Regulation of SIGIRR expression mechanism in human renal tubular epithelial cells by nuclear transcription factor NF - κB
10.19405/j.cnki.issn1000-1492.2023.03.003
- Author:
Keguo Jiang
1
,
2
;
Li Zhu
3
;
Wenman Zhao
3
;
Rui Shi
3
;
Deguang Wang
3
Author Information
1. Dept of Nephrology，The Second Affiliated Hospital of Anhui Medical University，Hefei 230601
2. Dept of Nephrology，The Third Affiliated Hospital of Anhui Medical University，Hefei 230001
3. Dept of Nephrology，The Second Affiliated Hospital of Anhui Medical University，Hefei 230601
- Publication Type:Journal Article
- Keywords:
human renal tubular epithelial cells;
Toll-like receptor;
nuclear transcription factor;
single immuno- globulin interleukin-1 receptor-related protein
- From:
Acta Universitatis Medicinalis Anhui
2023;58(3):358-365
- CountryChina
- Language:Chinese
-
Abstract:
Objective :To investigate the molecular mechanisms underlying the feedback regulation of single immunoglobin interleukin-1 related receptor ( SIGIRR) expression by the nuclear factor kappa-κB ( NF-κB) in human renal tubular epithelial cells ( HKC) ．
Methods :The pLNCX2-G418-SIGIRR overexpression vector was constructed by molecular cloning，and the SIGIRR overexpression cells and control cells were constructed by infecting HKC cells after packaging with PT67 cells．Using IL-1 β induction，Western blot verified that overexpression of SIGIRR inhibited NF-κB activation．After using NF-κB blocker and interfering with NF-κB activity ，immunofluorescence assay verified that activated NF-κB regulated SIGIRR expression． Online tools predicted the presence of NF-κB binding sites in the SIGIRR promoter region．The SIGIRR promoter sequence containing the binding site was obtained from within human genomic DNA by molecular cloning，ligated to the luciferase vector pGL3-Luc，constructed pGL3-Luc-SIGIRR , and mutated the binding site．The luciferase reporter gene assay and chromatin immunopre- cipitation technique ( ChIP) were used to jointly verify that activated NF-κB could bind to the SIGIRR promoter region to regulate SIGIRR gene expression.
Results :The results showed that the constructed pLNCX2-G418-SIGIRR retroviral vector was verified by enzymatic digestion and sequencing to be identical to the coding sequence of the SIGIRR gene for comparison，the recombinant and control vectors were transferred into HKC cells after viral packaging，and the HKC / SIGIRR experimental and HKC / Co control cell lines were successfully constructed at the mRNA and protein levels of SIGIRR expression differences were statistically significant (P＜0．05，P＜0. 001) ．Overexpression of SIGIRR cell groups reduced IL-1 β-induced NF-κB activation compared to control cells (P＜0. 001) . SIGIRR expression was downregulated after inhibition of NF-κB activation and interference with NF-κB expression. After extracting human genomic DNA ，the SIGIRR target promoter sequence was obtained by molecular cloning method and linked to the vector，and the pGL3-Luc-SIGIRR luciferase vector was successfully constructed and targeted to mutate the vector，which was verified to be identical to the target sequence by digestion and sequencing. The luciferase reporter gene assay and CHIP assay confirmed that NF-κB could bind to SIGIRR promoter region and regulate SIGIRR expression．
Conclusion:It has been verified that SIGIRR can influence the activation of NF-κB in HKC cells，and activated NF-κB can bind to the promoter region of SIGIRR and regulate the gene expression changes of SIGIRR , forming a feedback system to control the over-activation of NF-κB.
- Full text:2024071610561368255核转录因子NF-κB调节人...SIGIRR表达机制的研究_蒋克国.pdf
