Calcitonin Gene-related Peptide Induces Interleukin-6 Expression Mediated By CircRNAs In Macrophages

Objective: To investigate the effect of CGRP on the expression of interleukin-6 and its upstream circularRNAs in RAW264.7 macrophages.Material and Methods: 1.Cells and drugs RAW264.7 macrophages in this study were incubated with high glucose DMEM media containing 10% fetal bovine serum.Cells were washed 2³ times by phosphate buffer solution before the replacement of media.Furthermore,α-CGRP was used in this study.2.Verification of optimum concentration and time course of CGRP on RAW264.7 cell lines In the concentration experiments,the cells were divided into such groups: 0.01 n M,0.1n M,1n M,10 n M,100 n M and the control group.Then cells were treated with CGRP at the corresponding concentration.After stimulation for designed time,the expression of IL-6 mRNA in each group was detected by quantitative polymerase chain reaction（qPCR）.Similarly,in the time course experiments,the cells were divided into such groups: 1h,2h,3h,4h and 5h.Then cells were treated with CGRP at certain concentraton.After the designed time,the expression of IL-6 mRNA in each group was detected by qPCR.We could ensure the optimum concentration and time course of CGRP.3.Preparation and Analysis of the circRNA microarrays The cells were incubated with some density.After stumilated by CGRP at certain concentration for designed time,the totalRNA of the subject group and the control group was isolated,collected and sent to the biotech firm to make circRNA microarray.According to the result of microarray,the circRNAs were selected by change fold or predicted miRNA binding sites.Then the selected circRNA were confirmed by qPCR and fluorescence in situ hybridization.4.Confirmation of the effect of circRNA on IL-6 expression 1)CircRNA interference The cells were incubated with some density.in 6 well plates.3 designed small interferingRNAs were used in the experiments at the same concentration.After 48 hours,the totalRNA of the subject group and the control group was isolated and collected.The expression of circRNA in each group was detected by qPCR and the optimum siRNA was selected by former experiments.2)Detection of IL-6 mRNA expression by qPCR The cells were incubated with some density.in 6 well plates.The cells were divided into 3 groups: the control group,CGRP group and CGRP+siRNA group.Before the stimulation of CGRP,cells in group CGRP+siRNA was incubated with the siRNA for 48 hours.Then the cells in CGRP group and CGRP+siRNA group were treated by CGRP at the same concentration.After stimulation for optimum time,the totalRNA of all groups was isolated and collected.The expression of IL-6 mRNA in each group was detected by qPCR.3)Detection of IL-6 expression by Western-blot The cells were incubated with some density.in 6 well plates.The cells were divided into 3 groups: the control group,CGRP group and CGRP+siRNA group.Before the stimulation of CGRP,cells in group CGRP+siRNA was incubated with the siRNA for 48 hours.Then the cells in CGRP group and CGRP+siRNA group were treated by CGRP at the same concentration.After stimulation for optimum time,the total protein of all groups was isolated and collected.The expression of IL-6 in each group was detected by Western-blot.5.Research of the effect of circRNA on the upstream miRNA of IL-6 1)Selection of miRNA According to the results of the circRNA microarray,there were 5 alternative target miRNAs of a circRNA.After the Selection via bioinformatic websites and software,the very miRNA involved in the inflammatory pathways related to IL-6 was ensured.2)Detection of the target miRNA expression by qPCR The cells were incubated with some density.in 6 well plates.The cells were divided into 3 groups: the control group,CGRP group and CGRP+siRNA group.Before the stimulation of CGRP,cells in group CGRP+siRNA was incubated with the siRNA for 48 hours.Then the cells in CGRP group and CGRP+siRNA group were treated by CGRP at the same concentration.After stimulation for optimum time,the totalRNA of all groups was isolated and collected.The expression of miRNA in each group was detected by qPCR.6.Confirmation of the effect of upstream miRNA on IL-6 expression 1)Detection of IL-6 mRNA expression by qPCR The cells were incubated with some density.in 6 well plates.The cells were divided into 4 groups: the control group,CGRP group,CGRP+mimics group and CGRP+inhibitor group.Before the stimulation of CGRP,cells in group CGRP+mimics were incubated with the overexpression reagent of the miRNA while cells in CGRP+inhibitor were incubated with the inhibition reagent of the miRNA for 24 hours.Then the cells in CGRP,CGRP+mimics group and CGRP+inhibitor group were treated by CGRP at the same concentration.After stimulation for optimum time,the totalRNA of all groups was isolated and collected.The expression of IL-6 mRNA in each group was detected by qPCR.2)Detection of IL-6 expression by Western-blot The cells were incubated with some density.in 6 well plates.The cells were divided into 4 groups: the control group,CGRP group,CGRP+mimics group and CGRP+inhibitor group.Before the stimulation of CGRP,cells in group CGRP+mimics were incubated with the overexpression reagent of the miRNA while cells in CGRP+inhibitor were incubated with the inhibition reagent of the miRNA for 24 hours.Then the cells in CGRP,CGRP+mimics group and CGRP+inhibitor group were treated by CGRP at the same concentration.After stimulation for optimum time,the total protein of all groups was isolated and collected.The expression of IL-6 in each group was detected by Western-blot..7.Statistical analysis The SPSS 19.0 program was used for the statistical analysis.The data of 2 groups were analyzed by the Student’s t test,with *p<0.05 and **p<0.01 indicating statistical significance.The results are reported as the mean±SEM.Results 1.Cell incubation It was proved that RAW264.7 macrophages with stable growth state could be obtained by the culture method in this experimen.The microscopic appearance of the cells indicated that they were mellow and bright as pearl.After invubation for a few hours,the cells became irregular and revealed pseudopodia around them.This result was accord with the biological characteristics of RAW264.7 macrophage.2.Verification of optimum concentration and time course of CGRP on RAW264.7 cell lines When the concentration of CGRP was 0.1n M or 1n M,the relative expression level of IL-6 mRNA in subject groups was significantly higher than that of the control group.Furthermore,the relative expression level of IL-6 mRNA was the highest in the 1n M group.When the stimulation time of CGRP were 2 hours or 3 hours,the relative expression level of mRNA IL-6 in subject groups was significantly higher than that of the control group.Furthermore,the relative expression level of mRNA IL-6 peaked at 2 hours.3.Preparation and Analysis of the circRNA microarrays According to the result of microarray,there were 173 circRNAs that were up-regulated more than 2 times while 179 circRNAs that were down-regulated more than 2 times.After the selection by fold change or predicted miRNA biding sites and confirmed by qPCR,mmu_circRNA₀07893 revealed a stable result which was consistent with that of the microarray.Then the circRNA was sequenced and observed by FISH experiments.The graphs showed that the circRNA mostly existed in cytoplasm which was accord with the known information.4.Validation of the effect of.circRNA on the expression of IL-6 1)circRNA interference experiment 3 siRNAs had significant inhibitory effect on the expression of mmu_circRNA₀07893,and the siRNA3 had the strongest inhibitory effect on mmu_circRNA₀07893 expression.2)Detection of IL-6 mRNA expression by qPCR Compared with the blank control group,the relative expression of IL-6 mRNA in the CGRP group was significantly higher.Furthermore,the expression of IL-6 mRNA in CGRP+siRNA group was significantly lower than that in the CGRP group.3)Detection of IL-6 expression by Western-blot Compared with the blank control group,the relative expression of IL-6 in the CGRP group was significantly higher.Furthermore,the expression of IL-6 in CGRP+siRNA group was significantly lower than that in the CGRP group.This result was accord with that of qPCR.5.Validation of the effect of.circRNA on downstream miRNA expression 1)miRNA selection According to mi RDB（www.mirdb.org）,David（david.ncifcrf.gov）and Target Scan （www.targetscan.org）,mmu-mi R-485-5p was most likely mediated by mmu_circRNA₀07893 and worked on IL-6 mRNA.Mmu-mi R-485-5p had at least 5 binding sites on the circRNA.2)Detection of mmu-mi R-485-5p expression by qPCR Compared with the blank control group,the relative expression of mmu-mi R-485-5p in the CGRP group was significantly lower.Furthermore,the expression of the miRNA in CGRP+siRNA group was significantly higher than that in the CGRP group.6.Validation of the effect of mmu-mi R-485-5p on the expression of IL-6 1)Detection of IL-6 mRNA expression by qPCR Compared with the control group,the relative expression of IL-6 mRNA significantly increased.However,in CGRP+mimics group,the relative expression of IL-6 mRNA was significantly lower than that in the CGRP group while the expression of IL-6 mRNA in CGRP+inhibitor group was significant higher than that in the CGRP group.2)Detection of IL-6 expression by Western-blot Compared with the control group,the relative expression of IL-6 significantly increased.However,in CGRP+mimics group,the relative expression of IL-6 was significantly lower than that in the CGRP group while the expression of IL-6 in CGRP+inhibitor group was significant higher than that in the CGRP group.This result was accord with that of qPCR.Conclusion: 1.When the concentration of CGRP was at 1n M,the relative expression level of IL-6 mRNA is the highest.2 When the stimulation time of CGRP was 2 hours,the relative expression level of IL-6 mRNA is the highest.3.Stimulation by CGRP at optimum concentration and time,the expression of mmu_circRNA₀07893 is significantly increased.4.Mmu_circRNA₀07893 mainly exists in the cytoplasm.5.Mmu_circRNA₀07893 has positive regulation effect on the expression of IL-6 mRNA and protein.6.Mmu-mi R-485-5p is one of downstream miRNA of mmu_circRNA₀07893.Mmu_circRNA₀07893 is a negative regulator of mmu-mi R-485-5p.7.Mmu_circRNA₀07893 can mediate the expression of IL-6 mRNA and protein by regulating the expression of mmu-mi R-485-5p.