Fractalkine Facilitates In The Epithelial-mesenchymal Transition Of HK2 Cells And In A Murine Model Of Lupus Nephritis Via The Wnt/β-catenin Pathway

Part Ⅰ Establishment of HK2 Cell Line Knockdown Fractalkine Gene Using Lentiviral Vectors: An Experimental StudyObjective: To establish a stable HK2 cell line knockdown FKN gene using lentiviral vectors,laying a basis for investigating the role of FKN gene in EMT remodeling.Methods: From June 2016 to December 2016,FKN gene sequence was ligated into the GV248 vector(hU6-MCS-Ubiquitin-EGFP-IRES-puromycin)via restriction enzyme digestion.After amplification,transformation,verification,sequencing and plasmid extraction,the GV248 vector plasmids carrying the target gene,together with lentiviral packaging plasmids Helper 1.0 and Helper 2.0 were used to co-transfect 293 T cells,and the lentiviral vector LV-FKN-RNAi was harvested after packaging and virus concentration test.After being infected by LV-FKN-RNAi for 72 h,the HK2 cells were screened for stable HK2/LV-FKN-RNAi cell lines.The expression of FKN was determined by western blotting.Results: Sequence analysis showed that the positive clone of LV-FKN-RNAi was consistent with the target gene FKN.The optimal conditions for HK2 cells to achieve 80% infectious efficiency are as follows: ENi.S medium,MOI of 10,infection time of 72 h.Western blotting showed that the abundance of FKN gene in HK2/LV-FKN-RNAi cells was low(P < 0.01).The cell viability of the FKN knockdown group was decreased by CCK-8(P < 0.01).The apoptosis rate of FKN knockdown group increased by flow cytometry(P < 0.01).Conclusion: In this study,we successfully established a lentiviral vector knockdown FKN;target genes with low expression levels could be obtained by LV-FKN-RNAi infected HK2 cells.Moreover,the activity of HK2 cells decreased after the knockdown of FKN,and the apoptosis rate increased.The screened HK2/LV-FKN-RNAi cell line can be used in further experimental studies.Part Ⅱ Fractalkine Participates in Angiotensin Ⅱ-induced Epithelial-mesenchymal Transition in HK2 cells via Wnt/β-catenin PathwayObjective: Taking fractalkine(FKN)as the key point of research,the mechanism of FKN in HK2 cells was further explored from the in vitro cell level.In vitro cell experiments were performed to observe FKN,Vimentin,α-smooth muscle actin(α-SMA),and E-cadherin in HK2 cells excess expression of FKN(Ex-FKN)and FKN-knockdown(FKN-KD).Changes in expression levels,and the specific mechanism of action of angiotensin(Ang)Ⅱ and XAV939 on HK2 cells,and explored the process of Wnt/β-cadherin signaling pathway in the process of epithelial-mesenchymal transition(EMT)The role and the specific effects of FKN on this signaling pathway,and a deep understanding of the mechanism of action of FKN in HK2 cells,provide a scientific theoretical basis for the prevention and treatment of lupus nephritis.Methods: HK2 cells were used as the research object,and HK2 cells were transfected with lentiviral FKN-KD vector and Ex-FKN vector.The cells were randomly divided into 9 groups: 1)normal control group;2)FKN-KD group 3)XAV939 group;4)FKN-KD + VAV939 group;5)Ex-FKN group;6)Ex-FKN + VAV939 group;7)Ang Ⅱ group;8)FKN-KD + Ang Ⅱ group;9)Ex-FKN + Ang Ⅱ group.The expressions of FKN,EMT-related factors and Wnt/β-cadherin signaling pathways were detected by western blotting and q RT-PCR.The apoptosis rate of each group was detected by flow cytometry.Results: FKN-KD group down-regulated the expression of EMT and Wnt/β-catenin in HK2 cells(P < 0.01).The expression of EMT and Wnt/β-catenin was significantly down-regulated after XAV939 interaction,and the apoptosis rate was significantly up-regulated(P < 0.01).Ex-FKN group was up-regulated in HK2 cells(P < 0.01).The expression levels of EMT and Wnt/β-catenin were increased in HK2 cells(P < 0.01);the expression of EMT and Wnt/β-catenin was significantly increased after interaction with Ang Ⅱ(P < 0.01),and the apoptosis rate was significantly down-regulated(P < 0.01).Conclusion: FKN may induce epithelial-mesenchymal transition of HK2 cells through Wnt/β-catenin signaling pathway under Ang Ⅱ.Part Ⅲ Fractalkine Participates in Renal Epithelial-mesenchymal Transition in a Murine Model of Lupus Nephritis via Wnt/β-catenin PathwayObjective: To research the role of FKN in renal pathological changes in lupus model MRL/lpr mice.To investigate the role of FKN in the regulation of Wnt/β-catenin signaling pathway in epithelial-mesenchymal transition in renal tissues of lupus model MRL/lpr mice.Methods: Fifteen 12-week-old female MRL/lpr mice were randomly divided into 4 groups: rFKN group,daily submitted with Recombinant Mouse CX3CL1/Fractalkine Chemokine Domain intraperitoneal injection;Anti-FKN group,daily given CX3CL1/fractalkine Chemokine Domain Antibody Intraperitoneal injection;Ig G group,daily intraperitoneal injection of Rat Ig G2 A Isotype Control;blank control group,daily intraperitoneal injection of normal saline,7 days after the collection of peripheral blood of each group of mice,24 hours of urine protein,kidney cortex.The levels of ANA,anti-ds DNA and anti-Sm antibodies in each group were detected by enzyme-linked immunosorbent assay(ELISA).The m RNA and protein expression levels of FKN,EMT and Wnt/β-catenin related factors were detected by q RT-PCR and western blotting.The pathological changes of renal tissues were observed.The expressions of FKN,EMT and Wnt/β-catenin related proteins in renal cortex were detected by immunohistochemical staining.Results: Compared with the blank control group,the expression levels of ANA,anti-ds DNA and anti-Sm in the rFKN group were significantly increased(P < 0.01),urine protein levels were increased(P < 0.01),and renal inflammatory lesions were aggravated.The expression levels of FKN,wnt-4,Vimentin,α-SMA,CCL22,F4/80,TGFβ,NF-κB-p65 protein and m RNA were significantly increased(P < 0.01),and the expression levels of E-cadherin protein and m RNA were significantly decreased(P < 0.01).In the Anti-FKN group,the expression levels of ANA,anti-ds DNA and anti-Sm were significantly decreased(P < 0.01),urine protein levels were decreased(P < 0.01),and renal inflammatory lesions were significantly improved.The expression levels of FKN,wnt-4,Vimentin,α-SMA,CCL22,F4/80,TGFβ,NF-κB-p65 protein and m RNA were significantly decreased(P < 0.01),and E-cadherin protein and m RNA expression levels were significantly increased(P < 0.01).Conclusion: FKN may induce epithelial-mesenchymal transition in renal tissues of MRL/lpr mice by lupus model via Wnt/β-catenin signaling pathway.