| Objectives:Keloid is abnormal scar whose pathological changes are mainly characterized by fibroblast proliferation and large amount of extracellular matrix.The lesions can spread to the surrounding normal skin and have the characteristics of invasion and easy recurrence similar to malignant tumors.At present,there is no unified conclusion on the specific molecular biological mechanism of keloid.Epithelial mesenchymal transition is a process in which epidermal epithelial cells transformed into mesenchymal cells.It exists in many malignant tumors and has been proved to be related to tumor invasion and metastasis.Some studies have confirmed the existence of epithelial mesenchymal transition in keloid tissue,and that hypoxia can promote the expression of HIF-la and the occurrence of epithelial mesenchymal transition in keratinocyte.LncRNAs are non-coding RNAs with a length greater than 200 nt.LncRNA can take part in gene expression regulation from multiple levels,thus participating in the occurrence and metastasis of various diseases.Studies have reported the abnormal expression of IncRNA in keloid tissues.However,the specific effects of abnormally expressed IncRNA on the development of keloid and the mechanism of its action are still not fully studied.In this study,LncPath Human EMT Pathway Microarrays were used to screen IncRNAs related to epithelial mesenchymal transition in keloid tissue.Then the molecular biological mechanism of IncRNA in keloid development was explored by culturing keratinocytes under hypoxia condition.Methods:In this study,we collected 15 pairs of keloid tissue and adjacent normal skin tissue samples.cDNAs were synthesized from total RNA extracted from 3 pairs of samples,and IncRNA microarrays was used to detect the expression profile of IncRNA related to epithelial mesenchymal transition.LncRNAs related to epithelial mesenchymal transition in keloid were screened out and verified by qPCR in the 3 pairs of samples.LncRNAs were selected based on the validation results and qPCR validation was performed in 12 pairs of expanded samples.HaCat cells were then divided into the normoxic group,1he hypoxic group,the sh-lncRNA group and the sh-control group.The normoxic group was cultured under normal oxygen condition,the hypoxic group was cultured under hypoxic condition,the sh-lncrna group was cultured xmder hypoxic condition after transfection with shRNA to inhibit the expression of IncRNA,and the sh-control group was cultured under hypoxic condition after transfection with sh-control.After the intervention treatment,expressions of HIF-1α,E-cadherin and Vimentin in keratinocytes were detected by qPCR and western blotting,apoptosis of keratinocytes was detected by flow cytometry,migration ability was detected by Transwell,and expression relationship between lncRNA and HIF-1α was detected by dual luciferase reporter gene.Results:LncPath Human EMT Pathway Microarrays chip was used to detect the IncRNA expression profiles of 3 pairs of keloid tissue and normal skin tissue.The relative expression level of>1.5 and P<0.05 were used as the standard to screen out 11 abnormal IncRNAs,among which 7 were up-regulated and 4 were down-regulated.Meanwhile,18 abnormal mRNAs were found,among which 14 were up-regulated and 4 were down-regulated.Eight abnormally expressed lncRNAs out of 11 were selected for qPCR verification in the 3 pairs of samples.The results showed that expression of ENST00000563754,NR002605,NR033321 and TCONS00002241 showed statistically significant differences.We selected NR002605,NR033321,TCONS00002241,ENST00000437232 and HOTAIR for qPCR verification in 12 pairs of samples,and the results showed that the differences in NR002605,NR033321 and TCONS00002241 were consistent with the microarray results and the differences were statistically significant.Under hypoxic condition,the expression of HIF-la and Vimentin in keratinocytes was increased,while the expression of E-cadherin was decreased,the cell migration ability was enhanced,and the apoptosis rate was decreased,while inhibiting the expression of lncRNA HOTAIR could inhibit this phenomenon.Double luciferase reporter assay showed that HOTAIR was involved in regulating HIF-1α expression.Conclusions:LncRNA related to epithelial mesenchymal transition in keloid tissues was detected by LncPath Human EMT Pathway Microarrays and qPCR verification.Besides,we found that lncRNA HOTAIR plays an important role in the process of keloid epithelial mesenchymal transition.HOTAIR may be involved in the regulation of epithelial mesenchymal transition of keratinocyte through HIF-la signaling pathway,thus further participating in the formation and invasive progression of keloid. |
