The Role And Its Mechanism Of ANRIL In Muc5ac Secretion Of Beas-2B Cell Induced By Atmospheric PM_2.5

Objective:siRNA technology was used to target silencing of ANRIL gene.NF-?B-mediated difference in expression levels of inflammatory factors and mucin Muc5ac were observed by comparing ANRIL gene silencing and normal Beas-2B cell after exposure to PM_2.5.The regulation and mechanism of ANRIL in mucus secretion induced by atmospheric PM_2.5 was explored.This study could provide a scientific basis for elucidating the pathogenesis of respiratory diseases caused by atmospheric PM_2.5 and provide new targets for effective interventions to prevent and control the occurrence and aggravation of respiratory diseases.Method:Beas-2B cell was cultured in vitro using DMEM medium containing 10%fetal bovine serum.The ANRIL siRNA fragment was synthesized and transfected into well-grown Beas-2B cell.The ANRIL silencing efficiency was verified by Q-PCR.Gene silencing and normal Beas-2B cell was respectively exposed to0?g/mL?blank control group?,100?g/mL?low dose group?,200?g/mL?middle dose group?and 400?g/mL?high dose group?atmospheric PM_2.5 for 6h,12h and 24 h,and the cell and culture supernatant were collected.The survival rate of Beas-2B cell was detected by MTT assay.The levels of IL-1?,TNF-?and Muc5ac in the culture supernatant were determined by ELISA.The expression levels of NF-?B gene family mRNA were determined by Q-PCR.The expression levels of I?B-?protein were determined by Western Blot.Statistical analysis was performed by IBM SPSS 24.0.Variance analysis was used to compare the differences among different groups.The LSD method was used to compare the two groups.The t test was used to compare the differences between gene silencing and normal cell groups.The rank sum test was used to compare the non-normality data.Differences between groups were considered statistically significant if P<0.05.Result:1.The cell viability of Beas-2B cell in each dose group was significantly lower than that of the blank control group after exposure to PM_2.5 for 6h,12h and 24h?P<0.05?,and after exposure to PM_2.5 for 12h and 24h,the cell survival rate decreased significantly with the increasing of exposure dose?P<0.05?.The cell viability of the medium and high dose groups exposed to PM_2.5.5 for 12h and 24h was significantly lower than those exposed to PM_2.5 for 6h?P<0.05?.2.After exposure to PM_2.5 for 6h,the levels of Muc5ac in the middle and high dose groups were significantly higher than those in the control group?P<0.05?.After exposure to PM_2.5 for 12h,the levels of Muc5ac in the high dose group were significantly higher than those in the other groups?P<0.05?.After exposure to PM_2.5for 24h,there was no significant difference of the levels of Muc5ac in each dose group?P>0.05?.The levels of Muc5ac in each dose group increased first and then decreased with the prolongation of PM_2.5.5 exposure time?P<0.05?.3.After exposure to PM_2.5 for 6h,12h and 24h,the levels of IL-1?in the supernatant of high dose group were significantly higher than those in the middle dose and control groups?P<0.05?.After exposure to PM_2.5.5 for 12h,the levels of IL-1?in the high dose group were significantly higher than those in the low dose group?P<0.05?.After exposure to PM_2.5.5 for 24h,the levels of IL-1?in the low dose group were significantly higher than those in the control group.There was no significant difference of the levels of IL-1?in each dose group with the prolongation of exposure time?P>0.05?.After exposure to PM_2.5 for 6h,the levels of TNF-?in the supernatant of the medium and high dose groups were significantly higher than those of the control group,and the levels of TNF-?in the high dose group were significantly higher than those in the low and middle dose groups?P<0.05?.After exposure to PM_2.5 for 24h,the levels of TNF-?in each dose group were significantly higher than those in the control group,the levels of TNF-?in the middle and high dose groups were significantly higher than those in the low dose group?P<0.05?.After exposure to PM_2.5 for 12h,the levels of TNF-?in each dose group were not statistically significant?P>0.05?.The levels of TNF-?in the each dose group after exposure to PM_2.5.5 for 24h were significantly higher than those after exposure to PM_2.5 for 6h and 12h?P<0.05?.4.After exposure to PM_2.5 for 6h,12h and 24h,the expression levels of NF-?B1,NF-?B2,RelA,RelB and Rel mRNA of Beas-2B cell in high dose group were significantly higher than those in the control group?P<0.05?.After exposure to PM_2.5for 6h,the expression levels of NF-?B1,NF-?B2 and RelA mRNA in middle dose group were significantly higher than those in the control group,while the expression levels of RelB mRNA was lower than those in the control group?P<0.05?.After exposure to PM_2.5 for 12h,the expression levels of Rel mRNA in middle dose group were significantly higher than those in the control group,while the expression levels of RelB mRNA was lower than those in the control group?P<0.05?.After exposure to PM_2.5 for 24h,the expression levels of NF-?B1,NF-?B2,RelA,RelB and Rel mRNA in middle dose group were significantly higher than those in the control group?P<0.05?.After exposure to PM_2.5 for 6h,the expression levels of NF-?B2,RelA and RelB mRNA in low dose group were significantly lower than those in the control group?P<0.05?.After exposure to PM_2.5 for 12h,the expression levels of RelA and RelB mRNA in low dose group were significantly lower than those in the control group,while the expression levels of Rel mRNA were significantly higher than those in the control group?P<0.05?.The expression levels of NF-?B1 and Rel mRNA in Beas-2B cell after exposure to PM_2.5.5 for 12h and 24h were significantly lower than those after exposure for 6h in each dose group?P<0.05?.The expression levels of NF-?B2 mRNA in middle and high dose group after exposure to PM_2.5.5 for 12h and 24h were significantly lower than those after exposure for 6h,while there was the opposite phenomenon in the low dose group?P<0.05?.The expression levels of RelA and RelB mRNA after exposure to PM_2.5.5 for 24h were significantly lower than those after exposure for 6h and 12h in each dose group?P<0.05?.5.The expression levels of I?B-?protein,NF-?B inhibitory protein,were decreased gradually with the increasing of PM_2.5 dose and prolonged exposure time,in Beas-2B cell?P<0.05?.6.The expression levels of ANRIL in high dose group were significantly higher than those in the control group after exposure to PM_2.5 for 6h,12h and 24h?P<0.05?.The expression levels of ANRIL were significantly higher in the middle dose group than those in the control group after exposure to PM_2.5 for 12h and 24h?P<0.05?.7.In the ANRIL gene silencing cell group,after exposure to PM_2.5 for 6 h,the levels of Muc5ac in the 400?g/mL group were significantly higher than those in the other groups?P<0.05?,The levels of Muc5ac in the middle dose group were significantly higher than those in the control group?P<0.05?.After exposure to PM_2.5for 24 h,the levels of Muc5ac in the middle and high dose groups were significantly higher than those in the low dose and control groups?P<0.05?.After exposure to PM_2.5 for 12h,the levels of Muc5ac in each dose group were not statistically significant?P>0.05?.After exposure to PM_2.5 for 12 h,the levels of Muc5ac in the middle and high dose groups of gene silencing group were significantly lower than those of normal cell?P<0.05?.8.In the ANRIL gene silencing cell group,after exposure to PM_2.5 for 6h,the levels of IL-1?in the middle dose group were significantly higher than those in the low and high groups,the levels of TNF-?in each dose group were significantly higher than those in the control group?P<0.05?.After exposure to PM_2.5 for 12h,the levels of IL-1?in the middle and high dose groups were significantly higher than those in the low dose group and control group,the levels of TNF-?in each dose group were significantly higher than those in the control group?P<0.05?.After exposure to PM_2.5for 24h,the levels of IL-1?in the middle and high dose groups were significantly higher than those in the low dose group,the levels of TNF-?in high dose group were significantly higher than those in the low and middle dose groups.After exposure to PM_2.5 for 6h,the levels of IL-1?in the high and low dose groups of gene silencing cell of were significantly lower than those of the normal cell?P<0.05?.After exposure PM_2.5 for 24h,the levels of IL-1?and TNF-?in the high dose group of gene silencing cell were significantly lower than those in the normal cell?P<0.05?.9.The difference of expression levels of NF-?B family genes between ANRIL gene silencing cell and normal cell groups?1?In the gene silencing cell group,after exposure to PM_2.5 for 6h,the expression levels of NF-?B1,Rel and RelB mRNA in high dose group were lower than those in normal cell?P<0.05?.The expression levels of NF-?B1 and NF-?B2 mRNA in middle dose group were significantly lower than those of normal cell?P<0.05?.The expression levels of NF-?B1,Rel,RelA and RelB mRNA in low dose group were significantly lower than those of normal cell?P<0.05?.?2?In the gene silencing cell group,after exposure to PM_2.5 for 12h,the expression levels of NF-?B1,NF-?B2,Rel and RelA mRNA in high dose group were lower than those in normal cell?P<0.05?.The expression levels of NF-?B1,NF-?B2,RelA and Rel mRNA in middle dose group were significantly lower than those of normal cell?P<0.05?.The expression levels of NF-?B1,Rel and RelB mRNA in low dose group were significantly lower than those of normal cell?P<0.05?.?3?In the gene silencing cell group,after exposure to PM_2.5 for 24h,the expression levels of NF-?B1,NF-?B2 and Rel mRNA in high dose group were lower than those in normal cell?P<0.05?.The expression levels of NF-?B2,RelB and Rel mRNA in middle dose group were significantly lower than those of normal cell?P<0.05?.The expression levels of NF-?B1,NF-?B2,RelA and Rel mRNA in low dose group were significantly lower than those of normal cell?P<0.05?.10.In the ANRIL gene silencing cell group,the expression levels of I?B-?in the high dose group gradually increased with the prolongation of PM_2.5 exposure time.Conclusion:1.Exposure to atmospheric PM_2.5 could reduce the survival rate of Beas-2B cell.2.Exposure to atmospheric PM_2.5 could stimulate Beas-2B cell to secrete more mucin.ANRIL silencing could reduce mucin secretion,indicating that ANRIL regulates the secretion of more mucin in Beas-2B cell induced by PM_2.5.3.Exposure to atmospheric PM_2.5 could stimulate Beas-2B cell to secrete more inflammatory factors.ANRIL silencing could reduce the secretion of inflammatory factors,indicating that ANRIL plays a regulatory role in Beas-2B cell inflammation induced by PM_2.5.4.Exposure to atmospheric PM_2.5 could up-regulate the expression levels of NF-?B family gene mRNA.ANRIL silencing could reduce the expression levels of NF-?B family gene mRNA,indicating that ANRIL plays a role in the expression of NF-?B family gene induced by PM_2.5.5.Exposure to atmospheric PM_2.5 could promote the activation of NF-?B signal pathway in Beas-2B cell.ANRIL silencing could attenuate the activation of NF-?B signal pathway,indicating that ANRIL plays a regulatory role in the enhancement of NF-?B signal pathwayin induced by PM_2.5.6.ANRIL may regulate the inflammatory response process of Beas-2B cell by regulating NF-?B signal pathway,and play a regulatory role in the increase of mucus secretion induced by PM_2.5.