Homeobox B7 Research Articles

HOXB8 mediates resistance to cetuximab in colorectal cancer cells through activation of the STAT3 pathway

Homeobox B8 (HOXB8) belongs to the HOX family and was essential to the development of colorectal carcinoma. Among the prevalent monoclonal antibodies for treating RAS/BRAF wild-type metastatic colorectal cancer (mCRC) patients, cetuximab stands out, but resistance to cetuximab frequently arises in targeted treatments. Currently, the role of HOXB8 in cetuximab-resistant mCRC remains unclear. By comparing drug-sensitive cell lines (SW48) with drug-resistant cell lines (HCT116, CACO2), we discovered that HOXB8 was substantially expressed in cetuximab-resistant cell lines, and furthermore, in drug-resistant cell lines (HCT116, CACO2), HOXB8 knockdown increased the cytotoxicity of cetuximab via blocking the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Conversely, the excessive expression of HOXB8 reduced the growth suppression in SW48 cells caused by cetuximab by triggering the STAT3 signaling pathway. Conclusively, we conclude that HOXB8 has played an essential role in cetuximab-resistant mCRC and that treating HOXB8 specifically may be a useful treatment approach for certain cetuximab-resistant mCRC patients.

Homeobox B9 promotes the invasion and metastasis of hepatocellular carcinoma cells via the EZH2–MIR203A–SNAI2 axis

BackgroundResearch has elucidated that homeobox B9 (HOXB9), an important transcriptional activator, plays a pivotal role in promoting the invasion and metastasis of hepatocellular carcinoma (HCC) cells. However, the mechanism by which HOXB9 promotes the invasion and metastasis of HCC cells is incompletely understood and needs further exploration.MethodsHOXB9 and snail family transcriptional repressor 2 (SNAI2) expression were analyzed using qRT-PCR and western blotting. The invasion and metastasis of hepatocellular carcinoma (HCC) cells were investigated using in vitro and in vivo assays. The H3K27me3 enrichment and HOXB9 interaction with microRNA 203a (MIR203A) or SNAI2 were detected using ChIP-qPCR. Transcriptional activities of SNAI2 and MIR203A promoter were detected using dual-luciferase reporter assays. Co-IP and GST pull-down assays were performed to confirm the binding between HOXB9 and EZH2.ResultsHOXB9 and SNAI2 were highly expressed in HCC tissues and their expression was positively intercorrelated and associated with poor prognosis in patients with HCC. In vitro and in vivo experiments confirmed that HOXB9 can upregulate the expression of SNAI2 to promote the invasion and metastasis of HCC cells. Furthermore, HOXB9 elevated SNAI2 expression by inhibiting MIR203A expression, a tumor suppressor gene, in HCC cells. Mechanistically, HOXB9 recruited enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) through interaction with its WD-binding domain, which increased EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) at the MIR203A promoter region, in turn repressing the transcriptional activity and expression of MIR203A and consequently increasing the SNAI2 level in HCC cells. Finally, empirical evidence from in vitro and in vivo studies confirmed that mitigation of the HOXB9-mediated enhancement of epigenetic silencing of MIR203A inhibited SNAI2 expression, impeding the invasion and metastasis of HCC cells.ConclusionsOur study reveals a novel mechanism by which HOXB9 promotes the invasion and metastasis of HCC cells and expands the understanding of the function of HOXB9 in tumor progression and provides a novel therapeutic strategy for curtailing HCC invasion and metastasis.

Microglia replacement by ER-Hoxb8 conditionally immortalized macrophages provides insight into Aicardi-Goutières Syndrome neuropathology.

Microglia, the brain's resident macrophages, can be reconstituted by surrogate cells - a process termed "microglia replacement." To expand the microglia replacement toolkit, we here introduce estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages, a cell model for generation of immune cells from murine bone marrow, as a versatile model for microglia replacement. We find that ER-Hoxb8 macrophages are highly comparable to primary bone marrow-derived (BMD) macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells. Furthermore, ER-Hoxb8 progenitors are readily transducible by virus and easily stored as stable, genetically manipulated cell lines. As a demonstration of this system's power for studying the effects of disease mutations on microglia in vivo, we created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi-Goutières Syndrome (AGS), an inherited interferonopathy that primarily affects the brain and immune system. We find that Adar1 knockout elicited interferon secretion and impaired macrophage production in vitro, while preventing brain macrophage engraftment in vivo - phenotypes that can be rescued with concurrent mutation of Ifih1 (MDA5) in vitro, but not in vivo. Lastly, we extended these findings by generating ER-Hoxb8 progenitors from mice harboring a patient-specific Adar1 mutation (D1113H). We demonstrated the ability of microglia-specific D1113H mutation to drive interferon production in vivo, suggesting microglia drive AGS neuropathology. In sum, we introduce the ER-Hoxb8 approach to model microglia replacement and use it to clarify macrophage contributions to AGS.

HOXB9 promotes osteosarcoma cell survival and malignancy under glucose starvation via upregulating SPP1 expression

Homeobox B9 (HOXB9) has been shown to play a critical role in several tumors. However, the precise biological mechanisms and functions of HOXB9 in osteosarcoma remain largely unknown. In this study, we found that HOXB9 was increased upon glucose starvation. Elevated HOXB9 suppressed osteosarcoma cell death and supported cell growth and migration under glucose starvation. Further mechanistic studies demonstrated that HOXB9 directly bound to the promoter of secreted phosphoprotein 1 (SPP1) and transcriptionally upregulated SPP1 expression which then led cell death decrease and cell growth increase under glucose deprivation environment. Clinically, HOXB9 was significantly upregulated in osteosarcoma compared with normal tissues and increase of HOXB9 expression was positively associated with the elevation of SPP1 in osteosarcoma. Overall, our study illustrates that HOXB9 contributes to malignancy in osteosarcoma and inhibits cell death through transcriptional upregulating SPP1 under glucose starvation.

Thalidomide suppresses migration and invasion of colorectal cancer cells by inhibiting HOXB7-mediated activation of the Wnt/β-catenin signaling pathway.

Heaps of studies have verified the effects of thalidomide (THA) on colorectal cancer (CRC). Howbeit, the corresponding mechanism awaits illustration, which is the foothold of this study. Following the treatment of 0, 1.94, 7.75, or 19.36 μM THA, CRC cell viability, apoptosis, migration, and invasion were evaluated by methyl tetrazolium, flow cytometry, wound-healing, and transwell assays. Homeobox B7 (HOXB7) expression in CRC was analyzed and detected by bioinformatics analysis, quantitative real-time PCR or western blot. After the corresponding transfection or treatment with inhibitor of catenin-responsive transcription-3 (iCRT-3), abovementioned CRC cell biological behaviors as well as expression levels of HOXB7 and β-catenin were evaluated. 7.75 and 19.36 μM THA dwindled CRC cell viability, migration, and invasion, and facilitated apoptosis. HOXB7 upregulation was detected in CRC cells, which promoted the viability, migration, invasion, and β-catenin expression, and weakened the apoptosis of CRC cells. Also, HOXB7 upregulation counteracted the effects of THA on CRC cells. iCRT-3 restrained β-catenin expression, viability, migration, and invasion, whereas promoting the apoptosis of CRC cells. In addition, iCRT-3 antagonized the effects of overexpressed HOXB7 on CRC cells. THA inhibits the migration and invasion of CRC cells, which is achieved by suppressing HOXB7-mediated activation of Wnt/β-catenin signaling pathway.

Downregulation of homeobox B8 in attenuating non-small cell lung cancer cell migration and invasion though the epithelial-mesenchymal transition pathway.

Homeobox (HOX) family genes have been identified as regulators of cancer development. No research exists concerning the mechanisms underlying homeobox B8 (HOXB8) activity in non-small cell lung cancer (NSCLC). In this study, we investigated expression and biological function in NSCLC to determine whether it is an important marker of patient prognosis. HOXB8 expression in NSCLC tissues was investigated using immunohistochemistry (IHC) and Western blot assays. In addition, HOXB8 was knocked down in NSCLC cells to assess its biological functions in this context. The invasive and migratory potential of cells was evaluated by using Transwell (BD, Franklin Lakes, NJ, USA) inserts with 8-µm pores. Furthermore, Western blotting was used to explore whether HOXB8 can influence epithelial-mesenchymal transition (EMT). HOXB8 was expressed at high levels in NSCLC tissues and cell lines compared with adjacent normal tissues. Patients with high HOXB8 expression had shorter survival time and worse prognosis. HOXB8 expression was associated with pathological grading, tumor size, and lymph node metastasis. HOXB8 was prognostic in patients with NSCLC. After knockdown of HOXB8 via small interfering RNA, the proliferation, migration and invasion ability of the cells were significantly reduced compared with the control group. Moreover, EMT was inhibited by the downregulation of HOXB8 expression, as the expressions of E-cadherin was upregulated and that of the N-cadherin, vimentin, matrix metalloproteinase 2 (MMP2), and twist were downregulated. HOXB8 is a member of the ANTP homeobox family and encodes a nuclear protein with a homeobox DNA-binding domain. It is included in a cluster of homeobox B genes located on chromosome 17. The encoded protein functions as a sequence-specific transcription factor that is involved in development. HOXB8 is highly expressed in NSCLC and may predict prognosis of patients with this type of cancer. Furthermore, HOXB8 may promote NSCLC progression through the regulation of the EMT process.

MIR143HG promotes methylation of transcription factor HOXB7 promoter by recruiting methyltransferase DNMT1 to prevent the progression of colon cancer.

In recent years, accumulating evidence has demonstrated the role of long noncoding RNAs (lncRNAs) in colon cancer. We aim to investigate the role of MIR143HG, also known as CARMN (Cardiac mesoderm enhancer-associated noncoding RNA) in colon cancer and explore the related mechanisms. An RNAseq data analysis was performed to screen differentially expressed lncRNAs associated with colon cancer. Next, MIR143HG expression was quantified in colon cancer cells. Moreover, the contributory roles of MIR143HG in the progression of colon cancer with the involvement of DNMT1 and HOXB7 (Homeobox B7) were evaluated after restored MIR143HG or depleted HOXB7. Finally, the effects of MIR143HG were investigated invivo by measuring tumor formation in nude mice. High-throughput transcriptome sequencing was employed to validate the specific mechanisms by which MIR143HG and HOXB7 affect tumor growth invivo. MIR143HG was found to be poorly expressed, while HOXB7 was highly expressed in colon cancer. MIR143HG could promote HOXB7 methylation by recruiting DNMT1 to reduce HOXB7 expression. Upregulation of MIR143HG or downregulation of HOXB7 inhibited cell proliferation, invasion and migration and facilitated apoptosis in colon cancer cells so as to delay the progression of colon cancer. The same trend was identified invivo. Our study provides evidence that restoration of MIR143HG suppressed the progression of colon cancer via downregulation of HOXB7 through DNMT1-mediated HOXB7 promoter methylation. Thus, MIR143HG may be a potential candidate for the treatment of colon cancer.

HOXB8 overexpression induces morphological changes in chicken mandibular skin: an RNA-seq analysis

The Huiyang beard chicken is a well-known Chinese local breed known for its elongated feathers gathered from both sides of the face (muffs) and below the beak (beard), as well as short wattles (SW). The muff and beard (Mb) mutation is caused by ectopic upregulation of the homeobox B8 (HOXB8) gene in the mandibular skin; and the chi-square test showed a significant correlation between SW and Mb genotypes. However, the underlying molecular mechanisms that regulate Mb and SW variations remain unclear. In this study, we investigated the transcriptomes of the mandibular skin and wattles of chickens with and without the Mb genotype to elucidate the molecular basis of these traits. Our results show that HOXB8 is expressed at significantly higher levels in both the mandibular skin and wattles of Mb chickens than in those of wild-type chickens, indicating that HOXB8 regulates both the Mb and SW phenotypes. Key genes for keratin synthesis were highly expressed in the mandibular skin of Mb chickens, suggesting that HOXB8 may play a role in feather development. In wattles, changes in the expression of extracellular matrix synthesis genes may contribute to SW traits. DNA-binding motif analyses revealed that differentially expressed genes were likely to be directly regulated by HOXB8 binding, indicating that HOXB8 may directly or indirectly regulate feather follicle development and wattle growth. Our study identified both known and novel targets, including several genes not previously implicated in feather development and mesenchymal formation. These findings provide insights into the molecular mechanisms of skin appendage variation in birds and offer potential applications in breeding poultry breeds with unique phenotypes.

Pan-cancer analysis of Homeobox B9 as a predictor for prognosis and immunotherapy in human tumors

Although several animal and cell studies have described the association between HOXB9 and cancers, there is no pan-cancer investigation of HOXB9. In this article, we explored the expression levels and prognosis of HOXB9 in pan-cancer. We evaluated the correlation of HOXB9 expression level with the efficacy of immunotherapy.We conducted a survival analysis of HOXB9 in various types of cancer using publicly available databases. We also examined the relationship between HOXB9 expression levels and several factors including prognosis, immune infiltration, immune checkpoint genes, tumor mutational burden, microsatellite instability, mismatch repair, and DNA methylation. TIMER2.0 tool was conducted to explore the immune cell infiltrations related to HOXB9 in this analysis.It was discovered through a comprehensive analysis of multiple public datasets that HOXB9 expression was highly expressed in most tumor tissues and cancer cell lines and that distinct associations exist between HOXB9 expression and tumor patient prognosis. Besides, HOXB9 expression was closely associated with immune cell infiltration and checkpoint genes in many cancers. Further, HOXB9 was associated with immune cell infiltration, TMB, MSI, MMR, and DNA methylation. It was also confirmed that HOXB9 was highly expressed in clinical GBM tissues. Experiments further revealed that knockdown of HOXB9 expression could suppress proliferation, migration, and invasion of glioma cells.The results revealed that HOXB9, a robust tumor biomarker, has a significant prognostic value. HOXB9 may act as a new predictor to assess cancer prognosis and therapeutic efficacy of the immune in various cancers.

Homeobox B9 Promotes Colon Cancer Progression by Targeting SRSF3.

Homeobox B9 (HOXB9) is one of the HOX family of transcription factors that are essential for cancer development and embryonic growth. However, the clinical importance and biological involvement of HOXB9 in colon cancer (CC) are not adequately understood. To investigate whether HOXB9 participates in the proliferation, invasion, and migration of CC. This study investigated the function and clinical significance of HOXB9 mRNA and protein expression in CC. Furthermore, overexpression and knockdown experiments of HOXB9 were developed to explore their effects on CC cell transwell and proliferation. Moreover, a molecular mechanism of HOXB9 regulate serine/arginine-rich splicing factor 3 (SRSF3) was explored. HOXB9 expression was higher in CC cells and tissues at both the mRNA and protein levels. Poor survival in CC patients was significantly connected with high HOXB9 expression, which was also strongly associated with the TNM stage and lymph node metastases. Furthermore, in vitro CC cell proliferation, transwell were markedly aided by HOXB9 overexpression. Contrarily, HOXB9 knockdown had the reverse result and inhibited the formation of xenograft tumors in naked mice. Gene set enrichment analysis (GSEA)revealed a correlation between high HOXB9 expression and spliceosomes. JASPAR and GEPIA2.0, in addition to CHIP and dual-luciferase reporting assays, confirmed that HOXB9 targets the promoter of SRSF3 to enhance its expression. We also found that SRSF3 knockdown eliminated HOXB9 from cell proliferation and transwell. We characterized the function and mechanism of HOXB9 in regulating colon cancer growth, suggesting a novel molecular approach for colon cancer-targeted therapy.

Circ_0068631 sponges miR-139-5p to promote the growth and metastasis of cutaneous squamous cell carcinoma by upregulating HOXB7.

Circular RNAs (circRNAs) are often dysregulated in cancers and closely related to cancer progression, including cutaneous squamous cell carcinoma (CSCC). However, the role and mechanism of circ_0068631 in CSCC progression have not been reported. The expression of circ_0068631, microRNA-139-5p (miR-139-5p), and homeobox B7 (HOXB7) was measured by real-time quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, and colony formation assay were used to measure cell proliferation. Cell apoptosis was assessed by flow cytometry. Cell migration was detected by transwell assay. The interaction between miR-139-5p and circ_0068631 or HOXB7 was confirmed by dual-luciferase reporter assay. A xenograft tumor model was established to confirm the function of circ_0068631 in vivo. Circ_0068631 was upregulated in CSCC tissues and cells, and its silencing could inhibit CSCC cell proliferation and metastasis while promoting apoptosis in vitro, as well as restrain CSCC tumor growth in vivo. Circ_0068631 acted as a sponge of miR-139-5p, and miR-139-5p inhibition reversed the repressive effect of circ_0068631 knockdown on CSCC cell progression. Furthermore, HOXB7 was a target of miR-139-5p, and miR-139-5p inhibited the malignant behaviors by downregulating HOXB7 expression in CSCC cells. Further, circ_0068631 sponged miR-139-5p to regulate HOXB7 expression. Circ_0068631 functioned as a novel oncogene in CSCC progression by regulating miR-139-5p/HOXB7 axis, suggesting that circ_0068631 may be a potential target for CSCC treatment. Circ_0068631 was overexpressed in CSCC tissues and cells. Circ_0068631 downregulation suppressed CSCC progression via miR-139-5p. Circ_0068631 regulated HOXB7 via sponging miR-139-5p.

Mitogen-activated protein kinase inhibition augments the T cell response against HOXB7-expressing tumor through human leukocyte antigen upregulation.

Homeobox B7 (HOXB7) is a master regulatory gene that regulates cell proliferation and activates oncogenic pathways. Overexpression of HOXB7 correlates with aggressive behavior and poor prognosis in patients with cancer. However, the expression and role of HOXB7 in head and neck squamous cell carcinoma (HNSCC) remain unclear. In this study, we observed that most samples from patients with oropharyngeal cancer and HNSCC expressed HOXB7. As no direct inhibitor has been reported, we identified a potent peptide epitope to target HOXB7-expressing tumors through immune cells. A novel HOXB7-derived peptide epitope (HOXB78-25 ) elicited antigen-specific and tumor-reactive promiscuous CD4+ T cell responses. These CD4+ T cells produced γ-interferon (IFN-γ) and had the direct ability to kill tumors through granzyme B. Notably, downregulation of HOXB7 using siRNA enhanced human leukocyte antigen class II expression on tumor cells by decreasing the phosphorylation of MAPK. Mitogen-activated protein kinase inhibition augmented IFN-γ production by HOXB7-reactive CD4+ T cell responses without decreasing the expression of HOXB7. These results suggest that combining HOXB7 peptide-based vaccine with MAPK inhibitors could be an effective immunological strategy for cancer treatment.

CARD9 contributes to ovarian cancer cell proliferation, cycle arrest, and cisplatin sensitivity

BackgroundOvarian cancer recurrence and chemotherapy resistance are still urgent issues, and exploring the mechanisms of metastasis and chemotherapy resistance is beneficial to the development of therapeutic methods. Caspase recruitment domain family member 9 (CARD9) and homeobox B5 (HOXB5) are related and both are upregulated in ovarian cancer. This study aimed to define their functions in ovarian cancer cell proliferation, migration, and cisplatin sensitivity.ResultsThe levels of CARD9 were detected in acquired ovarian cancer tissues and cell lines. CARD9 was indeed abnormally upregulated in them. CARD9 knockdown significantly suppressed cell proliferation, colony formation, migration, cycle arrest, and cisplatin sensitivity. HOXB5 bound to the CARD9 promoter, and HOXB5 overexpression reversed the regulation by CARD9 knockdown in cells, as well as the activation of NF-κB signaling. This indicated that CARD9 was positively regulated by HOXB5 in ovarian cancer cells.ConclusionTogether, CARD9 is involved in ovarian cancer cell proliferation, migration, and cisplatin sensitivity via NF-κB signaling after transcriptional activation by HOXB5.

Homeobox B9 Promotes the Progression of Hepatocellular Carcinoma via TGF-β1/Smad and ERK1/2 Signaling Pathways.

Objectives Homeobox B9 (HOXB9), a homeodomain-containing transcription factor, may play a role in hepatocellular carcinoma (HCC) progression. However, the exact mechanisms underlying its action remain unclear. Materials and methods. Immunohistochemistry was used to investigate the expression of HOBX9 and its prognostic values in HCC patients. HCC cells were transfected with pBabe-HOXB9 and shHOXB9 plasmids, and MTT assay, Transwell assays, and xenograft mouse models were employed to determine the effects of HOXB9 on HCC cell proliferation, migration, and invasion in vitro and in vivo. The biological mechanisms involved in the role of HOXB9 were determined with Western blot and RT-qPCR methods. Results HOXB9 expression was significantly increased in HCC tissues and cell lines. Patients with higher HOXB9 levels were associated with poor prognosis. Overexpression of HOXB9 in BEL-7405 cells promoted proliferation, migration, and invasion, whereas knockdown of HOXB9 in HepG2 cells significantly reduced cell proliferation, migration, and invasion abilities. Mechanically, a positive correlation was found between HOXB9 expression and transforming growth factor-β1 (TGF-β1) and extracellular signal-regulated kinase (ERK)1/2 pathway in HCC tissues. HOXB9 overexpression stimulated TGF-β1/Smads signaling pathway in BEL-7405 cells. In contrast, HOXB9 knockdown inhibited the TGF-β1/Smads signaling pathway in HepG2 cells. In addition, the treatment with TGF-β1 inhibitor, LY364947, significantly reserved HOXB9 overexpression-induced cell proliferation, migration, and invasion abilities. Conclusions These findings validated that HOXB9 promoted proliferation, migration, and invasion in HCC cells by stimulating the TGF-β1/Smads and ERK1/2 signaling pathway. HOXB9 could be a promising prognostic biomarker and a potential therapeutic target in HCC.

HOXB5-activated ANGPT2 promotes the proliferation, migration, invasion and angiogenic effect of esophageal cancer cells via activating ERK/AKT signaling pathway.

Esophageal cancer, which is the eighth most common cancer worldwide, has a poor prognosis and high mortality rate. The present study was designed to investigate the proliferation, migration, invasion and angiogenic effect of the homeobox B5 (HOXB5)/angiopoietin-2 (ANGPT2) interplay in esophageal cancer. The relative expression of ANGPT2 and HOXB5 in esophageal cancer and the association between gene expression was evaluated using data from Gene Expression Profiling Interactive Analysis databases. Following transduction of short hairpin RNA-ANGPT2#1/2 plasmids, ANGPT2 was silenced. Viability, proliferation and invasion of esophageal cancer cells were assessed using CCK-8, 5-EdU, colony formation, wound healing and Transwell assays, respectively. Moreover, the transcriptional activity of ANGPT2 and angiogenesis were detected with luciferase reporter, chromatin immunoprecipitation (CH-IP) and tube formation assays. The results of the present study indicated that ANGPT2 was upregulated, both in esophageal cancer cell lines and tissue and there was an association between the ANGPT2 upregulation and the poor patient prognosis. In addition, ANGPT2 silencing suppressed esophageal cancer cell proliferation, migration, invasion and angiogenesis. The HOXB5 expression was also increased in esophageal cancer, and transcriptionally activated ANGPT2. Moreover, HOXB5 overexpression reversed the effects of ANGPT2 silencing in esophageal cancer cells. Furthermore, ANGPT2 silencing inactivated ERK/AKT signaling, whereas the HOXB5 overexpression blocked this effect. In conclusion, ANGPT2, which was transcriptionally activated by HOXB5, activated the ERK/AKT signaling pathway to promote proliferation, metastasis and angiogenesis of esophageal cancer cells.

Circ_0020123 enhances the cisplatin resistance in non-small cell lung cancer cells partly by sponging miR-140-3p to regulate homeobox B5 (HOXB5)

ABSTRACT Cisplatin (DDP) therapy is widely used for the treatment of non-small cell lung cancer (NSCLC), but the curative effect is limited by chemoresistance. This study was designed to explore circ_0020123 function in DDP resistance of NSCLCDDP. Expression detection for circ_0020123, microRNA-140-3p (miR-140-3p) and homeobox B5 (HOXB5) was performed by real-time polymerase chain reaction (qRT-PCR). Half inhibitory concentration (IC50) of DDP and cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. Colony formation ability was assessed using colony formation assay. Cell migration and invasion were evaluated via transwell assay. Cell apoptosis was examined by flow cytometry. Protein analysis was conducted by Western blot. Dual-luciferase reporter assay was used to affirm target interaction. Circ_0020123 expression was upregulated in DDP-resistant NSCLC cells. DDP resistance was reduced by downregulation of circ_0020123 in NSCLC cells. Circ_0020123 was identified as a miR-140-3p sponge. The effect of si-circ_0020123 on DDP resistance was partly associated with miR-140-3p upregulation. HOXB5 was a downstream target for miR-140-3p. Overexpression of HOXB5 mitigated miR-140-3p-induced inhibition of DDP resistance in NSCLC cells. Circ_0020123 upregulated the level of HOXB5 partly via sponging miR-140-3p. Also, circ_0020123 promoted tumor growth in NSCLC/DDP xenografts by regulating miR-140-3p and HOXB5 levels at least in part. These results revealed that circ_0020123 promoted DDP resistance in NSCLC cells partly by targeting miR-140-3p/HOXB5 axis, indicating that circ_0020123 might be used as a molecular target in DDP treatment for NSCLC.

HOXB9 blocks cell cycle progression to inhibit pancreatic cancer cell proliferation through the DNMT1/RBL2/c-Myc axis

Homeobox B9 (HOXB9) is involved in the occurrence and development of malignant tumors. However, the functions and underlying molecular mechanisms of HOXB9 in pancreatic cancer have yet to be identified. In this study, we find that both HOXB9 mRNA and protein levels are down-regulated in pancreatic cancer tissues and cell lines. Kaplan–Meier survival plots of 150 pancreatic cancer cases show that higher expression of HOXB9 in pancreatic cancer patients is associated with higher survival rates. We also find that over-expression of HOXB9 inhibits pancreatic cancer cell proliferation both in cell lines and the nude mouse xenograft as well as PDX models. Applying cell cycle PCR array analysis, Flow CytoMetry, ChIP-qPCR, and luciferase experiments, we observe that HOXB9 blocks cell cycle progression in the G0/G1 phase via up-regulating RBL2 and inhibiting c-Myc, and we further find that DNMT1 inhibits the expression of HOXB9 in pancreatic cancer by promoting the methylation of its promoter. Our findings highlight a novel mechanism of the DNMT1/HOXB9/RBL2/c-Myc pathway in regulating the cell cycle and proliferation of pancreatic cancer cells and provide a research basis for the prognosis and therapeutic application of HOXB9 in pancreatic cancer.

Identification of the minimum requirements for successful haematopoietic stem cell transplantation.

SummaryHistorically, defining haematopoietic subsets, including self‐renewal, differentiation and lineage restriction, has been elucidated by transplanting a small number of candidate cells with many supporting bone marrow (BM) cells. While this approach has been invaluable in characterising numerous distinct subsets in haematopoiesis, this approach is arguably flawed. The haematopoietic stem cell (HSC) has been proposed as the critical haematopoietic subset necessary for transplantation. However, due to the presence of supporting cells, the HSC has never demonstrated sufficiency. Utilising the homeobox B5 (Hoxb5)‐reporter system, we found that neither long‐term (LT) HSCs nor short‐term (ST) HSCs alone were sufficient for long‐term haematopoietic reconstitution. Critically, reconstitution can be rescued by transplanting combined LT‐ and ST‐HSCs, without supporting cells; a fraction we term the ‘Minimum Subset for Transplantation’ (MST). The MST accounts for only 0·005% of nucleated cells within mouse BM, and this MST can be cultured, expanded and genetically modified while preserving its rapid haematopoietic engraftment potential. These results support the consideration of an MST approach for clinical translation, especially for gene therapy approaches that require HSC compartment modification.

Comprehensive bioinformatics analyses identified Homeobox B9 as a potential prognostic biomarker and therapeutic target for gastric cancer.

The Homeobox B (HOXB) family promotes tumor progression, but the mechanism of its action in gastric cancer (GC) is unclear. We sought to identify the HOXB family members that are critical to the prognosis of GC patients. The Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), cBioPortal, UALCAN, Kaplan-Meier plotter, and the GeneMANIA databases were used to analyze the messenger RNA (mRNA) expression levels, prognostic value, and gene-gene interaction network of the HOXB9 family members in GC. The expression of HOXB9 in GC and its relationship with various clinicopathological parameters and the prognosis of patients were verified by immunohistochemistry. The expression of HOXB3, HOXB5, HOXB6, HOXB7, HOXB9, and HOXB13 mRNA was significantly upregulated in GC. There was a significant correlation between the upregulation of HOXB3, HOXB5, and HOXB9 mRNA and a low overall survival (OS) rate. The high expression of HOXB7, HOXB9, and HOXB13 mRNA was closely correlated to tumor grade and stage. HOXB9 was the HOXB family member most closely related to the occurrence and development of GC. A further analysis showed that HOXB9 might be involved in deoxyribonucleic acid repair and division regulation. A validation study showed that the advanced cancer group had a higher level of HOXB9 expression than the early cancer group. The high expression of HOXB9 in gastric tissue plays an important role in the survival and prognosis of GC patients. HOXB family members have different degrees of abnormal expression in GC. High HOXB9 expression in GC tissues was significantly correlated with a worse prognosis. Thus, HOXB9 is a potential novel biomarker and therapeutic target for GC.

WT1 regulates HOXB9 gene expression in a bidirectional way

The homeoboxB9 (HOXB9) gene is necessary for specification of the anterior-posterior body axis during embryonic development and expressed in various types of cancer.Here we show that the Wilms tumor transcription factor WT1 regulates the HOXB9 gene in a bidirectional manner. Silencing of WT1 activates HOXB9 in Wt1 expressing renal cell adenocarcinoma-derived 786-0 cells, mesonephric M15 cells and ex vivo cultured murine embryonic kidneys. In contrast, HOXB9 expression in U2OS osteosarcoma and human embryonic kidney (HEK) 293 cells, which lack endogenous WT1, is enhanced by overexpression of WT1. Consistently, Hoxb9 promoter activity is stimulated by WT1 in transiently transfected U2OS and HEK293 cells, but inhibited in M15 cells with CRISPR/Cas9-mediated Wt1 deletion. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrate binding of WT1 to the HOXB9 promoter in WT1-overexpressing U2OS cells and M15 cells. BASP1, a transcriptional co-repressor of WT1, is associated with the HOXB9 promoter in the chromatin of these cell lines. Co-transfection of U2OS and HEK293 cells with BASP1 plus WT1 prevents the stimulatory effect of WT1 on the HOXB9 promoter.Our findings identify HOXB9 as a novel downstream target gene of WT1. Depending on the endogenous expression of WT1, forced changes in WT1 can either stimulate or repress HOXB9, and the inhibitory effect of WT1 on transcription of HOXB9 involves BASP1. Consistent with inhibition of Hoxb9 expression by WT1, both transcripts are distributed in an almost non-overlapping pattern in embryonic mouse kidneys. Regulation of HOXB9 expression by WT1 might become relevant during kidney development and cancer progression.