International Immunopharmacology 14 (2012) 217–223
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International Immunopharmacology
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Altered let-7 expression in Myasthenia gravis and let-7c mediated regulation of IL-10by directly targeting IL-10 in Jurkat cells
Lin Jiang a, Zhuoan Cheng a, Shaobo Qiu b, Zujun Que a, Wenjing Bao c, Chao Jiang b, Fangyuan Zou a,Ping Liu b,⁎, Jianwen Liu a,⁎a State Key Laboratory of Bioreactor Engineering & Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237,PR Chinab Longhua Hospital Affiliated to Shanghai University of traditional Chinese Medicine, Shanghai 200032, PR Chinac The China Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Liaoning 110032, PR China
Article history:Received 26 May 2012Received in revised form 4 July 2012Accepted 10 July 2012Available online 24 July 2012
Keywords:Myasthenia gravislet-7let-7cIL-10
Myasthenia gravis (MG) is a T cell-dependent and B cell-mediated autoimmune disease of neuromuscularjunctions and cytokines may play a crucial role in the pathogenesis and perpetuation of MG. MicroRNAs(miRNAs) have been implicated as fine-tuning regulators controlling diverse biological processes at thelevel of posttranscriptional repression. Dysregulation of miRNAs has been described in various disease states.In this study, miRNA microarrays identified let-7 family to be decreased in peripheral blood mononuclearcells (PBMCs) fromMG patients compared to the healthy controls. We next demonstrated the differential ex-pression of let-7 family in larger samples by quantitative real-time PCR. Using a combination of bioinformat-ics and molecular approaches, we confirmed IL-10 as a target for let-7c. IL-10 expression also showed anegative correlation with let-7c expression in PBMCs from MG patients. Further experiments revealed thatinduced levels of IL-10 were inversely related to let-7c levels. We also showed that let-7c could regulateIL-10 expression in Jurkat cells. In summary, our results suggest that abnormal expression/regulation ofmicroRNAs may contribute to or be indicative of the initiation and progression of MG.
Myasthenia gravis (MG) is a T cell-dependent and B cell-mediatedautoimmune disease of neuromuscular junctions [1]. Muscle weak-ness and fatigue (hallmarks of MG) are functions of improper signal-ing between T- and B-cells which result in the elicitation of anantibody-mediated autoimmune response against the acetylcholinereceptor (AChR) located at neuro-muscular junctions [2]. The mecha-nisms that contribute to the development or pathogenesis of MG arevery complicated. It is reported that T-helper lymphocytes and cyto-kines probably participate in the development of MG [3]. It has beenshown that IL-10 level was significantly higher in MG patients thanin controls and correlated well with clinical severity [4]. In addition,the number of immune cells secreting IL-2, IL-4, interferon (IFN)-γ,and IL-10 has been shown to be significantly higher in patients withMG than in healthy individuals [5–7].
MicroRNAs (miRNAs) are small (∼22 nucleotides long), non-codingRNAs that mediate posttranscriptional silencing of target genes. Inanimals, miRNAs usually bind to complementary sites in the 3′untranslated region (UTR) of target genes and regulate target gene ex-pression by either translational inhibition, mRNA degradation, or both[8]. let-7 (lethal-7) was found as a second microRNA, first discoveredin Caenorhabditis elegans and conserved functionally from lower inver-tebrates to higher mammals. Its members have been shown to be cru-cial in various cancerous conditions [9]. let-7i‐mediated regulation ofToll-like receptor 4 expression suggests its involvement in the inflam-matory response [10]. Dysregulation of miRNAs by several mechanismshas also been described in various autoimmunity disease states, includ-ing psoriasis, rheumatoid arthritis (RA), systemic lupus erythematosus(SLE) and so on [11–14]. However, to the best of our knowledge, studiesrelevant to miRNAs and MG are still lacking.
In this study, we used both miRNA microarray- and quantitativeRT-PCR‐based approaches to assess miRNA expression in PBMCsfrom patients with MG. Focusing on the significantly downregulatedmiRNAs (let-7 family) in disease, we subsequently demonstratedthat let-7c can medicate regulation of IL-10 by directly targetingIL-10 3′UTR in Jurkat cell. Since IL-10 is a key orchestrator of the im-mune system [15], these results therefore suggest that abnormalexpression/regulation of microRNAs in MG may contribute to or beindicative of the initiation and progression of MG.
Fig. 1. Expression of let-7 family is down-regulated in PBMCs from patients with MG. A, Heat map of 44 dysregulated miRNAs in PBMCs isolated from normal controls and patients.B–E, PBMC total RNA was isolated from healthy control individuals ( n=10) and MG-patients (n=34) and relative expression levels of (B) let-7a, (C) let-7b, (D) let-7c and (E)let-7d were analyzed by qRT-PCR using U6 RNA as an internal control. Bars indicate median, *pb0.05, **pb0.01, ***pb0.001 as determined by Mann–Whitney test for panels (B–E).
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2. Material and method
2.1. Patient samples
We studied clinical samples from thirty-four patients with MGwho were followed at Longhua Hospital Affiliated to Shanghai Uni-versity of Traditional Chinese Medicine and the China AffiliatedHospital of Liaoning University of Traditional Chinese Medicine. Diag-nosis of MG was based on standard criteria including symptoms offluctuating muscle weakness supported by an electromyographic pat-tern of neuromuscular transmission dysfunction by repetitive stimu-lation. Ten gender-matched healthy donors with no history ofautoimmune disease were included as control. This study was ap-proved by the Institutional Review Board of the Longhua Hospitaland written permission was obtained from all who participated inthe study.
Peripheral blood samples were collected from each subject intubes containing acid citrate dextrose formula A and PBMCs were iso-lated using the Leukolock filter system (Ambion, Austin, TX).
2.2. miRNA microarray analysis
Total RNA was extracted from PBMCs and purified using mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, US) following themanufacturer's instructions and RNA quality was assessed with anAgilent 2100 bioanalyzer (Agilent, Palo Alto, CA), and only sampleswith RNA integrity number>8 were used.
We then submitted the samples to Shanghai Biotechnology Corpo-ration for array hybridization on an Agilent Human miRNA array(v.12.0). Each microarray chip was hybridized with a single samplelabeled with either Cy3 or Cy5. Background subtraction and normali-zation were performed. We selected miRNAs whose expression levelsbetweenMG and healthy controls differed by at least 2-fold (pb0.05).
In subsequent studies, miRNA qRT-PCR was performed using theOne Step PrimeScript® miRNA cDNA Synthesis Kit (TaKaRa, Dalian,China) and the SYBR® Premix Ex Taq™ II (Takara, Dalian, China) forthe quantification of let-7 was used. Normalization was performedwith U6 RNA endogenous controls for human, respectively. Compara-tive real-time PCR was performed in triplicate, including no-templatecontrols. Relative expression was calculated with the comparativecycle threshold method.
The primers used for real-time PCR were as follows: let-7a(5′-CGGTGAGGTAGTAGGTTGTATAGTT-3′), let-7b (5′-CGGTGAGGTAGTAGGTTGTGTGGTT-3′), let-7c (5′-CGGTGAGGTAGTAGGTTGTATGGTT-3′),and let-7d (5′-CGGAGAGGTAGTAGGTTGCATAGTT-3′).
2.3. miRNA mimics and inhibitors
miRNA mimics, hairpin inhibitors and scrambled control werepurchased from Biomics Biotechnologies (Nantong, China).miRNA mimics are dsRNA oligonucleotides and miRNA hairpininhibitors are single-stranded oligonucleotides. The combination ofgain-of-function (mimic-induced downregulation) and loss-of-function(inhibitor-induced upregulation) experiments has demonstrated themiRNA–target relationships and allows miRNA functional analysis.
2.4. Cell culture and transfection
Jurkat, a T cell leukemia line, was cultured in RPMI 1640 medium(Gibco Industries, Inc., Carlsbad, CA) with 10% fetal bovine serum(Gibco Industries, Inc.) at37 °C in a humidified atmosphere with 5%CO2. Cultured Jurkat cells were transfected in 24-well plates usingLipofectamine 2000 (Invitrogen). A hundred thousand (105) cells weretransfected with 50 to 200 nmol/L let-7c mimic or 100 nmol/L let-7cinhibitor oligonucleotides (Dharmacon), incubated for 24 h before
stimulation with phorbol 12-myristate 13-acetate (PMA:50 ng/mL)and PHA (5 μg/mL) and further incubated for 24 h.
2.5. Real-time PCR
RNA sampleswere reverse transcribed into cDNAusing PrimeScript®RT reagent Kit (Takara, Dalian, China). To determine the quantity ofmRNA, the cDNA was amplified by real-time PCR with SYBR® PremixEx Taq™ II (Takara, Dalian, China) and GAPDH was used as the internalcontrol. The SYBR Green assays were performed in duplicate or triplicateon an iCycler® thermal cycler (Bio-Rad, Hercules, USA). The relative ex-pression levels were calculated using the 2-ΔΔCt method.
The primers used were: IL‐10 5′-ACCAAGACCCAGACATCA-3′ (for-ward) and 5′-ATTCTTCACCTGCTCCAC-3′ (reverse). GAPDH 5′-GGTCGGAGTCAACGGA TTTG-3′ (forward) and 5′-ATGAGCCCCAGCCTTCTCCAT-3′ (reverse).
2.6. Plasmid construction
The IL‐10 3′UTR containing the let-7c binding site was amplifiedfrom cDNA using primers containing XbaI sites: 5′-CGGGGTACCTCAGGGTGGCGACTCTAT-3′ (forward) and 5′-CCGCTCGAGGGTCAGGCTTGGAATGGA-3′ (reverse). The amplicon was cloned into the XbaI site ofthe pGL3 promoter vector (Promega, Madison, WI). Quickchangesite-directed mutagenesis kit (Stratagene) was used to induce muta-tions into the seed region. Wild-type and mutant inserts were con-firmed by sequencing.
2.7. Luciferase activity assay
Twenty-four hours after transfection, cells were seeded into96-well plates (8×103 viable cells/well) and allowed to attach over-night. Two hundred nanograms of luciferase vector plus 50 ngpRL-TK (Promega) were transfected alone or in combination withscrambled control oligonucleotide (final concentration of 200 nM)or mimic (50, 100, 200 nM) using Lipofectamine 2000 (Invitrogen)according to the manufacturer's protocol. Luciferase activity wasmeasured 48 h after transfection using the Dual Luciferase ReporterAssay System (Promega). Firefly luciferase activity was normalizedto Renilla luciferase activity for each transfected well. Three indepen-dent experiments were performed in triplicate.
2.8. ELISA
Jurkat cells were stimulated with PMA (50 ng/mL) and PHA(5 μg/mL), for indicated time points and the supernatant was usedto perform ELISA for IL-10 (R&D Systems) per the manufacturer'sprotocol. Results were expressed in picograms and normalized byprotein concentrations.
2.9. Statistical analysis
Statistical analyses were performed using Prism 5.0 (GraphPadSoftware, San Diego, CA). Mann–Whitney was used to determine sig-nificant differences in miRNA expression between patients and con-trols, and pb0.05 was considered significant. Student's t test wasused for all other experiments unless indicated otherwise.
3. Results
3.1. let-7 family expression is decreased in MG patients compared withhealthy controls
Because miRNAs are evolutionarily conserved and play critical rolesin essential physiological events, we reasoned that a common set ofmiRNAs might be involved in the development of MG. To identify
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miRNAs potentially involved in MG, we first examined the miRNAexpression profiles with Agilent Human miRNA array (v.12.0). Priorto t-test, microRNA datasets were quantile-normalized and the invari-ant microRNAs removed. A microRNA was considered statisticallysignifican if its t-test p value was b0.05. False detection rates helpedto control for multiple testing. Six independent samples wereperformed, three with MG patients and three with healthy controls.The microarray result showed that after normalization with the endog-enous control, 21 miRNAs were significantly overexpressed (foldchange>2, pb0.05) in MG patients compared with healthy controlswhile 23 miRNAs were underexpressed significantly (fold change>2,pb0.05) (Fig. 1A). The miRNA profiling data indicate that miRNAs arefrequently dysregulated in MG and led us to the hypothesis thatthese miRNAs may play certain roles in regulating MG.
Of the 866 human miRNAs assayed, we selected let-7c for furtherinvestigation for the following reasons: first, it was observed that thelet-7 family (except mir-98 and let-7e) were all significantlyunderexpressed in MG patients (Table 1). Second, let-7c was thebest representative miRNA among the group of decreased miRNAsin MG. Third, previous reports have revealed their critical roles inthe establishment of autoimmunity [10,16].
To confirm the differential expression of let-7 family in PBMCs ofMG, we next examined the expression of these miRNAs in larger sam-ples obtained from PBMCs of 34 patients with MG and 10 healthycontrols. As expected, the levels of the let-7a, let-7b, let-7c andlet-7d were remarkably decreased in MG PBMCs relative to those inhealthy PBMCs (Fig. 1B–E). The miRNA expression levels were calcu-lated by miRNA/U6 expression ratio (i.e., 2−ΔCt). As shown inFig. 1B, the median expression level of let-7a was 41.4-fold lowerfor MG patients than for healthy controls (pb0.0001 as determinedby Mann–Whitney test). let-7b expression was 28.9-fold lower inMG patients (Fig. 1C, pb0.0001). let-7c expression was 52.2-foldlower in MG patients (Fig. 1D, pb0.0001) and let-7d expression wasalso decreased 33.5‐fold in MG patients (Fig. 1E, pb0.0001).
Put together, the results confirmed that these miRNAs are intrinsi-cally downregulated in patients with MG.
Fig. 2. let-7c targets IL-10 3′UTR. A, Expression patterns of IL-10 with let-7c in PBMCsfrom MG patients (p=0.011, spearman correlation). B, Schema of the WT and theMUT IL-10 3′UTR reporter vector, indicating the interaction sites between let-7c andthe 3′UTR of IL-10. C, Dual luciferase assay of HeLa cells cotransfected with the reportervectors containing the IL-10 WT or MUT 3′UTR or IL-10 controls. The Renilla luciferaseactivity was normalized to the firefly luciferase activity. The data were shown as rela-tive luciferase activity of IL-10 transfected cells with respect to the controls from threeindependent experiments. *pb0.05, **pb0.01, ***pb0.001.
3.2. let-7c targets IL-10 3′UTR
Generally, miRNAs repress protein-coding gene expressionthrough sequence-specific base pairing with the 3′UTRs of targettranscripts. We next aimed to investigate the potential gene targetsof let-7c using prediction tools, including miRanda, PicTar andTargetScan. Hundreds of different targets were predicted, thosegenes involved in immune system and regulation of immune func-tions may be the relevant targets with respect to the biological func-tions of let-7c. Of the predicted targets, IL-10 was chosen as a goodcandidate. IL-10 is a pleiotropic cytokine produced by both T/B cellsand macrophages and possesses both anti-inflammatory and immu-nosuppressive properties [17]. The B-cell-stimulating property of
List of significantly downregulated let-7 family in PBMCs from MG patients comparedto healthy controls.
IL-10 is thought to be the basis of several antibody-mediated autoim-mune disorders [18].
Based on the bioinformatics analysis we asked whether let-7c hada suppressive effect on IL-10 expression. To first test our hypothesis,we analyzed the expression of let-7c and IL-10 in PBMCs fromMG pa-tients. The result showed a negative correlation between the levels oflet-7c and IL-10 mRNA in these cells (p=0.011, Fig. 2A).
To obtain further direct evidence that IL-10 is a target of let-7c,we investigated the binding site of let-7c in the 3′UTR of IL-10mRNA (Fig. 2B). We constructed a luciferase reporter in which thenucleotides of the IL-10 3′UTR complementary to let-7c (position140–146 of IL-10 3′UTR) were inserted into the 3′UTR ofPGL3-promoter vector (IL‐10-WT). Correspondingly, we also
Fig. 3. A possible inverse correlation between let-7 and IL-10 levels in response to PHAand PMA stimulation. A and B, Effect of time on let-7c levels in culture supernatants ofPMA/PHA-stimulated Jurkat cells (real-time PCR) and IL-10 protein (ELISA). The relativeexpression levels of let-7c were calculated using the 2-ΔΔCt method. Data were plottedas means±SD of 3 independent experiments.* pb0.05, **pb0.01, ***pb0.001.
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generated both a mutant reporter (IL‐10-Mut), in which the se-quence in the let-7c seed-region complementary sites was changed,and a control reporter (IL‐10-Ctrl), which contained a non-relatedfragment of cDNA. let-7c mimics were co-transfected with IL‐10-WT, IL‐10-Mut or IL‐10-Ctrl into HeLa cells. We observed a re-markable reduction of the wild-type 3′UTR reporter gene expressionin the presence of let-7c. In contrast, no obvious change in the mu-tant 3′UTR reporter plasmid expression was observed (Fig. 2C).Therefore, we concluded that the inserted fragment of IL-10 (posi-tion 140–146 of IL‐10 3′UTR) was the target of let-7c.
3.3. let-7c regulates IL-10 secretion in Jurkat cells
The results described above demonstrated that IL‐10 is a directtarget of let-7c and they presented a negative correlation in PBMCsfrom MG patients. Therefore, we next investigated the effect oflet-7c on levels of endogenous IL-10 mRNA and protein in Jurkat cells.
It has been reported that the levels of let-7c expression can bechanged after stimulation with PMA and PHA in T cells [16]. So wenext examined whether the expression of let-7c in Jurkat cells couldbe changed after stimulation with PMA and PHA for indicated timepoints. It was observed that stimulation with PMA/PHA can decreaselet-7c levels in a time-dependent manner (by using real time PCR)(Fig. 3A). Interestingly, the time kinetics of IL-10 protein expressionalso indicated a monophasic response to PMA/PHA induction. IL-10levels remained unchanged as late as 8 h after stimulation and thenincreased suddenly (Fig. 3B). Put together, these results suggested apossible inverse correlation between let-7 and IL-10 levels in re-sponse to PHA and PMA stimulation.
When Jurkat cells were transfected with let-7c followed by stimu-lation with PHA and PMA for 24 h, it was observed that let-7c reducedthe expression of IL-10, as detected in the culture supernatant(Fig. 4A). In contrast, transfection with a random oligonucleotide(used as scrambled control) had no effect on the IL-10 expression(Fig. 4A). However, a saturation effect was observed at high doses(200 nmol/L in Fig. 4A), which might be due to system-level factorssuch as cellular concentrations of the target transcripts and smallRNAs loaded in RNA-induced silencing complex, which determinesthe kinetics of the regulation [19].
To checkwhether let-7c affects the stability of IL‐10mRNA, real timePCRwas performed by using RNA prepared from Jurkat cells transfectedwith increasing concentration of let-7c mimics. It was observed that itdown-regulated IL‐10 mRNA in a concentration-dependent manner(Fig. 4B), whereas a scrambled control had no effect. Thus, it is likelythat let-7c decreases IL-10 expression by degrading its mRNA.
When Jurkat cells were transfected with let-7c inhibitor and IL-10levels were measured in the culture supernatant, an increase in thelevels of IL-10 was observed (Fig. 4C). In contrast, a scrambled controlhad no effect on IL-10 expression. Thus, these experiments demon-strate that let-7c regulates IL-10 expression.
4. Discussion
As for many autoimmune diseases, the triggering events involvedin MG are not clearly defined. Complex disease pathogenesis of MGhas hindered the advancement of our understanding on disease initi-ation, thus delaying the identification and treatment of susceptible in-dividuals. miRNAs participate in keeping the balance of generegulating networks that determine the cells' fate. Bioinformaticsanalysis reveals that miRNAs can control the expression of one-thirdof the human proteome [20].
Recent evidence showing miRNA as a micromanager of variousstages of immune regulations has generated interest in the involvementof miRNAs in autoimmune disorders. Ever since the discovery ofmiRNAs, tremendous effort has been devoted to determining thebiological functions of miRNAs and their relevance to diseases.
Dysregulation of miRNAs has been associated with certain human dis-eases, such as leukemia and heart disease [21]. Although still at anearly stage in understanding their impact on immunity, miRNAs arechanging the way we think about the development of the immune sys-tem and regulation of immune functions [22]. However, no report onthe biological consequences of miRNA dysregulation in MG has beencharacterized.
Our work, for the first time, investigated the miRNA expression pro-files of MG patients and found that MG patients had significantly de-creased levels of let-7 family in comparison with healthy controls byusing miRNA array. The miRNA array result of let-7 down-regulationin MG was further confirmed by testing PBMCs from 34 MG patientsand 10 healthy controls using real-time PCR.
Previous studies have reported that let-7 microRNAs are a principalregulator that controls major cell functions in various physiological andpathological processes. let-7 microRNAs belong to a highly conservedmicroRNA family consisting of 12 genes encoding for 9 differentmicro-RNAs (let-7a to let-7i) [9]. The let-7 family have been reportedto act as tumor suppressors because their levels are down-regulatedin various cancerous conditions and they also target oncogenes such
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as RAS and HMGA2 [23,24]. Most let-7 family members areposttranscriptionally regulated by Lin28, which prevents let-7 matura-tion by binding to its precursor and inhibits Drosha processing [25,26].Nuclear factor κB, an important transcription factor during the initialphase of inflammation is known to reduce let-7 levels by upregulatingLin28 levels. Lin28-mediated downregulation of let-7 was shown tobe crucial in inflammation and cancer [27].
Our further work extends the role of let-7c in the pathogenesis ofautoimmune diseases with an emphasis on its regulation of IL-10 inJurkat cells. miRNAs post-transcriptionally regulate gene expressionby binding to the 3′UTR of target mRNAs. Binding leads to the deg-radation of target mRNAs and reduces translation of target proteins.Therefore, we predicted that let-7c had a potential binding site inthe 3′UTR of IL‐10 by employing the miRNA target predictionsoftware. There was a negative correlation between the levels oflet-7c and IL-10 mRNA in PBMCs from MG patients. Thedual-luciferase activity assay showed that the relative lucifease ac-tivity was down-regulated when cells were treated with let-7c. In-terestingly, we also found a possible inverse correlation betweenendogenous let-7c and IL-10 expression in response to PHA andPMA stimulation. Furthermore, it was shown that let-7c could mod-ulate IL-10 levels in Jurkat cells. This is also the first study to showthat the IL-10 is negatively regulated by let-7c via a specific targetsite within the 3′UTR.
Cytokines exert an important role in the pathogenesis and thepathophysiology of MG, by modulating autoantibody productionand interfering with cell-mediated immunity [28]. Several cytokineshave been shown to be associated with MG, for example, IL-10, IL-6,interferon (IFN)-γ, IL-12 and interferon-γ inducible protein (IP)-10[29–32]. Matusevicius et al. found that the number of AChR-reactiveIL-10 mRNA-expressing PBMCwas higher in MG patients than in con-trols with non-inflammatory neurological diseases [33]. Huang et al.reported that the number of IL-10-secreting cells tended to be higherin patients with generalized MG than in patients with ocular MG [34].These findings support that interleukin-10 might play a crucial role inthe pathogenesis and perpetuation of MG.
Collectively, the above-described evidence suggests that de-creased let-7c levels may play a role in MG pathogenesis by regulat-ing IL-10 production.
In conclusion, we provided evidence for a previously unknownkey regulatory mechanism by discovering that dysregulation oflet-7c was involved in MG, which inhibited IL-10 expression in Jurkatcells. To the best of our knowledge, this is the first report onregulation of IL-10 by let-7c. And all the results suggest a possiblelink between the miRNA-mediated mechanisms and the pathogenesisof MG.We speculate that increasing the levels of let-7c in MG patientsmight partially reverse the symptoms of MG. This hypothesis needs tobe validated by further studies on the experimental model of MG.
Acknowledgments
This work was supported by Shanghai Science and TechnologyCommission (No. 10JC1414500) and Shanghai Committee of Scienceand Technology (grant 11DZ2260600).
Fig. 4. let-7c modulates IL-10 levels in Jurkat cells. (A and C) Jurkat cells were transfectedwithout orwith increasing concentrations of let-7cmimics or scramble or let-7c inhibitor,and IL-10 level was measured as described in Material and method. (B) Jurkat cells weretransfected with or without increasing concentrations of let-7c mimics or scramble,mRNA was prepared followed by real-time PCR by using IL‐10 specific primers. GAPDHwas performed as loading control. Fig. 4A and C, were presented as means±SD of 3 inde-pendent experiments. Fig. 4, B, the relative expression levels were calculated using the2-ΔΔCt method.
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