Sine Oculis Research Articles

SIX4 activates Akt and promotes tumor angiogenesis

Angiogenesis plays important roles in solid tumors progression. Growth factors such as vascular endothelial growth factors (VEGFs) can induce angiogenesis and hypoxia promotes the expression of VEGFs through activating hypoxia-inducible factor 1 (HIF-1α). However, the regulation of HIF-1α still not been fully understood. Here, we demonstrate that the Sine Oculis Homeobox Homolog 4 (SIX4) is up-regulated in colorectal cancer (CRC) and high expression of SIX4 predicts a poor prognosis. Overexpression of SIX4 enhances tumor growth and angiogenesis in vitro and in vivo, while knockdown of SIX4 inhibits tumor growth and angiogenesis. Furthermore, we show that SIX4 increases the expression of VEGF-A by coordinating with the HIF-1α. Mechanically, we explore that SIX4 up-regulates the expression of HIF-1α depending on Akt activation. Collectively, we demonstrate that SIX4 is functional in regulating tumor angiogenesis and SIX4 might be used as anti-angiogenic therapy in CRC.

Abstract 1733: Role of the SIX1 oncofetal protein in endometrial basal cell metaplasia and carcinogenesis following neonatal exposure to diethylstilbestrol

Abstract Early-life exposure to estrogenic chemicals can increase cancer risk, likely by disrupting normal patterns of cellular differentiation. In a model of this process, female mice exposed to the potent synthetic estrogen diethylstilbestrol (DES) on postnatal days 1-5 develop endometrial adenobasal carcinoma as adults. Neoplastic glands are comprised of abnormal populations of basal cells (cytokeratin (CK)14+), luminal cells (CK18+), and low numbers of "mixed” basal/luminal cells (CK14+/18+), all of which express the oncofetal protein sine oculis homeobox 1 (SIX1). We hypothesized that DES-induced SIX1 expression is necessary for aberrant endometrial differentiation patterns and carcinogenesis. To test this hypothesis, a conditional knockout model was generated in which floxed Six1 was excised in the uterus using progesterone receptor (Pgr) cre. The most prominent change in DES-exposed SIX1 knockout (DES-Six1d/d) mice was the absence of basal cells in the uterine horns. There was a greater than 10-fold decrease in CK14 labeling in the uterine horns of DES-Six1d/d mice compared to DES-exposed SIX1 wildtype (DES-Six1+/+) mice as determined by quantitative image analysis. However, DES-Six1d/d mice exhibited a 42% increase in cancer incidence compared to DES-Six1+/+ mice at 6 months of age (16/18 DES-Six1d/d vs. 7/15 DES-Six1+/+). Interestingly, mixed cells were still present in DES-Six1d/d mice. These findings demonstrate that SIX1 is a cellular differentiation factor necessary for DES-induced basal cells but not mixed cell development or cancer. Furthermore, these data suggest that DES-induced SIX1 expression decreases endometrial carcinogenesis by facilitating basal cell differentiation. Studies investigating the mixed cell population as a putative cancer progenitor cell population are ongoing. Interestingly, mixed cells were present in 35% (63/181) of malignant human endometrial tissue biopsies and in 0% (0/29) of normal endometrial tissue biopsies, suggesting that the DES mouse model has significant similarities to human endometrial cancer. Citation Format: Alisa A. Suen, Wendy N. Jefferson, Charles E. Wood, Anna C. Kenan, Carmen J. Williams. Role of the SIX1 oncofetal protein in endometrial basal cell metaplasia and carcinogenesis following neonatal exposure to diethylstilbestrol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1733.

Upregulation of sine oculis homeobox homolog 3 is associated with proliferation, invasion, migration, as well as poor prognosis of esophageal cancer.

Esophageal cancer (EC) is a common cancer worldwide. Sine oculis homeobox homolog (SIX3) is a human transcription factor that regulates the progression of vertebrate eye and fetal forebrain. However, studies on the function of SIX3 in human tumorigenesis remain rare. In this study, we aim to evaluate the role and the significance of SIX3 in EC. The TCGA database and clinical samples were used to assess the expression of SIX3 in EC patients. The Kaplan-Meier method and Cox's proportional hazards model were performed to analyze the correlations between SIX3 expression and EC clinical outcomes. The expressions of SIX3 in EC cells were measured by quantitative reverse transcription PCR analysis. The cell proliferation was detected using cell counting kit-8 and colony formation assay. The migration and invasion capacity of EC cells were evaluated using wound healing and Transwell methods. Western blot assay was used to measure the alterations in some important protein expression levels in the PI3K/Akt signaling pathway. We found that SIX3 was highly expressed in the EC tissues and cells. In addition, high expression of SIX3 was related to poor survival. The knockdown of SIX3 significantly inhibited the proliferation, migration, and invasion of ECA109 cells. A similar pattern was also found in the proliferation and migration of SKGT-4 cell line. The expression levels of some key proteins in the PI3K/Akt signaling pathway were obviously decreased after cells were transfected with si-SIX3, possibly resulting in PI3K/AKT signaling inactivation. In addition, E-cadherin and N-cadherin showed some change. Collectively, the results shed light on a potentially promoting role of SIX3 in human EC. Thus, SIX3 might be considered a novel prognostic biomarker and therapeutic target for EC patients.

SIX4 acts as a master regulator of oncogenes that promotes tumorigenesis in non-small-cell lung cancer cells

A number of homeobox genes are implicated in the malignancy of various cancers. Here, we investigated the role of the homeobox gene SIX4 in non-small-cell lung cancer (NSCLC). The sine oculis homeobox (SIX4) gene was found to be highly expressed at both mRNA and protein levels in NSCLC tumor tissues as compared with matching normal counterparts. In this study, the SIX4 gene of two human NSCLC cell lines (A549 and PC9) was overexpressed or silenced using the lentiviral system. We evaluated the malignancy-associated phenotype of transfected cells, which demonstrated that exogenous expression of the SIX4 gene greatly enhanced the proliferation, migration, and invasion of NSCLC cells. The opposite was true in the SIX4-silenced cells. Transcriptomic profiling analysis revealed that the SIX4 gene modulated the expression of hundreds of downstream target genes in a cell context-dependent manner. Most notably, the SIX4 gene controls the expression of crucial genes with evidently oncogenic function. We conclude that SIX4 plays an oncogenic role and may be potentially utilized as a diagnostic and therapeutic marker for NSCLC.

MicroRNA-23b suppresses cervical cancer biological progression by directly targeting six1 and affecting epithelial-to-mesenchymal transition and AKT/mTOR signaling pathway.

To elucidate the effects and mechanism of microRNA-23b (miR-23b) in cervical cancer (CC) progression. Fifty-six pairs of CC tissue samples and matched para-carcinoma tissue samples were collected. Meanwhile, human normal cervical epithelial cell and CC cell lines were cultured. The abilities of cell proliferation and migration were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays and transwell assays. The correlation between sine oculis homeobox 1 (six1) and miR-23b was clarified by dual-luciferase reporter assay. The relative protein and mRNA expression were detected by quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and Western blot. In addition, Xenograft tumor formation assay was performed in this study. MiR-23b was remarkably down-regulated in CC and the low miR-23b expressions were associated with the poor prognosis and worse OS of CC patients. Additionally, the functional assays demonstrated that miR-23b overexpression obviously repressed CC cell proliferation, invasion and migration abilities through the regulation of the AKT/mTOR pathway and the epithelial-to-mesenchymal transition (EMT) progress. Moreover, the luciferase reporter assay indicated that six1 was one functional target for miR-23b in CC cells, indicating that the inhibitory functions of miR-23b in CC cells were partially regulated by six1. Moreover, miR-23b restoration could prominently repress tumor growth in vivo. MiR-23b suppressed CC progression via directly targeting six1 and affecting AKT/mTOR signaling pathway as well as EMT progress. Therefore, miR-23b/six1 may be promising biomarkers for CC diagnosis and therapy.

Six1 regulates leukemia stem cell maintenance in acute myeloid leukemia.

Molecular genetic changes in acute myeloid leukemia (AML) play crucial roles in leukemogenesis, including recurrent chromosome translocations, epigenetic/spliceosome mutations and transcription factor aberrations. Six1, a transcription factor of the Sine oculis homeobox (Six) family, has been shown to transform normal hematopoietic progenitors into leukemia in cooperation with Eya. However, the specific role and the underlying mechanism of Six1 in leukemia maintenance remain unexplored. Here, we showed increased expression of SIX1 in AML patients and murine leukemia stem cells (c‐Kit+ cells, LSCs). Importantly, we also observed that a higher level of Six1 in human patients predicts a worse prognosis. Notably, knockdown of Six1 significantly prolonged the survival of MLL‐AF9‐induced AML mice with reduced peripheral infiltration and tumor burden. AML cells from Six1‐knockdown (KD) mice displayed a significantly decreased number and function of LSC, as assessed by the immunophenotype, colony‐forming ability and limiting dilution assay. Further analysis revealed the augmented apoptosis of LSC and decreased expression of glycolytic genes in Six1 KD mice. Overall, our data showed that Six1 is essential for the progression of MLL‐AF9‐induced AML via maintaining the pool of LSC.

MiR-150-5p regulates melanoma proliferation, invasion and metastasis via SIX1-mediated Warburg Effect

Aerobic glycolysis is a hallmark of cancer. Sine oculis homeobox 1 (SIX1), a key transcription factor in terms of regulating aerobic glycolysis (the Warburg Effect), plays a critical role in tumorigenesis of various cancer types, including breast cancer, liver cancer, and lung cancer. However, the upstream regulating mechanisms of SIX1 in melanoma remain to be determined. MicroRNAs (miRNAs) have emerged as key regulators in tumorigenesis and progression. Here, we initially showed that microRNA-150-5p (miR-150-5p) inhibits SIX1 expression by directly targeting its 3′-UTR in melanoma cells. miR-150-5p suppressed melanoma cell proliferation, migration, and invasion through inhibition of SIX1. Mechanistically, miR-150-5p dampens glycolysis by decreasing the glucose uptake, lactate production, ATP generation, and extracellular acidification rate (ECAR), and increasing oxygen consumption rate (OCR) by targeting SIX1. Importantly, glycolysis regulated by miR-150-5p/SIX1 axis is critical for its regulation of melanoma growth and metastasis both in vitro and in vivo. Collectively, our study demonstrates the importance of miR-150-5p/SIX1 axis in melanoma, which could be a promising therapeutic target in melanoma.

MiR-140-5p induces cell apoptosis and decreases Warburg effect in chronic myeloid leukemia by targeting SIX1.

microRNAs (miRNA), as tumor suppressors or oncogenes, are involved in modulating cancer cell behavior, including cell proliferation and apoptosis. The miR-140-5p acts as a tumor suppressor in several tumors, but the role of miR-140-5p in chronic myeloid leukemia (CML) remains unclear. Here, we investigated the suppression of miR-140-5p in CML patients and CML cell lines using quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH). Overexpression miR-140-5p in CML cells significantly inhibited cell proliferation as revealed by the CCK-8 assay and promoted cell apoptosis as revealed by flow cytometry. Moreover, the sine oculis homeobox 1 (SIX1) gene had been confirmed as a direct target of miR-140-5p using bioinformatics analysis and luciferase reporter assays. Overexpression of miR-140-5p decreased the SIX1 protein level in CML cells. SIX1 mRNA and protein levels were significantly up-regulated in CML patients and CML cell lines. Knockdown of SIX1 expression significantly inhibited CML cell proliferation and promoted cell apoptosis. Furthermore, SIX1 as a transcriptional factor positively regulated pyruvate kinase isozyme type M2 (PKM2) expression and played an important role in the Warburg effect. In addition, these findings indicated that miR-140-5p functions as a tumor suppressor and plays a critical role in CML cell apoptosis and metabolism by targeting SIX1. Moreover, the miR-140-5p/SIX1 axis may be a potential therapeutic target in CML.

A NIK-SIX signalling axis controls inflammation by targeted silencing of non-canonical NF-κB.

SUMMARYThe non-canonical NF-κB signaling cascade is essential for lymphoid organogenesis, B-cell maturation, osteoclast differentiation, and inflammation in mammals1,2, whereas dysfunction of this system is associated with human diseases, including immunological disorders and cancer3–6. While controlled expression of NF-κB Inducing Kinase (NIK) is the rate-limiting step in non-canonical NF-κB activation2,7, mechanisms of inhibition remain largely unknown. Here, we report the identification of the sine oculis homeobox homolog family transcription factors SIX1 and SIX2 as essential inhibitory components of the non-canonical NF-κB signaling pathway. The developmentally silenced SIX-proteins are reactivated in differentiated macrophages by NIK-mediated suppression of the ubiquitin proteasome pathway. Consequently, SIX1 and SIX2 target a subset of inflammatory gene promoters and directly inhibit RelA and RelB trans-activation function in a negative feedback circuit. In support of a physiologically pivotal role for SIX-proteins in host immunity, human SIX1 transgene suppressed inflammation and promoted the recovery of mice from endotoxic shock. In addition, SIX1 and SIX2 protected RAS/p53-driven lung adenocarcinoma cells from inflammatory cell death induced by SMAC-mimetic chemotherapeutic agents, small-molecule activators of the non-canonical NF-κB pathway. Collectively, our study reveals a NIK-SIX signaling axis that fine-tunes inflammatory gene expression programs under both physiological and pathological conditions.

Six6 and Six7 coordinately regulate expression of middle-wavelength opsins in zebrafish

Color discrimination in the vertebrate retina is mediated by a combination of spectrally distinct cone photoreceptors, each expressing one of multiple cone opsins. The opsin genes diverged early in vertebrate evolution into four classes maximally sensitive to varying wavelengths of light: UV (SWS1), blue (SWS2), green (RH2), and red (LWS) opsins. Although the tetrachromatic cone system is retained in most nonmammalian vertebrate lineages, the transcriptional mechanism underlying gene expression of the cone opsins remains elusive, particularly for SWS2 and RH2 opsins, both of which have been lost in the mammalian lineage. In zebrafish, which have all four cone subtypes, rh2 opsin gene expression depends on a homeobox transcription factor, sine oculis homeobox 7 (Six7). However, the six7 gene is found only in the ray-finned fish lineage, suggesting the existence of another evolutionarily conserved transcriptional factor(s) controlling rh2 opsin expression in vertebrates. Here, we found that the reduced rh2 expression caused by six7 deficiency was rescued by forced expression of six6b, which is a six7-related transcription factor conserved widely among vertebrates. The compensatory role of six6b was reinforced by ChIP-sequencing analysis, which revealed a similar pattern of Six6b- and Six7-binding sites within and near the cone opsin genes. TAL effector nuclease-induced genetic ablation of six6b and six7 revealed that they coordinately regulate SWS2 opsin gene expression. Mutant larvae deficient for these transcription factors showed severely impaired visually driven foraging behavior. These results demonstrate that in zebrafish, six6b and six7 govern expression of the SWS2 and RH2 opsins responsible for middle-wavelength sensitivity, which would be physiologically important for daylight vision.

Role of the PRC2-Six1-miR-25 signaling axis in heart failure

The reduced expression of cardiac sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2a) is a hallmark of heart failure. We previously showed that miR-25 is a crucial transcriptional regulator of SERCA2a in the heart. However, the precise mechanism of cardiac miR-25 regulation is largely unknown. Literatures suggested that miR-25 is regulated by the transcriptional co-factor, sine oculis homeobox homolog 1 (Six1), which in turn is epigenetically regulated by polycomb repressive complex 2 (PRC 2) in cardiac progenitor cells. Therefore, we aimed to investigate whether Six1 and PRC2 are indeed involved in the regulation of the miR-25 level in the setting of heart failure. Six1 was up-regulated in the failing hearts of humans and mice. Overexpression of Six1 led to adverse cardiac remodeling, whereas knock-down of Six1 attenuated pressure overload-induced cardiac dysfunction. The adverse effects of Six1 were ameliorated by knock-down of miR-25. The epigenetic repression on the Six1 promoter by PRC2 was significantly reduced in failing hearts. Epigenetic repression of Six1 is relieved through a reduction of PRC2 activity in heart failure. Six1 up-regulates miR-25, which is followed by reduction of cardiac SERCA2a expression. Collectively, these data showed that the PRC2-Six1-miR-25 signaling axis is involved in heart failure. Our finding introduces new insight into potential treatments of heart failure.

Sine oculis homeobox 1 promotes proliferation and migration of human colorectal cancer cells through activation of Wnt/β-catenin signaling.

Sine oculis homeobox 1 (Six1) is a homeodomain transcription factor that is aberrantly expressed in a variety of human cancers, including colorectal cancer (CRC). Six1 has been reported to play a key role in the proliferation and migration of CRC cells but the underlying molecular mechanisms are still poorly characterized. In the present study, we found that Six1 overexpression promoted the proliferation and migration of CRC cells. Consistently, Six1 knockdown (KD) significantly inhibited proliferation and migration of CRC cells. In addition, we showed that Six1 promoted proliferation and migration of CRC cells through activation of Wnt/β‐catenin signaling, as evidenced by promotion of nuclear localization of β‐catenin. Silencing of β‐catenin expression with siRNA or inhibiting Wnt signaling with a specific inhibitor, xav939, significantly blocked Six1‐induced nuclear localization of β‐catenin and mitigated Six1‐promoted proliferation and migration of CRC cells. We further confirmed the involvement of β‐catenin in Six1‐promoted proliferation and migration of CRC cells by activation of Wnt signaling with lithium chloride (LiCl) in Six1 KD CRC cells and results showed that LiCl restores defective β‐catenin nuclear localization and proliferation and migration of CRC cells. Taken together, these results suggest that Six1 homeoprotein promotes the proliferation and migration of CRC cells by activating the Wnt/β‐catenin signaling pathway, and strategies targeting Six1 may be promising for the treatment of CRC.

Regulation of cancer stem cell properties by SIX1, a member of the PAX-SIX-EYA-DACH network.

The PAX-SIX-EYA-DACH network (PSEDN) is a central developmental transcriptional regulatory network from Drosophila to humans. The PSEDN is comprised of four conserved protein families; including paired box (PAX), sine oculis (SIX), eyes absent (EYA), and dachshund (DACH). Aberrant expression of PSEDN members, particularly SIX1, has been observed in multiple human cancers, where SIX1 expression correlates with increased aggressiveness and poor prognosis. In conjunction with its transcriptional activator EYA, the SIX1 transcription factor increases cancer stem cell (CSC) numbers and induces epithelial-mesenchymal transition (EMT). SIX1 promotes multiple hallmarks and enabling characteristics of cancer via regulation of cell proliferation, senescence, apoptosis, genome stability, and energy metabolism. SIX1 also influences the tumor microenvironment, enhancing recruitment of tumor-associated macrophages and stimulating angiogenesis, to promote tumor development and progression. EYA proteins are multifunctional, possessing a transcriptional activation domain and tyrosine phosphatase activity, that each contributes to cancer stem cell properties. DACH proteins function as tumor suppressors in solid cancers, opposing the actions of SIX-EYA and reducing CSC prevalence. Multiple mechanisms can lead to increased SIX1 expression, including loss of SIX1-targeting tumor suppressor microRNAs (miRs), whose expression correlates inversely with SIX1 expression in cancer patient samples. In this review, we discuss the major mechanisms by which SIX1 confers CSC and EMT features and other important cancer cell characteristics. The roles of EYA and DACH in CSCs and cancer progression are briefly highlighted. Finally, we summarize the clinical significance of SIX1 in cancer to emphasize the potential therapeutic benefits of effective strategies to disrupt PSEDN protein interactions and functions.

Installation of a Cancer Promoting WNT/SIX1 Signaling Axis by the Oncofusion Protein MLL-AF9

The oncofusion protein MLL-AF9 promotes acute myeloid leukemia (AML) by altering histone methylation and DNA accessibility to transcriptional machinery. Using antagonists of WNT signaling and genetic ablation of the WNT chaperone Wntless (WLS), we identified the homeobox domain gene sine oculis homeobox homolog 1 (Six1) to be a WNT-controlled target gene in MLL-AF9-transformed leukemic initiating cells (LICs). Wls or Six1 disruption in MLL-AF9 LICs, or exposure of these cells to WNT pathway inhibitors results in extended survival in LIC-transplanted recipient mice suggesting that WNT/SIX1 signaling is critical to MLL-AF9 transformation. Profiling of chromatin accessibility changes in MLL-AF9 LICs using ATAC-seq reveals chromatin remodeling in Six1 DNA regions that support binding of the transcriptional effector TCF7L2. Finally we show human AML cell lines with MLL-rearrangements exhibit elevated Six1 expression and are sensitive to a WNT inhibitor in a Six1-dependent fashion. Our data suggests de novo installation of a WNT/SIX1 signaling axis is critical to the development of some forms of AML and reveals how epigenetic alterations can rewire cell fate regulatory pathways with cancer-promoting consequences. Funding Statement: This work was supported by the Welch Foundation (I-1665; LL), CPRIT (RP130212; LL and CC), the National Cancer Institute (1R01 CA168761; JK), and P50-CA70907 Declaration of Interests: L. Lum, J. Lu, H. Shi, C. Chen, and L.S. Zhang are named inventors on patents covering various WNT inhibitors including those targeting PORCN. Ethics Approval Statement: All animal experiments were performed with the approval of the Institutional Animal Care & Use Committee (IACUC) at the University of Texas Southwestern Medical Center (UTSWMC).

Changes in cell fate determine the regenerative and functional capacity of the developing kidney before and after release of obstruction.

Congenital obstructive nephropathy is a major cause of chronic kidney disease (CKD) in children. The contribution of changes in the identity of renal cells to the pathology of obstructive nephropathy is poorly understood. Using a partial unilateral ureteral obstruction (pUUO) model in genetically modified neonatal mice, we traced the fate of cells derived from the renal stroma, cap mesenchyme, ureteric bud (UB) epithelium, and podocytes using Foxd1Cre, Six2Cre, HoxB7Cre, and Podocyte.Cre mice respectively, crossed with double fluorescent reporter (membrane-targetted tandem dimer Tomato (mT)/membrane-targetted GFP (mG)) mice. Persistent obstruction leads to a significant loss of tubular epithelium, rarefaction of the renal vasculature, and decreased renal blood flow (RBF). In addition, Forkhead Box D1 (Foxd1)-derived pericytes significantly expanded in the interstitial space, acquiring a myofibroblast phenotype. Degeneration of Sine Oculis Homeobox Homolog 2 (Six2) and HoxB7-derived cells resulted in significant loss of glomeruli, nephron tubules, and collecting ducts. Surgical release of obstruction resulted in striking regeneration of tubules, arterioles, interstitium accompanied by an increase in blood flow to the level of sham animals. Contralateral kidneys with remarkable compensatory response to kidney injury showed an increase in density of arteriolar branches. Deciphering the mechanisms involved in kidney repair and regeneration post relief of obstruction has potential therapeutic implications for infants and children and the growing number of adults suffering from CKD.

SIX1 is upregulated in gastric cancer and regulates proliferation and invasion by targeting the ERK pathway and promoting epithelial-mesenchymal transition.

Sine oculis homeobox homologue 1 (SIX1) is a Six class homeobox gene conserved throughout many species. It has been reported to act as an oncogene and is overexpressed in many cancers. However, the function and regulatory mechanism of SIX1 in gastric cancer (GC) remains unclear. In our study, we detected protein levels of SIX1 via immunohistochemistry (IHC) and its proliferation and invasion effects via CCK8 and transwell assays. Additionally, expression of cyclin D1, MMP2, p-ERK, and EMT-related proteins was measured by western blotting. We found that SIX1 had significantly higher expression in GC tissues and that it could promote GC cell proliferation and invasion. Also, overexpression of SIX1 increased the expression of cyclin D1, MMP2, p-ERK, and EMT-related proteins, which could all be inhibited by knocking down SIX1. In conclusion, SIX1 is upregulated in GC tissues. It can promote GC cell proliferation by targeting cyclin D1, invasion via ERK signalling, and EMT pathways by targeting MMP2 and E-cadherin. SIGNIFICANCE OF THE STUDY: Our study showed that SIX1 was upregulated in GC tissues, and promoted GC cell proliferation by targeting cyclin D1, invasion via ERK signalling, and EMT pathways by targeting MMP2 and E-cadherin. These results suggested the potential regulatory mechanism of SIX1 in proliferation and invasion of gastric cancer.

MiR‑23b‑3p promotes the apoptosis and inhibits the proliferation and invasion of osteosarcoma cells by targeting SIX1.

Osteosarcoma (OS) is the most common primary malignant bone tumor and the third most common cancer that occurs during childhood and adolescence. Increasing evidence has suggested that microRNA (miR)‑23b‑3p has an important role in OS tumorigenesis; however, the underlying molecular mechanisms remain unknown. The aim of the present study was to investigate the expression levels of miR‑23b‑3p and sine oculis homeobox homolog 1 (SIX1) in OS tissues and cell lines (MG‑63, SaOS‑2 and U2OS), as well as to observe the effects of miR‑23b‑3p on U2OS cell viability, cell cycle, apoptosis and invasive ability. The results revealed that the expression levels of miR‑23b‑3p were significantly decreased in OS tissues and cell lines compared with tumor‑adjacent normal tissues and a non‑cancerous human fetal osteoblastic cell line (hFOB1.19). To investigate the underlying mechanisms of miR‑23b‑3p in OS tumorigenesis and progression, human U2OS cell lines over‑ or under expressing miR‑23b‑3p were established. The effects of miR‑23b‑3p on U2OS cell viability, cell cycle, apoptosis and invasion properties were determined by performing Cell Counting Kit‑8, flow cytometry and Transwell invasion assays. miR‑23b‑3p was revealed to suppress cell viability, proliferation and invasion, and to enhance the levels of cell apoptosis. Furthermore, SIX1 mRNA and protein expression levels in OS tissues and cell lines were significantly upregulated when compared with tumor‑adjacent normal tissues and hFOB 1.19 cells, which suggested that SIX1 expression levels may be inversely associated with miR‑23b‑3p levels in OS. Luciferase reporter system analysis demonstrated that miR‑23b‑3p binds to the SIX1 3'‑untranslated region. miR‑23b‑3p downregulation contributed to SIX1 upregulation, which facilitated the potentiation of cyclin D1 and vascular endothelial growth factor‑C expression levels, as well as the inhibition of caspase‑3 expression. Collectively, these results suggested that miR‑23b‑3p is downregulated and SIX1 is upregulated in OS cells, and that miR‑23b‑3p inhibition may suppress the proliferation and invasion of OS cells, and contribute to cell apoptosis via negative regulation of SIX1. miR‑23b‑3p/SIX1 may therefore represent a potential target for the treatment of OS.

MiR-448-5p inhibits TGF-β1-induced epithelial-mesenchymal transition and pulmonary fibrosis by targeting Six1 in asthma.

MicroRNAs (miRNAs) are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation. The aim of this study was to explore how miR-448-5p affects airway remodeling and transforming growth factor-β1 (TGF-β1)-stimulated epithelial-mesenchymal transition (EMT) by targeting Sine oculis homeobox homolog 1 (Six1) in asthma. Asthmatic mice models with airway remodeling were induced with ovalbumin solution. MiRNA expression was evaluated using quantitative real-time polymerase chain reaction. Transfection studies of bronchial epithelial cells were performed to determine the target genes. A luciferase reporter assay system was applied to identify whether Six1 is a target gene of miR-448-5p. In the current study, we found that miR-448-5p was dramatically decreased in lung tissues of asthmatic mice and TGF-β1-stimulated bronchial epithelial cells. In addition, the decreased level of miR-448-5p was closely associated with the increased expression of Six1. Overexpression of miR-448-5p decreased Six1 expression and, in turn, suppressed TGF-β1-mediated EMT and fibrosis. Next, we predicted that Six1 was a potential target gene of miR-448-5p and demonstrated that miR-448-5p could directly target Six1. An SiRNA targeting Six1 was sufficient to suppress TGF-β1-induced EMT and fibrosis in 16HBE cells. Furthermore, the overexpression of Six1 partially reversed the protective effect of miR-448-5p on TGF-β1-mediated EMT and fibrosis in bronchial epithelial cells. Taken together, the miR-448-5p/TGF-β1/Six1 link may play roles in the progression of EMT and pulmonary fibrosis in asthma.

Brain region-dependent gene networks associated with selective breeding for increased voluntary wheel-running behavior.

Mouse lines selectively bred for high voluntary wheel-running behavior are helpful models for uncovering gene networks associated with increased motivation for physical activity and other reward-dependent behaviors. The fact that multiple brain regions are hypothesized to contribute to distinct behavior components necessitates the simultaneous study of these regions. The goals of this study were to identify brain-region dependent and independent gene expression patterns, regulators, and networks associated with increased voluntary wheel-running behavior. The cerebellum and striatum from a high voluntary running line and a non-selected control line were compared. Neuropeptide genes annotated to reward-dependent processes including neuropeptide S receptor 1 (Npsr1), neuropeptide Y (Npy), and proprotein convertase subtilisin/kexin type 9 (Pcsk9), and genes implicated in motor coordination including vitamin D receptor (Vdr) and keratin, type I cytoskeletal 25 (Krt25) were among the genes exhibiting activity line-by-region interaction effects. Genes annotated to the Parkinson pathway presented consistent line patterns, albeit at different orders of magnitude between brain regions, suggesting some parallel events in response to selection for high voluntary activity. The comparison of gene networks between brain regions highlighted genes including transcription factor AP-2-delta (Tfap2d), distal-less homeobox 5 gene (Dlx5) and sine oculis homeobox homolog 3 (Six3) that exhibited line differential expression in one brain region and are associated with reward-dependent behaviors. Transcription factors including En2, Stat6 and Eomes predominated among regulators of genes that differed in expression between lines. Results from the simultaneous study of striatum and cerebellum confirm the necessity to study molecular mechanisms associated with voluntary activity and reward-dependent behaviors in consideration of brain region dependencies.

Meningioma transcription factors link cell lineage with systemic metabolic cues.

Tumor cells recapitulate cell-lineage transcriptional programs that are characteristic of normal tissues from which they arise. It is unclear why such lineage programs are fatefully maintained in tumors and if they contribute to cell proliferation and viability. Here, we used the most common brain tumor, meningioma, which is strongly associated with female sex and high body mass index (BMI), as a model system to address these questions. We screened expression profiling data to identify the transcription factor (TF) genes which are highly enriched in meningioma, and characterized the expression pattern of those TFs and downstream genes in clinical meningioma samples as well as normal brain tissues. Meningioma patient-derived cell lines (PDCLs) were used for further validation and characterization. We identified 8 TFs highly enriched in meningioma. Expression of these TFs, which included sine oculis homeobox 1 (SIX1), readily distinguished meningiomas from other primary brain tumors and was maintained in PDCLs and even in pulmonary meningothelial nodules. In meningioma PDCLs, SIX1 and its coactivator eyes absent 2 (EYA2) supported the expression of the leptin receptor (LEPR), the cell-surface receptor for leptin (LEP), the adipose-specific hormone that is high in women and in individuals with high BMI. Notably, these transcriptional regulatory factors, LEPR and LEP, both contributed to support meningioma PDCLs proliferation and survival, elucidating a survival dependency on both a core transcriptional program and a metabolic cell-surface receptor. These findings provide one rationale for why lineage TF expression is maintained in meningioma and for the epidemiological association of female sex and obesity with meningioma risk.