rheumatic heart disease ( rhd ) , the primarily autoimmune sequelae of acute rheumatic fever ( arf ) , is an autoimmune disease that arises following infection by s. pyogenes in people ages 319 years .
the disease remains a major cause of cardiovascular disability in the young , and imposes a heavy burden on public health in the developing world .
rhd , the most serious complication , occurs in 3045% of rf patients and leads to chronic valvular lesions .
the pathogenesis of rf / rhd is complex and autoimmune reactions triggered by pathogen infection are thought to be the main factor .
genetic factors that predispose a person to the development of autoimmune reactions were also considered to be related to rhd . in recent years , much attention has been paid to toll - like receptors ( tlr ) and their roles in autoimmune diseases .
tlrs are a class of proteins that play a key role in the innate immune system .
tlrs are a type of pattern recognition receptor ( prr ) ; they recognize molecules that are broadly shared by pathogens but distinguishable from host molecules , collectively referred to as pathogen - associated molecular patterns ( pamps ) .
currently , 11 mammalian tlrs have been identified , each of which responds to a specific class of pamp .
tlr2 can be activated by a variety of microbial components , including lipoproteins , peptidoglycan , lipoteichoic acid , and , especially , antigens of gram - positive bacteria , including streptococci .
microrna ( mirna ) is a short non - coding rnas that suppress the expression through targeting the 3 untranslated regions ( utrs ) of messenger rnas ( mrnas ) . according to the predicted results of bioinformatics
, there may be more than 60% of protein coding genes with the expression regulated by mirna . in the present study
, we detected 11 selected mirnas expression in clinical samples to investigate the relations between mirnas and rhd pathogenesis .
the inclusion criteria for the experimental group were : ( i ) every patient diagnosed with rheumatic mitral valve insufficiency with or without mitral stenosis , and scheduled for mitral valve replacement ; ( ii ) normal preoperative erythrocyte sedimentation rate and anti - streptolysin o to eliminate rheumatism in active stage ; ( iii ) all patients in new york heart association ( nyha ) functional class ii 
iii ; and ( iv ) no other complications ( patients with acute heart failure were excluded ) .
the inclusion criteria of the control group were : ( i ) every patient diagnosed with mitral valve prolapse because of mitral chordae tendineae fracture and mitral insufficiency , and scheduled for mitral valve replacement .
the other conditions are the same as criteria iii  v of the experimental group .
rhd cases and their controls were well matched based on the following details : ( i ) same sex , ( ii ) difference of age < 5 years old , ( iii ) difference of left ventricular ejection fraction ( ef ) < 5% , ( iv ) difference of left ventricular end - diastolic diameter ( lvedd ) < 10% of the larger of the 2 ; and ( v ) other physiological indexes .
left ventricular papillary muscle was transferred from resected mitral valve to physiological saline and liquid nitrogen and then moved to 80c refrigerator for storage .
the study protocol was approved by the ethics committee of the affiliated hospital of qingdao university , and informed consent was obtained from all subjects . a549 and
hek293 t cells were cultured in dulbecco s modified eagle medium ( hyclone , logan , ut , usa ) containing 10% fetal bovine serum , 100 iu / ml penicillin , and 10 mg / ml streptomycin .
the thp1 cell line was maintained in rpmi 1640 medium ( hyclone , logan , ut , usa ) supplemented with 10% heat - inactivated fetal bovine serum , 2 mm l - glutamine , 100 u / ml penicillin , and 100 g / ml streptomycin .
for analysis of thp-1 monocyte response to microbial ligand in vitro , log - phase cells were seeded at 510 cells / ml in a 24-well plate .
cells were stimulated with different doses of pgn ( derived from staphylococcus aureus ) ( 0.011 g / ml ) ( sigma - aldrich ) for 24 h. the cell culture supernatants were collected for cytokines detection .
quantitative rt - pcr analysis was used to detect the relative expression of selected mirnas .
total rna was extracted from tissue samples using trizol reagent ( invitrogen , carlsbad , ca , usa ) , following the manufacturer s instructions .
single - stranded cdna was synthesized by using taqman microrna specific reverse transcript primer ( applied biosystems , foster city , ca , usa ) and then amplified by using mirna specific primer and taqman mgb probes ( applied biosystems , foster city , ca , usa ) .
the full length of 828bp erbb2 3utr was cloned into pmirglo plasmid ( promega , madison , wi , usa ) following the firefly luciferase coding sequence to generate reporter vector . for dual luciferase reporter assays ,
mir-101 mimic or inhibitor was co - transfected with luciferase reporter vector by using lipofectamine 2000 ( invitrogen , carlsbad , ca usa ) .
cells were harvested at 48 h post - transfection and the luciferase activity was measured by using the dual - luciferase assay system ( promega , madison , wi , usa ) .
whole - cell protein extracts were boiled in sds/-mercaptoethanol sample buffer , separated by sodium dodecyl sulfate ( sds)-polyacrylamide gel electrophoresis , and then transferred to pvdf membranes ( amersham pharmacia biotech , st .
rabbit anti - tlr2 monoclonal antibody ( abcam , cambridge , ma , usa ) and mouse anti--actin monoclonal antibody ( santa cruz biotechnology inc . , santa cruz , ca , usa ) were both commercially obtained .
the specific protein - antibody complex was detected by using horseradish peroxidase conjugated rabbit anti - mouse or goat anti - rabbit igg .
cytokine concentrations in cell culture supernatants and serum samples were measured by elisa using cytokine kits ( abnova ) following the manufacturers instructions .
all the results were analyzed by using spss 16 software package and the t test was used .
the inclusion criteria for the experimental group were : ( i ) every patient diagnosed with rheumatic mitral valve insufficiency with or without mitral stenosis , and scheduled for mitral valve replacement ; ( ii ) normal preoperative erythrocyte sedimentation rate and anti - streptolysin o to eliminate rheumatism in active stage ; ( iii ) all patients in new york heart association ( nyha ) functional class ii 
iii ; and ( iv ) no other complications ( patients with acute heart failure were excluded ) .
the inclusion criteria of the control group were : ( i ) every patient diagnosed with mitral valve prolapse because of mitral chordae tendineae fracture and mitral insufficiency , and scheduled for mitral valve replacement .
the other conditions are the same as criteria iii  v of the experimental group .
rhd cases and their controls were well matched based on the following details : ( i ) same sex , ( ii ) difference of age < 5 years old , ( iii ) difference of left ventricular ejection fraction ( ef ) < 5% , ( iv ) difference of left ventricular end - diastolic diameter ( lvedd ) < 10% of the larger of the 2 ; and ( v ) other physiological indexes .
left ventricular papillary muscle was transferred from resected mitral valve to physiological saline and liquid nitrogen and then moved to 80c refrigerator for storage .
the study protocol was approved by the ethics committee of the affiliated hospital of qingdao university , and informed consent was obtained from all subjects .
a549 and hek293 t cells were cultured in dulbecco s modified eagle medium ( hyclone , logan , ut , usa ) containing 10% fetal bovine serum , 100 iu / ml penicillin , and 10 mg / ml streptomycin .
the thp1 cell line was maintained in rpmi 1640 medium ( hyclone , logan , ut , usa ) supplemented with 10% heat - inactivated fetal bovine serum , 2 mm l - glutamine , 100 u / ml penicillin , and 100 g / ml streptomycin .
for analysis of thp-1 monocyte response to microbial ligand in vitro , log - phase cells were seeded at 510 cells / ml in a 24-well plate .
cells were stimulated with different doses of pgn ( derived from staphylococcus aureus ) ( 0.011 g / ml ) ( sigma - aldrich ) for 24 h. the cell culture supernatants were collected for cytokines detection .
quantitative rt - pcr analysis was used to detect the relative expression of selected mirnas .
total rna was extracted from tissue samples using trizol reagent ( invitrogen , carlsbad , ca , usa ) , following the manufacturer s instructions .
single - stranded cdna was synthesized by using taqman microrna specific reverse transcript primer ( applied biosystems , foster city , ca , usa ) and then amplified by using mirna specific primer and taqman mgb probes ( applied biosystems , foster city , ca , usa ) .
the full length of 828bp erbb2 3utr was cloned into pmirglo plasmid ( promega , madison , wi , usa ) following the firefly luciferase coding sequence to generate reporter vector . for dual luciferase reporter assays ,
mir-101 mimic or inhibitor was co - transfected with luciferase reporter vector by using lipofectamine 2000 ( invitrogen , carlsbad , ca usa ) .
cells were harvested at 48 h post - transfection and the luciferase activity was measured by using the dual - luciferase assay system ( promega , madison , wi , usa ) .
whole - cell protein extracts were boiled in sds/-mercaptoethanol sample buffer , separated by sodium dodecyl sulfate ( sds)-polyacrylamide gel electrophoresis , and then transferred to pvdf membranes ( amersham pharmacia biotech , st .
rabbit anti - tlr2 monoclonal antibody ( abcam , cambridge , ma , usa ) and mouse anti--actin monoclonal antibody ( santa cruz biotechnology inc . , santa cruz , ca , usa ) were both commercially obtained .
the specific protein - antibody complex was detected by using horseradish peroxidase conjugated rabbit anti - mouse or goat anti - rabbit igg .
cytokine concentrations in cell culture supernatants and serum samples were measured by elisa using cytokine kits ( abnova ) following the manufacturers instructions .
all the results were analyzed by using spss 16 software package and the t test was used .
we collected left ventricular papillary muscle samples from rhd patients and detected the expression of 11 candidate mirnas by real - time rt - pcr .
as shown in figure 1 , the expression of mir-101 was significantly down - regulated in the rhd patients , suggesting a role of mir-101 in rhd pathogenesis . to further investigate the biological function of mir-101
, we first predicted the feasible targets of mir-101 by using an online bioinformatics tool , targetscan ( http://www.targetscan.org ) . to our surprise ,
tlr2 , an important sensor for pathogen infection , is a predicted target gene of mir-101(figure 2a ) . to understand whether the expression of tlr2 was impressed by mir-101 , full lengths of 828 bp 3-utr from tlr2 were cloned into the downstream of the firefly luciferase gene in pmirglo vector to generate pmirglo - tlr2 reporter vector .
t cells were co - transfected with pmirglo - tlr2 and mir-101 mimics or inhibitor . at 48 h after transfection ,
as shown in figure 2b , the luciferase activity was significantly suppressed by the ectopically expressed mir-101 by about 27% ( p<0.05 ) when compared with the mirna control .
furthermore , the luciferase activity was up - regulated by the mir-101 inhibitor by about 24% ( p<0.05 ) compared with the anti - mir control .
these results indicate that mir-101 targets the 3-utr of tlr2 , leading to the change in firefly luciferase translation . to further identify the binding region of mir-101 ,
we used a vector containing a putative mir-101 binding region in the 3-utr of tlr2 with 3 mutant nucleotides ( designated as pmirglo - tlr2-mu ) .
the histogram in figure 2e shows that the enzyme activity was not significantly reduced in cells transfected with mir - control compared with mir-101 mimic ( p>0.05 ) .
these data indicate that mir-101 may suppress tlr2 expression through targeting the seed sequence at the 3-utr of tlr2 . to further examine whether endogenous tlr2 expression is suppressed by mir-101
as shown in figure 2c , the level of tlr2 protein level was significantly suppressed by mir-101 mimics and up - regulated by mir-101inhibitor compared with corresponding controls in a549 cells .
the tlr2 mrna levels were not significantly changed in these 4 groups ( figure 2d ) .
these results indicate that mir-101 repressed endogenous tlr2 expression by directly suppressing tlr2 mrna translation .
thp-1 cells were seeded in a 24-well plate and then transfected with mir-101 inhibitor or corresponding control . at 48 h after transfection ,
cells were stimulated with various doses of pgn ( 0.011 g / ml ) for 24 h. the cell culture supernatants were collected for cytokines detection . as shown in figure 3 , the concentration of tnf- , il-1 , and il-6 displayed a dose - dependent relationship with pgn .
meanwhile , the mir-101 knockdown cells showed a significantly more radical response when stimulated by 0.5 and 1 g / ml than the control groups , with higher cytokines concentration .
these results are in accordance with the data from clinical samples . as shown in figure 4a , the serum cytokines ( tnf- , il-1 , and il-6 ) level was significantly higher than the controls , and cardiac tissue samples also have higher level cytokines ( figure 4b )
. we also detected the tlr2 expression in cardiac tissue samples in both protein and mrna levels .
as shown in figure 4c , almost all the rhd clinical tissue samples had a higher tlr2 expression in protein levels . however ,
the mrna level of tlr2 was not significantly changed in rhd patient samples ( figure 4d ) .
rhd is an autoimmune disease that arises following infection by s. pyogenes in young people .
nt - probnp is a well - known biomarker for congenital and acquired heart disease , which is also confirmed to be significantly elevated in children with acute rhd , but many limitations reduced its accuracy .
research indicates that soluble st2 ( suppression of tumorigenicity 2 ) is a potential early marker for cardiovascular disease , but the relationship between st2 and rhd is not understood yet .
recently , reports indicated that mirnas participate in the pathogenesis of many kinds of cardiovascular disease . but to date
, only the work of 1 group has shown that disturbed mirnas expression contributes to rhd . in the present study , to investigate the roles of mirna in the pathogenesis of rhd , we first detected 11 selected mirnas expression in cardiac tissue samples of rhd patients .
we found mir-101 had a significantly down - regulated expression in patients , which may play a role in modulating the immune system by disturbing tlr2 expression .
subsequently , by employing dual - luciferase assay and western blot , we first identified that tlr2 is a direct target gene of tlr2 and mir-101 knock - down is related to over - stimulated immune response in pgn - activated thp-1 cells .
finally , we detected the concentration and mrna level of tnf- , il-1 , and il-6 in patient serum samples and found these cytokines levels were significantly higher in patients . furthermore , the mrna and protein levels of tlr2 were also detected in cardiac tissue samples .
we found that tlr2 had a higher expression in patients , mainly in the protein level rather than the mrna level , meaning that post - transcriptional regulation factors like mirna play a crucial role in regulating tlr2 expression .
tlr2 was considered to be activated by streptococci , which is the major pathogen for rheumatic fever , which further contributed to rhd .
although arg753gln polymorphism of the tlr2 gene was confirmed to be related to acute rheumatic fever susceptibility in child , 2 research groups have found that there were no relations between this arg753gln polymorphism and rhd pathogenesis .
this study may provide clues for genetics researchers . since nucleotides variations can alter mature mirnas expression or disturb the mirna - mrna interaction , mutation sites in the mirna coding region may contribute to susceptibility to rhd [ 1113 ] .
herein , we showed that mir-101 functions as an immune inhibitor through targeting tlr2 in rhd patients .
however , zhu et al . indicated that mir-101 acted as an immune response activator by suppressing mkp1 , which is a negative regulatory factor of the tlr4 pathway .
this contradiction may be caused by the complexity of mirna function pattern and tissue specificity . since
a single mirna may have hundreds of targeting genes and a single gene may be regulated by tens of mirnas in at the same time , the function of mir-101 in the immune system needs to be fully studied in the future .
in conclusion , the present study confirmed for the first time that mir-101 targeted tlr2 3utr and repressed tlr2 expression .
this work also found an association between down - regulated mir-101 and rheumatic heart disease , which sheds light on the mechanism of rhd pathogenesis and may create an opportunity to approach the diagnosis and treatment of rhd .
 backgroundrhd is an autoimmune disease that arises following infection by s. pyogenes and imposes a heavy burden on public health.material/methodswe detected 11 selected mirnas expressed in the cardiac tissues of 11 rhd patients and 11 controls . by employing dual - luciferase assay and western blot 
 , we identified the relationship between tlr2 and mir-101 and mir-101 . 
 we used elisa to test the concentration of tnf- , il-1 , and il-6.resultsin cardiac tissue of rhd patients , mir-101 was significantly down - regulated ( p=0.011 ) . 
 ectopically expressed mir-101 repressed the luciferase activity by 27% through targeting tlr2 3utr . 
 combined with the results of western blot , we confirmed that tlr2 is a direct target gene of mir-101 . mir-101 
 knock - down is related to over - stimulated immune response in pgn - activated thp-1 cells . 
 we detected a significantly higher concentration of tnf- ( p=0.0017 ) , il-1 ( p=0.015 ) , and il-6 ( p=0.014 ) in serum samples . 
 tlr2 had a higher expression in patients in the protein level rather than the mrna level , indicating that post - transcriptional regulation factors play a crucial role in regulating tlr2 expression.conclusionsthe present study confirmed that mir-101 targets tlr2 3utr and represses tlr2 expression . 
 this work also found an association between down - regulated mir-101 and rheumatic heart disease . 
Background
Material and Methods
Sample collection
Cell culture
RT-qPCR
Dual luciferase assay
Western blotting
ELISA
Statistical analysis
Results
Discussion
Conclusions