Study On The Expression And Function Of OPN3 In Langerhans Cell Histiocytosis

Objective:Langerhans cell histiocytosis（LCH）is a group of rare diseases characterized by pathological Langerhans cell clonal proliferation.According to clinical manifestations and the extent of disease infiltration,LCH is divided into two types:single system LCH and multi-system LCH.Liver,bone marrow,and spleen involvement belong to the high-risk type,while other organs,including skin involvement,belong to the low-risk type.At present,the pathogenesis of LCH is unclear.In 2010,Badalian Very G et al.found a mutation in the BRAF^V600Egene in LCH,suggesting that the RAS/RAF/MEK/ERK pathway may be involved in the pathogenesis of LCH.Opsins（OPNs）are a kind of light sensitive G protein coupled receptor superfamily proteins,and more than 1000 kinds have been found so far.In humen,OPNs are divided into seven categories,including opsin 1（OPN1）,opsin 2（OPN2）,opsin 3（OPN3）,opsin 4（OPN4）,opsin 5（OPN5）,and two light isomerase,named retinal G protein coupled receptor（RGR）and periopsin,which are the core molecules of human visual light perception.In recent years,research has found the expression and subcellular localization of opsins in normal skin tissues,normal skin cells（melanocytes,keratinocytes,fibroblasts,vascular endothelial cells）,skin tumor tissues and cells（pigmented nevus,malignant melanoma,skin squamous cell carcinoma,basal cell carcinoma）.It has ben confirmed that OPN3 sense UVA to induce the formation of melanin cap structure,OPN3 plays a key role in melanocyte apoptosis,pigment synthesis and angiogenesis.It is also found that OPN3 can regulate melanogenesis of human congenital melanocytic nevus through interaction with BRAF^V600Egene.However,there is no relevant report on the expression and function of OPN3 in Langerhans cell histiocyte.Therefore,this study collected data on tissue samples of langerhans cell histiocytosis,primary dendritic cells from human blood,and the Langerhans cell histiocytosis ELD-1 cell line to detect the expression and function of OPN3.The function and mechanisms of OPN3 on human Langerhans cell histiocytosis were studied by using cell molecular biology,immunohistochemistry,bioinformatics and other technologies.Methods:（1）Detection of OPN3 and BRAF^V600Ein tissue samples of human Langerhans cell histiocytosis:using a retrospective survey method,clinical and pathological information,including gender,age,location,and pathological diagnosis.The 31 tissue samples of Langerhans cell histiocytosis were collected by Department of Pathology,Affiliated Hospital of Guizhou Medical University from 2007 to 2022.The expression and cellular localization of OPN3 in Langerhans cell histiocytosis tissues and normal tissues adjacent to the lesion were detected by immunohistochemistry,tissue immunofluorescence,and RNA Scope detection methods.Extracting deoxyribonucleic acid（DNA）from the wax tissue of the lesion and using Sanger sequencing to detect mutations of the BRAF^V600Egene.（2）Isolation,cultivation,and identification of human peripheral blood dendritic cells,as well as detection of transcription and protein levels of OPNs and CD86before and after induction of maturation:Collect 15 ml of fresh peripheral anticoagulant from normal people,dilute and mix with PBS 1:1,and then put it into a50 ml centrifuge tube.Add lymphocyte separation solution gradient to separate cells,take the middle ring of milky white lymphocyte layer cells,and resuscitate the cells with 3 ml of RPMI 1640 complete medium containing 10%fetal bovine serum,2 m M L-glutamine,100 U/ml penicillin and 100μg/ml streptomycin.Then press 1×10⁶cells/ml in a 6-well plate and obtain mononuclear cells through adhesion method.Induce factors r GM-CSF（1000 U/ml）,r IL-4（500 U/ml）were added and cultured at37℃and 5%CO₂level.The cell culture medium were changed in every 2 days to maintain a consistent final concentration of cytokines.After 6 days of induction,mature inducing factors r TNF-α（10 ng/ml）、IL-1β（10 ng/ml）、IL-6（10 ng/ml）and PGE2（1μg/ml）were added to incubate for another 2 days.Observing cell morphology through an inverted microscope and determining cell phenotype by using flow cytometry.Collecting cell precipitates before and after induction,and using real-time fluorescence quantitative polymerase chain reaction（q RT-PCR）and Western blot（WB）to detect the transcriptional and protein expression changes of OPNs and CD86 before and after induction.（3）Screening,identification and transcriptome sequencing of ELD-1 cell OPN3knockdown and overexpression stable strains:by using lentivirus transfection technology,we constructed ELD-1 cell line with OPN3 knockdown and overexpression,and screened OPN3 stable expression strain through puromycin.The cells were collected,and the expression efficiency of OPN3 was detected by q RT-PCR and Western blot,and then transcriptome sequencing was performed.（4）After knocking down or overexpressing OPN3,detect the proliferation,migration,antigen presentation,cell cycle,and apoptosis of ELD-1 cells:Using q RT-PCR method to detect the expression changes of OPN3 and CD86 transcription levels in ELD-1 cells,validating the protein expression changes of OPN3 and CD86 using WB method,detecting the changes in proliferation activity of ELD-1 cells using CCK-8 and EDU methods.The ability of ELD-1 cell migration was detected by Transwell method,the apoptosis of ELD-1 cells was detected using propidium iodide（PI）staining method and cell cycle of ELD-1was detected by flow cytometry.The Opsin3 protein structure and CD86 protein structure were predicted by Alphafold2,and the protein protein docking tool HDOCK was used for molecular docking to analyze and predict the protein interaction between OPN3 and CD86.（5）After knocking down or overexpressing OPN3,the expression changes of the MAPK signaling pathway in ELD-1 cells were detected:The WB method was used to verify the expression changes of MAPK（p38,JNK,and ERK）signaling pathway proteins and their phosphorylated proteins p38/p-p38,JNK/p-JNK,and ERK/p-ERK in ELD-1 cells.Results:（1）Among 31 cases of human Langerhans cell histiocytosis,there were 20 males and 11 females,including 16 cases aged 0-10（52%）,4 cases aged 11-20（13%）,3cases aged 21-30（10%）,2 cases aged 31-40（6%）,2 cases aged 41-50（6%）,and 4cases aged over 50（13%）.Among them,6 cases were skin involvement;single（84%）and multiple（16%）involvement.There were 13 BRAF^V600Emutations（42%）and 18BRAF^V600Ewild-type（58%）.（2）The results of OPN3 and CD1a tissue immunofluorescence double staining showed that OPN3 was expressed on the cell membrane and cytoplasm in CD1a positive Langerhans cells.The semi quantitative analysis of RNA Scope showed that compared with the langerhans cells adjacent to the lesion,the expression of OPN3 in langerhans cell histiocytosis tissue lesions was significantly increased,with a statistically significant difference（P<0.05）.The immunohistochemical staining results showed that there was no statistically significant difference in the expression of OPN3between the wild-type and mutant BRAF^V600E.（3）The transcriptome sequencing results showed that 496 differentially expressed genes were detected after down-regulation of OPN3,of which 343 genes were up-regulated and 153 genes were down-regulated.The differentially expressed genes were mainly enriched to be related to cellular immune function and MAPK cell signaling pathway.（4）Primary dendritic cells from human blood were isolated and cultured in vitro.Immature dendritic cells were found to be in a semi adherent and semi suspended state.Under light microscopy,cells were circular in shape,while mature dendritic cells were found to be semi adherent and semi suspended.Under light microscopy,adherent cells were dendritic in shape,and the suspended cells had good refractive index and were oval in shape.After inducing the maturation of human primary dendritic cells,q RT-PCR and WB detection results showed a significant increase in the transcription and protein levels of OPN3,with statistical significance（P<0.05）,consistent with the expression changes of CD86;（5）The q RT-PCR and WB detection results showed that compared with the control group,the overexpression of OPN3 increased the transcription and protein levels of CD86,while the knockdown of OPN3 group decreased the transcription and protein levels of CD86,with statistical significance（P<0.05）.The CCK-8 detection results showed that the overexpression of OPN3 increased cell proliferation activity,while the knockdown of OPN3 decreased cell proliferation activity,with a statistically significant difference（P<0.05）.The EDU fluorescence and flow cytometry results showed that compared with the control group,the overexpression of OPN3 increased the proportion of cell proliferation,while the knockdown of OPN3 decreased the proportion of cell proliferation,with a statistically significant difference（P<0.05）;The results of cell migration experiment showed that,compared with the control group,after 24h,the number of migrating cells in the overexpression OPN3 group increased significantly,and the number of migrating cells in the knockdown OPN3group decreased significantly（P<0.05）.The results of flow cytometry showed that compared with the control group,the proportion of S phase cells in the overexpression OPN3 group was significantly increased,while the proportion of S phase cells in the knockdown OPN3 group was significantly decreased,with a statistically significant difference（P<0.05）.The flow cytometry detection results showed that compared with the control group,there was no significant difference in the proportion of apoptotic cells in the overexpression OPN3 group and knockdown OPN3 group.The molecular docking results show that there are a total of 100 possible docking configurations between CD86 and Opsin3,with a docking score of-385.24 kcal/mol for top1 and a confidence level of 0.991.The folded region of CD86 is bound to the helical region of Opsin3 through hydrogen bonding,π-πstacking,and van der Waals contact.Analysis shows that the side chain amino groups of Asn93 on CD86（A-chain）form hydrogen bonds with the backbone carbonyl groups of Thr96 on Opsin3,the charge centers of the aromatic rings of His155 and Phe93 formπ-πstacking interactions,and the backbone carbonyl groups of Ser224 form hydrogen bonds with the amino groups on the syncycle of Trp155 on Opsin3.（6）The WB test results showed that compared with the control group,the overexpression of OPN3 increased the expression of phosphorylated JNK,P38,and ERK,while the knockdown of OPN3 group decreased the expression of phosphorylated JNK,P38,and ERK,with a statistically significant difference（P<0.05）.Compared with the control group,there was no significant change in the expression of JNK,P38,and ERK in the overexpression OPN3 group,while there was no significant change in the expression of JNK,P38,and ERK in the knockdown OPN3 group.Conclusions:（1）OPN3 was expressed in Langerhans cell histiocytosis tissues,and the expression of OPN3 was higher in focal tissues than in matching normal tissues.（2）OPN3 was expressed in both human primary dendritic cells and LCH cell line ELD-1 cells.OPN3 could regulate the proliferation,migration,antigen presentation,apoptosis and cell cycle of Langerhans cell histiocytosis cells.（3）OPN3 regulates the function of ELD-1 cells through the MAPK pathway.Our study may reveal molecular targets for clinical treatment of Langerhans cell histiocytosis.