The role of innate lymphoid cells in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a connective tissue disorder that affects various body systems. Both the innate and adaptive immunity contribute to the onset and progression of SLE. The main mechanism of SLE is an excessive immune response of immune cells to autoantigens, which leads to systemic inflammation and inflammation-induced organ damage. Notably, a subset of innate immune cells known as innate lymphoid cells (ILCs) has recently emerged. ILCs are pivotal in the early stages of infection; participate in immune responses, inflammation, and tissue repair; and regulate the immune function of the body by resisting pathogens and regulating autoimmune inflammation and metabolic homeostasis. Thus, ILCs dysfunction can lead to autoimmune diseases. This review discusses the maturation of ILCs, the potential mechanisms by which ILCs exacerbate SLE pathogenesis, and their contributions to organ inflammatory deterioration in SLE.

A murine Skint6 W168X allele contributes to autoimmune disease in a transgenic model.

BACKGROUND: The genetic factor is a great driver of systemic lupus erythematosus. A Skint6 W168X allele was previously identified in the murine lupus susceptibility rec1d1 sublocus. The purpose of this study is to investigate the pathogenic role and mechanism of the Skint6 W168X allele in lupus autoimmune disease. METHODS: The gene-editing CRISPR/Cas9 system was used to generate transgenic models with the Skint6 W168X allele. PCR and Sanger's sequencing techniques were applied to mRNA quantification and DNA sequence detection. Flow cytometry was adopted for immunophenotyping. Pathological evaluation of kidneys and lungs was performed using several immunopathological approaches. RESULTS: The transgenic models with the Skint6 W168X allele were created, including B6.Skint6X/X and B6.lpr.Skint6X/X strains. The B6.lpr.Skint6X/X mice showed bigger spleen and lymph nodes, more lymphocytes and effector T cell populations, more severe nephritis with more IgG and C3 deposit in glomeruli as well as worse proteinuria, and more severe lung inflammation than control B6.lpr mice. In addition, a skint6 receptor binding Skint6 peptide was identified from T and B lymphocytes. B6.Skint6X/X mice have lower percentages of skint6 receptor+ T and B cells in spleen than B6 mice. CONCLUSION: The Skint6 W168X allele in murine lupus rec1d1 sublocus was validated to be a pathogenic mutant gene and contributes to autoimmune disease through producing a truncated Skint6 peptide of binding the skint6 receptors on lymphocytes.

The current status of anti-citrullinated protein antibodies and citrullinated protein-reactive B cells in the pathogenesis of rheumatoid arthritis.

Anti-citrullinated protein antibodies are a hallmark of rheumatoid arthritis. It is widely acknowledged that the presence of ACPAs is the result of the interaction of genes, the environment and epigenetic modifications. The mechanism by which the factors, especially citrullination and ACPA glycosylation, affect ACPAs is still unclear. In this article, we review the presence of the ACPAs in RA and their relationship with clinical manifestations. The pathogenicity of ACPAs and B cells in RA was also summarized. A growing body of evidence has shown that ACPA-positive patients have more serious bone erosion and destruction and poor clinical prognosis than ACPA-negative patients. Recently, with the direct study of citrullinated protein-reactive B cells, their role in the development of rheumatoid arthritis has been further understood. It indicates that further understanding of the mechanism of ACPAs and CP-reactive B cells would beneficial in the prevention and treatment of RA.

A Variant of sNASP Exacerbates Lymphocyte Subset Disorder and Nephritis in a Spontaneous Lupus Model Sle1.Yaa Mouse.

A variant of somatic nuclear autoantigenic sperm protein (sNASP) was identified from the murine lupus susceptibility locus Sle2c1 by whole exome sequencing (WES). Previous studies have shown that mutant sNASP could synergize with the Faslpr mutation in exacerbating autoimmunity and aggravating end-organ inflammation. In the current study, the sNASP mutation was introduced into Sle1.Yaa mice to detect whether it has a synergistic effect with Sle1 or Yaa loci. As expected, compared with Sle1.Yaa mice, Sle1.Yaa.ΔsNASP mice showed enlarged lymph nodes, aggravated renal inflammation, and shortened survival time. The proportions of CD3+ T cells, activated CD19+CD86+ B cells, Th1 cells in the spleen and lymph nodes, and Th17 cells in lymph nodes in Sle1.Yaa.ΔsNASP mice were increased compared to those in Sle1.Yaa mice. The levels of IFN-γ and TNF-α in the serum of Sle1.Yaa.ΔsNASP mice were higher than those of Sle1.Yaa mice. The above results show that mutant sNASP can interact with different lupus susceptibility genes and promote the disease process of systemic lupus erythematosus.

Chimeric specific antigen epitope-carrying dendritic cells induce interleukin-17(+) regulatory T cells to suppress food allergy.

BACKGROUND: The on-purpose-modulated dendritic cells (DCs) have shown charming effects on restoring immune regulatory functions in subjects with immune diseases. OBJECTIVE: This study aims to construct DCs carrying chimerical antigen (Ag) peptides (CAP-DCs) to induce interleukin (IL)-17+ inducible Tregs (iTregs) to alleviate food allergy (FA) in a murine model. METHODS: In this study, we constructed CAP-DCs. The CAP is a fusion protein, consisting of a segment of recombinant scFv of anti-DEC205 antibody and an ovalbumin (OVA) epitope (IC). A murine OVA-FA model was developed to test the effects of CAP-DCs on suppressing the allergic response in the intestine. RESULTS: The CAP-DCs are characterized as that a complex of scFv-IC is presented on the surface of the cells, moderately express CD80 and CD86 as well as IL-6, IL-23, transforming growth factor (TGF)-ß and CCR9. After being passively transferred with CAP-DCs or injection of scFv-IC, Ag-specific IL-17+ Foxp3+ iTregs were induced in the intestinal lamina propria of FA mice. The iTregs showed immune suppressive effects on Ag-specific Th2 response. FA mice were adoptively transferred with the CAP-DCs or scFv-IC injection, which resulted in a significant decrease in the number of Ag-specific Th2 cells and suppression of FA response in an Ag-specific manner. CONCLUSIONS AND CLINICAL RELEVANCE: CAP-DCs can ameliorate FA response by inducing Ag-specific IL-17+ Foxp3+ iTregs and suppressing Ag-specific Th2 response. To generate CAP-DCs has the translational potential in the treatment of FA.

Molecular cloning, expression, and anti-tumor activity of a novel serine protease from Arenicola cristata.

Arenicola cristata, a marine annelid, is a well-known and prized traditional Chinese medicine. However, the serine protease gene of A. cristata has not been cloned yet. In this study, a novel protease of A. cristata was cloned, sequenced, and expressed in Escherichia coli, and the functions of this recombinant protease were also investigated. The whole complementary DNA (cDNA) of this novel protease was of 980 bp in length and consisted of an open reading frame of 861 bp encoding 286 aa. Sequence analysis of the deduced amino acid sequence revealed that the protease belongs to the serine protease family. The active enzyme of the proposed A. cristata protease is composed of a signal peptide, a propeptide, and a mature polypeptide. The molecular weight of the recombinant mature protein was ~26 kDa after over-expression in E. coli. The recombinant protein significantly inhibited cell growth and induced cell apoptosis of esophageal squamous cell carcinoma (ESCC) in vitro, and reduced tumorigenicity in vivo. Furthermore, administration of the recombinant protein led to the activation of caspase-9 as well as down-regulation of Mcl-1 and Bcl-2. Taken together, our findings indicated that the recombinant serine protease of A. cristata could inhibit ESCC cell growth by mitochondrial apoptotic pathway and might act as a potential pharmacological agent for ESCC therapy.

Therapeutic landscape in systemic lupus erythematosus: mtDNA activation of the cGAS-STING pathway.

Mitochondrial DNA (mtDNA) serves as a pivotal immune stimulus in the immune response. During stress, mitochondria release mtDNA into the cytoplasm, where it is recognized by the cytoplasmic DNA receptor cGAS. This activation initiates the cGAS-STING-IRF3 pathway, culminating in an inflammatory response. The cGAS-STING pathway has emerged as a critical mediator of inflammatory responses in microbial infections, stress, autoimmune diseases, chronic illnesses, and tissue injuries. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by connective tissue involvement across various bodily systems. Its hallmark is the production of numerous autoantibodies, which prompt the immune system to target and damage the body's own tissues, resulting in organ and tissue damage. Increasing evidence implicates the cGAS-STING pathway as a significant contributor to SLE pathogenesis. This article aims to explore the role of the mtDNA-triggered cGAS-STING pathway and its mechanisms in SLE, with the goal of providing novel insights for clinical interventions.

[Administration of a single chain variable fragments chimeric protein (SD) of ovalbumin epitopes internalizing receptor DEC-205 antibody inhibits food allergy in mice].

Objective To investigate the preventive therapeutic effect and possible mechanism of single chain variable fragments chimeric protein (SD) of ovalbumin epitopes internalizing receptor DEC-205 antibody on food allergy in mice. Methods Mice were randomly divided to five groups (control, PBS, scFv DEC 100 µg, SD 50 µg, SD 100 µg) and treated for 24 hours before OVA administration. After challenge, the serum level of OVA-specific IgE, IgG1, IgG2a and IL-4 were detected by ELISA. Infiltration of eosinophils and mast cells in the jejunum was observed by HE staining and toluidine blue staining respectively. The bone marrow of tibia and femur was isolated and cultured to obtain immature dendritic cells(BMDCs), which were further treated with LPS (10 ng/mL), TSLP (50 ng/mL), scFv DEC protein (1000 ng/mL) and SD protein (10,100,1000)ng/mL for 24 hours, and the IL-10 level of supernatant was assayed by ELISA. Results Compared with PBS group, the number of SD-treated mice with diarrhea was markedly reduced. The difference in rectal temperature and the levels of serum OVA-specific IgE, IgG1, IgG2a and IL-4 decreased significantly after prophylactic administration of SD; The number of eosinophils and mast cells in jejunum also decreased significantly while the IL-10 level in the supernatant of BMDCs increased significantly after SD intervention. Conclusion SD mitigates experimental FA response by fosters the immune tolerance property of dendritic cells.

sNASP Mutation Aggravates to the TLR4-Mediated Inflammation in SLE by TAK1 Pathway.

Genetic factors play an important role in the pathogenesis of systemic lupus erythematosus (SLE), and abnormal Toll-like receptor (TLR) signaling pathways are closely related to the onset of SLE. In previous studies, we found that the mutant somatic nuclear autoantigenic sperm protein (sNASP) gene in the mouse lupus susceptibility locus Sle2 can promote the development of lupus model mice, but the mechanism is still unclear. Here, we stimulated mouse peritoneal macrophages with different concentrations of lipopolysaccharide. The results showed that sNASP gene mutations can promote the response of the TLR4-TAK1 signaling pathway but have no significant effect on the TLR4-TBK1 signaling pathway. sNASP mutations enhanced TLR4-mediated nuclear factor-κ-gene binding and mitogen-activated protein kinase activation and IL-6, tumor necrosis factor secretion in murine peritoneal macrophages. Collectively, our study revealed the impact of sNASP gene mutation on the sensitivity of TLR4 receptors in mouse peritoneal macrophages and shed light on potential mechanisms underlying inflammation in autoimmune diseases.

Updated insight into the role of Th2-associated immunity in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with multiple organs involvement, abundant autoantibodies, complement activation, and immune complexes depositions. By regulating inflammation and immune homeostasis, cytokines have been well documented to participate in the pathogenesis of SLE. A number of studies have shown that T helper 2 (Th2)-associated immunity plays an important role in autoimmune diseases, including SLE. Key molecules underlying Th2-related immunity are expected to serve as promising targets for the diagnosis and targeted treatment of SLE. Current progress in SLE pathogenesis and biological treatment strategies has been reviewed, focusing on the latest development in Th2-associated immunity.

The signaling pathways regulated by KRAB zinc-finger proteins in cancer.

Kruppel-associated box (KRAB) zinc-finger proteins (KRAB-ZFPs) are the largest transcriptional/transcription-regulatory factor family in mammalian cells. The amino-terminal KRAB domain, which recruits other transcription-regulating proteins, and the carboxyl-terminal C2H2 zinc-finger motifs, which bind to specific DNA sequences, are the typical structural characteristics of KRAB-ZFPs. Many KRAB-ZFPs are abnormally expressed in several cancer types and involved in many cancer-related signaling pathways and bioprocesses, including cell proliferation, apoptosis, migration, invasion, and metastasis. In this review, we summarize the protein structure and mechanisms involved in transcriptional regulation, and focus on multiple key signaling pathways regulated by KRAB-ZFPs, including the p53, Wnt/ß-catenin, and NF-κB pathways, highlighting the oncogenic and tumor-suppressive roles of KRAB-ZFPs in different cancers. We also discuss the mechanisms regulating KRAB-ZFP expression. The further elucidation of the oncogenic and tumor-suppressive roles of KRAB-ZFPs and their targeting for multiple synergistic signaling pathways may be valuable for effective cancer therapy.

[Hepatic fibrosis aggravation in nuclear autoantigenic sperm protein (NASP) mutant mice induced by concanavalin A].

Objective To investigate how mutation of nuclear autoantigenic sperm protein (NASP) gene affects mouse liver fibrosis induced by concanavalinA (ConA) and its mechanism. Methods The wild-type B6 (B6-WT) mice were used as a control group, and the NASP mutant B6 (B6-NASPM) mice as an experimental group. The mice were injected with ConA via tail vein once a week for 8 weeks to establish the model of liver fibrosis. The histopathological changes were observed by HE staining, the collagen fiber deposition by Masson staining, smooth muscle alpha-actin (α-SMA) expression in liver tissue by immunohistochemical staining. The levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by microplate assay. The serum tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) were measured by ELISA. The mRNA expressions of type I collagen (Col1) and Col3 in liver tissue were determined by real-time quantitative PCR, and the changes of T lymphocyte subsets in liver tissue were detected by flow cytometry. Results Compared with the B6-WT group, B6-NASPM group had disordered liver structure and no significant changes in mRNA expression of Col 1 and Col3, but the collagen fiber hyperplasia and α-SMA expression in liver tissue were more obvious, and the levels of serum ALT and TNF-α were significantly increased. In addition, the proportion and number of CD4+CD44hi T lymphocyte subsets in liver tissue were markedly decreased. Conclusion Mutation of NASP gene aggravates mouse liver fibrosis by increasing the release of TNF-α, changing the proportion of T lymphocyte subsets in liver tissue and promoting the activation of hepatic stellate cells.

Apolipoprotein A-I Mimetic Peptide L-4F Suppresses Granulocytic-Myeloid-Derived Suppressor Cells in Mouse Pancreatic Cancer.

L-4F is an apolipoprotein A-I (ApoA-I) mimetic peptide, it was engineered to imitate the anti-inflammatory and anti-oxidative activity of ApoA-I. In this paper, H7 cell was used to construct a mouse model of pancreatic cancer in situ, and the mice were treated with L-4F. Then, the development of pancreatic cancer and myeloid-derived suppressor cells (MDSCs) infiltration were investigated in vivo. After L-4F treatment, the differentiation, proliferation and apoptosis of MDSCs were detected in vitro. Moreover, we test its effects on the immunosuppressive function of MDSCs ex vivo. The results show that L-4F significantly reduced the tumorigenicity of H7 cells. L-4F suppressed granulocytic myeloid-derived suppressor cells (PMN-MDSCs) differentiation and inhibited the accumulation of PMN-MDSCs in the mouse spleen and tumor tissue. L-4F weakened the immunosuppressive function of MDSCs, resulting in decreased production of ROS and H2O2 by MDSCs, and increased T cell proliferation, interferon γ and tumor necrosis factor ß secretion, and CD3+CD4+ T and CD3+CD8+ T cell infiltration into the mouse spleen and pancreatic cancer tissue. Furthermore, L-4F significantly down regulated the STAT3 signaling pathway in PMN-MDSCs. These results indicated that L-4F exerts an effective anti-tumor and immunomodulatory effect in pancreatic cancer by inhibiting PMN-MDSCs.

[Mutation of nuclear autoantigenic sperm protein (NASP) gene aggravates autoimmune response in induced lupus model mice].

Objective To prepare inducible lupus model mice and investigate the effect of nuclear autoantigenic sperm protein (NASP) gene mutation on the autoimmune response of mice with induced systemic lupus erythematosus (SLE). Methods The 3-month wild-type B6 (B6-WT) mice were used as a control group and the NASP mutant B6 (B6-NASPM) mice as an experimental group. Mouse spleen lymphocytes were activated with concanavalin A (ConA), and the DNA was extracted as autoantigen. B6-WT mice and B6-NASPM mice were immunized three times. Serum anti-double stranded DNA (dsDNA) IgG levels were detected by ELISA. Renal lesions were detected by HE staining. Immunohistochemical staining was performed to detect the deposition of IgG and complement C3 in the renal tissues. Flow cytometry was applied to compare the spleen lymphocyte subsets in B6-WT and B6-NASPM mice and to explore the mechanism of NASP gene mutation affecting the immune response in mice. Results Compared with B6-WT mice, B6-NASPM mice showed no significant changes in body weight, kidney index and spleen index; serum anti-dsDNA IgG levels significantly increased; glomerular cell proliferation was obvious and the deposition of IgG and C3 in the renal tissues increased. The proportion of spleen CD3+ T cells and natural killer (NK) cells decreased, while the proportion of CD19+ B cells and regulatory B cells (Breg) increased. Conclusion Mutation in the NASP gene can increase the levels of anti-dsDNA IgG antibodies, promote cell proliferation in the glomerulus of the kidney, deposition of IgG antibodies and complement C3, alter the proportion of immune cells in the spleen and aggravate the autoimmune response in lupus model mice.

A Variant of the Histone-Binding Protein sNASP Contributes to Mouse Lupus.

The Sle2c1rec1c (rec1c) sublocus is derived from the mouse lupus susceptibility 2 (Sle2) locus identified in the NZM2410 model. Our current study dissected the functional characters and the genetic basis of the rec1c locus relative to lupus when co-expressed with the Fas lpr mutation, an established inducer of autoimmunity. The rec1c.lpr mice exhibited mild expansion of lymph nodes and had a normal T cell cellularity, but developed significantly kidney and lung inflammation, indicating that the rec1c amplifies lpr-induced autoimmune pathogenesis. A variant of somatic nuclear autoantigenic sperm protein (sNASP) was identified from the rec1c interval as a substitution of two consecutive amino acid residues in the histone-binding domain, resulting in an increased binding affinity to histone H4 and H3.1/H4 tetramer. To determine the role of the sNASP rec1c allele in mouse lupus, a novel strain was generated by introducing the rec1c mutations into the B6 genome. In this transgenic model, the sNASP allele synergized with the lpr mutation leading to moderate autoimmune phenotypes and aggravating inflammatory pathology alterations in kidney and lung that were similar to those observed in the rec1c.lpr mice. These results establish that the sNASP allele is a pathogenic genetic element in the rec1c sublocus, which not only promotes autoimmunity, but also exacerbates the inflammation reaction of end organs in mouse lupus pathogenesis. It also shows the complexity of the Sle2c locus, initially mapped as the major locus associated with B1a cell expansion. In addition to Cdkn2c, which regulates this expansion, we have now identified in the same locus a protective allele of Csf3r, a variant of Skint6 associated with T cell activation, and now a variant of sNASP that amplifies autoimmunity and tissue damage.

[Experimental BCSC-1 gene therapy on nasopharyngeal carcinoma mediated by adenovirus].

OBJECTIVE: To explore the therapeutic value of BCSC-1 in tumor gene therapy. METHODS: Recombinant adenovirus Ad5-BCSC-1 was prepared. Cell proliferation was assayed using CellTiter 96 Aqueous one solution cell proliferation assay kit. Ad5-BCSC-1 was injected into tumor with Ad5-egfp or with PBS injection as controls. The injections were repeated one weak later. After another 2 weeks, the mice were sacrificed and the tumors were excised and weighed. RESULTS: The growth of the CNE-2L2 cell infected with Ad5-BCSC-1 in vitro was remarkably slower than that of the controls, the wild type cell and the cell infected with Ad5-egfp. In contrast to the controls, the cells infected with Ad5-BCSC-1 aggregated and formed huge messes in the culture. The average weight of the CNE-2L2 tumors in mice was (2.28 +/- 0.73), (2.07 +/- 0.40), and (0.58 +/- 0.32) g for the tumors injected with PBS, Ad5-egfp, and Ad5-BCSC-1, respectively (Ad5-BCSC-1 vs. PBS or Ad5-egfp, P<0.05). CONCLUSION: Intra-tumor injection of Ad5-BCSC-1 can suppress the growth of CNE-2L2 tumor in nude mice, suggesting that BCSC-1 gene therapy may be effective for tumors with low or no expression of BCSC-1 gene.

Association of BCSC-1 and MMP-14 with human breast cancer.

Breast cancer suppressor candidate-1 (BCSC-1) is a candidate tumor suppressor gene that was identified recently. Decreased levels of BCSC-1 have been detected in a variety of cancer types in previous studies. Matrix metalloproteinase (MMP)-14 is a membrane-type MMP that plays an important role in tumor progression and prognosis. Previous research has indicated that MMP-14 is highly expressed in different cancer types and promotes tumor invasion or metastasis by remodeling the extracellular matrix. However, there have been few reports on BCSC-1 and MMP-14 in human breast cancer in recent years. In the present study, the association of BCSC-1 and MMP-14 with human breast cancer was investigated. The immunohistochemical analysis results revealed reduced expression of BCSC-1 and overexpression of MMP-14 in breast cancer tissues compared with adjacent normal breast tissues. Quantitative polymerase chain reaction and western blot analyses also showed that BCSC-1 was expressed at significantly lower levels, and that MMP-14 was expressed at significantly higher levels in breast cancer tissues compared with healthy breast tissue. Furthermore, decreased expression of BCSC-1 and overexpression of MMP-14 were associated with tumor cellular differentiation, lymph node metastasis and distant metastasis. A correlational analysis between BCSC-1 and MMP-14 was also conducted, and the results indicated a negative correlation between the two. In conclusion, the current findings indicate that BCSC-1 is downregulated, while MMP-14 is overexpressed in human breast cancer. These two genes may play important roles during the process of human breast cancer development.

BCSC-1 suppresses human breast cancer metastasis by inhibiting NF-κB signaling.

Breast cancer suppressor candidate-1 (BCSC-1; also termed von Willebrand factor A domain containing 5A and LOH11CR2A) is a newly identified candidate tumor suppressor gene that has been implicated in several types of cancer in previous studies. However, there have been few reports about the association between BCSC-1 and human breast cancer in recent years. In the present study, the expression of BCSC-1 in breast cancer was determined by immunohistochemistry (IHC) staining of tissue microarrays and clinical tissue specimens. Subsequently, BCSC-1 gene expression was evaluated in different breast cancer cell lines by quantitative polymerase chain reaction and the MDA-MB-231 cell line was selected for further use in subsequent experiments, due to its low BCSC-1 expression. An MDA-MB-231 cell line with stable overexpression of BCSC-1 was established through transfection with plasmid containing the BCSC-1 gene, and then screening for G418 resistance. Wound-healing, migration and invasion assays were conducted to detect the effect of BCSC-1 on MDA-MB-231 cells. Furthermore, changes in matrix metalloproteinases (MMPs), osteopontin (OPN) and the nuclear factor-κB (NF-κB) pathway were detected in the current study. Additionally, stable silencing of BCSC-1 expression in MCF-7 cells was performed using a lentivirus. The results of IHC indicated that BCSC-1 is expressed at low levels in breast cancer tissues compared with in normal breast tissue. Results of the wound healing, migration and invasion assays demonstrated that BCSC-1 overexpression reduced the metastasis ability of MDA-MB-231 cells in vitro. Further research confirmed that the BCSC-1 overexpression reduced the expression levels of MMP7, MMP9 and OPN, and the phosphorylation of NF-κB p65. Furthermore, inhibition of BCSC-1 via lentivirus-mediated RNA interference revealed that the downregulation of BCSC-1 increased the invasive ability of MCF-7 cells. In summary, the results demonstrated that BCSC-1 is expressed at low levels in breast cancer tissues, and that it can suppress human breast cancer cell migration and invasion, potentially altering the expression of MMP7, MMP9, OPN, and the activity of the NF-κB pathway. Therefore, BCSC-1 may be useful as a biomarker for the treatment of breast cancer in the future.

KRAB-containing zinc finger protein ZNF496 inhibits breast cancer cell proliferation by selectively repressing ERα activity.

KRAB-containing zinc finger proteins (KZNF) constitute the largest family of transcriptional regulators in humans and play critical roles in normal development and tumorigenesis. However, the function and mechanism of most KZNFs remain unclear. Here, we report that ZNF496, a KZNF family member, interacts with the DNA binding domain (DBD) of estrogen receptor alpha (ERα) via its C2H2 domain. This interaction decreases ERα binding to chromatin DNA and results in the repression of ERα transactivation, the selective suppression of ERα target genes, and ultimately in a reduction of ERα-positive cell growth in the presence of E2. An analysis of clinical data revealed that the downregulation of ZNF496 expression is observed only in ERα-positive and not in ERα-negative breast cancer tissues when compared with that in matched adjacent tissues. Lastly, we also observed that the downregulation of ZNF496 is associated with poor recurrence-free survival among patients with breast cancer. Collectively, our findings demonstrate that ZNF496 is a novel ERα-binding protein that acts as a target gene-specific ERα corepressor and inhibits the growth of ERα-positive breast cancer cells.

[Reduction of CD44 expression results in growth inhibition of human nasopharyngeal carcinoma cell CNE-2L2 in vitro].

OBJECTIVE: To study the effect of the inhibition of CD44 gene expression on the growth of human nasopharyngeal carcinoma cell CNE-2L2 in vitro. METHODS: CD44 gene expression in cells was suppressed by siRNA which was introduced into cells through retrovirus infection. Integration of siRNA into genomic DNA was examined by genomic PCR. CD44 gene expression in cells was detected by Western blot analysis. Cell growth in vitro was assayed using Cell Titer 96 AQueous One Solution Cell Proliferation Assay kit Promega. Cells were stained with propidium iodium and cell DNA content was detected upon a flow cytometer. RESULTS: siRNA was integrated into genomic DNA of host cells. The 4 cell pools integrated with one of the 4 siCD44s showed a significant inhibition of CD44 gene expression comparing to the controls, the wild type cell and the cell pool integrated with siegfp. The cell pools integrated with siCD44-1 or siCD44-2 showed the most profound inhibition. Growth of these 2 cells in vitro was compared to that of the controls and was found to be significantly inhibited. Cell DNA content analysis indicated 44.4%, 45.5%, 53.9%, and 53.3% in G0/G1 phase; 39.3%, 40.0%, 27.1%, and 28.2% in S phase; and 16.3%, 14.5%, 19.0%, and 18.5% in G2/M phase for the wild type cell, the cell pool integrated with siegfp, the cell pools integrated with siCD44-1, and the cell pools integrated with siCD44-2, respectively. CONCLUSION: Reduction in CD44 expression inhibit the growth of CNE-2L2 cell and affects the development of cells from G0/G1 into S phase, but may somehow promote cells to develop from S into G2/M phase.