The Role Of KLF2Regulate S1PR1in Neutrophil-like Cells Migration

Objective and meaning: Bronchial asthma is a chronic airwaysinflammatory disease, about50%of asthma related to eosinophilicinflammation, whereas most of the rest of asthma accompanied by anincrease of neutrophils. In sputum of patients with severe asthma, theproportion of neutrophils increased significantly. The increasingneutrophils is related with reducing and delaying apoptosis, but also withincreasing migration through increasing surface adhesion molecule. Thisarticle is to investigate whether exist KLF2regulate S1PR1on neutrophil-like cells migration progress in cellular level.Method:（1）HL-60cells which differentiated into neutrophil-like cells inducedby1.3%DMSO for five days. It can be checked by diff-quik cell stain ofcell morphology and the expression of mRNA of neutrophils differentiationmarker CD11b were tested by Real-time PCR. Both of these was to determinewhether the induction was successful.（2）The neutrophil-like cells was infected with the unloaded andKLF2-RNAi lentiviral vectors.Transfection efficiency identified byfluorescence microscopy, KLF2knock-down rate identified by western blotand Realtime PCR.(3) The expressions of KLF2and S1PR1mRNA in three groups(the blank group,unload group and siRNA intervention group) were detected by Realtime PCR.The expressions of KLF2and S1PR1protein in three groups were detectedby western blot. The expressions of KLF2protein in three groups weredetected by immunofluorescence.Results1.The HL-60cells were induced into neutrophil-like cells successfully.The HL-60cells were cultured with1.3%DMSO for5days, then cellswere dyed by diff-quik cell stain, we found the cells were smaller thanthe HL-60cell, and the nuclear was renal nuclear. The expression of mRNAof CD11b were tested by RT-PCR, the result was neutrophil-like cell CD11bexpression was significantly increased (P <0.01).2. lentivirus transfection①Observation of infection efficiency of KLF2knocded down group and emptygroup by the fluorescence microscope, both of the two groups’infectionefficiency were over90%.②KLF2expressionReal-timePCR：the expression of KLF2mRNA of siRNA intervention group was lower than the blank group and unload group significantly (P <0.01),KLF2mRNA expression of the blank group and unload group was basicallysame (P>0.05),the rate of knockdown was80%.Western blot：the expressionof KLF2protein of siRNA intervention group was lower than the blank groupand unload group significantly (P <0.01), KLF2protein expression of theblank group and unload group was basically same (P>0.05), the rate ofknockdown was81%. The KLF2gene expression was successfully intervened.Immunofluorescence：the expression of KLF2protein of siRNA interventiongroup was lower than the blank group and unload group significantly (P<0.01), KLF2protein expression of the blank group and unload group wasbasically same (P>0.05).3. S1PR1mRNA expressionRealtime PCR results showed that the S1PR1mRNA expression of siRNAintervention group was lower than the blank group and unload group (P<0.01).4. S1PR1protein expressionWestern blot results showed the S1PR1protein expression of siRNAintervention group was lower than the blank group and unload group (P<0.01).5.Transwell experiments:Select0.1μM S1P performed transwell experiments,the migration rate ofsiRNA intervention group was little than the blank group and unload groupsignificantly (P <0.01).Conclusion:KLF2upregulated S1PR1expression, then promoted neutrophil-like cellsmigration to S1P attractant.