Detection And Effect Study Of Pathogenesis Related MicroRNAs In Infantile Hemangioma

Infantile hemangiomas(IH)are the most common benign tumor of infancycharacterized by a unique pattern of rapid proliferation that occurs in the first monthsof life, followed by slow involution that may take years to complete. It is reported thatthe incidence rate is4-12%at Caucasia and about1%in Asian. Female infants arefour to six times as likely to have hemangiomas as compared to male infants, andthere is an increased incidence of premature and low birth weight babies.There is littleskin damage left in majority patients，but about20%of them need interventiontreatment because of rapid proliferation or threat normal physiological function andlife with obstructing orbit or airway.For difficult to decide which patient with an IH isat higher risk and which will need and benefit most from referral for specialty care,early intervention is getting more general.But the accurate mechanism of IH is stilluncovered,and clinic treatment is still part in blindness.It is necessary to study themechanism more deeply.The present study show that, IH is most probably derivedfrom hemangioma stem cells,which differentiate into the majority cells in IHincluding endothelial cells.Rapid proliferation of the na ve endothelial cells is themain reason of the character in proiferation period.The high activaty of VEGFR2signal pathway is key to proliferation and migration of endothelial cells,and NF-kBsignal pathway and Notch signal pathway maybe share the VEGF signal pathway intakeing part in the proliferation and migration of endothelial cells. Glucocorticoid andpropranolol maybe is also play the therapy effect on IH through VEGF signalpathway.There is still little known ahout the spontaneous involution of IH,maybe theapoptosis of endothelial cells play important role in it.But the post-transcriptionalregulation element, microRNA is still little known in IH.In this study,we makesure therole of some microRNAs in the IH.The main sduty is as followed: Part one:microRNAs microarray and screening on infantile hemangiomatissues from different periods.Method Three typical IH tissues were collected after surgery,and the totalRNA were extracted.Affymetrix miRNA3.0microarray was used to analyse all thesamples. Diversity microRNA’s target gene were predicted from miRanda date baseand targetscan date base.GO-Analysis was taken to list the function order,based onwhich gene regulation networks was made. Nine IH samples were collected forrealtimeRCR screening,included4in ealy proliferating phase,3in involutin phase and2in involuted phase. Stem ring microRNA primers were designed for hsa-miR-29a-3p，hsa-miR-130b-3p，hsa-miR-206，hsa-miR-371a-5p，hsa-miR-373-3p andhsa-miR-455-5p. SuperReal PreMix(SYBRGreen)was taken for realtimePCR.All thedates were analysed by2-ΔΔCT.Result Total38microRNAs were picked out, include22up regulated and16down regulated.After GO-analysis and functions chose,540genes were elected,whichmade up the gene regulation networks. hsa-miR-29a-3p，hsa-miR-130b-3p，hsa-miR-206，hsa-miR-371a-5p，hsa-miR-373-3p and hsa-miR-455-5p were the maincenter of the network. There were significant difference between different period inhsa-miR-29a-3p，hsa-miR-206and hsa-miR-455-5p. There were no significantdifference between different period in hsa-miR-130b-3p，hsa-miR-371a-5p，hsa-miR-373-3p.Part Two:Culture and identify of endothelial cells derived from infantilehemangiomaMethod Specimens of proliferating IH were obtained from the Department ofPlastic Surgery at Changhai Hospital and Xinhua Hospital. Single cell suspensionswere prepared from the proliferating phase specimens which treated by dispase I24hand collagenase II40min in37℃.HemECs were selected using anti-CD31-coatedmagnetic beads.Growth curve,LDL endocytosis analysis,tube formation analysis weretaken to identify the cell,while CD31and vWF’s immunocytofluorescent were also be taken.Result HemECs could be separated under the condition we designed. It wasidentified by LDL endocytosis analysis,tube formation analysis andimmunocytofluorescent contorled by HUVEC.Part Three:The effect of hsa-miR-29a-3p，hsa-miR-206and hsa-miR-455-5pon HemECs.Method The mimics or inhibitor of hsa-miR-29a-3p，hsa-miR-206andhsa-miR-455-5p were trasfected into HemECs,transfected control by fam-miR67andnegative controled by the transfection reagent without miRNA.Proliferationanalysis,apotosis analysis and invasion analysis were taken.Result Well transfection rate can be got in the condition we used.Comparedwith control,48h after trasfected by hsa-miR-29a-3p mimics could induce HemECssignificant apoptosis but no obvious change in proliferation and invation. Comparedwith control,48h after trasfected by hsa-miR-206mimics could induce HemECs lowerproliferation and apoptosis,24h after trasfected by hsa-miR-206mimics could induceweaker invation ability. Compared with control,48h after trasfected by hsa-miR-206inhibitor could induce HemECs stronger invation ability. Compared with control, noobvious change had been found trasfected by hsa-miR-455-5p mimics or inhibitor48hlater.Conclusion The level of miR-29a-3p，miR-206and miR-455-5p is differentin different period of IH. miR-206maybe take part in modulate IH through changingthe proiferation,apoptosis and invation ability of HemECs. miR-29a-3p possibly takepart in modulate IH through changing the apoptosis ability of HemECs.