To study the function of KLF14, we used the genome editing technology TALEN to establish a KLF14 gene knockout mouse models. The results showed that the levels of blood cholesterol, HDL-C, LDL-C, and TG in KLF14-/- mice significantly decreased compared with the KLF14+/+ group. The pathological results showed that the KLF14 knockout could aggravate glomerular proliferation. Similar to the histological manifestations, the number of glomerular nucleated cells increased more noticeably in the KLF14-/- mice.
To investigate the role of KLF14 in mouse kidneys, the differences in mRNA expression profiles between the kidney tissues of KLF14+/+ and KLF14-/- mice were analyzed using RNA-SEq. Compared with wild-type mice, we observed significant changes in mRNA expression in the kidneys of KLF14-/- mice. Among these differentially expressed mRNAs, most mRNAs were down-regulated, and a few mRNAs were up-regulated. Biological processes that involved these differentially expressed genes (DEGs) focused mainly on the regulation of proliferation, lipid metabolic process, immune system process and immune response, and response to corticosteroid.
To gain further insights into the role of KLF14 in kidneys, the target genes of KLF14 in kidneys were determined using ChIP-SEq. The biological function and pathways of these targets were analyzed. The results revealed that the peak related genes in kidneys mainly regulated the pathways or genes associated with metabolism, cell proliferation, immune pathways, and extracellular matrix (ECM) degradation. The combined analysis of both ChIP-Seq and RNA-Seq results showed that KLF14 might play very important roles in the regulation of proliferation and cancer, lipid metabolic process, and immune system process and immune response.
KLF14 has been verified as an important regulator in the lipid metabolism [18, 23]. GWAS has suggested that a single nucleotide polymorphism (SNP) in one of the KLF14 locus is associated with low HDL-C and TGs in European populations . The possible mechanism underlying the function of KLF14’s regulating effect of lipid metabolism may be related with transcriptional activation of the SK1 promoter ,which can lead to the up-regulation of sphingosine-1-phosphate (S1P) lipid signaling . In addition, KLF14 regulates plasma HDL-C levels and cholesterol efflux capacity by mediating hepatic apoA-I production . Our study results showed that cholesterol, HDL-C, LDL-C, and TGs were significantly decreased in KLF14-/- mice, which was partially consistent with previous study results. The reasons for this difference might be associated with the different functions of KLF4 between mice and humans; it might also be due to the housing environment and timing differences between mice and humans.
In addition to the important role in lipometabolism pathways, KLF14 may significant ly influence the immune functions. The latest development has shown that KLF14 is of high expression in the lymphatic organs, which revealed that KLF14 may play an important part in driving T cell differentiation s.The underlying mechanism may be that KLF14 can bind to the Treg-specific demethylation region (TSDR) enhancer locus of FOXP3, thereby epressing the expression of the immune gene FOXP3 . The knockdown of KLF14 has been shown to greatly decrease the levels of some inflammatory cytokines, such as IL-6, TNF-α and MCP-1 in apoe-/- mice fed a high-fat diet. However, KLF14 over-expression clearly suppressed the secretion of those cytokines in RAW264.7 cells. The effect of KLF14 on Ac-LDL-induced inflammatory cytokine secretion in macrophages at least partly concerns the p38 MAPK and extracellular signal-regulated kinase 1 (ERK1)/2 signaling pathways . Our study also revealed that some inflammatory genes, such as Ccl28, Socs2, Jchain, and Cfd, were directly or indirectly regulated by KLF14. The level of Socs2 is closely related to KLF14, but the dual-luciferase reporter assay of Socs2 has failed to find that KLF14 can directly regulate the expression of Socs2. The mechanism of KLF14 regulation of Socs 2 needs to be further clarified.
The function of KLF14 in proliferation and tumorigenesis has also been reported. Previous studies have demonstrated that KLF14-induced expression of alk-1may be involved in the TGF-β1-mediated cell proliferation in Leydig cells . A recent study revealed that KLF14 knockout mice showed an induction pf centrosome amplification, chromosome aneuploidy, and spontaneous tumorigenesis, which was mainly caused by transcriptional inhibition of Plk4, while Plk4 overexpression could induce centrosome overduplication . Our study showed that glomerular proliferation was significantly aggravated in the kidneys of KLF14-/- compared with wild-type mice. Similar to the histological manifestations, the number of glomerular nucleated cells increased more noticeably in the KLF14-/- mice. Furthermore, cell experiments also confirmed that KLF14 overexpression in renal mesangial cells could significantly reduce the rate of EdU positivity, suggesting that KLF4 had an anti-proliferative function in the kidneys.
To further confirm the target genes of KLF14, ChIP-Seq was used to screen its target genes. The ChIP-Seq and RNA-Seq results in KLF14-/- mice were combined for analysis. The results revealed five overlapping target genes between the RNA-Seq and Chip-Seq results. These target genes were Socs2, Btg2, Hdc, Ler2, and Akr1b3, among which, Btg2 was associated with the regulation of proliferation. We further performed bioinformatics analyses, the dual-luciferase reporter assay, and the EMSA assay to confirm that Btg2 was a target gene of KLF14, which is a member of the BTG/TOB family. Recent studies have indicated that all TOB/BTG family members are tumor suppressor genes and that this family is closely related to other tumor suppressor genes such as p53 . Btg2 is located on band 3, region 2 of the long arm of chromosome 1 and is a protein consisting of 158 amino acids. Btg2 is an immediate early response gene. When cells are stimulated by genotoxic drugs, oxidative stress, and ionizing radiation, Btg2 expression is up-regulated through p53-dependent or independent pathways to act on a series of downstream target molecules, causing cells to arrest at G1/S phase, which is the major functional pathway of the Btg2 gene. A large number of studies have confirmed that Btg2 expression is down-regulated in many tumors. As a tumor suppressor gene, Btg2 is widely inloved in differentiation, proliferation, DNA damage repair, and apoptosis of various tumor cells [29–31].
To clarify whether KLF14 inhibited the proliferation of mouse renal mesangial cells by regulating the target gene Btg2, a Btg2 gene siRNA was synthesized and transfected into the cells. The results showed that the anti-proliferative effect of KLF14 on mesangial cells was significantly diminished after transfection of Btg2 siRNA. This result indicated that Btg2 activation was dispensable for KLF14-induced anti-proliferation.
In short, the transcriptome sequencing and ChIP-Seq results in this study confirmed that KLF14 mainly regulated metabolism, proliferation, and inflammation-related processes. Expression of the Btg2 gene in mouse kidneys was regulated by KLF14, and Btg2 gene expression decreased after KLF14 knockout. The combined results of the luciferase reporter gene assay and EMSA herein confirmed that Btg2 was a target gene of KLF14. In vitro studies showed that KLF14 could inhibit renal mesangial cell proliferation by regulating the activity of Btg2. Thus, we can get the conclusion that KLF14 might exert its function in kidney tissues by regulating metabolism, proliferation, and inflammation signaling pathways in cells. KLF14 inhibited the proliferation of primary renal mesangial cells by promoting expression of its target gene Btg2. This study provides a basis for further studies of the mechanism of KLF14 in the development of kidney diseases, especially in mesangial proliferative glomerulonephritis, metabolic nephropathy, and kidney tumors.