circFOXO3 Induced by KLF16 Modulates Clear Cell Renal Cell Carcinoma Growth and Natural Killer Cell Cytotoxic Activity through Sponging miR-29a-3p and miR-122-5p.

Renal cell carcinoma (RCC) is one of the most common urological malignancies with high incidence and metastatic relapse. Clear cell RCC (ccRCC) comprises nearly 70% of all RCC cases and is responsible for the majority of morbidity and mortality of RCC. Due to the poor diagnosis strategy and unsatisfactory clinical intervention, ccRCC causes a huge economic burden and poor patient quality of life; therefore, novel diagnostic or therapeutic targets for ccRCC are urgently needed. This study investigated the biological role of circFOXO3 in ccRCC development, showing that circFOXO3 is highly expressed in RCC cells and tissues and inhibits the viability of ccRCC cells. circFOXO3 dysregulation regulates NK cell cytotoxicity towards RCC cells by directly sponging miR-29a-3p and miR-122-5p. Overexpression of miR-29a-3p or miR-122-5p attenuated NK cell toxicity towards RCC cells and the transcriptional factor Kruppel-Like Factor 16 (KLF16) regulates circFOXO3 expression in RCC cells. In conclusion, this study has partially elucidated the function of circFOXO3 in ccRCC development, providing potential novel therapeutic targets for ccRCC.

Circular RNA-0007059 protects cell viability and reduces inflammation in a nephritis cell model by inhibiting microRNA-1278/SHP-1/STAT3 signaling.

BACKGROUND: Increasing evidence has indicated that circular RNAs (circRNAs) play a role in various diseases. However, the influence of circRNAs in nephritis remains unknown. METHODS: Microarray analysis and RT-qPCR were used to detect the expression of circRNA. Type I IFN were administrated to RMC and HEK293 cells to establish a nephritis cell model. CCK-8, MTT assay, and flow cytometry were used to assess cell proliferation, viability, and apoptosis of cells. Bioinformatics analysis and dual luciferase reporter assay detect the interaction of circ_0007059, miRNA-1278, and SHP-1. Glomerulonephritis was performed in a mouse model by administration of IFNα-expressing adenovirus. IHC staining showed the pathogenic changes. RESULTS: In the present study, the expression of circ_0007059 in type I interferon (IFN)-treated renal mesangial cells (RMCs), lupus nephritis (LN) specimens, and HEK293 cells was downregulated compared with that in normal healthy samples and untreated cells. Circ_0007059 overexpression resulted in increased cell proliferation, cell viability, apoptosis, and inflammation-associated factors (CXCL10, IFIT1, ISG15, and MX1) in RMCs and HEK293 cells. In addition, circ_0007059 overexpression significantly restored cell proliferation and viability and inhibited IFN-induced apoptosis. Further, the increased expression resulted in reduced inflammation and the downregulation of CXCL10, IFIT1, ISG15, and MX1 in RMCs and HEK293 cells. Circ_0007059 serves as a sponge for miR-1278 so that the latter can target the 3'-untranslated region of SHP-1. Overexpressed circ_0007059 inhibited miR-1278 expression and elevated SHP-1 expression, subsequently reducing STAT3 phosphorylation. Meanwhile, miR-1278 was upregulated and SHP-1 was downregulated in LN samples and IFN-treated cells. The restoration of miR-1278 counteracted the effect of circ_0007059 on viability, apoptosis, and inflammation as well as on SHP-1/STAT3 signaling in RMCs and HEK293 cells. We also investigated the role of SHP-1 overexpression in IFN-treated RMCs and HEK293 cells; SHP-1 overexpression resulted in a similar phenotype as that observed with circ_0007059 expression. CONCLUSIONS: The study indicates that circ_0007059 protects RMCs against apoptosis and inflammation during nephritis by attenuating miR-1278/SHP-1/STAT3 signaling.

Transcriptome Profiling Reveals B-Lineage Cells Contribute to the Poor Prognosis and Metastasis of Clear Cell Renal Cell Carcinoma.

Although immune therapy can improve the treatment of clear cell renal cell carcinoma (ccRCC) significantly, there are still a large proportion of ccRCC patients who progress to metastasis. Targeting the pro-metastatic immune cell in the ccRCC microenvironment could provide a solution to this problem. In this study, B cells in ccRCC biopsies were identified by using scRNA-seq and flow cytometry. The findings indicated the presence of a pro-metastatic B cell type which could be further classified into 3 subpopulations, MARCH3, B2M and DTWD1, based on their large-scaled genetic profiles, rather than traditional Immature/Mature ones. Although all of the 3 subpopulations appeared to contribute to distant metastasis, B cell (B2M) was deemed to be the most essential. Moreover, STX16, CLASRP, ATIC, ACIN1 and SEMA4B, were genes found to be commonly up-regulated in the 3 subpopulations and this was correlated to a poor prognosis of ccRCC. Furthermore, the heterogeneity of plasma cells in ccRCC was also found to contribute to metastasis of the disease. This study offers potential novel therapeutic targets against distant metastasis of cancers, and can help to improve the therapeutic efficiency of ccRCC patients.

CYR61, regulated by miR-22-3p and MALAT1, promotes autophagy in HK-2 cell inflammatory model.

BACKGROUND: Renal tubular epithelial cells play an important role in renal function and are a major site of injury from inflammation. Emerging evidence suggests that CYR61 is involved in the regulation of autophagy. However, there are few studies on CYR61 in nephropathy and associated inflammation. This study aimed to clarify how CYR61 regulates autophagy in human renal epithelial cells while in an inflammatory state and regulates the upstream pathway of CYR61 levels. METHODS: The human renal tubular epithelial cells (HK-2) cell line treated by lipopolysaccharide (LPS) was used as an inflammatory model of human epithelial cells. Short hairpin RNA (shRNA) was used to down-regulate CYR61, and the changes in the transcription and expression levels of related molecules, as well as the morphological changes of HK-2 cells, were detected by quantitative real time-PCR (qRT-PCR), western blot (WB), and transmission electron microscopy. Either CYR61 or MALAT1 were up-regulated by overexpression vectors, or MALAT1 was down-regulated by miR-22-3p mimics. Subsequently, the levels of CYR61, MALAT1, related inflammatory factors, and autophagy factors were measured by qPCR, WB, and enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was detected by flow cytometry and acridine-orange assay. RESULTS: We observed that down-regulation of CYR61 could down-regulate 1B-light chain 3 (LC3) level and inhibit autophagy in the LPS-induced inflammation model of HK-2 cells. The expression levels of CYR61, Beclin1, Atg5, LC3, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) were significantly increased by upregulating CYR61 or MALAT1 by overexpression vector, while the expression level of p62 was significantly decreased, intracellular reactive oxygen species (ROS) content was increased, and the proportion of autophagy and apoptosis was increased. The use of miR-22-3p mimics significantly reversed the changes induced by up-regulation of CYR61 or MALAT1 at the molecular and cellular levels. CONCLUSIONS: Our data indicated that CYR61 positively regulates autophagy of HK-2 cells under an inflammatory state, and was negatively regulated by miR-22-3p, while miR-22-3p and MALAT1 were negatively regulated by each other.

Upregulated fractalkine levels in Chinese patients with lupus nephritis.

OBJECTIVE: To investigate the expression levels of fractalkine (FKN) mRNA in peripheral blood mononuclear cells (PBMCs) and FKN protein in serum of patients with lupus nephritis (LN) from China, and to evaluate the associations between the expression of FKN and systemic lupus erythematosus disease activity index 2000 (SLEDAI-2 K), anti-double-stranded DNA and complement proteins in LN patients. METHODS: Real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay were used to detect the expression levels of FKN mRNA in PBMCs and FKN protein in serum separately from 105 patients with LN and 52 healthy controls. RESULTS: Serum level and mRNA level of FKN were significantly increased in LN patients when compared to controls (P < 0.001). Higher FKN levels were found in active LN patients and LN patients with renal damage when compared with inactive LN patients and LN patients without renal damage (P < 0.001). Higher serum FKN levels were detected in inactive LN patients in comparison with healthy controls (Z = -7.165, P < 0.001). The FKN expression levels were positively correlated with SLEDAI-2 K, and was associated with the presence of autoantibodies and negatively correlated with complement proteins C3 and C4 in LN patients. CONCLUSIONS: The results suggest that upregulation of FKN is associated with the pathogenesis and activity of LN in Chinese patients.

Role of MiR-155 Signal Pathway in Regulating Podocyte Injury Induced by TGF-ß1.

BACKGROUND/AIMS: Transforming growth factor beta 1 (TGF-ß1) plays a critical role in the pathogenesis of glomerulosclerosis. The purpose of this study was to examine the effects of inhibition of miR-155 on podocyte injury induced by TGF-ß1 and to determine further molecular mediators involved in the effects of miR-155. METHODS: Conditionally immortalized podocytes were cultured in vitro and they were divided into four groups: control; TGF-ß1 treatment; TGF-ß1 with miR-155 knockdown [using antisense oligonucleotides against miR-155 (ASO-miR-155)] and TGF-ß1 with negative control antisense oligonucleotides (ASO-NC). Real time RT-PCR and Western blot analysis were employed to determine the mRNA and protein expression of nephrin, desmin and caspase-9, respectively. Flow cytometry was used to examine the apoptotic rate of podocytes and DAPI fluorescent staining was used to determine apoptotic morphology. In addition, we examined the levels of miR-155, TGF-ß1, nephrin, desmin and caspase-9 in glomerular tissues of nephropathy induced by intravenous injections of adriamycin in rats. RESULTS: mRNA and protein expression of desmin and caspase-9 was increased in cultured TGF-ß1-treated podocytes, whereas nephrin was decreased as compared with the control group. Importantly, miR-155 knockdown significantly attenuated upregulation of desmin and caspase-9, and alleviated impairment of nephrin induced by TGF-ß1. Moreover, the number of apoptotic podocytes was increased after exposure to TGF-ß1 and this was alleviated after miR-155 knockdown. Knocking down miR-155 also decreased an apoptosis rate of TGF-ß1-treated podocytes. Note that negative control antisense oligonucleotides failed to alter an increase of the apoptosis rate in TGF-ß1-treated podocytes. Consistent with in vitro results, expression of miR-155, TGF-ß1, desmin and caspase-9 was increased and nephrin was decreased in glomerular tissues with nephropathy in vivo experiments. CONCLUSIONS: TGF-ß1 impairs the protein expression of nephrin and amplifies the protein expression of desmin and caspase -9 via miR-155 signal pathway. Inhibition of miR-155 alleviates these changes in podocytes-treated with TGF-ß1 and attenuated apoptosis of podocytes. Our data suggest that miR-155 plays a role in mediating TGF-ß1-induced podocyte injury via nephrin, desmin and caspase-9. Results of the current study also indicate that blocking miR-155 signal has a protective effect on podocyte injury. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of podocyte injury observed in glomerulosclerosis.

Methylprednisolone attenuates lipopolysaccharide-induced Fractalkine expression in kidney of Lupus-prone MRL/lpr mice through the NF-kappaB pathway.

BACKGROUND: Fractalkine (FKN) is involved in the occurrence and development of human lupus nephritis. It is known to be upregulated by lipopolysaccharide (LPS) as a stimulus in vivo. MRL/lpr mice have been used as an in vivo model to study lupus nephritis. Methylprednisolone (MP) is used widely in the clinical treatment of progressive glomerular diseases such as lupus nephritis. The aim of this study is to explore the mechanism of LPS induced FKN expression and to determine whether other molecular mechanisms contribute to the signaling pathway of MP action in MRL/lpr mice. METHODS: Forty-eight female MRL/lpr mice at 12 weeks of age were randomly distributed into six groups. Each group received various treatments for 8 weeks by receiving twice weekly intraperitoneal injections of (1) MP (MP-treated mice), of (2) SC-514 (SC-514-induced mice), of (3) normal saline and a single injection of LPS (LPS-induced mice), of (4) MP and a single injection of LPS (LPS + MP mice), of (5) SC-514 and a single injection of LPS (LPS + SC mice) and of (6) normal saline (control mice). One-way ANOVA was used for data analysis and P value <0.05 was considered statistically significantly. RESULTS: The expression of FKN and NF-kappaB p65 mRNA was detected by qPCR. The expression of FKN protein and the activation of NF-kappaB p65 were detected by immunohistochemistry and western blots respectively. The expression of FKN in the kidney of LPS induced mice was significantly increased and this was mediated by increased expression of NF-κB p65 and an increase in NF-kappaB phospho-p65. MP reduced proteinuria and ameliorated the renal damage in MRL/lpr mice. MP as well as the NF-kappaB inhibitor, SC-514, inhibited the LPS-induced increase of expression of FKN and the activation of NF-kappaB. CONCLUSIONS: The results indicate that MP attenuates LPS-induced FKN expression in kidney of MRL/lpr mice through the NF-kappaB pathway.

MicroRNA-155 deficiency promotes nephrin acetylation and attenuates renal damage in hyperglycemia-induced nephropathy.

MiR-155 has been reported to be involved in both innate and adaptive immune responses. But the role of miR-155 in hyperglycemia-induced nephropathy is still unknown. In our current study, 3-month-old male wild-type C57 mice and Mir-155(-/-) mice were used to establish hyperglycemia-induced nephropathy. In our hyperglycemia-induced nephropathy model, the expression of podocyte injury marker desmin was markedly increased in the diabetes group when compared with control. Diabetes also significantly decreased the levels of nephrin and acetylated nephrin, whereas the expression of miR-155 was markedly increased in diabetes group when compared with control. MiR-155(-/-) mice showed significantly increased expression of nephrin, acetylated nephrin, and Wilm's tumor-1 protein (WT-1) when compared with wild-type control. MiR-155 deficiency results in significantly decrease in IL-17A expression both in vivo and in vitro. And the increased expression of WT-1, nephrin, and ac-nephrin was reversed with additional treatment of rmIL-17. Furthermore, we found that the inhibited Th17 differentiation induced by miR-155 deficiency was dependent on increased expression of SOCS1. In conclusion, miR-155 deficiency promotes nephrin acetylation and attenuates renal damage in hyperglycemia-induced nephropathy. This was associated with inhibited IL-17 production through enhancement of SOCS1 expression.

[T cell receptor constant alpha chain gene +1592C/T polymorphism may not be associated with IgA nephropathy].

OBJECTIVE: To investigate the relationship between T cell receptor constant alpha chain (TCRCα) gene +1592C/T polymorphism and IgA nephropathy. METHODS: TCRCα +1592C/T genotypes were identified by PCR-RFLP and direct sequencing in 244 Chinese Han patients with IgA nephropathy, who were classified according to their genotype into CC (188 cases), CT (54 cases) and TT (2 cases) groups. The clinical and pathological data of the patients were analyzed in relation to the TCRCα +1592C/T genotypes. RESULTS: No significant differences in the clinical and biochemical indices were found in these patients with different TCRCα gene +1592C/T genotypes. TCRCα +1592C/T polymorphism was not found to contribute to severity or manifestations of renal pathology. CONCLUSIONS: TCRCα+1592C/T polymorphism may not be associated with the susceptibility to IgA nephropathy in the Chinese Han population.