| Objective(s):ERBB receptor feedback inhibitor 1(ERRFI1)is a cell function regulator that can be activated by a wide range of stimuli and has been linked to cancers and other illnesses.However,limited research exists on the physiological and pathological roles of ERRFI1 in the nervous system.Our previous study indicated that astrocytes in the hippocampus of epileptic mice exhibited considerably higher levels of ERRFI1 expression,suggesting that ERRFI1 might contribute to the onset and progression of epilepsy by controlling astrocyte activity.In this study,the effects of ERRFI1 on astrocyte proliferation,apoptosis,and cytokine expression were explored in vitro,laying a foundation for clarifying the mechanism of epilepsy and other central nervous system diseases related to astrocyte abnormalities.This study may provide new targets for the treatment of these diseases.Methods:1.We established overexpressed and knockdown cell lines for ERRFI1 in mouse astrocyte C8-D1 A and rat astrocyte TNC1.We validated these cell lines using Real-time PCR and Western Blot.2.To evaluate changes in cell proliferation,we used the CCK-8 assay,crystal violet staining assay,and Ed U proliferation assay.We used the Propidium Iodide cell cycle staining method with flow cytometry to examine changes in cell cycle.The Annexin 5 PE/7-ADD apoptosis staining assay was employed to count apoptotic cells by flow cytometry.Western Blot was used to quantify the amounts of cleaved Caspase-3 and phosphorylated EGFR protein.3.To establish an astrocytes inflammation model,we treated astrocytes with LPS or media supernatant from LPS-stimulated microglial cells.Then,we measured the expression of inflammatory factors using Real-time PCR to determine the method of astrocytes inflammatory model.4.Finally,we evaluated cytokine expression in the overexpression and down-regulated ERRFI1 cell lines derived from C8-D1 A and TNC1.We used Real-time PCR,Western Blot,and ELISA to measure cytokine expression levels in untreated cells and cells treated with media supernatant from LPS-stimulated microglial cells.Additionally,we detected phosphorylated changes of EGFR protein by Western Blot and tried to inhibite EGFR receptors to verify the conclusions,Results:1.We cloned and inserted the ERRFI1 gene into pc DNA3.1-flag and PLJM1-flag vectors to obtain overexpression vectors successfully.The inserted fragments aligned with the ERRFI1 CDS reference sequence.Meanwhile,a 20 bp oligonucleotide was side-linked to the NGG PAM sequence of Lenticrispr-V2.We compared the sequencing results with the ERRFI1 sequence to obtain the ERRFI1 knockout vector.2.After transfecting the overexpression vectors of pc DNA3.1-flag-ERRFI1 and PLJM1-flag-ERRFI1 into TNC1 and C8-D1 A cells,the expressions of ERRFI1 were increased significantly at m RNA level(24 h)and protein level(48 h)(P<0.05).Besides,Western Blot confirmed the presence of flag fusion protein,demonstrating the effectiveness of the overexpression vectors.3.The expression of ERRFI1 was too low to detect in resting state TNC1.Therefore,we performed the ERRFI1 knockdown experiment in TNC1 derived cells which overexpress ERRFI1 Steadily.After transfection of Lenticrispr-ERRFI1 and si RNA-ERRFI1,the expression of ERRFI1 decreased at the m RNA level(24 h)and protein level(48 h)significantly in TNC1-PLJM1-flag-ERRFI1 and C8-D1A(P<0.05).4.In TNC1 and C8-D1 A,compared with the control group,overexpression of ERRFI1 resulted in decreased numbers of viable cells,reduced DNA replication,increased the number of cells in the G1 phase of the cell cycle,increased the proportion of apoptotic cells,and increased the portion of cleaved caspase3(P<0.05).Knockdown of ERRFI1 resulted in opposite results(P<0.05).5.After LPS treatment,m RNA levels of TNF-α and IL-6 were down-regulated in TNC1 cells within 4 hours(P<0.05);in C8-D1 A,m RNA expression levels of TNF-αand IL-6 were down-regulated after 0.5 and 2 hours of LPS treatment(P<0.05)but there was no significant difference after 4 hours(P>0.05).In contrast,media supernatant from 6 hours LPS stimulated microglial cell line BV-2 upregulated the expression of pro-inflammatory factors TNF-α and IL-6 in TNC1 and C8-D1 A cells treated for 2 hours(P<0.05),and the expression of ERRFI1 was up-regulated also(P<0.05),which proved the inflammation model of astrocytes was constructed successfully.6.In TNC1 and C8-D1 A,after treating overexpressed ERRFI1 astrocyte with media supernatant from LPS-stimulated BV-2 cells,the expression of IL-6 and TNF-αmeasured by Realtime PCR was down-regulated in C8-D1 A and TNC1 cells significantly(P<0.05).However,there were no changs of IL-6 and TNF-α in knockdown cells significantly(P>0.05).But Western Blot results showed a significant decrease(P<0.05)of active TNF-α portion in overexpressed C8-D1 A and TNC1 cells,while it increased significantly in knockdown cells(P<0.05).Realtime PCR was also used to measure changes in m RNA expression levels of TGF-β,ICAM-1,CCL2,and CXCL1.After ERRFI1 overexpression,we treated astrocytes with media supernatant from LPS-stimulated BV-2 cells.The expression difference was not statistically significant in C8-D1 A for TGF-β,ICAM-1,CCL2,and CXCL1(P>0.05).In TNC1,there were no statistically significant changes in TGF-β and CXCL1 expression levels(P>0.05),but the expression of ICAM-1 and CCL2 was significantly down-regulated(P<0.05).After knockdown of ERRFI1,the expression difference in treated C8-D1 A for TGF-β and CXCL1 was not statistically significant(P>0.05),and the expression of ICAM-1 and CCL2 was significantly down-regulated(P<0.05).In TNC1 cells,there was no statistically significant difference in the expression levels of TGF-β and ICAM-1(P>0.05),but the expression levels of CCL2 and CXCL1 were significantly up-regulated(P<0.05).Thus,ERRFI1 has an inhibitory effect on CCL2 m RNA in TNC1,but not in C8-D1 A.7.The phosphorylation level of EGFR did not change significantly after overexpression or knockdown of ERRFI1 in TNC1 and C8-D1 A cells(P>0.05).However,treating cells with media supernatant from LPS-stimulated BV-2 cells resulted in a significant increase in the phosphorylation level of EGFR in both TNC1 and C8-D1 A cells(P<0.05).In ERRFI1-overexpressed TNC1 and C8-D1 A cells,the level of EGFR phosphorylated protein was significantly reduced(P<0.05),while the result after knockdown was opposite(P<0.05).Conclusion(s):1.ERRFI1 inhibit the proliferation and promote the apoptosis of murine astrocytes.2.ERRFI1 inhibit the expression of pro-inflammatory factor TNF-αby inhibiting EGFR receptor phosphorylation in astrocytes probably.Additionally,ERRFI1 has a species-specific inhibitory effect on CCL2 in astrocytes. |
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