| Background:Psoriasis is a chronic inflammatory skin disease induced by a variety of factors such as heredity,environment,immunity and drugs.A variety of factors work together on keratinocytes,which eventually lead to their excessive proliferation.Long non-coding RNA(lncRNA)is a class of eukaryotic non-coding RNA molecules with a length of more than 200nt,which has been confirmed to play a role in promoting tumor growth in a variety of cancers.LncRNA AGAP2 antisense RNA1(AGAP2-AS1)was discovered in recent years.It has been found that it can be involved in many diseases such as lung cancer,liver cancer,pancreatic cancer and scleroderma.However,no studies have shown that its role in psoriasis.N6-methyladenosine(m6A)is the most common methylation modification in eukaryotes,which could be dynamic regulated by methyltransferase,m6A-binding proteins and demethylase.This study aimed to probe whether METTL3 regulates the lncRNA AGAP2-AS1 in an m6A-dependent manner,thereby promoting the pathogenesis of psoriasis.Methods:1.Expression location of AGAP2-AS1 was detected in 3 psoriatic tissues and 3 healthy normal tissues by RNAscope(?) assay.HaCaT and NHEK cells were stimulated with interleukin 22(IL-22),and the expression level of AGAP2-AS1 was detected by quantitative real-time PCR(qRT-PCR)experiments.5-ethynyl-2'-deoxyuridine(EdU)and wound healing experiments were used to detect the effects of AGAP2-AS1 on the proliferation and migration ability of keratinocytes.2.The expression level of METTL3 was detected in 14 psoriasis tissues and 12 healthy normal tissues,and the correlation between METTL3 and AGAP2-AS1 was analyzed.Methylated RNA immunoprecipitation(MeRIP)assay was performed to detect the m6A modification level of AGAP2-AS1 and the effect of METTL3 on the m6A modification level of AGAP2-AS1.The stability of AGAP2-AS1 after knockdown of METTL3 was detected by RNA stability assay.3.RNA Immunoprecipitation(RIP)assay was used to detect whether AGAP2-AS1 could interact with YTH domain family 2(YTHDF2)protein as well as the regulation function of METTL3 on the interaction between AGAP2-AS1 and YTHDF2.The stability of AGAP2-AS1 after knockdown of YTHDF2 was detected by RNA stability assay.Results:1.In the dataset GSE13355 we found that AGAP2-AS1 was highly expressed in psoriasis tissues,and was also upregulated in psoriasis tissues we collected.It was mainly expressed in the spinous layer of corneum.IL-22 could increase the expression of AGAP2-AS1 in keratinocytes.Both the proliferation ability and migration ability of HaCaT and NHEK cells were inhibited in AGAP2-AS1 knockdown cells.2.In the dataset GSE13355 we found that METTL3 was expressed lower in psoriasis tissues,and was also decreased in psoriatic skin lesions.METTL3 was negatively correlated with the expression of AGAP2-AS1.Furthermore,m6A modification on the AGAP2-AS1 was predicted in the m6AVar database and the sequence-based RNA adenosine methylation site predictor(SRAMP)site.It was verified that there was m6A modification in AGAP2-AS1.After knockdown of METTL3,the m6A modification level of AGAP2-AS1 was decreased,whereas the expression of AGAP2-AS1 was increased,with improved RNA stability of AGAP2-AS1.Knockdown of METTL3 increased the migration capacity of HaCaT and NHEK cells while the proliferation ability of the cells was not affected.3.Through browsing m6AVar database,we found that the m6A modification site of AGAP2-AS1 can be coveraged by YTHDF2 binding site.In HaCaT and NHEK cells,there was an interaction between AGAP2-AS1 and YTHDF2 protein,which could be weakened by knockdown of METTL3.At last,knockdown of YTHDF2 can improve the expression level and stability of AGAP2-AS1.Conclusion:In psoriasis,METTL3 regulates the expression of AGAP2-AS1 and promotes keratinocyte migration in a YTHDF2-m6A-dependent manner.In addition,AGAP2-AS1 can affect keratinocyte proliferation and participate in the pathogenesis of psoriasis independently of METTL3 regulation. |
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