Functional And Mechanistic Studies Of Long Noncoding RNAs Identified By Microarray Analysis In Infantile Hemangioma

Part 1 Expression profile of lncRNA in infantile hemangioma identified by microarray analysisInfantile hemangioma is one of the most common benign vascular tumors of infancy,the incidence of infantile hemangioma is varying from 3%to 10%.Clinically infantile hemangioma is divided into three phases including proliferating phase,involuting phase and involuted phase.Infantile hemangioma is featured of the rapid proliferating in the first year after birth,which is called the proliferating phase.The infantile hemangioma regresses spontaneously after the first year,and turns into the involuting phase.After three to five years,the infantile hemangioma comes upon the stage of involuted phase.In proliferating phase,infantile hemangioma is composed of immature hemangioma endothelial cells with rapid proliferating ability,meanwhile immature microvascular tubes are formed accordingly.In the involuting phase,the immature hemangioma endothelial cells grow mature gradually with limited proliferating potential,and the microvascular tubes become distinct.The hemangioma.endothelial cells become spindle in shape in the involuting phase of infantile hemangioma compared with the ovary shape in the proliferating phase of infantile hemangioma.And in the involuted phase of infantile hemangioma,the hemangioma endothelial cells and the vascular tubes are gradually replaced by the fatty tissues and the connective tissues accordingly.Recently,several researches figured out the cellular components of infantile hemangioma already.Infantile hemangioma is composed of hemangioma stem cells,hemangioma endothelial cells,hemangioma pericytes and mast cells.Because the previous researches have already studied the cellular components in infantile hemangioma clearly.Furthermore,the hemangioma stem cells are discovered and succesfully seperated.The vivo assays demonstrate that the hemangioma stem cells transplantation in nude mice could mimic the typical three phases life span of infantile hemangioma,which indicating that infantile hemangioma might be derived from hemangioma stem cells.This feature provides new options for the pathogenesis of the origin of infantile hemangioma.The rapid proliferating phase in early months of infancy and the spontaneously regression later on,might be caused by the early proliferation of hemangioma stem cells and the subsequent differentiation of hemangioma stem cells.The pathological mechanism of infantile hemangioma is still unclear.Several hypothesis are proposed according to the previous studies.These hypothesis include hypothesis of placental endothelial cell embolism,hypothesis of vascular formation or enhanced ability of vascular formation,and hypothesis of tissue hypoxia.The significant signaling pathways in infantile hemangioma includes VEGF/VEGFR signaling pathway,Notch signaling pathway,? adrenaline receptor signaling pathway,HIF-la mediated signaling pathway,PI3K/Akt/mTOR signaling pathway,and PDGFB/PDGFR-? signaling pathway.Although the mechanism of pathophysiology of IH remains unknown at the moment,but the pathological angiogenesis which is featured of hemangioma endothetial cells hyperproliferation plays crital roles in the pathology of infantile hemangioma.Insufficient vessel growth and regression occurs in some ischemic diseases generally,such as myocardial infarction,stroke,or obesity-associated disorders.The excess vessel growth promotes tumorigenesis,inflammatory disorders,and ocular disorders.The exploring of the angiogenic mechanism of infantile hemangioma will pave the way to unveil the mechanism of pathological angiogenesis.Long non-coding RNAs(lncRNAs)are the RNAs which contains more than 200 nucleotides with limited or non-protein coding potential.LncRNAs are involved in a large amount of biological functions,including chromosome imprinting,epigenetical regultion,cell cycle regulation,cell apoptosis regulation,pluripotent stem cell regulation and so on.Several studies found out that,IncRNA could regulate the pathological and biological functions in tumor,cardiovascular,neurogenesis and so on.In terms of angiogenesis,IncRNAs are involved in the function of pathological,physiological angiogenesis or vasculogenesis,for example,knockdown of lncRNA MALAT1 in vascular endothelial cell promoted cell migration and inhibited cell proliferation.In diabetes associatied renal microvacular dysfunction,IncRNA RNCR3 inhibition attenuated the renal microvascular dysfunction.In diabeted related retinapathy,lncRNA MIAT knockdown alleviates the diabetes induced microvascular dysfunction.These results indicate that lncRNAs could play an critical role in angiogenesis.So far,no research concerning the function and expression profile in terms of lncRNAs in infantile hemangioma has been published yet.We investigated the expression profile of IncRNAs in infantile hemangioma,and explored the effects and mechanisms of IncRNAs in terms of pathological angiogenesis.This study will investigate the mechanism in pathophysiology of infantile hemangima.In the meantime,it will also provide strong evidence for the mechanism of pathological angiogenic diseases.Purpose:To study the IncRNA expression profile in infantile hemangioma,IncRNA microarray analysis was adopted.Bioinformatics analysis was employed to analyze the differential expression of molecules.Target prediction program was used to select the target molecules relative to angiogenesis.QPCR analysis was adopted to validate the expression level of target genes in IH,then the function of molecules will be discussed briefly.Our studies will provide outbreaks for the researches of pathology in infantile hemangioma.Methods:Infantile hemangiomas of proliferative phase and adjacent non-tumor tissues were obtained surgically between June 2013 and June 2016 in the department of plastic surgery at the Shandong Provincial Hospital.Specimens were snap-frozen in liquid nitrogen after excision.All samples were assessed for further validation by two independent pathologists.Total RNAs were extracted from frozen tissue blocks using the TRIzol reagent according to the manufacturer's instructions strictly.The expression profile of the IncRNAs and the mRNAs in infantile hemangioma were screened by microarray analysis,and compared to the adjacent non-tumorous tissues.Bioinformatics analysis was employed to analyze the differential expression of lncRNAs and mRNAs in infantile hemangioma.Target molecules were selected according to bioinformatics analysis in combine with literatures.Afterwards,proliferating phase of infantile hemangioma and the adjacent nontumorous tissues were obtained for further verification by QPCR analysis.QPCR analysis was employed to verify the expression level of our target molecules,in order to check if the expression level was in accordance with the microarray analysis or not.Results:Proliferative phase infantile hemangioma tissues were obtained,and RNA were extracted for microarray analysis.The analyzing of the raw microarray data,showed that 1469 mRNAs were upregulated in hemangioma,while 1184 mRNAs were downregulated.1259 IncRNAs were upregulated in hemangioma,while 857 lncRNAs were downregulated,at a cutoff fold change of 2.0(p<0.05).These data had been submitted to GEO dataset(GSE78811).According to KOBAS 2.0 analysis,the top 30 significantly enriched GO terms included vasculature development,blood vessel development,blood vessel morphogenesis,cell migration,endothelium development,cardiovascular system development,angiogenesis and tissue migration.Meanwhile,the top 30 significant enriched pathway terms comprised Nitric oxide stimulates guanylate cyclase,epidermal growth factor receptor(EGFR)interacts with phospholipase C-gamma,Signaling by vascular endothelial growth factor(VEGF),vascular smooth muscle contraction,signaling by nerve growth factor(NGF),Non-integrin membrane-extracellular matrix(ECM)interactions and Rho GTPase cycle.An IncRNA-mRNA co-expression network displayed the associations of MEG3,MEG8,FENDRR,and Linc00152 with their related mRNAs.One lncRNA could be associated with one to tens of mRNAs and vice versa.According to the bioinformatics analysis,p29066,p33867,p44557_v4,and p8683 were selected as target molecules.MALAT1,MEG3,MEG8,FENDRR,Linc00152,EGFL7 and FOXF1 were chosen based on the literatures.Microarray data were validated by QPCR in expanded samples of infantile hemangioma.QPCR results obtained from 9 lncRNAs and 2 mRNAs validated the microarray findings.Conclusion and Significance:1.The expression profiles of IncRNAs in infantile hemangioma in compare with the adjacent non-tumorous tissues were carried out for the first time.It showed that 1469 mRNAs were upregulated in hemangioma,while 1184 mRNAs were downregulated,1259 IncRNAs were upregulated in hemangioma,while 857 IncRNAs were downregulated,at a cutoff fold change of 2.0(p<0.05).These data implied that lncRNAs and mRNAs were significantly differentially expressed in infantile hemangioma.2.The bioinformatics analysis indicated that the top 30 significantly enriched GO terms included vasculature development,blood vessel development,blood vessel morphogenesis,cell migration,endothelium development,cardiovascular system development,angiogenesis and tissue migration.The correlation network analysis showed the relationship of MEG3,MEG8,FENDRR and Linc00152 and mRNAs,indicating that differential expressions of IncRNAs could be related to the pathological angiogenesis of infantile hemangioma.3.Among the differential expression of IncRNAs,we chose the MALAT1,MEG3.MEG8,FENDRR,Linc00152 based on the literatures review,and p29066,p33867,p44557_v4 and p8683 according to bioinformatics analysis.FOXF1 and EGFL7 were selected as targets among differential mRNAs.QPCR analysis was employed to verify the data of microarray.It indicated that those differential expressed IncRNAs may be involved in the pathogenesis of infantile hemangioma.Part 2 Expression and function of MALAT1 in infantile hemangiomaPurpose:Our previous work indicated that lncRNAs were differential expressed in infantile hemangioma,specifically,the star IncRNA MALAT1 was upregulated in infantile hemangioma compared with the adjacent non-tumorous tissues.As to angiogenesis,lncRNA MI AT and MALAT1 promoted angiogenesis in diabetes induced retinopathy.In addition,infantile hemangioma is featured for pathological angiogenesis.We aimed to study if the excess expressed MALAT1 would promote angiogenesis in infantile hemangioma.Methods:Firstly,QPCR analysis was employed to study the expression level in expanded pairs of infantile hemangioma tissues and adjacent non-tumorous tissues.Secondly,after HUVECs were infected by MALATI shRNA lentivirus,MTT analysis was used to assess whether MALAT1 could promote proliferation.Cytometry analysis was used to assess the effect of cell cycle and apoptosis of MALAT1.Tube formation analysis was employed to assess the effect of angiogenesis when MALAT1 was inhi,bited.Results:MALAT1 was significantly upregulated in expanded pairs of proliferating phase infantile hemangioma tissues compared with adjacent nontumorous tissues.HUVECs were infected by MALAT1 shRNA lentivirus,QPCR analysis was employed to estimate the efficiency of MALAT1 knockdown.The expression level of MALAT1 in knockdown groups was reduced to 41%compare with the control groups.MTT assays indicated that knockdown of MALAT1 inhibited the cell proliferation.Cytometry analysis indicated that knockdown of MALAT1 induced S phase prolongation.Apoptosis analysis showed that inhibition of MALAT1 promoted apoptosis.In vitro angiogenesis analysis indicated that MALAT1 knockdown inhibited angiogenesis.Conclusion and significance:MALAT1 was highly expressed in proliferating phase infantile hemangioma;knockdown of MALAT1 in HUVECs inhibited proliferation,promoted apoptosis,induced S phase arrest,and then promoted in vitro angiogenesis.As the result,the overexpression of MALAT1 might play critical roles in the rapid proliferation and angiogenesis in the proliferative phase of infantile hemangioma.Part 3 Expression and mechanism analysis of FENDRR and FOXF1 in infantile hemangiomaPurpose:LncRNAs expression profiles of infantile hemangioma showed the differential expression of IncRNAs in infantile hemangioma compared with the adjacent non-tumorous tissue.Specifically lncRNA FENDRR and its adjacent transcription factor FOXF1 was significantly upregulated in infantile hemangioma.Moreover,IncRNA FENDRR was the most upregulated IncRNA molecule.LncRNA FENDRR was essential for the embryonic proper vasculature,heart and body wall development.Transcription factor FOXF1 was required for the formation of embryonic vasculature by regulating endothelial genes critical for vascular development and vascular endothelial growth factor signaling.FOXF1 promoted angiogenesis in vascular endothelial cells.Infantile hemangioma is featured for pathological angiogenesis.LncRNA FENDRR is upregulated in infantile hemangioma,however.the function and mechanism of IncRNA FENDRR and transcription factor FOXF1 in infantile hemangioma remains unclear.We aimed to explore the function of IncRNA FENDRR in infantile hemangioma,and the regulatory mechanism of transcription factor FOXF1 and IncRNA FENDRR.Methods:First,QPCR analysis was employed to explore the expression level of IncRNA FENDRR and transcription factor FOXF1 in expanded pairs of infantile hemangioma tissues compared with the adjacent non-tumorous tissues.Then,after HUVECs was infected by IncRNA FENDRR shRNA lentivirous,MTT analysis was used to assess whether IncRNA FENDRR could promote endothelial cell proliferation.Cytometry analysis was used to assess the effect of cell cycle regulation and apoptosis when IncRNA FENDRR was inhibited.Tube formation analysis was employed to assess the effect of angiogenesis when IncRNA FENDRR was inhibited.Dual luciferase reporter gene assays were employed to explore whether FOXF1 could activate the transcription of IncRNA FENDRR.QPCR analysis was adopted to assess the expression level of transcription factor FOXF1,when IncRNA FENDRR was inhibited in HUVECs.QPCR analysis was employed to discover the expression level of IncRNA FENDRR after the overexpression of transcription factor FOXF1 in HUVECs.The gene expression profile assays were adopted to explore the indirect downstream mechanism of IncRNA FENDRR,bioinformatics analysis and literature review analysis was used to find out the target proteins of IncRNA FENDRR.And western blot assay was adopted to assess whether the expression level of target proteins was in accordance with the data from the microarray analysis.Results:1.According to the data of QPCR analysis,transcription factor FOXF1 and IncRNA FENDRR was significantly upregulated in proliferating phase infantile hemangioma compared with the adjacent non-tumorous tissues,and the expression level of IncRNA FENDRR was positively correlated with that of FOXF1 in infantile hemangioma.2.The functional assays in vitro discovered that inhibition of LncRNA FENDRR in HUVECs could significantly inhibit the proliferation of HUVECs,and also induced apoptosis.Cytometry analysis indicated that knockdown of FENDRR induced G2/M phase arrest in HUVECs.In vitro angiogenesis assay showed that inhibition of LncRNA FENDRR attenuated angiogenesis.3.Dual luciferase reporter gene assay was adopted to find out whether transcription factor FOXF1 could activate the transcription of LncRNA FENDRR,and the data showed that transcription factor FOXF1 could activate the transcription of IncRNA FENDRR.In addition,overexpression of FOXF1 in HUVECs could upregulate the expression level of IncRNA FENDRR;knockdown of IncRNA FENDRR could induce the low expression level of FOXF1 in HUVECs,hinting that negative feedback regulatory mechanism may exist among the transcription factor FOXF1 and lncRNA FENDRR.4.Gene expression profile assay was adopted to explore the downstream mechanism of IncRNA FENDRR.By comparing with the control groups,knockdown of FENDRR in HUVECs upregulated 364 mRNAs,and downregulated 207 mRNAs(fold change over 1.5,the p value below 0.05).The IPA analysis of differential expression of mRNAs indicated that the EIF2 signaling pathway was significantly inhibited,and the AMPK signaling pathway was significantly activated.As shown in the previous studies,the functional assay indicated that inhibition of lncRNA FENDRR attenuated cell proliferation and angiogenesis,and also induced G2/M phase arrest.We chose target genes associated with these functions,including PLK1,KIAA0101 and CCND2.Western blot analysis was adopted to assess the expression level of these molecules.And PLK1,KIAA0101 and CCND2 was significantly downregulated according to the western blot analysis,indicating that inhibition of lncRNA FENDRR could significantly inhibit the expression of PLK1,KIAA0101 and CCND2.Finally,the transcriptional factor FOXF1 induced upregulation of lncRNA FENDRR may promote proliferation and angiogenesis via upregualtion of PLK1,KIAA0101 and CCND2.Conclusion and significance:1.Transcription factor FOXF1 and lncRNA FENDRR were significantly upregulated in proliferating phase infantile hemangioma,and the expression level of FENDRR was positively correlated with that of FOXF1 in infantile hemangioma.2.The transcription factor FOXF1 induced upregulated lncRNA FENDRR may promote endothelial cell proliferation and angiogenesis by activating PLK1,KIAA0101 and CCND2.3.Unveiling the angiogenic mechanism of lncRNA FENDRR may provide opportunity to disclose the possible mechanism of lncRNAs in infantile hemangioma,which potentially offer new therapeutic options for other angiogenic diseases.Part 4 Conclusion and innovationConclusion:1.Expression profiles of proliferative phase infantile hemangioma were identified by microarray analysis.The raw microarray data were analyzed.As a result,1469 mRNAs were upregulated in hemangioma,while 1184 mRNAs were downregulated,1259 IncRNAs were upregulated in hemangioma,while 857 lncRNAs were downregulated,at a cutoff fold change of 2.0(p<0.05).Among these molecules,9 LncRNAs and 2 mRNAs were validated the expression levels by QPCR,and the expression levels were in accordance with the microarray data.2.LncRNA MALAT1 was significantly upregulated in proliferating phase infantile hemangioma compared with non-tumorous tissues.In vitro functional analysis discovered that inhibition of lncRNA MALAT1 attenuated cell proliferation,angiogenesis and induced S phase arrest and apoptosis.3.LncRNA FENDRR was significantly upregulated in proliferating phase infantile hemangioma compared with the adjacent non-tumorous tissues.In vitro functional analysis discovered that inhibition of FENDRR attenuated cell proliferation,angiogenesis and induced G2/M phase arrest and apoptosis.4.Mechanism studies found out that transcription factor FOXF1 activated the transcription of IncRNA FENDRR.Inhibition of IncRNA FENDRR may attenuate angiogenesis and proliferation,induced apoptosis and G2/M phase arrest by the downregulation of PLK1,KIAA0101 and CCND2.Innovations:1.For the first time,expression profiles of lncRNAs in infantile hemangioma were identified by microarray analysis.The differentially expressed lncRNAs were analyzed by bioinformatics combined with literature reviews,then the target molecules were selected.Our research provides strong evidence for the functional and mechanistic study lncRNA in infantile hemangioma.LncRNAs play critical roles in pathological angiogenesis and biological angiogenesis,and infantile hemangioma is featured of pathological angiogenesis.As a result,by exploring the functions and mechanisms in terms of pathological angiogenesis of those differentially expressed IncRNA molecules screened from infantile hemangioma,we would obtain strong evidences for other pathological angiogenic diseases.2.For the first time,we discovered that lncRNA MALAT1 was upregulated in proliferating phase infantile hemangioma compared with the adjacent non-tumorous tissues.LncRNA MALAT1 promoted endothelial cell proliferation,and also promoted angiogenesis,indicating that IncRNA MALATI may have a function to promote cell proliferation and angiogenesis.The highly expressed IncRNA MALAT1 may promote hemangioma endothelial cell proliferation and angiogenesis in infantile hemangioma.LncRNA MALAT1 could be a new therapeutic target for treatment of excessive angiogenesis related diseases.This study also provides straightforward evidences for the mechanism and therapeutic studies of other angiogenesis related diseases.3.For the first time,we discovered that transcription factor FOXF1 induced overexpression of IncRNA FENDRR promoted endothelial cell proliferation and promoted angiogenesis through upregulation of PLK1,KIAA0101,and CCND2.As a result,IncRNA FENDRR can promote cell proliferation and also promote angiogenesis,so we speculated that IncRNA FENDRR is likely to be involved in the pathogenesis of infantile hemangioma.And IncRNA FENDRR may become a novel therapeutic target for the treatment of angiogenesis related diseases.Specific goals of this study are to explore the functions and the mechanisms of IncRNA FENDRR and related molecular pathways in terms of the pathological angiogenesis in infantile hemangioma.Firstly,this research contributes to the studies of pathogenesis in infantile hemangioma.which even opens up a new field concerning IncRNAs and infantile hemangioma.Secondly,from the respect of molecular biology,this study will provide a theoretical basis for the researches of new intervention approaches in infantile hemangioma in the future.Thirdly,this study will provide important reference in the field of angiogenesis intervention approaches for other angiogenesis related diseases such as cancer,diabetes mellitus related microvascular dysfunctional diseases and so on.