PHLDA3 activated by BARX2 transcription, suppresses the malignant development of esophageal squamous cell carcinoma by downregulating PI3K/AKT levels.

BACKGROUND: Low pleckstrin homology-like domain family A, member 3 (PHLDA3) expression has been reported to be associated with cancer specificity and disease-free survival in esophageal squamous cell carcinoma (ESCC), and was an independent predictor of postoperative recurrence. However, the specific mechanisms involved are still unclear. This paper aimed to explore the role and its mechanisms of PHLDA3 in ESCC. MATERIALS AND METHODS: PHLDA3 and BarH-like homeobox 2 (BARX2) expressions in ESCC were predicted by Gene Expression Profiling Interactive Analysis (GEPIA) analysis and determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western Blot. Western blot detected the expression of proteins associated with migration, angiogenesis and phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway. The University of California Santa Cruz Genomics Institute (UCSC) database predicted that the relationship of BARX2 and PHLDA3 promoter and JASPAR identified the possible binding sites. Dual luciferase gene reporter verified PHLDA3 promoter activity, and the relationship of both was determined by chromatin immunoprecipitation (CHIP). Cell counting kit (CCK)-8, 5-ethynyl-2'-deoxyuridine (EDU) and colony formation were used to assess cell proliferation. Wound healing and transwell were used to detect cell migration and invasion ability. Tube formation assay was applied to assess angiogenesis. Mice were injected with transfected KYSE30 cells under the right axilla. Body weight and tumor volume and mass were recorded for each group of mice. Immunohistochemistry was performed to detect KI67 level in tumor tissues. RESULTS: Both PHLDA3 and BARX2 were downregulated in ESCC. The upregulated PHLDA3 suppressed PI3K/AKT expression. In addition, BARX2 bound to the PHLDA3 promoter and transcriptionally activated PHLDA3. PHLDA3 overexpression inhibited ESCC cell proliferation, migration, invasion and angiogenesis, but this effect was reversed by BARX2 knockdown. In addition, BARX2 overexpression inhibited ESCC cell proliferation, migration, invasion and angiogenesis, but this effect was reversed by PHLDA3 knockdown. CONCLUSION: PHLDA3 was transcriptionally activated by BARX2 and inhibited malignant progression of ESCC by downregulating PI3K/AKT levels.

Tet2 regulates Barx2 expression in undifferentiated and early differentiated mouse embryonic stem cells.

The methylcytosine oxidase TET proteins play important roles in DNA demethylation and development. In developing embryos, TET2 are upregulated during pre-implantation development, and significantly expressed in the trophectoderm and inner cell mass. In this study, we identified Barx2 as a new target of Tet2. Tet2 bound and demethylated the promoter of Barx2 in mouse embryonic stem cells (mESCs) to maintain the expression of Barx2. During mESC differentiation, Tet2 bound the promoter of Barx2 in day 4 embryonic bodies but not in day 8 EBs. However, Barx2 expression remained unchanged. Thus, Tet2 functioned as a demethylase and maintained the expression of Barx2 in undifferentiated and early differentiated mESCs.

miR-942 promotes tumor migration, invasion, and angiogenesis by regulating EMT via BARX2 in non-small-cell lung cancer.

Epithelial-mesenchymal transition (EMT) has an important function in cancer. Recently, microRNAs have been reported to be involved in EMT by regulating target genes. miR-942 is considered a novel oncogene in esophageal squamous cell carcinoma. However, its role in non-small-cell lung cancer (NSCLC) has not been investigated. In this study, the expression of miR-942 in NSCLC patients tumor and paired adjacent tissues were assessed by quantitative real-time polymerase chain reaction and in situ hybridization. Transwell, wound healing, tube formation, and tail vein xenograft assays were conducted to assess miR-942's function in NSCLC. Potential miR-942 targets were confirmed using dual-luciferase reporter assays, immunohistochemistry, immunoblot, and rescue experiments. The results showed miR-942 is relatively highly expressed in human NSCLC tissues and cells. In vitro assays demonstrated that overexpression of miR-942 promoted cell migration, invasion, and angiogenesis. Tail vein xenograft assays suggested that miR-942 contributed to NSCLC metastasis in vivo. Three bioinformatics software was searched, and BARX2 was predicted as a downstream target of miR-942. Direct interaction between them was validated by dual-luciferase assays. Rescue experiments further confirmed that BARX2 overexpression could reverse functional changes caused by miR-942. Moreover, miR-942 increased EMT-associated proteins N-cadherin and vimentin by inhibiting BARX2, while E-cadherin expression is reduced. In summary, this study reveals that miR-942 induces EMT-related metastasis by directly targeting BARX2, which may provide a potential therapeutic strategy for NSCLC.

Down-regulation of Barx2 predicts poor survival in colorectal cancer.

Human BarH-like homeobox 2 (Barx2), a homeodomain factor of the Bar family, has an important role in controlling the expression of cell adhesion molecules and has been reported in an increasing array of tumor types except colorectal cancer (CRC). The purpose of the current study was to characterize the expression of Barx2 and assess the clinical significance of Barx2 in CRC. First, we analyzed the expression of Barx2 in two independent public datasets from Oncomine. Subsequently, we evaluated Barx2 mRNA and protein expression by quantitative real-time PCR and western blotting, respectively. It was determined that Barx2 expression was lower in tumor tissues than in adjacent non-tumorous colorectal tissues of CRC patients, consistent with results from the public datasets. Subsequently, a tissue microarray containing 196 CRC specimens was evaluated for Barx2 expression by immunohistochemical staining. It was found that low expression of Barx2 significantly correlated with TNM stage, AJCC stage, differentiation, and relapse in patients with CRC. Patients with lower levels of Barx2 expression showed reduced disease-free survival and overall survival. Furthermore, a trend toward shorter overall survival in the patient group with Barx2-negative tumors independent of advanced AJCC stage and poor differentiation was determined by Kaplan-Meier survival analysis. Based on univariate and multivariate analyses, Barx2 expression was an independent prognostic factor for determining CRC prognosis. Taken together, low Barx2 expression was associated with the progression of CRC and could serve as a potential independent prognostic biomarker for patients with CRC.

Low expression of BARX2 in human primary hepatocellular carcinoma correlates with metastasis and predicts poor prognosis.

AIM: The homeobox gene Barx2 was recently identified as a regulator of ovarian and breast cancer; however, the expression level of BARX2 and its significance in hepatocellular carcinoma (HCC) remain unknown. METHODS: Protein and mRNA expression levels of Barx2 were examined using Western blotting and real-time PCR respectively, in paired HCC tissue and matched adjacent non-cancerous tissue from 12 patients. The expression levels of epithelial-mesenchymal transition (EMT) markers were also detected in relation to BARX2 expression. Lastly, immunohistochemistry for BARX2 was also performed on a tissue microarray containing 231 HCC tissue samples. RESULTS: We observed that BARX2 expression was lower in HCC tissues compared to matching adjacent non-cancerous tissue. The low expression level of BARX2 was significantly correlated with metrics of tumor size, tumor differentiation, clinical stage, metastasis and relapse. Furthermore, the patients with low BARX2 expression had adverse survival outcomes. Importantly, multivariate Cox regression analysis revealed that low BARX2 expression was an independent marker for lower overall survival (P = 0.007). Moreover, a significant negative relationship was observed between the expression of BARX2 and markers of EMT. CONCLUSION: These findings provide evidence that the low expression level of BARX2 in HCC is significantly correlated with tumor metastasis, and that BARX2 may be an independent prognostic biomarker for patients with HCC.

Effect of Zbed6 Single-Allele Knockout on the Growth and Development of Skeletal Muscle in Mice.

ZBED6, a key transcription factor, plays an important role in skeletal muscle and organ growth. ZBED6 knockout (ZBED6-/-) leads to the upregulation of IGF2 in pig and mice muscle, thereby increasing muscle mass. However, the effects and mechanism of Zbed6 single-allele knockout (Zbed6+/-) on mice muscle remain unknown. Here, we reported that Zbed6+/- promotes muscle growth by a new potential target gene rather than Igf2 in mice muscle. Zbed6+/- mice showed markedly higher muscle mass (25%) and a markedly higher muscle weight ratio (18%) than wild-type (WT) mice, coinciding with a larger muscle fiber area (28%). Despite a significant increase in muscle growth, Zbed6+/- mice showed similar Igf2 expression with WT mice, indicating that a ZBED6-Igf2-independent regulatory pathway exists in Zbed6+/- mice muscle. RNA-seq of muscle between the Zbed6+/- and WT mice revealed two terms related to muscle growth. Overlapping the DEGs and C2C12 Chip-seq data of ZBED6 screened out a potential ZBED6 target gene Barx2, which may regulate muscle growth in Zbed6+/- mice. These results may open new research directions leading to a better understanding of the integral functions of ZBED6 and provide evidence of Zbed6+/- promoting muscle growth by regulating Barx2 in mice.

BarH-like homeobox 2 represses the transcription of keratin 16 and affects Ras signaling pathway to suppress nasopharyngeal carcinoma progression.

Nasopharyngeal carcinoma (NPC) refers to a malignancy initiating from the superior mucosal epithelium of the nasopharynx. Optimal therapies for NPC are still needed. In this investigation, we attempted to explore whether BarH-like homeobox 2 (BARX2), a well-known tumor suppressor, had anti-cancer properties on NPC, and the possible mechanisms. After searching for NPC-related databases, we determined BARX2 as one of the core genes in NPC. The results of RT-qPCR and immunohistochemistry or Western blot demonstrated that BARX2 was reduced in NPC patients and cells. Ectopic expression of BARX2 reverted the malignant phenotype of NPC cells. Mechanistically, BARX2 bound to the keratin 16 (KRT16) promoter to downregulate its expression. In addition, BARX2 was found to reduce the phosphorylation levels of MEK and ERK. Further KRT16 upregulation in cells overexpressing BARX2 promoted malignant aggressiveness of C666-1 and HNE3 cells and activated the Ras signaling pathway. BARX2 inhibited the growth and metastasis of tumors and suppressed the Ras signaling pathway in vivo. In conclusion, our findings indicate that BARX2 reverts malignant phenotypes of NPC cells by downregulating KRT16 in a Ras-dependent fashion. BARX2 might act as a possible therapeutic regulator for NPC.

A circadian rhythm-related biomarker for predicting prognosis and immunotherapy efficacy in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) remains a major reason of cancer-associated mortality globally, and there exists a lack of indicators for survival in LUAD patients. Therefore, it is clinically required to obtain a novel prognostically indicator for guiding clinical management. In this study, we established a circadian rhythm (CR) related signature by a combinative investigation of multiple datasets. The newly-established signature showed an acceptable ability to predict survival and could serve as an independent indicator for prognosis. Moreover, the newly-established signature was critically associated with tumor malignancy, including proliferation, invasion, EMT and metastasis. The newly-established signature was predictive of response to immune checkpoint blockade. Collectively, we established a CR-related gene signature that could forecast survival, tumor malignancy and therapeutic response; our findings could help guiding clinical management.

BARX2/FOXA1/HK2 axis promotes lung adenocarcinoma progression and energy metabolism reprogramming.

Background: Metabolic reprogramming is an emerging cancer feature that has recently drawn special attention since it promotes tumor cell growth and proliferation. However, the mechanism of the Warburg effect is still largely unknown. This research aimed to reveal the effects of BarH-like homeobox 2 (BARX2) in regulating tumor progression and glucose metabolism in lung adenocarcinoma (LUAD). Methods: Expression of BARX2 was measured by quantitative real-time polymerase chain reaction (qRT-PCR) in LUAD cell line and tissues, and the tumor-promoting function of BARX2 in LUAD cells was detected in vitro and in vivo xenograft models. The metabolic effects of BARX2 were examined by detecting glucose uptake, the production levels of lactate and pyruvate, and the extracellular acidification rate (ECAR). Chromatin immunoprecipitation (ChIP) assay and luciferase reporter gene assay were used to identify the underlying molecular mechanism of BARX2 regulation of HK2. Further studies showed that transcription factor FOXA1 directly interacts with BARX2 and promotes the transcriptional activity of BARX2. Results: BARX2 was remarkably up-regulated in LUAD tissues and positively linked to advanced clinical stage and poor prognosis. In vitro and in vivo data indicated ectopic expression of BARX2 enhanced cell proliferation and tumorigenesis, whereas BARX2 knockdown suppressed these effects. Metabolic-related experiments showed BARX2 promoted the reprogramming of glucose metabolism. Mechanistically, the BARX2/FOXA1/HK2 axis promoted LUAD progression and energy metabolism reprogramming. Conclusions: In summary, our research first defined BARX2 as a tumor-promoting factor in LUAD and that it may act as a novel prognostic biomarker and new therapeutic target for the disease.

The miR-187 induced bone reconstruction and healing in a mouse model of osteoporosis, and accelerated osteoblastic differentiation of human multipotent stromal cells by targeting BARX2.

BACKGROUND: Multiple microRNAs (miRNAs) have been proven to regulate osteogenic differentiation by affecting the Runx2 signaling pathway. The intervention of miRNA can delay the progress of osteoporosis (OP) and induce fracture repair by affecting bone regeneration. However, the function and mechanism of miR-187 in osteoporotic fractures are still unknown. METHODS: We first established the OP mouse model. Next, the BMD value was certified by iDXA. The miR-187 level in the OP mice and serum of OP patients was identified through qRT-PCR. Bone repair and bone healing were assessed through toluidine blue staining and X-ray, and BARX2 expression was also confirmed. Osteogenesis-related proteins, ALP activity, and the matrix mineralization state were evaluated by western blot, ALP staining, and Alizarin Red staining in hMSCs after transfection with miR-187 mimics, miR-187 inhibitor, or human BarH-like homeobox 2 (BARX2) siRNA. Moreover, the interplay between miR-187 and BARX2 was identified through the dual-luciferase reporter. RESULTS: The BMD value was notably reduced in the OP mice, and miR-187 was markedly downregulated in the OP mice and serum of OP patients. Meanwhile, we proved that miR-187 induced bone reconstruction and healing, and downregulated BARX2 in the OP mouse model. We also proved that BARX2 was a direct target of miR-187, and could be significantly downregulated by miR-187. Furthermore, miR-187 induced osteogenic differentiation of hMSCs by targeting BARX2. CONCLUSIONS: The miR-187 might have a significant therapeutic effect in osteoporotic fractures. miR-187 accelerated osteogenic differentiation of hMSCs by directly regulating BARX2.

circFBXW7 attenuates malignant progression in lung adenocarcinoma by sponging miR-942-5p.

BACKGROUND: As a type of non-coding RNA, circular RNAs (circRNAs) are considered to be functional molecules associated with human cancers. An increasing number of circRNAs have been verified in malignant progression in a number of cancers. The circRNA, circFBXW7, has been proven to play an important role in tumor proliferation and metastasis. However, whether circFBXW7 influences progression in lung adenocarcinoma (LUAD) remains unclear. METHODS: Quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to verify circFBXW7 in LUAD cell lines and LUAD tissues. Kaplan-Meier analysis was then used to compare the disease-free survival (DFS) and overall survival (OS) of these LUAD patients. The biological function of circFBXW7 was examined by overexpression and knockdown of circFBXW7 using MTT assay, EdU assay, wound-healing assay, and Transwell in vitro assays. To explore the mechanism of the circFBXW7, RNA pull-down assay, dual luciferase reporter assay, and RNA immunoprecipitation (RIP) assay were employed to examine the interaction between circFBXW7 and miR-942-5p. Western blot was used to study the fundamental proteins associated with the epithelial-mesenchymal transition (EMT) pathway. In vivo studies with BALB/c nude mice subcutaneously injected with cells stably overexpressing circFBXW7 were performed to further validate the in vitro results. RESULTS: circFBXW7 was downregulated in LUAD cell lines and tissues, and LUAD patients with lower levels had shorter DFS and OS. The in vitro study showed that circFBXW7 overexpression inhibited proliferation and migration of A549 and HCC2279 cell lines. These results were confirmed by circFBXW7 knockdown, which showed the reverse effect. The in vivo model showed that the circRNA levels influenced the tumor growth. Finally, we determined that circFBXW7 target miRNA-942-5p which regulates the EMT gene BARX2. The modulation of circFBXW7 levels produced significant changes in EMT genes in vitro and in vivo. CONCLUSIONS: Our findings showed that circFBXW7 inhibits proliferation and migration by controlling the miR-942-5p/BARX2 axis in LUAD cell lines and its levels correlates with patient survival suggesting that regulating circFBXW7 could have therapeutic value in treating LUAD patients.

BARX2 expression is downregulated by CpG island hypermethylation and is associated with suppressed cell proliferation and invasion of gastric cancer cells.

BarH­like homeobox 2 (BARX2), a homeobox gene, is associated with several types of cancers. The present study aimed to determine whether DNA methylation downregulates BARX2 expression and whether BARX2 is associated with suppression of gastric carcinogenesis. BARX2 protein expression in normal and cancerous gastric tissues and various gastric cancer (GC) cell lines was detected using immunohistochemical and western blot assays. BARX2 mRNA levels were detected using both reverse transcription­polymerase chain reaction (RT­PCR) and quantitative PCR (qPCR). Promoter hypermethylation in GC cells was detected using methylation­specific PCR or bisulfite DNA sequencing PCR. Effects of BARX2 expression on GC cell proliferation, clonal formation, and migration were evaluated after lentivirus­BARX2 transfection. The effect of stable BARX2 transfection on tumor formation was assessed in a nude xenograft mouse model. BARX2 was strongly expressed in the normal gastric mucosa, but weakly or not expressed in GC tissues and most GC cell lines. BARX2 expression was negatively correlated with DNMT (a marker for DNA methylation) expression in the gastric tissues. The BARX2 promoter fragment was hypermethylated in the GC cell lines. Overexpression of BARX2 significantly inhibited GC cell proliferation, clonal formation, and migration. Stable BARX2 transfection inhibited tumor formation in xenograft mice, which was correlated with decreased expression of E­cadherin, proliferation markers, and matrix metalloproteinases. In conclusion, BARX2 expression is aberrantly reduced in GC, which is associated with increased DNA methylation of its promoter. BARX2 inhibits GC cell proliferation, migration, and tumor formation, suggesting that BARX2 acts as a tumor suppressor in gastric carcinogenesis.

Downregulation of BarH-like homeobox 2 promotes cell proliferation, migration and aerobic glycolysis through Wnt/ß-catenin signaling, and predicts a poor prognosis in non-small cell lung carcinoma.

BACKGROUND: Human BarH-like homeobox 2 (Barx2), a homeodomain factor of the Bar family, plays a critical role in cell adhesion and cytoskeleton remodeling, and has been reported in an increasing array of tumor types except non-small cell lung carcinoma (NSCLC). The purpose of the current study was to characterize the expression of Barx2 and assess the clinical significance of Barx2 in NSCLC. METHODS: Quantitative real-time polymerase chain reaction, immunohistochemistry and western blot analysis were used to examine mRNA and protein expression, respectively. The relationships between Barx2 expression and clinicopathological variables were analyzed. Cell Counting Kit-8 and plate colony formation assay were used to detect cell proliferation. Transwell assay was used to examine cell migration ability. Glucose uptake, lactate, adenosine triphosphate, and lactate dehydrogenase assays were used to detect aerobic glycolysis. RESULTS: Barx2 is downregulated in NSCLC tissues compared with para-carcinoma. Furthermore, Barx2 expression shows a negative correlation with advanced TNM stage and a high level of Ki-67. Survival analysis reveals that Barx2 level is an independent prognostic factor for NSCLC patients. The Barx2 (low) Ki-67 (high) group had the worst prognosis. Furthermore, the data indicate that downregulation of Barx2 expression promotes cell proliferation, migration, and aerobic glycolysis, including increased lactate dehydrogenase activity, glucose utilization, lactate production, and decreased intracellular adenosine triphospahte level. Furthermore, Barx2 acts as a negative regulator of the canonical Wnt/ß-catenin pathway. Reactivation of Wnt/ß-catenin pathway by LiCl can reverse the inhibiting effect of Barx2. CONCLUSIONS: These findings reveal that Barx2 serving as a tumor suppressor gene could decrease cell proliferation, migration, and aerobic glycolysis through inhibiting the Wnt/ß-catenin signaling pathway, and predicts a good prognosis in NSCLC.

Up-regulation of miR-187 modulates the advances of oral carcinoma by targeting BARX2 tumor suppressor.

Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. Aberrations in miRNA regulation are known to play important roles in OSCC pathogenesis. miR-187 was shown to be up-regulated in head and neck malignancies in our previous screening. This study further investigated the oncogenic potential, clinical implications, and targets of miR-187 in OSCC. We observed that miR-187 increased oncogenicity, particularly migration, of OSCC cells. miR-187 expression increased the xenografic tumorigenicity and metastasis in mice. In addition, metastatic human OSCC had higher miR-187 expression than did non-metastatic tumors. Through vigorous screening, we confirmed BarH-like Homeobox 2 (BARX2) gene as an miR-187 target. BARX2 expression suppressed the migration, invasion, anchorage-independent colony formation, and orthotopic tumorigenesis of OSCC cells. The migratory phenotype and neck metastasis induced by miR-187 was rescued by BARX2 expression. BARX2 expression was down-regulated in the vast majority of OSCC, and this down-regulation was particularly conspicuous in tumors with advanced nodal metastasis. In addition, plasma miR-187 was significantly higher in OSCC patients than in normal individuals. This study highlights the roles of miR-187-BARX2 in driving the carcinogenesis of OSCC. The results suggest that miR-187 is a potential serological marker for OSCC and that targeting of miR-187 might prove effective in attenuating nodal metastasis.

Downregulation of homeobox gene Barx2 increases gastric cancer proliferation and metastasis and predicts poor patient outcomes.

Barx2 is a Bar family homeodomain transcription factor shown to play a critical role in cell adhesion and cytoskeleton remodeling, key processes in carcinogenesis and metastasis. Using quantitative real-time PCR, Western blotting, and immunohistochemistry, we found that Barx2 is expressed at lower levels in human gastric cancer (GC) tissues than in adjacent normal mucosa. In a multivariate analysis, Barx2 expression emerged as an independent prognostic factor for disease-free and overall survival. Kaplan-Meier survival analysis showed a trend toward even shorter overall survival in the patient group with Barx2-negative tumors, independent of advanced UICC stage and tumor relapse. Using in vitro and in vivo assays, we demonstrated that under normal conditions Barx2 inhibited GC cell proliferation and invasiveness through inhibition of the Wnt/ß-catenin signaling pathway. These findings indicate that reduction or loss of Barx2 dis-inhibits GC cell proliferation and invasion, and that reduction in Barx2 could serve as an independent prognostic biomarker for poor outcome in GC patients.

Immunohistochemical localization of barx2 in the developing fetal mouse submandibular glands.

The development of mouse submandibular gland (SMG) begins at embryonic day 11.5-12 (E11.5-12), during which successive rounds of epithelial clefting and branching create complex epithelial tree-like structures. Homeobox genes regulate place-dependent morphogenesis, including epithelial-mesenchymal interactions, and control the expression patterns of signaling molecules. The Barx2 containing Homeobox exerts several key roles in development. Some studies have shown that the Barx2 plays important roles in the epithelial-mesenchymal interactions of organogenesis. However, the mechanisms of Barx2 associated with the development of SMG are obscure. In this study, we demonstrated for the first time the exact spatial and temporal Barx2 expression pattern in SMG epithelial tissue during development using immunohistochemical staining and Real-Time quantitative PCR. Barx2 was expressed in the nucleus of the epithelial cells located in the proliferative and differentiative regions of the developing SMG during the early development stages (E11.5-E13.5). After the E14.5-time period, the expression gradually decreased, and at E16.5, expression mostly disappeared despite the fact that evidence of cytodifferentiation, such as the appearance of proacinar cells, distinct lumen formation, and secretory products, was beginning to be observed. Results of Real-Time PCR demonstrated that the amount of Barx2 mRNA expression in SMG was maximal on E14.5, and gradually decreased by E18.5. These results indicate that Barx2 is associated with early stage epithelial tissue development, and can be a useful epithelial marker of the SMG during early developmental stages.