| Objective: Polycyclic aromatic hydrocarbons(PAHs)have become the focus of environmental health research due to their high emission density,prolonged presence,high carcinogenicity,mutagenicity,and teratogenicity.Studies have shown that PAHs can induce invasion and migration of associated cells through the epithelial-mesenchymal transition(EMT),leading to the development of many diseases.However,there are few studies on low-molecularweight PAHs(LMW-PAHs)and EMT.Long non-coding RNA(lncRNA)Long have become a hot spot for research due to their greater number and broader pattern of regulation of gene expression.lncRNA plays fundamental roles in EMT by working as a competing endogenous RNA(ce RNA)to sponge miRNAs to modulate gene expression.In our previous studies,hsa＿circ＿0039929/hsa＿miR-15b-3p＿R-1/FGF2 was found that can participate in and regulate EMT through PI3K/AKT signaling pathway in LMW-PAHs-treated A549 cells.Therefore,this study aims to further explore the role of LINC01376/miR-15b-3p＿R-1/FGF2 axis on EMT in LMW-PAHs-treated A549 and H1299 cells.It provides a reference for studies related to PAHs exposure,as well as an experimental and scientific basis for the early diagnosis and comprehensive treatment of EMT-related diseases caused by LMWPAHs.Methods:(1)The bioinformatics analysis was applied to screen lncRNA upstream of miR-15b-3p＿R-1and predict target binding sites between LINC01376 and miR-15b-3p＿R-1 and between miR-15b-3p＿R-1 and FGF2,respectively.(2)Determination of gene expression of LINC01376 in A549 and H1299 cells induced by LMW-PAHs(experimental group: 200 μM,400 μM and 600 μM of Phe and Flu mixture)using real-time fluorescence quantitative PCR(RT-q PCR).(3)LINC01376 siRNA(experimental group: LMW-PAHs group,LMW-PAHs+NC group and LMW-PAHs+si-LINC01376 group)was transfected into A549 and H1299 cells exposed to LMWPAHs(400μM Phe and Flu mixture)knockdown of LINC01376,and the effect of LINC01376 on key EMT proteins was detected by Western blot,and the effect of LINC01376 on the migration and invasion ability of LMW-PAHs-treated A549 and H1299 cells was detected by wound healing assay and transwell assay.(4)The target binding relationship between LINC01376 and miR-15b-3p＿R-1 was verified using a dual luciferase reporter assay.(5)Transfection of miR-15b-3p＿R-1 mimic overexpression of miR-15b-3p＿R-1 in LMWPAHs-treated(400μM Phe and Flu mixture)A549 and H1299 cells(experimental group: LMWPAHs group,LMW-PAHs+NC group and PAHs+miR-15b-3p＿R-1 mimic group),and the effect of miR-15b-3p＿R-1 on EMT key proteins was detected by Western blot,and the effect of miR-15b-3p＿R-1 on cell migration and invasion ability was detected by wound healing assay as well as transwell assay.(6)The target binding relationship between miR-15b-3p＿R-1 and FGF2 was verified using a dual luciferase reporter assay.(7)Western blot,wound healing and transwell rescue assay were performed to explore the effect of LINC01376/miR-15b-3p＿R-1/FGF2 axis on EMT migration,and invasion in A549 and H1299 cells treated by LMW-PAHs(experimental group: NC+inhibitor NC group,siLINC01376+Inhibitor NC group,si-LINC01376+miR-15b-3p＿R-1 inhibitor group and siLINC01376+miR-15b-3p＿R-1 inhibitor+si FGF2 group).Results:(1)Using bioinformatics analysis,we screened 64 up-regulated lncRNAs and 31 downregulated lncRNAs that were differentially expressed based on the previous microarray data.Then the DIANA Tools was used to predict lncRNAs that might bind to miR-15b-3p＿R-1,and the venn diagram showed the aim gene is LINC01376 by the overlapping of lncRNA between sequencing data and DIANA Tools.(2)The RT-q PCR experiments showed that compared to the control group,LINC01376 expression levels were significantly increased in A549 and H1299 cells treated with 200μM,400μM and 600μM LMW-PAHs(P<0.05),and FGF2 expression levels were significantly increased in the400μM and 600μM LMW-PAHs treated groups(P<0.05),whereas the expression levels of miR-15b-3p＿R-1 were significantly down-regulated(P<0.05).(3)Knockdown of LINC01376 by transfection with LINC01376 siRNA,the results of Western blot assay demonstrated that in LMW-PAHs-treated A549 and H1299 cells,knockdown of LINC01376 resulted in a significant increase in E-cadherin protein expression level(P<0.05)and significant decreases in Vimentin and α-SMA protein levels(P<0.05).The results of wound healing assay and transwell assay revealed that knockdown of LINC01376 inhibited the migration and invasion of A549 and H1299 cells treated by LMW-PAHs.(4)The result of dual luciferase reporter assay showed that overexpression of miR-15b-3p＿R-1 in 293 T cells decrease luciferase activity of LINC01376-WT(P<0.05),but did not affect the luciferase activity of LINC01376-MUT(P>0.05).(5)Overexpression of miR-15b-3p＿R-1 by transfection with miR-15b-3p＿R-1 mimic,the results of Western blot assay showed that overexpression of miR-15b-3p＿R-1 in LMW-PAHstreated A549 and H1299 cells could result in a significant increase in E-cadherin protein expression level(P<0.05)and significant decreases in Vimentin and α-SMA protein levels(P<0.05).The results of wound healing assay as well as transwell assay showed that overexpression of miR-15b-3p＿R-1inhibited the migration and invasion of A549 and H1299 cells treated by LMW-PAHs.(6)The result of dual luciferase reporter assay showed that overexpression of miR-15b-3p＿R-1 in 293 T cells decrease luciferase activity of FGF2-3’UTR-WT(P<0.05),but did not affect the luciferase activity of FGF2-3’UTR-MUT(P>0.05).(7)The role of LINC01376/miR-15b-3p＿R-1/FGF2 axis in EMT,migration and invasion of A549 and H1299 cells treated by LMW-PAHs were tested in rescue assays.Firstly,Western blot rescue assay showed that knockdown of LINC01376 alone could reduce LMW-PAHS-treated expression of EMT-related proteins(Vimentin and α-SMA levels)and increase E-cadherin protein levels,while miR-15b-3p＿R-1 inhibitor significantly attenuated the inhibitory effect of LINC01376 siRNA on LMW-PAHS-treated EMT(P<0.05).In addition,silencing of FGF2 could further inhibite the expression of Vimentin and α-SMA protein levels and promote upregulation of E-cadherin protein expression levels(P<0.05).The results of wound healing assay and transwell assay showed that LINC01376 knockdown reduce LMW-PAHS-treated migration and invasion,which was alleviated by miR-15b-3p＿R-1 inhibitor.Compared to the si-LINC01376+miR-15b-3p＿R-1inhibitor group,si FGF2 further inhibited LMW-PAHS-treated migration and invasion of A549 and H1299 cells(P<0.05).Conclusion: In this study,LINC01376 and FGF2 expression were significantly upregulated and miR-15b-3p＿R-1 expression was significantly downregulated in LMW-PAHs-treated A549 and H1299 cells.After knockdown of LINC01376 or overexpression of miR-15b-3p＿R-1,EMT,migration and invasion were significantly inhibited in LMW-PAHs-treated A549 and H1299 cells.The possible mechanism was that LINC01376 could act as a molecular sponge to competitively bind miR-15b-3p＿R-1 and inhibit the regulatory effect of miR-15b-3p＿R-1 on FGF2,which in turn regulated EMT,migration and invasion of cells caused by LMW-PAHs。... |
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