Identification Of Critical Genes In Secondary Hair Follicle Cycling And Functional Analysis Of LHX2 And MiR-144 In Dermal Papilla Cells Of Shanbei Cashmere Goat

Cashmere is famous as “soft gold” and a kind of high-grade textile material produced by secondary hair follicles.Furthermore,secondary hair follicles are a good model for studies on the mechanisms of hair regeneration.We intend to study on the molecular regulation mechanism of the secondary hair follicle in Shanbei cashmere goat,which not only provide a theoretical foundation for improving the quantity and quality of cashmere,but also a reference for the study of on hair regeneration.In order to explore the regeneration rhythm of hair follicle cycle,and screen the key genes regulating hair follicle cycle,including non-coding and coding genes,and reveal the regulatory role of non-coding genes in hair follicle cycle.The mainresearch contents include the following four parts: 1)To reveal a regular point in time in secondary hair follicle circulation,we measured two morphological indexes including the length of secondary hair follicles and the width of their hair bulbs in twelve different months.2)Screen the key regulator genes which forces anagen-catagen-telogen-anagen shift in turn by further analysis of mi RNA and m RNA profiling data.3)Explore the molecular mechanism of the transfer factors from the anagen to the catagen by whole transcriptomics.4)Verify the targeting relationship between mi R-144-3p and LHX2 through dual-luciferase array system,and construct mi R-144 adenovirus,and LHX2-/-dermal papilla cell lines,and then verify their function on SDP.The main research results are as follows:1.We found the secondary hair follicle depth ranges from 1070.16 ?m to 2267.61 ?m in the whole year,and the width of secondary hair follicle bulb ranges from 44.05 ?m to108.61 ?m by microscopic measurement;According to the changes in the whole year,we came to the conclusion that,the SHFs growth phase ranges from May to October,regression phase ranges from November to February,and the rest phase ranges from March to April.2.Integrating mi RNA and m RNA expression profiling data of Shanbei cashmere goat skin in anagen,catagen and telogen.There were 5694 network terms which were constructed by 104 different expressed mi RNAs and 683 different expressed m RNAs in anagen vs.catagen group,and there were 4185 network terms which were constructed by 92 different expressed mi RNAs and 583 different expressed m RNAs in anagen vs.telogen group,and there were 574 network terms which were constructed by 57 different expressed mi RNAs and 125 different expressed m RNAs in catagen vs.telogen group.We found there were 48 enrichment terms by GO analysis for the mi RNA target genes,and these items were according with hair follicle development in each growth status.We also found some outstanding pathways,such as Focal adhesion,Protein digestion and absorption,ECM-receptor interaction,Cardiomyopathy related pathway,Regulation of actin cytoskeleton and PPAR signaling pathway,and these pathways provided a clear direction for our further researches.We also found several candidate mi RNAs(mi R-1,mi R-125 b,mi R-144,mi R-145 and mi R-206)and their participating networks,which might have a crucial role in hair growth cycle.3.Through the whole transcriptomic analysis comparing the anagen to the catagen of Shanbei cashmere goat skins,we found 403 novel lnc RNA transcripts and 350 TUCP transcripts.We identified 3500 differently expressed coding m RNA transcripts(including 3357 genes)and 172 differently expressed lnc RNA transcripts.We also identified 411 known mi RNAs and 307 novel mi RNAs,as well as 72 differently expressed mi RNAs.We found several outstanding enrichment pathways enriched by lnc RNA-mi RNA target genes,such as pathways in cancer,systemic lupus erythematosus and Transcriptional mis-regulation in cancer.Besides these there were some pathways associated with fat deposition(PPAR signaling pathway)and environmental information processing(ECM-receptor interaction and Notch signaling pathway)and cellular process(Focal adhesion).4.We found the expression level of LHX2 were significantly expressed in skin,liver and brain(p<0.01),and the expression of LHX2 was higher in October than that in April(p<0.01).Interestingly,we also found the expression of mi R-144 possessed the opposite expression pattern for LHX2,and was significantly higher in telogen than in other growth phases(p<0.01).5.We successfully isolated and cultured SDP.The molecular markers showed positive results in SDPs,and the SDPs still possessed strong agglomeration in high passages(P8).6.There were at least three mi R-144-3p target sites in LHX2 3' UTR,and the mi R-144 adenovirus increased the expression of LHX2 in SDP,while inhibited post-transcriptional translation of which.The adenovirus could significantly down regulate the expression of BMP2?BMP4 and NOG,while up-regulated the expression of FGF7,?-Catenin and TGF?1.7.Then we obtained LHX2-/-SDP cell lines.In the following works,we found the expression of FGF7,FGF10,TGF?2 and TGF?3 were significantly up-regulated,this might promote the proliferation of SDP and hair Mx proliferation.8.We found the expression of FGF7 and ?-catenin had a similar expression pattern between the two treatments.In summary,we summarized SHF growth characteristics of Shanbei white cashmere goats according to quantifiable indexes of their morphology in different seasons in this study,for the first time.We had screened the candidate genes for validation through integrating lnc RNA-mi RNA and m RNA into analysis,and there was a synergy between mi R-144-3p and LHX2 on the proliferation of SDP and Mx cells.Thus,it provides a novel insight into SHF cycle and novel clues for further studies by integrating analysis.