Regulatory Role Of PPP1R26-AS1 In Monocytes Immime Response Against Heat-killed Cryptococcus Neoformans And Clinical Significance

Part 1 Screen and identity long noncoding RNA involved in human peripheral monocytes immune response against heat-killed Cryptococcus NeoformansIn this part,we aim to identity the long noncoding RNA involved in human peripheral monocytes immune response against heat-killed Cryptococcus Neoformans.First,peripheral blood mononuclear cells were obtained through density gradient centrifugation,and monocytes were acquired using CD 14 microbeads kit.Monocytes were stimulated with heat-killed Cryptococcus Neoformans for 12 hours.The key different expression genes were identified by the next-generation sequencing technology and validated by real time quantitative PCR.The long noncoding RNA expression was determined in twenty cryptococcosis patients and healthy controls respectively,admitted from Shanghai ChangHai hospital and ChangZheng hospital.Compared to unstimulated group,839 genes were upregulated and 349 genes were downregulated.Among them,701 genes belonged to Biological Process,and 698 genes belonged to Molecular Function,and 751 genes belonged to Cellular Component.Compared to unstimulated group,PPP1R26-AS1 was downregulated in stimulated group(P<0.05).Furthermore,compared to healthy controls,PPP1R26-AS1 was upregulated in cryptococcosis patients.This part results indicated that PPP1R26-AS1 may be involved in the pathogenesis of cryptococcosis via regulating monocytes immune response.Part 2 PPP1R26-AS1 can downregulate proinflammatory cytokine produnction during monocytes immune response against heat-killed Cryptococcus NeoformansIn this part,we aim to explore the role of PPP1R26-AS1 in regulating monocytes immune response against heat-killed Cryptococcus Neoformans,PPP1R26-AS1 overexpressed adenovirus vector was constructed and transfected to human healthy monocytes.mRNA expression profiles in ADV-PPP1R26-AS1,ADV-NC,BLANK group were determined by Agilent Whole Human Genome Oligo Microarray respectively and immune-related signal parameters were detected through real time quantitative PCR.The key molecules were analyzed in cryptococcosis patients and healthy controls.Compared to control group,IL-1B and CCL3 expressions were both decreased in ADV-PPP1R26-AS1 group(P<0.05).Furthermore,IL-1B protein level was also statistically decreased in serum from cryptococcosis patients compared to healthy controls(P<0.05),not CCL3.Our results suggested that PPP1R26-AS1 can downregulate IL-1B expression during monocytes immune response against heat-killed Cryptococcus Neoformans.Part 3 PPP1R26-AS1 can downregulate monocytes IL-1B production via TLR2 after heat-killed Cryptococcus Neoformans exposureIn this part,we aim to explore the mechanism in monocytes IL-1B production induced by heat-killed Cryptococcus Neoformans.Toll-like receptors were detected through real time quantitative PCR in healthy human monocytes transfected with PPP1R26-AS1 overexpression vector prior to heat-killed Cryptococcus Neoformans stimulation.TLR2 signal molecules were validated using real time quantitative PCR and western blot in peripheral blood mononuclear cells from cryptococcosis patients and healthy controls.By gain and loss of function experimrat,IL-1B expression was analyzed after heat-killed Cryptococcus Neoformans exposure.Compared to control group,TLR2 was statistically decreased in ADV-PPP1R26-AS1 groiq)(P<0.05),not TLR4.Compared to healthy controls,TLR2 mRNA and protein,MyD88 and TRAF6 mRNA expression were reduced in cryptococcosis group(P<0.05).Blocking TLR2 can downregulate monocytes IL-1B production induced by heat-killed Cryptococcus Neoformans,and TLR2 agonist can partially reduce the inhibitory effect of PPP1R26-AS1 on monocytes IL-1B production after heat-killed Cryptococcus Neoformans exposure.