Effect Of Thalidomide On OSMR-related Pathway In Keratinocytes

Introduction:Primary cutaneous amyloidosis(PCA)is refractory pruritus skin disease.Its typical histopathological changes are thickening and hyperkeratosis of epidermal acanthocyte layer,and a large amount of amyloid deposits can be seen in dermal papilla.The formation of amyloid material is closely related to the apoptosis of keratinocytes.Most cases of primary cutaneous amyloidosis are sporadic,but familial aggregation of familial primary cutaneous amyloidosis(FPCA)has been reported many times in South America and Taiwan.The pathogenic gene of familial primary cutaneous amyloidosis is located in OSMR gene.The OSMR gene encodes OSMR beta protein.The latter is a component of OSM type two receptor and IL31 receptor.These two receptors are combined with two cytokines OSM and IL31,and complete the phosphorylation and signal transduction through JAK-STAT and MAPK pathway.When the OSMR gene mutated,keratinocytes more susceptible to apoptosis,resulting in deposition of keratin in the superficial dermis;on the other hand,OSMR beta/IL31RA signaling pathway disorder decreased inhibitions of the apoptosis of keratinocytes,the apoptosis of keratinocytes increases,and the degenerated keratinized substances are deposited in the superficial layer of the dermis,which eventually leads to the occurrence of amyloidosis in the skin.Thalidomide is effective in a variety of intractable pruritus dermatoses,which may be through different mechanisms of action.Primary cutaneous amyloidosis is resistant to treatment,and traditional therapies such as anti-histamine,glucocorticoid,and narrow band UVB are limited.Thalidomide has been reported to be used in the treatment of light chain amyloidosis.We used thalidomide to treat a case of familial primary cutaneous amyloidosis,and found that thalidomide could relieve skin pruritus,while clinical and histological signs were also improved.In the previous experiments,we identified the OSMR gene knockout cell lines by whole proteomics,and obtained the expression of multiple differential proteins,the function of differential proteins and the results of KEGG enrichment.The up-regulated expression of TC-PTP was the most significant.At the same time,the Jak-STAT pathway was most significant in KEGG pathway.T cell protein tyrosine phosphatase(TC-PTP,encoded by PTPN₂)is the first recognized member of PTP family,specifically expressed in embryonic and adult tissue cells,especially in hematopoietic system.TC-PTP participates in the regulation of various biological functions,including cell cycle regulation and apoptosis.It regulates various pathways,including JAK1,JAK3,Stat1,Stat3 and Stat5.TC-PTP plays a negative role in regulating JAK/Stat pathway.Scholars constructed PTPN₂ knockout mice,extracted epithelial cells from skin tissue of mice,and detected the level of P-AKT and P-Stat3 by Western Blot method.The results showed that both expressions increased.Ultraviolet radiation increases the phosphorylated level of Stat3 and increases the expression of TC-PTP.Scholars constructed PTPN₂ gene knockout and overexpression cell lines with different durations of ultraviolet radiation intervention,it is concluded that TC-PTP inhibit Stat3 mediated proliferation of mouse keratinocytes under ultraviolet irradiation,TC-PTP plays an important role in regulating cell proliferation and apoptosis.Whether OSMR gene affect the pathogenesis of amyloidosis in the skin by up-regulation of the expression of TC-PTP.Whether thalidomide affects the occurrence of amyloidosis in the skin by acting on the Stat3 pathway is the focus of this experiment.Experimental materials and methods:1.Experimental subjectHaCaT cells were selected for the experimental subject,which purchased from Jiangsu keygen biotechnology Limited by Share Ltd.2.siRNA transfectionCells were seeded the day before transfection in antibiotics-free Medium.LipofectamineTM RNAiMAX was used as siRNA transfection reagent according to the manufacturer's instructions.Cells grown in medium only and cells treated with Silencer?Select negative control siRNA 1 were used as negative controls.A final concentration of 10 nmol of siRNA was used after optimization.3.Screening of thalidomide concentration by Icelligence methodThe cells were seeded on the Icelligence cell culture plate,and the concentrations of thalidomide were 0,10nM,100nM,1000nM and 10000nM,respectively.The best concentration of drugs on cell proliferation was determined by cell growth curve.4.The effect of si OSMR on cell proliferation before and after the use of thalidomide by MTS detectionCell viability was measured by using the MTS assay following the manufacturer's instructions.The transfected siOSMR cells group,siOSMR cells after adding thalidomide group and negative control group cells were seeded in 96-well plates at a density of 5000 cells/well,and the cell viability was determined by measuring the absorbance at 490 nm at day 0,day 1,day 2,day3 and day4,when the cell density reach100%of confluence.All assays were completed in octuplicates and repeated three times.5.The effect of si OSMR on cell apoptosis before and after the use of thalidomide by flow cytometryThe effect of siOSMR on apoptosis was detected by flow cytometry before and after the addition of thalidomide.The transfected siOSMR cells group,siOSMR cells after adding thalidomide group and negative control group cells were seeded in 6-well plates,with the changes of apoptotic cells were determined by FITC V Annexin apoptosis kit and PI kit.6.Using lentivirus transfection technique to establish OSMR gene knockout stable cell lineThe cells were inoculated on 24 orifice plates,the MOI value of Hacat cells was determined to be 100,and the amount of lentivirus transfection was determined.The transfected efficiency was observed at 24,48 and 72 hours after transfection by fluorescence microscope.The transfected cells were screened by adding puromycin.The method of real-time quantitative PCR detection and Western blot detection was used to verify the transfected efficiency of the cells.7.Quantitative proteomic profiling for OSMR-deficient Stable CellsThe cells were divided into lentivirus control group and OSMR knockout group,and human cell samples were identified by whole protein quantitative proteomics,including differential protein expression detection,differential protein functional classification,functional enrichment and cluster analysis.8.The effect of thalidomide on the expression of Stat1,3,5 and Akt pathway proteins by Western blotThe expression level of Stat1,3,5,Akt pathway proteins was detected by Western blot method before and after the addition of thalidomide.The protein in cells was extracted according to the protein extraction method;BCA was used to detect protein concentration;the protein expression was observed by making glue,running glue,transferring membrane,blocking,incubated with primary antibody overnight at 4°C,and incubated with a HRP-conjugated secondary antibody and detected by ECL Substrate on the following day.9.Statistical analysisStatistical analysis was performed using GraphPad Prism.For more than two groups of experiments,the differences between each group were analysed by one-way analysis of variance(ANOVA).A p value of less than 0.05 was considered statistically significant.Result:1.Effect of OSMR knockout on the proliferation of keratinocytesMTS method was used to detect the effect of OSMR knockout on the proliferation of keratinocytes.The results showed that OSMR gene knockout could significantly inhibit cell proliferation,and the difference was statistically significant.(p<0.001)2.Effect of OSMR knockout on apoptosis of keratinocytesThe effect of OSMR knockout on the apoptosis of keratinocytes was detected by flow cytometry.The results showed that OSMR gene knockout could increase the apoptosis of keratinocytes and repeat three experiments,the difference was statistically significant.(p<0.05)3.The effect of thalidomide on the proliferation of OSMR knockout keratinocytesMTS method was used to detect the effect of thalidomide on the proliferation of OSMR gene knockout keratinocytes.The results showed that thalidomide could increase the proliferation ability of OSMR gene knockout keratinocytes,and the difference was statistically significant.(p<0.05)4.The effect of thalidomide on the apoptosis of OSMR knockout keratinocytesThe effect of thalidomide on the apoptosis of OSMR gene knockout keratinocytes was detected by flow cytometry.The results showed that thalidomide decreased the apoptosis of OSMR gene knockout keratinocytes and repeated three experiments,the difference was statistically significant.(p<0.05)5.The effect of thalidomide on the expression of OSMR related pathway Western blot method was used to detect the effect of thalidomide on the expression of OSMR related pathway proteins.The results showed that the expression of P-Stat3 in OSMR gene knockout keratinocytes decreased,and the expression of P-Stat3 increased after thalidomide added.OSMR knockout had no significant effect on the expression of Stat3.The expression of Stat1 and P-Stat1 in OSMR knockout keratinocytes decreased,and the decrease of P-Stat1 was more obvious.After the addition of thalidomide,the expression of Stat1 and P-Stat1 was not significantly increased.OSMR knockout had no significant effect on the expression of Stat5 and P-Stat5 in keratinocytes.OSMR knockout had no significant effect on the expression of Akt and P-Akt in keratinocytes.6.Quantitative proteomic identification of human cell samplesThe OSMR knockout stable cell line and lentivirus transfection control group were used to identify the whole protein quantitative proteome of human cell samples.The results showed that OSMR knockout could lead to many differential protein expression,of which the up-regulated TC-PCP expression was the most significant.The KEGG pathway enrichment results showed that the up-regulated protein enrichment in the Jak-STAT pathway was the most significant.7.Effects of PTPN₂ knockout on the proliferation of keratinocytesMTS method was used to detect the effect of PTPN₂ knockout on the proliferation of keratinocytes.The results showed that PTPN₂ gene knockout could significantly promote cell proliferation,and the difference was statistically significant.(p<0.05)8.The effect of thalidomide on the proliferation of PTPN₂ overexpressed keratinocytesMTS method was used to detect the effect of thalidomide on the proliferation of PTPN₂overexpressed keratinocytes.The results showed that thalidomide could increase the proliferation ability of PTPN₂ overexpressed keratinocytes,and the difference was statistically significant.(p<0.05)9.The effect of PTPN₂ on the expression of OSMR related pathway(1)The effect of thalidomide on the expression of Stat3 and P-Stat3 in the PTPN₂ gene knockout keratinocytes.The expression of P-Stat3 in PTPN₂ knockout keratinocytes increased significantly,and the expression of P-Stat3 decreased after adding thalidomide.This is opposite to the effect of OSMR knockout on P-Stat3,suggesting that OSMR gene and PTPN₂ may be located on the same signal pathway,OSMR gene is located upstream of PTPN₂,and OSMR gene inhibits PTPN₂.The knockout of PTPN₂ gene had no significant effect on the expression of Stat3.(2)PTPN₂ gene knockout had no effect on the expression of P-Stat5 and Stat5 in keratinocytes,and had no effect on the expression of Akt and P-Akt.(3)The effect of thalidomide on the expression Stat3 and P-Stat3 in PTPN₂ gene over-expressed keratinocytes.The expression of P-Stat3 in PTPN₂ over-expressed keratinocytes was reduced,and the expression of P-Stat3 increased after adding thalidomide.This is in the same direction as OSMR knockout to P-Stat3.It is proved again that OSMR gene and PTPN₂ may be located on the same signal pathway,OSMR is located upstream of PTPN₂,and OSMR gene inhibits PTPN₂.The overexpression of PTPN₂ gene has no significant effect on the expression of Stat3.(4)PTPN₂ overexpression has no effect on the expression of P-Stat5 and Stat5 in keratinocytes,and no effect on the expression of Akt and P-Akt.10.Effects of Stat3 inhibitors on the proliferation of PTPN₂ and OSMR knockout cellsAfter the addition of STA-21,the proliferation of PTPN₂ knockout and OSMR knockout keratinocytes were significantly inhibited.This shows that the effect of PTPN₂ gene and OSMR gene on cell proliferation is mediated by the Stat3 pathway.STA-21 can significantly inhibit the proliferation of two groups of gene knockout cells after adding thalidomide.Thus,it can be seen that thalidomide may affect the proliferation of PTPN₂knockout and OSMR knockout cells through the Stat3 pathway.Conclusion:1.OSMR gene knockout reduces the proliferation of keratinocytes and increases the level of apoptosis.2.Thalidomide increases the proliferation ability of OSMR gene deficient keratinocytes,reduces the level of apoptosis and increases the expression of P-Stat3.3.OSMR gene up-regulated the expression of TC-PTP in keratinocytes and acted on the Stat3 pathway.4.Thalidomide may act on the Stat3pathway,and exert an influence on the expression of OSMR related pathway proteins and affected the biological functions of cells through the action on TC-PTP or its upstream molecules.