| Part ?. Research BackgroundAging is one of the most important etiological factors of ED(erectile dysfunction,ED).Obviously high prevalence of ED in the aged has been reported.Aging and the degree of ED is closely related.With the increasing number of aged population,the number of old men with aging-related ED is growing.But many of them still need sexual activity to some extent.Therefore,aging-related ED has become a social problem which needs to be emphasized.Nowadays,the medical treatment of aging-related ED is limited.Main reason for this falls on its complicated pathogenesis.Aging-related ED is a disease which involves multiple etiological factors,such as neurogenous,endocrine,and vascular endothelial factors.This may take responsibility for the poor response to the current treatments.Indeed,researches had manifested that the PDE5(phosphodiesterase-5)inhibitor was not always efficient in the treatment of aging-related ED.As a result,the mechanism and strategy of aging-related ED have become to be very important topics in Andrology and need to be soloved.MicroRNA,which generally exists in the eukaryote,is a strongly conservative noncoding small RNA with 19-23nt length.It can regulate gene expression by binding completely or partially to the 3'UTR(3' untranslated region)of target gene related mRNA for posttranscriptional inhibition.It has been proved that miRNAs modulate a variety of functions including proliferation,differentiation,apoptosis,and senescence.Importantly,a series of aging-related genes which could be regulated by many miRNAs could also involve in the pathogenesis of ED.Thus,we hypothesize that miRNAs might be involved in the regulation of various genes' expression in the corpus cavernosum in aged male,and thus involved in the process of genesis and development of aging-related ED.The present research is to investigate the mechanism of miRNA's involvement in the pathogenesis of aging-related ED.Part ?.Research on the microRNA profile in aged rats with erectile dysfunctionAim:To investigate the miRNA profile in the corpus cavernosum of aged rats with ED,and to analyze the target genes and signaling pathways regulated by the dysregulated miRNAs.Methods:According to the apomorphine test,the experimental male Sprague-Dawley rats were divided into different groups.Intracavernous pressure response to electric stimulation of the cavernous nerve and content of corpus cavernosum were tested to validate the grouping.At last,the experimental animals were divided into three groups:aging rats with ED(group AE),aging rats with normal erectile function(group AN),and young rats as normal controls(group YN).The tissues from corpus cavernosum were collected to extract RNA,and the High-throughput sequencing technology was used to detect the dysregulated miRNAs.Quantitative real-time polymerase chain reaction(qRT-PCR)was performed to testify the dysregulated miRNAs,and then bioinformatic analysis of the miRNAs was conducted.Results:Accompanied with significantly decreased erectile function,the content of smooth muscle and endothelium within the corpus cavernosum of rats with aging-related ED were significantly decreased compared with both AN group and YN group.The miRNA profile of corpus cavernosum in group AE was significantly different from that of other groups.Four miRNAs,miR-1,miR-206,miR-200a,and miR-203,were found and validated up-regulating with over twofold change in AE group by qRT-PCR.According to the bioinformatics analysis of the four miRNAs,their most related pathways in the pathogenesis of aging-related ED were eNOS/NO/PKG and PGE1/PKA pathways,which involved 13 genes predicted to be regulated by the four miRNAs.Further bioinformatic analysis was performed to the 13 genes.Finally,three genes,CAV1(encode Caveolin-1,CAV-1),SIRT1(encode Sirtuin 1,SIRT1),and CALM(encode Calmodulin,CALM),were found to be more related to the dysregulated miRNAs in corpus cavernosum of the AE group.Conclusion:The miRNA regulated mechanism was first introduced into the pathogenesis of aging-related ED by the present study.Four miRNAs(miR-1,miR-200a,miR-203,and miR-206)were found up-regulated significantly in the corpus cavernosum of rats with aging-related ED.Their most related pathways in the pathogenesis of aging-related ED were eNOS/NO/PKG and PGE1/PKA pathways,and their main target genes probably were CAV1,SIRT1,and CALM.Part ?.Research on the regulated relationship between the dysregulated miRNAs and their targets in corpus cavernosum of aged rats with EDAim:To explore the expression of three target proteins(CAV-1,SIRT1,and CALM)in the corpus cavernosum of aged rats with ED,and to testify the regulated relationship between the dysregulated miRNAs and the three targets.Methods:The corpus cavernosum from the three groups in previous experiment were grouped as before.Firstly,Western Blotting was used to detect the expression of related three proteins in corpus cavernosum of the three groups.RT-PCR and agarose gel electrophoresis were used to test the expression of the related mRNAs.And then the regulated relationship between the miRNA and its target was firstly identified.Secondly,Luciferase report experiment was performed to detect the regulated relationship.Finally,over-expression and inhibition of miRNA in vitro were conducted to testify the regulated relationship.The miRNAs were detected by qRT-PCR,and the changed expressions of three proteins were detected by Western Blotting.Results:The three proteins were significantly decreased in the corpus cavernosum of AE group,while their mRNA levels were similar when compared with that of YN or AN group.The results of Luciferase report experiment manifested that miR-1/206 could bind to the 3'UTR of CALM,miR-200a could bind to the 3'UTR of SIRT1,and miR-203 could bind to the 3'UTR of CAV1.Over-expression of miRNAs in cultural cells could lead to distinguished decrease of their target proteins.Finally,according to the bioinformatic re-analysis,dysregulated expression of miR-1/206 could down-regulate CALM,and might mainly influence the function of eNOS.Dysregulated expression of miR-200a could down-regulate SIRT1,and might also mainly influence the function of eNOS.And dysregulated expression of miR-203 could down-regulate CAV-1,and might mainly influence the function of eNOS and sGC.As a result,the four miRNAs could influence the process of smooth muscle relaxation in the corpus cavernosum,and also the erectile function.Conclusion:Three target proteins of the four miRNAs in the corpus cavernosum of aged rats with ED were significantly decreased.The four dysregulated miRNAs(miR-1,miR-206,miR-200a,and miR-203)could down-regulate the three target genes(CALM,SIRT1,and CAV1).The four miRNAs might mainly influence the eNOS/NO/PKG pathway from the eNOS/NO/PKG and PGE1/PKA pathways via regulating the three targets,and then involved in the pathogenesis of aging-related ED.Part ?.Research on the regulated relationship between the dysregulated miRNAs and their most related pathways in corpus cavernosum of aged rats with EDAim:To study the regulated relationship between the dysregulated miRNAs and their mainly influenced pathways in the corpus cavernosum of aged rats with ED.Methods:The corpus cavernosum from the three groups in previous experiment were grouped as before.The endothelial cells were isolated from different groups to perform cell experiment in vitro.There were 6 experimental groups in this part,young rats as normal controls(group YN),aging rats with normal erectile function(group AN),aging rats with ED(group AE),group AE+negative control RNA(NC),group AE+miRNA mimics,and group AE+miRNA inhibitor.Over-expression and inhibition of miRNAs were performed via cell transfection.The efficiency of transfection was identified by qRT-PCR.And then the expression of target proteins and molecules in eNOS/NO/PKG pathways were detected to testify the regulated relationship between the dysregulated miRNAs and their most related pathways in corpus cavernosum of aged rats with ED.Results:Transfections of four miRNAs were conducted after isolating of endothelial cells from different groups.The efficiency of transfection was tested by qRT-PCR.The expression of miRNAs were significantly increased after over-expression,with the miR-1 increased over 100 folds,the miR-206 increased over 70 folds,the miR-200a increased nearly 30 folds,and the miR-203 increased nearly 30 folds,respectively.After inhibition,the expression of miRNAs decreased nearly 1.5 folds.When compared with AE group,the expression of relative target proteins were significantly decreased in the group AE+miRNA mimics,and were increased in the group AE+miRNA inhibitor,but without significance.The expression of eNOS,NO,and cGMP in eNOS/NO/PKG pathway were significantly decreased in the group AE+miRNA mimics.Among them,over-expression of miR-1/206 could lead to the decrease of eNOS with most significance;over-expression of miR-203 could lead to the decrease of eNOS and cGMP with most significance;while over-expression of miR-200a could lead to all of the detected molecules in the eNOS/NO/PKG pathway with distinguished significance.After inhibition of miR-1/206,and miR-203,the expression of eNOS,NO,and cGMP were all increased,but with no statistical significance when compared with AE group.Importantly,inhibition of miR-200a could lead to significant increase of the expression of eNOS,NO,and cGMP when compared with AE group,although the expression was still under the level of that in the normal control group.Conclusion:Four up-regulated miRNAs were expressed in the corpus cavemosum of aged rats with ED.They mainly regulated three targets,and then probably mainly influence the eNOS/NO/PKG pathway involved in the pathogenesis of aging-related ED.The regulated relationship included miR-1/206-CALM-eNOS,miR-200a-SIRT1-eNOS/NO/cGMP,and miR-203-CAV-1-eNOS/cGMP.With the results of corpus cavernosum endothelial cells' experiment in vitro of rats with A-ED,inhibition of miR-200a could result in significant increase of the expression of eNOS,NO,and cGMP when compared with that before the interference. |
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