at 24 h before I/R model induction. The final concentration was 100 nM. Mice were separately transfected with si-STINGs and its control (Genepharma, China) by Lipofectamine 6000 at 24 h prior to the I/R model establishment, of which the final concentration was 50 nM.
2.6. Luciferase activity Dual luciferase reporter assay was conducted following the direction. Briefly, 293T cells were co-transfected with pmirGLOSTING 30 UTR WT or pmirGLO-STING 30 UTR-Mut with miR-24-3p control or miR-24-3p mimic and pRL-TK (Promega, USA) using Lipofectamine 6000 (Beyotime, China). The final concentration was 10nM. After culture for 48 h, the relative luciferase activity of each well was determined. 2.7. Enzyme-linked immuno sorbent assay Serum of mice were collected and diluted, then, enzyme-linked immuno sorbent assay (ELISA) (Thermo Fisher Scientific, USA) were conducted according to the manufacturer’s introduction. 2.8. HE staining Liver tissues were harvested and fixed with paraffin, then 4 mm per section was cut and stained with hematoxylin and eosin (HE). The liver tissue injury was observed at 200 magnification under microscope and evaluated by SUZUKI’s criteria [18]. 2.9. TUNEL staining Apoptosis of liver tissue was assessed by In Situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol. The number of TUNEL-positive cells per field at 200 magnification was counted to estimate the level of liver tissue apoptosis. 2.10. Statistical analysis All data were analyzed by SPSS 21.0 and expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparisons tests were conducted for multiple-group comparisons, while the Student’s ttest for two groups. Pearson was performed to confirm the correlation between two groups. In all analyses, differences at P < 0.05 were considered statistically significant. 3. Results 3.1. The STING mRNA level was negatively correlated with miR-243p level in I/R treated liver tissue To determine the level of STING mRNA and miR-24-3p in liver tissue of I/R treated mice, qRT-PCR assay was performed. Our data revealed that after ischemia 1 h, STING mRNA increased in liver with extending the reperfusion time, and peaked at 9 h as compared to the sham group(p < 0.001) (Fig. 1A). While level of miR-24-3p decreased as early as at 6 h after reperfusion in liver of I/ R group as compared to the sham group, and reached to the bottom after reperfusion for 9 h, respectively (p < 0.05 and p < 0.01) (Fig. 1B). Moreover, the Pearson analysis showed that the STING mRNA level has negative correlation with the level of miR-24-3p in the I/R liver of mice (p < 0.0001, r ¼ 0.8706) (Fig. 1C). Meanwhile, as shown in Fig. 1D and E, the serum aminotransferase levels in mice with 1 h ischemia and 3, 6, 9, 12 h reperfusion were demonstrated. After I/R, the serum level of AST and ALT were increased and peaked at I1R9 (both are p < 0.001). Therefore, I1R9 was chosen as the standard time of liver I/R in the following experiment. 3.2. STING was targeted by miR-24-3p directly According to the TargetScan 7.2, we identified the 30 -UTR of
Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
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Fig. 1. miR-24-3p negatively correlated with STING during hepatic I/R in mice. A. Relative mRNA level of STING in liver of mice; B. Relative mRNA level of miR-24-3p in liver of mice; C. Pearson correlative analysis between miR-24-3p and STING mRNA; D and E. Serum level of AST and ALT, respectively. All experiments were repetitively conducted at least three times. (n ¼ 6). *P < 0.05, * *P < 0.01, * * *P < 0.001, n.s. P > 0.05 vs. Sham. Sham, sham operation; I1R3, ischemia for 1 h and reperfusion for 3 h; I1R6, ischemia for 1 h and reperfusion for 6 h; I1R9, ischemia for 1 h and reperfusion for 9 h; I1R12, ischemia for 1 h and reperfusion for 12 h.
STING was the potential binding site of miR-24-3p (Fig. 2A). Meanwhile, luciferase reporter gene assay was also conducted to confirm miR-24-3p binding to the 30 -UTR of STING. The wild-type and mutant gene sequence of STING were shown in Fig. 2B. When co-transfection of 293T cells with miR-24-3p mimic and wildtype STING, the luciferase activity was significantly downregulated as compared to the miR-24-3p control cells (p < 0.01)
(Fig. 2C). However, the luciferase activity nearly the same between 293T cells after co-transfection of miR-24-3p mimic or miR-24-3p control and mutant STING (p > 0.05) (Fig. 2D). 3.3. STING was a key regulator in liver I/R injury To explore the role of STING in the I/R-induced livers of mice,
Fig. 2. STING was the target gene of miR-24-3p. A. TargetScan 7.2 predicted that STING was the target gene of miR-24-3p; B. The base-pairing interaction between the miR-24-3p and 30 UTR of STING mRNA; C and D. Dual-luciferase reporter assay demonstrated that miR-24-3p repressed luciferase activity of STING 30 UTR-WT in 293T cells. All experiments were repetitively conducted at least three times. * *P < 0.01, n.s. P > 0.05. WT, wild type; MUT, mutant.
Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
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mice separately received caudal vein injection with siSTINGs and its control before subjecting to liver I/R. The STING mRNA level was obviously decreased in livers by the siSTING1 and siSTING2 precondition as compared to the I/R group (both are p < 0.001) (Fig. 3A). Due to the same inhibiting effect between siSTING1 and siSTING2 injection, we selected one of them to perform the following test. We observed that STING and p-IRF3 protein level increased in the I/R group as compared to the sham group (both are p < 0.001), which was reversed by the siSTING pretreatment (both are p < 0.01) (Fig. 3BeD). Furthermore, the level of proinflammation cytokines IL-6 and TNF-a decreased in the I/ R þ siSTING group as compared to the I/R group (both are p < 0.001) (Fig. 3E and F). So as to the aminotransferase level in serum of mice (both are p < 0.001) (Fig. 3G and H). 3.4. miR-24-3p protected against apoptosis and inflammation in liver I/R mice by modulating STING To investigate the role of miR-24-3p in the liver I/R injury, mice received injection with Ago-miR-24-3p mimic and Ago-miR-24-3p control before subjecting to I/R, respectively. By qRT-PCR, the AgomiR-24-3p mimic significantly increased miR-24-3p level in the liver tissue as compared to the I/R group (p < 0.001) (Fig. 4A). Moreover, liver function was evaluated by measurement of serum ALT and AST levels in each group. The results showed that the level of ALT and AST decreased in the I/R þ Ago-miR-24-3p mimic group as compared to the I/R group (both are p < 0.001) (Fig. 4B and C). Furthermore, the serum level of IL-6 and TNF-a was suppressed by the Ago-miR-24-3p mimic pretreatment as compared to the I/R group (both are p < 0.001) (Fig. 4D and E). To determine the antiapoptotic effect of miR-24-3p on the I/R mice, TUNEL staining was conducted. We observed that the number of TUNEL positive cells reduced in the I/R þ Ago-miR-24-3p group as compared to the I/R group (p < 0.001) (Fig. 4F and G). The HE staining data showed that exogenous Ago-miR-24-3p alleviated cell necrosis and inflammatory cell infiltration in liver tissue, and decreased the SUZUKI’s score as compared to the I/R group (p < 0.001) (Fig. 4H and I). In addition, exogenous Ago-miR-24 mimic administration
can reverse the upregulation of STING and p-IRF3 protein expression induced by liver I/R in mice (both are p < 0.01) (Fig. 4J-L). 4. Discussion In this current study, we identified miR-24-3p as a critical modulator of inflammation and apoptosis in liver I/R process. Our work demonstrates that miR-24 may exert its protective effects in hepatic I/R model in vivo by modulating the STING pathway. The STING pathway is emerging as an important regulator of innate immune cell behavior [19]. Activation of STING pathway has been reported to correlate with better clinical outcomes in human cancer patients [20e23]. Growing evidence has shown that STINGIRF3 pathway activation mediated inflammation and cellular apoptosis, which participates in several disease progression [24e26]. It has been reported that STING-IRF3 pathway activation promotes ER stress and hepatocyte apoptosis in early alcoholic liver disease [12]. Furthermore, overexpression of STING and p-IRF3 in the livers of an HFD-induced obese mouse model can lead to cell metabolic disorders, apoptosis and inflammation [11]. Moreover, the STING signaling pathway probably arises to detect aberrant cytosolic DNA species from an infectious agent or leakage of selfDNA from the nucleus of damaged cells [27]. Considering the importance of STING-IRF3 in innate immune response, we investigated whether STING activation occurs in the liver I/R process. We measured the STING and p-IRF3 protein expression in vivo and found that their protein expression significantly elevated in the I/R liver tissue, paralleling by upregulated serum level of proinflammatory cytokines and aminotransferase. Furthermore, to determine whether STING activation contributes to the pathology of liver I/R, we transfected mice with STING siRNA to downregulate STING expression in liver of mice. Under I/R condition, STING knocking down can significantly decrease IL-6 and TNF-a releasing and attenuate the aminotransferase alterations, which is similar to the previous reports [11]. These results indicate that STING activation participates in the I/R induced liver dysfunction and inflammation response. In recent decades, several miRNAs have been indicated as bio-
Fig. 3. STING play a crucial role in liver I/R injury regulation in mice. A. Selection and verification of silence RNA of STING; B-D. Relative protein expression of STING and p-IRF3 in liver of mice by western blot assay; E and F. Serum level of TNF-a and IL-6 by ELISA, respectively; G and H. Serum level of AST and ALT, respectively. All experiments were repetitively conducted at least three times. (n ¼ 6). * *P < 0.01, * * *P < 0.001, n.s. P > 0.05.
Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
Fig. 4. miR-24-3p may alleviate hepatic I/R injury via STING pathway. A. mRNA expression of miR-24-3p in liver of mice; B and C. Serum level of AST and ALT, respectively; D and E. Serum level of TNF-a and IL-6 by ELISA, respectively; F and G. TUNEL staining and evaluation of liver tissue apoptosis during hepatic I/R in mice (200 ); H and I. HE staining (200 ) and SUZUKI’s grades of liver tissue injury during hepatic I/R in mice; J-L. Protein expression of STING and p-IRF3 of liver in mice by western blot assay. All experiments were repetitively conducted at least three times. (n ¼ 6). * *P < 0.01, * * *P < 0.001, n.s. P > 0.05.
Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182
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marker or innovative therapeutic target in the I/R elicited organ injury [28]. However, it remains unknown whether STING activation in liver I/R process results from some miRNA molecule modulation. According to TargetScan 7.2 prediction, miR-24-3p is identified to bind the 3’ UTR site of STING, thereby downregulating STING mRNA. Also, our luciferase results confirmed this. It has been widely documented that miR-24-3p plays a key role in inflammation, apoptosis and tumor growth [29e32]. In this current study, we firstly determined the miR-24-3p alteration during the liver I/R process. By qRT-PCR assay, miR-24-3p level downregulated in the I/ R liver tissue, which was negatively related to the STING mRNA. To further investigate the role of miR-24 in the pathological alteration and liver dysfunction during I/R, exogenous miR-24 was utilized in I/R mice model. We found that miR-24-3p can interact with STING thereby reducing the inflammatory factor releasing, downregulating serum AST and ALT, and alleviating hepatic tissue injury and apoptosis. Similar results were showed in the previous research [16,31,33,34]. In conclusion, we indicate that STING-IRF3 activation is involved in the pathological progression of hepatic I/R injury, which can be attenuated by exogenous miR-24-3p. Our findings suggest that modulation of the STING pathway in the liver by administering miR-24-3p might be a potential therapeutic strategy for the liver I/R injury. Funding This study was funded by grants from the National Natural Science Foundation of China (No. 81873592) and the graduate tutor team construction project of Chongqing Municipal Education Commission Foundation, China (No. dstd201801). Acknowledgements We thank the Laboratory Research Center in the First Affiliated Hospital of Chongqing Medical University for their technical support and guidance. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.182. References [1] H.K. Eltzschig, T. Eckle, Ischemia and reperfusion–from mechanism to translation, Nat. Med. 17 (2011) 1391e1401. [2] S.P. Monga, Lipid metabolic reprogramming in hepatic ischemia-reperfusion injury, Nat. Med. 24 (2018) 6e7. [3] D. Ni, H. Wei, W. Chen, Q. Bao, Z.T. Rosenkrans, T.E. Barnhart, C.A. Ferreira, Y. Wang, H. Yao, T. Sun, D. Jiang, S. Li, T. Cao, Z. Liu, J.W. Engle, P. Hu, X. Lan, W. Cai, Ceria nanoparticles meet hepatic ischemia-reperfusion injury: the perfect imperfection, Adv. Mater. 31 (2019), e1902956. [4] C. Peralta, M.B. Jimenez-Castro, J. Gracia-Sancho, Hepatic ischemia and reperfusion injury: effects on the liver sinusoidal milieu, J. Hepatol. 59 (2013) 1094e1106. [5] J. Evankovich, S.W. Cho, R. Zhang, J. Cardinal, R. Dhupar, L. Zhang, J.R. Klune, J. Zlotnicki, T. Billiar, A. Tsung, High mobility group box 1 release from hepatocytes during ischemia and reperfusion injury is mediated by decreased histone deacetylase activity, J. Biol. Chem. 285 (2010) 39888e39897. [6] K.M. Quesnelle, P.V. Bystrom, L.H. Toledo-Pereyra, Molecular responses to ischemia and reperfusion in the liver, Arch. Toxicol. 89 (2015) 651e657. [7] Y. Zhai, R.W. Busuttil, J.W. Kupiec-Weglinski, Liver ischemia and reperfusion injury: new insights into mechanisms of innate-adaptive immune-mediated tissue inflammation, Am. J. Transplant. 11 (2011) 1563e1569. [8] H. Ishikawa, G.N. Barber, STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling, Nature 455 (2008) 674e678. [9] H. Ishikawa, Z. Ma, G.N. Barber, STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity, Nature 461 (2009) 788e792.
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Please cite this article as: A. Shen et al., MicroRNA-24-3p alleviates hepatic ischemia and reperfusion injury in mice through the repression of STING signaling, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.182