Hypoxia-induced exosomes facilitate lung pre-metastatic niche formation in hepatocellular carcinoma through the miR-4508-RFX1-IL17A-p38 MAPK-NF-κB pathway.

Background: Hypoxia plays an important role in the lung metastasis of hepatocellular carcinoma (HCC). However, the process by which hypoxia promotes the formation of a pre-metastatic niche (PMN) and its underlying mechanism remain unclear. Methods: Exosomes derived from normoxic and hypoxic HCC cells were collected to induce fibroblast activation in vitro and PMN formation in vivo. The micro RNA (miR) profiles of the exosomes were sequenced to identify differentially expressed miRNAs. Gain- and loss-of-function analyses were performed to investigate miR-4508 function. Dual-luciferase, western blotting, and real-time reverse transcription-PCR analyses were used to identify the direct targets of miR-4508 and its downstream signaling pathways. To demonstrate the roles of hypoxic tumor-derived exosomes (H-TDEs) and miR-4508 in the lung metastasis of liver cancer, H22 tumor cells were injected through the tail vein of mice. Blood plasma-derived exosomes from patients with HCC who underwent transarterial chemoembolization (TACE) were applied to determine clinical correlations. Results: We demonstrated that H-TDEs activated lung fibroblasts and facilitated PMN formation, thereby promoting lung metastasis in mice. Screening for upregulated exosomal miRNAs revealed that miR-4508 and its target, regulatory factor X1 (RFX1), were involved in H-TDE-induced lung PMN formation. Moreover, miR-4508 was significantly upregulated in plasma exosomes derived from patients with HCC after TACE. We confirmed that the p38 MAPK-NF-κB signaling pathway is involved in RFX1 knockdown-induced fibroblast activation and PMN formation. In addition, IL17A, a downstream target of RFX1, was identified as a link between RFX1 knockdown and p38 MAPK activation in fibroblasts. Conclusion: Hypoxia enhances the release of TDEs enriched with miR-4508, thereby promoting lung PMN formation by targeting the RFX1-IL17A-p38 MAPK-NF-κB pathway. These findings highlight a novel mechanism underlying hypoxia-induced pulmonary metastasis of HCC.

Regulatory factor X1 induces macrophage M1 polarization by promoting DNA demethylation in autoimmune inflammation.

Abnormal macrophage polarization is generally present in autoimmune diseases. Overwhelming M1 macrophage activation promotes the continuous progression of inflammation, which is one of the reasons for the development of autoimmune diseases. However, the underlying mechanism is still unclear. Here we explore the function of Regulatory factor X1 (RFX1) in macrophage polarization by constructing colitis and lupus-like mouse models. Both in vivo and in vitro experiments confirmed that RFX1 can promote M1 and inhibit M2 macrophage polarization. Furthermore, we found that RFX1 promoted DNA demethylation of macrophage polarization-related genes by increasing APOBEC3A/Apobec3 expression. We identified a potential RFX1 inhibitor, adenosine diphosphate (ADP), providing a potential strategy for treating autoimmune diseases.

RXR agonist, Bexarotene, effectively reduces drug resistance via regulation of RFX1 in embryonic carcinoma cells.

Aberrant expression of multidrug resistance (MDR) proteins is one of the features of cancer stem cells (CSCs) that make them escape chemotherapy. A well-orchestrated regulation of multiple MDRs by different transcription factors in cancer cells confers this drug resistance. An in silico analysis of the major MDR genes revealed a possible regulation by RFX1 and Nrf2. Previous reports also noted that Nrf2 is a positive regulator of MDR genes in NT2 cells. But we, for the first time, report that Regulatory factor X1 (RFX1), a pleiotropic transcription factor, negatively regulates the major MDR genes, Abcg2, Abcb1, Abcc1, and Abcc2, in NT2 cells. The levels of RFX1 in undifferentiated NT2 cells were found to be very low, which significantly increased upon RA-induced differentiation. Ectopic expression of RFX1 reduced the levels of transcripts corresponding to MDRs and stemness-associated genes. Interestingly, Bexarotene, an RXR agonist that acts as an inhibitor of Nrf2-ARE signaling, could increase the transcription of RFX1. Further analysis revealed that the RFX1 promoter has binding sites for RXRα, and upon Bexarotene exposure RXRα could bind and activate the RFX1 promoter. Bexarotene, alone or in combination with Cisplatin, could inhibit many cancer/CSC-associated properties in NT2 cells. Also, it significantly reduced the expression of drug resistance proteins and made the cells sensitive towards Cisplatin. Our study proves that RFX1 could be a potent molecule to target MDRs, and Bexarotene can induce RXRα-mediated RFX1 expression, therefore, would be a better chemo-assisting drug during therapy.

Prognostic risk factor analysis and nomogram construction for primary liver cancer in elderly patients based on SEER database.

OBJECTIVE: To evaluate the risk factors and construct a nomogram model for the prognosis of primary liver cancer in the elderly based on the data from the US SEER database. METHODS: The latest data of patients with primary liver cancer were extracted from the SEER database using SEER*STAT software, and the required variables were included. The data were screened and then divided into a training cohort and a validation cohort. A nomogram model was constructed by screening the variables through univariate and multivariate Cox analysis. The C-Index, ROC and calibration curves were used for model evaluation. RESULTS: A total of 10 824 eligible cases from 2004 to 2017 were extracted, among which, 7757 cases were included in the training cohort and 3247 in the validation cohort. The C-Index of the model was 0.747 (in the training cohort) and 0.773 (in the validation cohort). The 3-year area under the curve (AUCs) of the training and the validation cohorts were 0.760 and 0.750, and the 5-year AUCs of the two cohorts were 0.761 and 0.748. The calibration curves showed an ideal calibration of the constructed model. CONCLUSIONS: The nomogram model constructed followed by Cox regression analysis showed moderate calibration and discrimination property, and can provide reference to a certain extent for furture clinical application of primary liver cancer in the elderly.

Early extrahepatic recurrence as a pivotal factor for survival after hepatocellular carcinoma resection: A 15-year observational study.

BACKGROUND: Surgical resection is one of the most widely used modalities for the treatment of hepatocellular carcinoma (HCC). Early extrahepatic recurrence (EHR) of HCC after surgical resection is considered to be closely associated with poor prognosis. However, data regarding risk factors and survival outcomes of early EHR after surgical resection remain scarce. AIM: To investigate the clinical features and risk factors of early EHR and elucidate its association with survival outcomes. METHODS: From January 2004 to December 2019, we enrolled treatment-naïve patients who were ≥ 18 years and underwent surgical resection for HCC in two tertiary academic centers. After excluding patients with tumor types other than HCC and/or ineligible data, this retrospective study finally included 779 patients. Surgical resection of HCC was performed according to the physicians' decisions and the EHR was diagnosed based on contrast-enhanced computed tomography or magnetic resonance imaging, and pathologic confirmation was performed in selected patients. Multivariate Cox regression analysis was performed to identify the variables associated with EHR. RESULTS: Early EHR within 2 years after surgery was diagnosed in 9.5% of patients during a median follow-up period of 4.4 years. The recurrence-free survival period was 5.2 mo, and the median time to EHR was 8.8 mo in patients with early EHR. In 52.7% of patients with early EHR, EHR occurred as the first recurrence of HCC after surgical resection. On multivariate analysis, serum albumin < 4.0 g/dL, serum alkaline phosphatase > 100 U/L, surgical margin involvement, venous and/or lymphatic involvement, satellite nodules, tumor necrosis detected by pathology, tumor size ≥ 7 cm, and macrovascular invasion were determined as risk factors associated with early EHR. After sub-categorizing the patients according to the number of risk factors, the rates of both EHR and survival showed a significant correlation with the risk of early EHR. Furthermore, multivariate analysis revealed that early EHR was associated with substantially worse survival outcomes (Hazard ratio, 6.77; 95% confidence interval, 4.81-9.52; P < 0.001). CONCLUSION: Early EHR significantly deteriorates the survival of patients with HCC, and our identified risk factors may predict the clinical outcomes and aid in postoperative strategies for improving survival.

Prognostic and clinicopathological significance of transcription factor c-Jun in hypopharyngeal squamous cell carcinoma: a 3-year follow-up retrospective study.

PURPOSE: To investigate the expression and prognostic value of c-Jun in hypopharyngeal squamous cell carcinoma (HPSCC). METHODS: A retrospective study was performed on a cohort of 99 HPSCC patients. The expression of c-Jun and phosphorylated-c-Jun (p-c-Jun) was evaluated via immunohistochemistry (IHC) staining. Overall survival (OS) and progression-free survival (PFS) were assessed using Kaplan-Meier method and multivariate Cox regression analysis. RESULTS: The high expression of c-Jun and p-c-Jun in HPSCC accounted for 60.61% and 16.16%, respectively. High expression of c-Jun was closely associated with cT stage (p = 0.0401), tumor size (p = 0.0276), number of lymph node metastases (p = 0.0205) and pathological differentiation (p = 0.0108). The expression of c-Junhigh (p = 0.0043), p-c-Junhigh (p = 0.0376) and c-Junhigh/p-c-Junhigh were closely associated with poor OS. The Cox proportional multivariate hazard model revealed that lymphovascular invasion and c-Jun expression were independent influencing factors of OS in HPSCC patients. CONCLUSION: Our findings suggest that c-Jun is a reliable prognostic factors in HPSCC patients.

HLA-DRB1: A new potential prognostic factor and therapeutic target of cutaneous melanoma and an indicator of tumor microenvironment remodeling.

Cutaneous melanoma (CM) is the most common skin cancer and one of the most aggressive cancers and its incidence has risen dramatically over the past few decades. The tumor microenvironment (TME) plays a crucial role in the occurrence and development of cutaneous melanoma. Nevertheless, the dynamics modulation of the immune and stromal components in the TME is not fully understood. In this study, 471 CM samples were obtained from TCGA database, and the ratio of tumor-infiltrating immune cells (TICs) in the TME were estimated using the ESTIMATE algorithms and CIBERSORT computational method. The differently expressed genes (DEGs) were applied to GO and KEGG function enrichment analysis, establishment of protein-protein interaction (PPI) network and univariate Cox regression analysis. Subsequently, we identified a predictive factor: HLA-DRB1 (major histocompatibility complex, class II, DR beta 1) by the intersection analysis of the hub genes of PPI network and the genes associated with the prognosis of the CM patients obtained by univariate Cox regression analysis. Correlation analysis and survival analysis showed that the expression level of HLA-DRB1 was negatively correlated with the Stage of the patients while positively correlated with the survival, prognosis and TME of melanoma. The GEPIA web server and the representative immunohistochemical images of HLA-DRB1 in the normal skin tissue and melanoma tissue from the Human Protein Atlas (HPA) database were applied to validate the expression level of HLA-DRB1. CIBERSORT analysis for the ratio of TICs indicated that 9 types of TICs were positively correlated with the expression level of HLA-DRB1 and only 4 types of TICs were negatively correlated with the expression level of HLA-DRB1. These results suggested that the expression level of HLA-DRB1 may be related to the immune activity of the TME and may affect the prognosis of CM patients by changing the status of the TME.

Identification of RFX5 as prognostic biomarker and associated with immune infiltration in stomach adenocarcinoma.

BACKGROUND: Regulatory factor X (RFX) gene family is a series of encodes transcription factors with a highly conserved DNA binding domain. RFXs played a vital role in the development and progression of cancer. However, the significance of RFXs in stomach adenocarcinoma (STAD) has not been fully clarified. METHODS: Online bioinformatics tools such as GSCALite, Kaplan-Meier Plotter, TIMER, LinkedOmics were used to explore the immunomodulatory function and clinical value of RFXs in STAD. RESULTS: The mRNA level of RFX1, RFX3, RFX4, RFX5, RFX7 and RFX8 was significantly elevated in STAD tissue versus adjacent normal tissue. We also summarize the copy number variation, single nucleotide variants and drug sensitivity of RFXs in STAD. Prognostic analysis indicated that STAD patients with high RFX5 and RFX7 expression had a better overall survival, first progression, and post-progression survival. Moreover, RFX5 expression was significantly associated with the abundance of immune cells, the expression of immune biomarkers and tumor mutational burden score in STAD. Functional enrichment analysis revealed that RFX5 and its related genes were mainly involved in T cell activation, antigen receptor-mediated signaling pathway, cell adhesion molecules, and Th17 cell differentiation. Validation study further verified the expression and prognosis of RFX5 in STAD. Further univariate and multivariate analyses suggested that pathological stage and RFX5 could be a potential independent prognostic factor for STAD. CONCLUSIONS: RFX5 was a candidate prognostic biomarker and associated with immune infiltration in STAD.

Hsa_circ_0030042 Ameliorates Oxidized Low-Density Lipoprotein-Induced Endothelial Cell Injury via the MiR-616-3p/RFX7 Axis.

Atherosclerosis (AS) is a common etiology of cardiovascular disease. As an emerging functional biomarker, circular RNAs (circRNAs) are involved in various diseases, including cardiovascular disease. However, the mechanism of action of circ_0030042 in AS has not been reported.Human umbilical vein endothelial cells (HUVECs) stimulated by ox-LDL served as a cellular model of AS. Gene expression was detected using quantitative real-time polymerase chain reaction. The influence of circ_0030042 on cell viability, proliferation, and apoptosis was verified using Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, and flow cytometry assays. An enzyme-linked immunosorbent assay was performed to measure the contents of tumor necrosis factor-α, interleukin (IL) -6, and IL-1ß. Western blot assay was utilized to determine the protein levels of Bax, Bcl-2, PCNA, and regulatory factor X 7 (RFX7). The interrelationship between miR-616-3p and circ_0030042 or RFX7 was validated using dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays.The expression of circ_0030042 was downregulated in ox-LDL-induced HUVECs. It was found that overexpression of circ_0030042 facilitated cell proliferation, repressed apoptosis, and reduced the level of inflammatory factors in HUVECs. Circ_0030042 and miR-616-3p had a targeting relationship, and the miR-616-3p mimic eliminated the effects of overexpressed circ_0030042 on ox-LDL-induced HUVECs. RFX7 was a downstream gene of miR-616-3p and was lowly expressed in ox-LDL-induced HUVECs. The miR-616-3p inhibitor stimulated cell proliferation, arrested apoptosis, and caused a decline in the levels of inflammatory factors, whereas knockdown of RFX7 abolished the effects.Circ_0030042 weakened ox-LDL-induced HUVEC injury by regulating the miR-616-3p/RFX7 pathway, thereby influencing AS progression. Circ_0030042 is likely to be a potential biomarker for the future treatment of patients with AS.

The Norwich trabeculectomy study: risk factor analysis for the development of adverse, thin cystic blebs.

PURPOSE: To investigate potential risk factors, particularly antimetabolite choice, with regard to the development of adverse bleb morphology in eyes that had undergone trabeculectomy surgery. METHODS: A single-centre, observational cohort study of 631 consecutive eyes, which had undergone trabeculectomy over an 11-year period. For each case, bleb morphology was recorded at 2 years, and its association with the per-operative antimetabolite as well as potential confounding risk factors was analysed using univariate (unadjusted) and multivariate (adjusted) logistical regression analyses to identify those that could contribute to the development of adverse blebs. A standard protocol for 5-fluorouracil and mitomycin-C utilization was employed in the majority of cases. RESULTS: When 5-fluorouracil was used (n = 257), 24% of patients formed cystic or partially cystic blebs, whereas with mitomycin-C (n = 299), only 12% formed such adverse blebs, the difference being statistically significant (OR = 3.54, p = 0.002 unadjusted; OR = 7.49, p = 0.00 adjusted). Of the other potential confounding factors, care within the private sector (OR = 0.30 p = 0.02) and a history of previous ocular surgery involving a conjunctival incision were identified as potential risk factors for the formation of adverse cystic blebs (OR = 0.28, p = 0.02). CONCLUSIONS: Modern use of mitomycin-C appeared to be better than 5-fluorouracil as an adjunctive antimetabolite used at the time of trabeculectomy, with respect to the development of preferable final bleb morphology. The only potential preoperative risk factors found to be significant with respect to adverse cystic bleb development were care in the private health sector and previous ocular surgery involving a conjunctival incision.

Induction of functional neutrophils from mouse fibroblasts by thymidine through enhancement of Tet3 activity.

Neutrophils are derived from bone marrow hematopoietic stem cells (HSCs) and are the largest population among circulating white blood cells in humans, acting as the first line of defense against invading pathogens. Whether neutrophils can be generated by transdifferentiation strategies is unknown. Here, we show that thymidine induces the conversion of mouse fibroblasts to neutrophils. Induced neutrophils (iNeus) showed antibacterial effects and did not undergo malignant transformation in vivo. Importantly, iNeu transplantation cured neutropenia in mice in vivo. Mechanistically, thymidine mediates iNeu conversion by enhancing Tet3 activity. Tet3 initiates the expression of the neutrophil fate decision factors Cebpδ and Rfx1 that drive the transdifferentiation of mouse fibroblasts to neutrophils. Therefore, the induction of functional neutrophils by chemicals may provide a potential therapeutic strategy for patients with neutropenia patients and infectious diseases.Fibroblasts; Neutrophils; Thymidine; Transdifferentiation; Tet3.

KMT5A downregulation participated in High Glucose-mediated EndMT via Upregulation of ENO1 Expression in Diabetic Nephropathy.

Diabetic nephropathy (DN) has become the common and principal microvascular complication of diabetes that could lead to end-stage renal disease. It was reported endothelial-to-mesenchymal transition (EndMT) in glomeruli plays an important role in DN. Enolase1 (ENO1) and Lysine Methyltransferase 5A (KMT5A) were found to modulate epithelial-to-mesenchymal transition in some situations. In the present study, we speculated KMT5A regulates ENO1 transcript, thus participating in hyperglycemia-induced EndMT in glomeruli of DN. Our study represented vimentin, αSMA and ENO1 expression elevated, and CD31 expression decreased in glomeruli of DN participants and rats. In vitro, high glucose induced EndMT by increase of ENO1 levels. Moreover, high glucose downregulated KMT5A levels and increased regulatory factor X1 (RFX1) levels. KMT5A upregulation or si-RFX1 decreased high glucose-induced ENO1 expression and EndMT. RFX1 overexpression- or sh-KMT5A-induced EndMT was attenuated by si-ENO1. Further, the association between KMT5A and RFX1 was verified. Furthermore, histone H4 lysine20 methylation (the direct target of KMT5A) and RFX1 positioned on ENO1 promoter region. sh-KMT5A enhanced positive action of RFX1 on ENO1 promoter activity. KMT5A reduction and RFX1 upregulation were verified in glomeruli of DN patients and rats. KMT5A associated with RFX1 to modulate ENO1, thus involved in hyperglycemia-mediated EndMT in glomeruli of DN.

RFX1-mediated CCN3 induction that may support chondrocyte survival under starved conditions.

Cellular communication network factor (CCN) family members are multifunctional matricellular proteins that manipulate and integrate extracellular signals. In our previous studies investigating the role of CCN family members in cellular metabolism, we found three members that might be under the regulation of energy metabolism. In this study, we confirmed that CCN2 and CCN3 are the only members that are tightly regulated by glycolysis in human chondrocytic cells. Interestingly, CCN3 was induced under a variety of impaired glycolytic conditions. This CCN3 induction was also observed in two breast cancer cell lines with a distinct phenotype, suggesting a basic role of CCN3 in cellular metabolism. Reporter gene assays indicated a transcriptional regulation mediated by an enhancer in the proximal promoter region. As a result of analyses in silico, we specified regulatory factor binding to the X-box 1 (RFX1) as a candidate that mediated the transcriptional activation by impaired glycolysis. Indeed, the inhibition of glycolysis induced the expression of RFX1, and RFX1 silencing nullified the CCN3 induction by impaired glycolysis. Subsequent experiments with an anti-CCN3 antibody indicated that CCN3 supported the survival of chondrocytes under impaired glycolysis. Consistent with these findings in vitro, abundant CCN3 production by chondrocytes in the deep zones of developing epiphysial cartilage, which are located far away from the synovial fluid, was confirmed in vivo. Our present study uncovered that RFX1 is the mediator that enables CCN3 induction upon cellular starvation, which may eventually assist chondrocytes in retaining their viability, even when there is an energy supply shortage.

Adeno-Associated Virus D-Sequence-Mediated Suppression of Expression of a Human Major Histocompatibility Class II Gene: Implications in the Development of Adeno-Associated Virus Vectors for Modulating Humoral Immune Response.

A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.

Downregulation of RFX1 predicts poor prognosis of patients with small hepatocellular carcinoma.

OBJECTIVE: Regulatory factor X1 (RFX1) deletion has been reported to be correlated with poor prognosis of some types of cancer. The present study aimed to investigate the prognostic value of RFX1 in HCC, especially in small hepatocellular carcinoma. METHODS: Immunohistochemical assay was used to investigate RFX1 expression in 221 HCC tissues and another validation cohort of 71 small HCC samples. We also performed in vitro experiments to investigate if RFX1 regulated invasive capacity of HCC cells and expression of epithelial-mesenchymal transition (EMT) markers. RESULTS: We found that RFX1 expression was significantly lower in HCC tissues compared to the corresponding non-tumor tissues. Further survival analysis suggested that the downregulation of RFX1 correlated with poor prognosis and a high recurrence risk in HCC patients, particularly in small HCC patients. Furthermore, another validation cohort of small HCC samples confirmed that downregulation of RFX1 in HCC tissues predicted high recurrence risk and poor prognosis for early stage HCC patients. In vitro studies suggested that knocking down RFX1 facilitated HCC cell invasion, while overexpression of RFX1 reduced the invasion of HCC cells. Western blot assays also indicated that RFX1 regulated expression of some EMT markers. Knocking down RFX1 decreased E-cadherin and increased vimentin expression, while RFX1 overexpression enhanced E-cadherin and decreased vimentin expression. CONCLUSIONS: Our study demonstrated that RFX1 downregulation is a new predictive marker of high recurrence risk and poor prognosis of HCC; It has potential to help guide treatment for postoperative HCC patients, especially for small HCC patients.

RFX1 participates in doxorubicin-induced hepatitis B virus reactivation.

Cytotoxic chemotherapy drugs, including doxorubicin, can directly promote hepatitis B virus (HBV) replication, but the mechanism has not been fully clarified. This study investigated the potential mechanism underlying the cytotoxic chemotherapy-mediated direct promotion of HBV replication. We found that HBV replication and regulatory factor X box 1 gene (RFX1) expression were simultaneously promoted by doxorubicin treatment. The amount of RFX1 bound to the HBV enhancer I was significantly increased under doxorubicin treatment. Furthermore, the activity of doxorubicin in promoting HBV replication was significantly attenuated when the expression of endogenous RFX1 was knocked down, and the EP element of HBV enhancer I, an element that mediated the binding of RFX1 and HBV enhancer I, was mutated. In addition, two different sequences of the conserved EP element were found among HBV genotypes A-D, and doxorubicin could promote the replication of HBV harboring either of the conserved EP elements. Here, a novel pathway in which doxorubicin promoted HBV replication via RFX1 was identified, and it might participate in doxorubicin-induced HBV reactivation. These findings would be helpful in preventing HBV reactivation during anticancer chemotherapy.

IL-6/STAT3 pathway induced deficiency of RFX1 contributes to Th17-dependent autoimmune diseases via epigenetic regulation.

Epigenetic modifications affect the differentiation of T cell subsets and the pathogenesis of autoimmune diseases, but many mechanisms of epigenetic regulation of T cell differentiation are unclear. Here we show reduced expression of the transcription factor RFX1 in CD4+ T cells from patients with systemic lupus erythematosus, which leads to IL-17A overexpression through increased histone H3 acetylation and decreased DNA methylation and H3K9 tri-methylation. Conditional deletion of Rfx1 in mice exacerbates experimental autoimmune encephalomyelitis and pristane-induced lupus-like syndrome and increases induction of Th17 cells. In vitro, Rfx1 deficiency increases the differentiation of naive CD4+ T cells into Th17 cells, but this effect can be reversed by forced expression of Rfx1. Importantly, RFX1 functions downstream of STAT3 and phosphorylated STAT3 can inhibit RFX1 expression, highlighting a non-canonical pathway that regulates differentiation of Th17 cells. Collectively, our findings identify a unique role for RFX1 in Th17-related autoimmune diseases.

RFX1 and RFX3 Transcription Factors Interact with the D Sequence of Adeno-Associated Virus Inverted Terminal Repeat and Regulate AAV Transduction.

Adeno-associated virus (AAV) transduction efficiency depends on the way in which cellular proteins process viral genomes in the nucleus. In this study, we have investigated the binding of nuclear proteins to the double stranded D (dsD) sequence of the AAV inverted terminal repeat (ITRs) by electromobility shift assay. We present here several lines of evidence that transcription factors belonging to the RFX protein family bind specifically and selectively to AAV2 and AAV1 dsD sequences. Using supershift experiments, we characterize complexes containing RFX1 homodimers and RFX1/RFX3 heterodimers. Following transduction of HEK-293 cells, the AAV genome can be pulled-down by RFX1 and RFX3 antibodies. Moreover, our data suggest that RFX proteins which interact with transcriptional enhancers of several mammalian DNA viruses, can act as regulators of AAV mediated transgene expression.

Regulatory factor X1 depresses ApoE-dependent Aß uptake by miRNA-124 in microglial response to oxidative stress.

Decreased proteolytic clearance of soluble amyloid ß (Aß) in microglia affects Aß accumulation on Alzheimer's disease progression. However, the potential molecular mechanism by which microglial Aß uptake is regulated remains unclear. In this study, we identified a microRNA, miR-124, that was down-regulated in aging with a function in regulating apolipoprotein E (ApoE)-dependent Aß uptake by targeting regulatory factor X1 (RFX1) transcripts on BV2 microglia cell. Decreased expression of miRNA-124 in BV2 cells exposed to mild hydrogen peroxide increased RFX1 protein level and decreased the expression of ApoE, a gene which has been suggested to enhance cellular Aß uptake in microglia. We also identified a miR-124 binding site in the 3'-UTR of RFX1 mRNA and a RFX1 binding site in the first intron of ApoE gene. Furthermore, interfering this signaling pathway by knocking down RFX1 significantly improved Aß uptake in BV2 cells. These data demonstrate the mechanism through which decreased miR-124 expression under oxidative stress slowed Aß uptake and suggest that RFX1 might be a target for improving Aß clearance during aging.

CCAR2 Is Required for Proliferation and Tumor Maintenance in Human Squamous Cell Carcinoma.

CCAR2 is a widely expressed protein involved in the regulation of a variety of transcriptional complexes. High expression of CCAR2 correlates with poor outcomes in many human tumor types such as squamous cell carcinoma (SCC). Paradoxically, loss of Ccar2 in the mouse results in an increased tumor burden, suggesting that CCAR2 may in fact function as a tumor suppressor. This tumor suppressor function is dependent on p53, a protein that is inactivated in the vast majority of SCC tumors, leaving the role of CCAR2 in p53-null tumors unclear. We sought to identify p53-independent CCAR2 functions in SCC and to examine its role in tumorigenesis. We found that CCAR2 is highly overexpressed in p53-deficient SCC cell lines compared with normal primary keratinocytes due to increased protein stability. We identify a role for CCAR2 in promoting the stability of the transcription factors RFX1 and CREB1, which are both required for proliferation. Finally, we show that CCAR2 is required for proliferation in vitro and in established SCC tumors in vivo. Our data suggest an important role for CCAR2 in maintaining cell cycle progression and promoting SCC tumorigenesis.