Accepted Manuscript Title: miR-22 contributes to endosulfan-induced endothelial dysfunction by targeting SRF in HUVECs Authors: Dan Xu, Yubing Guo, Tong Liu, Shuai Li, Yeqing Sun PII: DOI: Reference:
S0378-4274(17)30026-7 http://dx.doi.org/doi:10.1016/j.toxlet.2017.01.014 TOXLET 9685
To appear in:
Toxicology Letters
Received date: Revised date: Accepted date:
26-11-2016 17-1-2017 21-1-2017
Please cite this article as: Xu, Dan, Guo, Yubing, Liu, Tong, Li, Shuai, Sun, Yeqing, miR-22 contributes to endosulfan-induced endothelial dysfunction by targeting SRF in HUVECs.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2017.01.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
miR-22 contributes to endosulfan-induced endothelial dysfunction by targeting SRF in HUVECs
Dan Xu, Yubing Guo, Tong Liu, Shuai Li, Yeqing Sun*
Institute of Environmental Systems Biology, Environment Science and Engineering College, Dalian Maritime University, Linghai Road 1, Dalian, 116026, P.R. China.
E-mail address: Dan Xu,
[email protected], Yubing Guo,
[email protected], Tong Liu,
[email protected], Shuai Li,
[email protected], *Correspondence should be addressed to Prof. Yeqing Sun,
[email protected], Tel: 86-411-84723633-888, Fax 86-411-84725675.
1
Graphical Abstract
endosulfan
miR-22 level SRF Endothelial dysfunction Apoptosis Inflammation
STAG2 Abnormal mitosis
Highlights 1. miR-22 was upregulated by endosulfan in HUVECs. 2. Anti-miR-22 attenuated endosulfan-induced endothelial dysfunction. 3. SRF and STAG2 were novel direct targets of miR-22. 4. SRF siRNAs caused apoptosis and inflammation. 5. Endosulfan suppressed SRF protein expression.
Abstract microRNAs (miRNAs) function in the posttranscriptional gene regulation, providing new insights into the epigenetic mechanism of toxicity induced by environmental pollutants. miR-22 was discovered to regulate cell proliferation and apoptosis in response to environmental toxicants. We have reported that endosulfan can cause endothelial toxicity in human umbilical vein endothelial cells (HUVECs). In the present study, we investigated the involvement of miR-22 in endosulfan-induced endothelial dysfunction. The expression level of miR-22 was increased in a dose-dependent manner by endosulfan exposure. Overexpression of miR-22 induced apoptosis and inflammation in HUVECs. Anti-miR-22 transfection significantly attenuated the increase in the percentage of apoptotic cells, caspase-3 activity and Interleukin (IL)-6, 8 mRNA levels in endosulfan-exposed HUVECs. Luciferase reporter assay confirmed that SRF and STAG2 were novel direct targets of miR-22. Endosulfan decreased mRNA expression of both SRF and STAG2, but only suppressed protein expression of SRF. Knockdown of SRF via siRNAs resulted in apoptosis and inflammation whereas STAG2 siRNAs only caused abnormal mitosis in HUVECs. Taken together, these findings will shed light on the role and mechanism of miR-22 in endosulfan-induced endothelial dysfunction via SRF in HUVECs. 2
Keywords: endosulfan; miR-22; apoptosis; inflammation; endothelial dysfunction
1. Introduction Endosulfan is one of the representative organochlorine pesticides (OCPs) commonly used in agriculture, which was classified into persistent organic pollutants (POPs) by the Stockholm Convention in 2011 (Weber et al., 2010). With long-term use of the pesticides, endosulfan has a widespread distribution in the environment (Jia et al., 2009). In fact, endosulfan has been detected in human blood, urine and even umbilical cord blood (Damgaard et al., 2006; Shen et al., 2007). Therefore, endosulfan is responsible for adverse effects on human health associated with a variety of human diseases including cardiovascular diseases. Endothelial dysfunction has been shown to be predictive of adverse cardiovascular events (Tang et al., 2014), characterized by endothelial cell apoptosis and inflammation response. Recently, it is reported that endosulfan exposure impaired vascular tissue in rats and caused mitochondrial damage in endothelial cells of rats due to oxidative stress and inflammation (Zhang et al., 2015a). Our previous study showed that endosulfan could induce endothelial dysfunction by inhibition of cell growth and induction of inflammation in human umbilical vein endothelial cells (HUVECs) (Li et al., 2016). We also utilized gene expression profile analysis to reveal the genetic mechanism in endothelial toxicity of endosulfan and potential relevant disease outcomes (Xu et al., 2016). However, it is still unknown what is the epigenetic control of gene expression when exposed to endosulfan. microRNAs (miRNAs) are an abundant class of small noncoding RNAs and function as 3
post-transcriptional regulators by base-pairing with the complementary sites in the 3'-untranslated region (3'-UTR) of the mRNA. miR-22 was originally identified from HeLa cells as a 22-nucleotide miRNA, and was subsequently shown to be ubiquitously expressed in various tissues (Lagos-Quintana et al., 2001; Neely et al., 2006). miR-22 has been connected to a great number of activities that encompass tumorigenesis, epigenetic modification, skeletal metabolism, senescence and so on (Iliopoulos et al., 2008; Jazbutyte et al., 2013; Liu et al., 2010; Xu et al., 2011). Recently, miR-22 has been implicated in cardiac pathology and vascular diseases (Dong and Yang, 2011; Huang and Wang, 2014; Qin and Zhang, 2011; Urbich et al., 2008). It is reported that miR-22 was prominently upregulated during cellular senescence and aging, involved in age-associated cardiac changes, such as cardiac fibrosis (Jazbutyte et al., 2013; Xu et al., 2011). miR-22 functions as an integrator of Ca2+ homeostasis and myofibrillar protein content during stress in the heart (Gurha et al., 2012). Microarray analysis of miRNA expression profiles showed that miR-22 was highly expressed in the peripheral blood of coronary heart disease patients and high-risk patients (Chen et al., 2015), implying the possible role of miR-22 in cardiovascular diseases. Endothelial dysfunction is associated with a variety of cardiovascular diseases, such as atherosclerosis and hypertension. However, the underlying mechanism of miR-22 in endothelial function was still obscure. Serum response factor (SRF) is a transcription factor that regulates the activity of many genes, and thereby participates in cell proliferation, apoptosis and cytoskeleton integrity in different cell types (Schratt et al., 2002; Schratt et al., 2004). SRF is a key endothelial cell regulator and contributes to endothelial dysfunction (Chen et al., 2015). HUVECs have considerably been used for the investigation of endothelial dysfunction (Chen et al., 2015; 4
Zhang et al., 2015b). In the present study, we provide evidence that miR-22 acts as a key regulator in endosulfan-induced apoptosis and inflammation in HUVECs. Furthermore, we identified that SRF was a novel target gene for miR-22 by luciferase reporter assay and knockdown of SRF could cause endothelial dysfunction. This study provides new insights into the miR-22-mediated mechanism in endosulfan-induced endothelial dysfunction. 2. Materials and Methods 2.1 Cell culture and endosulfan exposure HUVECs (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 medium (KeyGen, Nanjing, China) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in culture incubator with 5% CO2. Endosulfan exposure experiments were performed as previously described (Li et al., 2016). Endosulfan (Jiangsu Anpon Electrochemical Co., Huaian, China) caused cytotoxicity in HUVECs at the concentrations of 20, 40 and 60 μM within the detected concentration range of endosulfan (0.69-176.2 g/ml) in human blood of people exposed to endosulfan (Singh et al., 2007). 2.2 Transient miRNA/siRNA transfection Negative control siRNA (NC) were purchased from Invitrogen (Carlsbad, CA, USA). siRNAs targeting SRF and STAG2, pre-miR-22 and anti-miR-22 were obtained from Biomics (Nantong, China). Cells were transfected with 10 nM siRNA or 40 nM miRNA using LipofectamineRNAiMax (Invitrogen) according to the manufacturer’s protocol. 2.3 Apoptosis analysis Cells were stained with Annexin-V-FITC and PI according to the manufacturer’s protocol of the Annexin V-FITC Apoptosis Detection Kit (KeyGen). The fluorescence intensity of cells 5
was evaluated by flow cytometry (BD Biosciences, San Jose, CA, USA) using quadrant statistics for necrotic and apoptotic cell populations. PI was used for the detection of late apoptosis and necrosis, and Annexin-V was consumed for the detection of early and late apoptosis. Caspase-3 activity was measured using the Caspase-3 Colorimetric Assay Kit (KeyGen) according to the manufacturer’s protocol. Briefly, cell lysates were prepared in cell lysis buffer. Total protein was quantified by the Bradford method (Sangon, Shanghai, China). The protein lysate was mixed with the reaction buffer and incubated at 37°C for 4 h in the dark. The colour developed was measured at 405 nm using a microplate reader (SpectraMax M5, Molecular Devices, CA, USA). 2.4 Real-time qPCR Total RNA was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The expressions of miRNAs and mRNAs were quantified by TaqMan miRNA assays (Applied Biosystems, Foster City, CA, USA) and SYBR green (Invitrogen), respectively. Real-time qRT-PCR was performed using an ABI PRISM 7300 system (Applied Biosystems). qRT-PCR reactions were performed in triplicate from all the samples in different groups. Primers used were listed in Table S1. The relative expression levels of miRNAs and mRNAs among samples were calculated using the comparative delta CT method (2−△△CT) after normalization with reference to the expression of U6 small nuclear RNA and GAPDH, respectively. 2.5 Luciferase reporter assay The 3’-UTR fragments of human SRF mRNA (NM_003131.3) contain three putative 6
miR-22 binding sites (3’UTR: 1160-1165, 1246-1251, 1310-1315). STAG2 mRNA (NM_001042749.2; 3’UTR: 1298-1305) or mutant sequence were cloned at the XhoI and NotI sites into the pmiR-RB-REPORTTM luciferase reporter vector (RiboBio Co.Ltd., Guangzhou, China). These constructs were named pmiR-SRF-WT and pmiR-SRF-mut; pmiR-STAG2-WT and pmiR-STAG2-mut. PCR primers and oligonucleotide sequences for constructs are provided in Table S2. All the constructs were further confirmed by sequencing. For luciferase activity assay, each construct was cotransfected with miR-22 mimics or miR-NC (RiboBio) in a 96-well plate using Lipofectamine 2000 (Invitrogen) for 48 h. Luciferase assays were performed with the Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer’s instructions. Luminescent signal was quantified by luminometer (Glomax; Promega), and luciferase activity was presented by relative hRlu/hluc ratio. The tests were repeated in triplicate. 2.6 Western blot analysis Total protein extraction and western blotting were performed as previously described (Li et al., 2016). Primary antibodies include STAG2 and SRF (Santa Cruz, Delaware, CA, USA), Bcl-2 and BAX (Keygentec, Nanjing, China). GAPDH (ZSGQ-BIO, Beijing, China) was used as a loading control (detailed information is available in Supplementary file). 2.7 Statistical analysis Data in this study are presented as means ± SD. Statistical comparisons were made between two groups with student’s t test, and among multiple groups by One-way ANOVA. Statistical significance was considered to be reached when *P < 0.05 or **P < 0.01.
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3. Results 3.1 Expression of miR-22 in HUVECs exposed to endosulfan Our previous study (Li et al., 2016) showed that endosulfan induced apoptosis and inflammation at the concentration of 40 μM, which can be regarded as a start concentration of endosulfan-induced endothelial dysfunction in HUVECs. Accordingly, we firstly examined the expression of miR-22 in HUVECs exposed to endosulfan (40 μM) at different time points. The expression of miR-22 was markedly elevated with the increase of endosulfan exposure time (Fig. 1A). Endosulfan significantly upregulated miR-22 expression in a dose-dependent manner when HUVECs were exposed to endosulfan at 20, 40 and 60 μM dose for 48 h (Fig. 1B), indicating that upregulation of miR-22 expression might be associated with endosulfan-induced endothelial dysfunction. 3.2 miR-22 induced apoptosis and inflammation in HUVECs We performed both gain-of-function and loss-of-function studies of miR-22 in HUVECs using pre-miR-22 and anti-miR-22 transfection methods, respectively. We firstly examined the effect of miR-22 on endothelial cell growth using a cell count assay, and found that overexpression of miR-22 significantly inhibited cell growth in HUVECs at 48 h after transfection (Fig. S1A). To investigate whether miR-22-induced cell growth inhibition was associated with cell cycle and apoptosis, we performed cell cycle analysis and estimated the apoptosis ratio of HUVECs by flow cytometry. The results showed that there was no statistically significant increase at the G1 or G2 phase for those cells transfected with pre-miR-22 or anti-miR-22 8
(Fig. S1B), indicating that miR-22 did not affect cell cycle distribution in HUVECs. miR-22 overexpression induced a significant percentage of cells to undergo apoptotic cell death, while anti-miR-22 had no effect on apoptosis in HUVECs (Fig. 2A and 2B). The main mediators of apoptosis are caspases, and the most critical executioner caspase is considered to be caspase-3 (Moghadamtousi et al., 2014). We found that the relative activity of caspase-3 in pre-miR-22 transfected group was about two-fold higher than that in the NC group (Fig. 2C). Further, Western blot results showed that pre-miR-22 transfection resulted in the decrease of anti-apoptotic protein BCL-2 expression (Fig. 2D). These results indicate that miR-22 could cause apoptosis in HUVECs. We further investigated the effect of miR-22 on the pro-inflammatory cytokines IL-6 and chemokine IL-8. qRT-PCR results showed that miR-22 overexpression upregulated mRNA expression of IL-8 but not IL-6 in HUVECs (Fig. 2E). These results indicate that miR-22 could induce apoptosis and inflammation response in HUVECs, resulting in endothelial dysfunction. 3.3 Effect of anti-miR-22 on endosulfan-induced apoptosis and inflammation in HUVECs To investigate the involvement of miR-22 in endosulfan-induced endothelial dysfunction, we transfected HUVECs with anti-miR-22 for knockdown of miR-22, followed by endosulfan (ES, 60 μM) exposure treatment. The results showed that endosulfan significantly induced apoptosis and elevated mRNA levels of IL-6, 8 in untreated control (C) and NC-transfected cells (Fig. 3A-D). In contrast, transfection with anti-miR-22 significantly reduced the percentage of apoptotic cells (Fig. 3A), attenuated the increase in caspase-3 activity (Fig. 3B), and decreased mRNA levels of IL-6, 8 in HUVECs exposed to endosulfan (Fig. 3C and 3D). These findings indicate that miR-22 indeed could be involved in endosulfan-induced 9
endothelial dysfunction. 3.4 SRF and STAG2 are direct targets of miR-22 To search for potential targets of miR-22 in endosulfan-exposed HUVECs, we used a consensus approach with three widely used softwares (miRanda, Targetscan, PicTar) to perform target prediction. After overlapping prediction analysis, we analyzed differentially expressed genes in gene expression profile to screen those genes that were down-regulated in HUVECs exposed to endosulfan and implicated in cell growth and/or apoptosis. Based on above all, SRF and STAG2 were selected to be putative targets for miR-22. The mRNA of STAG2 contains one putative binding site for miR-22 in the 3’-UTR, whereas SRF mRNA contains three miR-22 binding sites in the 3’-UTR (Fig. S2). To address those genes directly regulated by miR-22, we performed luciferase reporter assay. We constructed luciferase reporters pmiR-SRF-WT and pmiR-STAG2-WT containing the complimentary seed sequence of miR-22 at the 3’-UTR region of SRF and STAG2 mRNA, respectively. pmiR-SRF-mut and pmiR-STAG2-mut contain the mutated seed sequence of the same fragment as pmiR-SRF-WT and pmiR-STAG2-WT, respectively. The results showed that miR-22 significantly reduced the luciferase activities of pmiR-SRF-WT and pmiR-STAG2-WT, compared with the miR negative control (miR-NC). In contrast, luciferase activities of mutant reporters were not repressed by cotransfection with miR-22 (Fig. 4A and 4B). These results provide experimental evidence that SRF and STAG2 are direct targets for miR-22. The up-regulation of miR-22 in endosulfan-exposed HUVECs prompted us to investigate whether SRF and STAG2 had reverse expression when exposed to endosulfan. The results 10
demonstrated that endosulfan significantly reduced the expression of SRF and STAG2 at mRNA levels (Fig. 4C and 4D), which is consistent with our previous results from gene expression profile. SRF protein expression was significantly downregulated by endosulfan in a dose-dependent manner, but STAG2 did not change at protein level when exposed to endosulfan (Fig. 4E). We further found that knockdown of STAG2 by siRNAs could lead to abnormal mitosis, but had no effect on cell proliferation in HUVECs (Fig. S3). These results indicate that SRF, not STAG2 may function in endosulfan-exposed HUVECs. 3.5 SRF siRNAs caused growth inhibition, apoptosis and inflammation in HUVECs To further verify SRF is directly regulated by miR-22, we examined the expression of SRF after transfection with miR-22 into HUVECs. Western blotting analysis showed that endogenous SRF protein expression was remarkably reduced in miR-22 overexpressed cells (Fig. 5A). To study the function of SRF in endothelial cells, we utilized three SRF siRNAs to investigate the changes in cell growth, apoptosis and inflammation in HUVECs. The results showed that SRF siRNAs significantly knocked down SRF expression at mRNA and protein levels, resulting in cell growth inhibition (Fig. S4). SRF siRNAs induced a significant percentage of cells to undergo apoptotic cell death (Fig. 5B). We also found that the relative activity of caspase-3 was significantly elevated by SRF siRNAs (Fig. 5C). Knockdown of SRF decreased the expression of Bcl-2, but increased BAX protein expression (Fig. 5D). BAX/Bcl-2 ratio in SRF siRNA transfected groups was over 7-fold higher than that in NC group (Fig. 5E), indicating the occurrence of apoptosis induced by SRF siRNAs. We investigated the effect of SRF on the expressions of IL-6 and IL-8. qRT-PCR results showed 11
that SRF siRNAs (si-1 and si-3) up-regulated both IL-6 and IL-8 mRNA expression levels in HUVECs (Fig. 5F), although SRF si-2 had no effect on expression of IL-6 and IL-8 possibly due to non-specific effect. Taken together, these results suggest that SRF siRNAs could cause endothelial dysfunction through inducing apoptosis and inflammation in HUVECs.
4. Discussion In the present study, we demonstrated for the first time that endosulfan exposure increased miR-22 expression and reduced miR-22 target gene SRF at both mRNA and protein levels in human endothelial cells. We identified SRF as a direct target for miR-22 in HUVECs. Overexpression of miR-22 and knockdown of SRF caused apoptosis and inflammation in HUVECs, implying that miR-22 contributes to endosulfan-induced endothelial dysfunction via SRF. Recently, it has been reported that miR-22 can play a crucial role in a variety of cellular processes including differentiation, control of growth and stress response (Liu et al., 2010; Pandey and Picard, 2009; Ting et al., 2010). Differentiation agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) induced miR-22 transcription and enforced miR-22 expression inhibited cell growth in the HL-60 leukemia cell line (Ting et al., 2010). miR-22 was highly expressed in transformed human bronchial epithelial cells induced by anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), concomitant with downregulation of tumor suppressor gene PTEN protein. Anti-miR-22 promoted cell apoptosis, decreased colony formation and reduced the motility of malignant cells (Liu et al., 2010). In the present study, we found that miR-22 expression was elevated when HUVECs were exposed to endosulfan. 12
Overexpression of miR-22 induced apoptosis and inflammation in human endothelial cells, indicating the role of miR-22 in endothelial dysfunction. miR-22 can suppress hundreds of mRNA targets, resulting in various changes in cellular phenotype. miR-22 induced cellular senescence by targeting SIRT1, Sp1 and CDK6 in human fibroblast cells (Xu et al., 2011). miR-22 acts as a tumor suppressor to inhibit cell growth by directly targeting the estrogen receptor a (ERa) in MCF7-SH cells (Pandey and Picard, 2009). miR-22 functions as a micro-oncogene to repress PTEN expression in human prostate cancer cells and transformed epithelial cells (Liu et al., 2010; Poliseno et al., 2010). In the present study, we identified that STAG2 and SRF were two novel target genes for miR-22. The STAG2 gene ecodes a subunit of the cohesin complex, playing an important role in the maintenance of genome stability and cell survival (Solomon et al., 2011). It is reported that loss of STAG2 could cause aneuploidy, but had no obvious effect on cell proliferation in normal human bladder cells (Li et al., 2015). In the current study, STAG2 siRNAs led to abnormal mitosis, but had no effect on cell proliferation (Fig. S3). STAG2 mRNA was downregulated by endosulfan, whereas STAG2 protein expression did not change in endosulfan-exposed HUVECs, which might be due to the complex biological processes including translational regulation and protein complex formation. As a known transcription factor, SRF can regulate Bcl-2 expression as a direct regulator of Bcl-2 transcription and promote cell survival during murine embryonic development (Schratt et al., 2004). SRF deficiency almost completely prevents Bcl-2 expression in differentiating embryonic stem cells. In the present study, we found that endosulfan exposure reduced SRF expression at both mRNA and protein levels in HUVECs. Knockdown of SRF by siRNAs 13
decreased BCL-2 protein expression, increased BAX protein expression and elevated caspase-3 activity, thus inducing apoptosis in HUVECs. SRF was reported to be a physiological target of miR-320a, implicated in cardiovascular diseases (Chen et al., 2015). We here identified that SRF was a novel target for miR-22, involved in endothelial dysfunction, a key event for atherogenesis. SRF regulates the expression of miR-1 and miR-133a, important for cardiac and skeletal muscles (Liu et al., 2008; Zhao et al., 2005). At least 169 miRNAs in mammalian genomes contain at least one SRF binding element CArG in their promoter region (Niu et al., 2008). On the other hand, some miRNAs can also regulate SRF synthesis (Chen et al., 2006), therefore forming intricate and complex regulatory web. Vascular endothelial cells play an important role in the initiation, amplification, and resolution of the inflammatory response (Stohl et al., 2013). Inflammatory molecules, such as MCP-1, IL-6, VEGF and IL-8, have been implicated in atherosclerosis (Schnittker et al., 2013). In the present study, we mainly focused on pro-inflammatory molecules cytokine IL-6 and chemokine IL-8, because they can be self-secreted by human endothelial cells (Lee et al., 2014) and play an important role in inflammatory reaction induced by endosulfan in HUVECs (Li et al., 2016). Here, our results showed that SRF siRNAs caused the increase in mRNA expression of IL-6 and IL-8 in HUVECs, which is consistent with the previous results from endosulfan exposure (Li et al., 2016). The expression of IL-8 but not IL-6 mRNA was significantly upregulated in HUVECs transfected with pre-miR-22 for 48 h, indicating that miR-22 only affected IL-8 expression in HUVECs. The difference between IL-6 and IL-8 can be attributed to their different function in inflammation. It is known that IL-6 was regarded as 14
a major mediator of the acute-phase response (Sato and Ohshima, 2000) and involved in inflammatory reaction, immune response and cellular proliferation (Liu et al., 2014). IL-8 is a chemokine and acts as a key mediator associated with inflammation. IL-8 is also known to be a potent promoter of angiogenesis and functions in immune surveillance. In conclusion, we found that miR-22 can cause apoptosis and inflammation in HUVECs via
SRF.
Importantly,
endosulfan
exposure
caused
miR-22
overexpression
and
downregulation of SRF, resulting in endothelial dysfunction. Aberrant expression of miR-22 is linked to endosulfan exposure, which might be associated with cardiovascular diseases.
Conflict of Interest Statement Our data has never been published and none of them are under consideration elsewhere until now. Copies of this paper or part of paper also are not in press or under consideration. All authors declare that they have no competing interests. Dan Xu, Yubing Guo, Tong Liu, Shuai Li, Yeqing Sun
Acknowledgments The present work was supported by the national natural science foundation of China (No. 21207012), the Fundamental Research Funds for the Central Universities (3132016330) and State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (No. KF2014-15).
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Figure legends Fig. 1 Effect of endosulfan on the expression of miR-22. (A) HUVECs were treated with 40 μM endosulfan (ES) or DMSO control (D) for 12 h, 24 h, 48 h. (B) HUVECs were treated with endosulfan at 20 ,40 and 60 μM dose for 48 h. Relative expression level of miR-22 was shown. *P<0.05, **P<0.01 versus DMSO. Fig. 2 miR-22 induced apoptosis and inflammation in HUVECs. HUVECs were untreated or transfected with pre-miR-22, anti-miR-22 or NC control for 48 h. (A) The percentage of early apoptosis and late apoptotic cells (B) Total apoptotic rates (C) Caspase-3 activity (D) Western blot analysis of BAX and Bcl-2 expression (E) Relative expression levels of IL-6 and IL-8 mRNA in different groups. *P<0.05 versus NC control. Fig. 3 miR-22 was involved in endosulfan-induced apoptosis and inflammation. HUVECs were treated with 60 μM endosulfan (ES) or DMSO control (D) for 48 h after cells were untreated (C) or transfected with anti-miR-22 or NC control for 24 h. (A) Total apoptotic rates (B) Caspase-3 activity (C and D) Relative expression levels of IL-6 and IL-8 mRNA in different groups. *P<0.05, **P<0.01 versus DMSO. ##P<0.05 versus NC control. Fig.4 Effect of endosulfan on the expression of SRF and STAG2. (A and B) Luciferase reporter assay was performed to confirm that SRF and STAG2 are direct target genes for miR-22. **P<0.01 versus miR-NC. (C and D) Relative expression levels of SRF and STAG2 mRNA were examined by qRT-PCR in HUVECs treated with endosulfan for 48 h. (E) Western blot analysis showed that endosulfan reduced SRF protein expression but had no effect on STAG2. *P<0.05, **P<0.01 versus DMSO. Fig. 5 SRF siRNAs caused endothelial dysfunction. (A) Western blot analysis showed that 22
SRF protein expression was down-regulated by pre-miR-22. (B-F) HUVECs were transfected with SRF siRNAs or NC control for 48 h. (B) Total apoptotic rates (C) Caspase-3 activity (D) Western blot analysis of SRF, BAX and Bcl-2 expression (E) BAX/Bcl-2 ratio (F) Relative expression levels of IL-6 and IL-8 mRNA in different groups. *P<0.05, **P<0.01 versus NC control.
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Fig. 1 A
B D
miR-22
5
* Relative expression level
Relative expression level
3 ES 2
1
0
miR-22
**
4 3
*
2 1 0
12 h
24 h
48 h
D
20
40
60
Endosulfan (µM)
Fig. 2 A
B
C
Caspase-3 activity
Apoptotic cells (%)
30 20 10 0
D
30 17 ratio 30 17 ratio 46 30
pre-miR-22 anti-miR-22 Bcl-2
1
0.36
1.21 BAX
1
1.05
1.17 GAPDH
* 2
1
0
E NC
3
*
40
A
B
Apoptotic cells (%)
D 25
**
5 ##
ES
20 15
Caspase-3 activity
30
*
*
10 5 0
C
NC
*
##
**
##
4
D ES
3 2 1
C
D
IL-6 mRNA *
**
0
anti-miR-22
Relative expression level
C
Relative expression level
Fig. 3
NC
anti-miR-22
IL-8 mRNA ** **
##
Relative Rluc/Luc ratio
B
**
1.2 1 0.8 0.6 0.4 0.2 0
miR-NC miR-22 pmiR-SRF-WT
C
**
1.2 1 0.8 0.6 0.4 0.2 0
miR-NC miR-22
miR-NC miR-22
pmiR-STAG2-WT pmiR-STAG2-Mut
pmiR-SRF-Mut
D
*
*
*
0.8 0.6 0.4 0.2 D
20
40
60
Endosulfan (µM)
D
1
ratio
*
0.8
20
40
60 STAG2
1
1.04
1.12
0.96
*
SRF ratio
1
0.55
0.41
0.27
0.6
GAPDH
0.4 **
0.2 0
0
C
STAG2 mRNA
1.2 Relative expression
Relative expression
1
Endosulfan (µM)
E
SRF mRNA
1.2
miR-NC miR-22
D
20
40
60
Endosulfan (µM)
1.5 Relative expression protein/GAPDH
A
Relative Rluc/Luc ratio
Fig. 4
STAG2 SRF
1 * *
0.5
*
0 D
20
40
60
Endosulfan (µM)
Fig. 5 A
B
C 2.5
80 58
SRF 1
0.39
1.23
46 30
GAPDH
*
12
*
Caspase-3 activity
NC
Apoptotic cells (%)
15 *
9 6 3
*
2 **
1 0.5 0
0 NC
si-1
si-2
NC
si-3
si-1
SRF siRNAs
E si-2
si-3 SRF
1
0.48
0.40
0.42 Bcl-2
1
0.26
0.28
0.15 BAX
1
1.51
1.62
4
18
1.58
BAX/Bcl-2 ratio
80 58 ratio 30 17 ratio 30 17 ratio 46 30
si-1
*
15
*
12
*
9 6 3 0
GAPDH
si-3
F
SRF siRNAs NC
si-2
SRF siRNAs
NC
si-1
si-2
si-3
SRF siRNAs
Relative expression level
D
*
1.5
IL-6
IL-8 *
3
* *
2
*
1 0 NC
si-1
si-2
si-3
SRF siRNAs