Heran Wang

Knockout of BRD7 results in impaired spermatogenesis and male infertility.

BRD7 was originally identified as a novel bromodomain gene and a potential transcriptional factor. BRD7 was found to be extensively expressed in multiple mouse tissues but was highly expressed in the testis. Furthermore, BRD7 was located in germ cells during multiple stages of spermatogenesis, ranging from the pachytene to the round spermatid stage. Homozygous knockout of BRD7 (BRD7−/−) resulted in complete male infertility and spermatogenesis defects, including deformed acrosomal formation, degenerative elongating spermatids and irregular head morphology in postmeiotic germ cells in the seminiferous epithelium, which led to the complete arrest of spermatogenesis at step 13. Moreover, a high ratio of apoptosis was determined by TUNEL analysis, which was supported by high levels of the apoptosis markers annexin V and p53 in knockout testes. Increased expression of the DNA damage maker λH2AX was also found in BRD7−/− mice, whereas DNA damage repair genes were down−regulated. Furthermore, no or lower expression of BRD7 was detected in the testes of azoospermia patients exhibiting spermatogenesis arrest than that in control group. These data demonstrate that BRD7 is involved in male infertility and spermatogenesis in mice, and BRD7 defect might be associated with the occurrence and development of human azoospermia.

miR-141 is involved in BRD7-mediated cell proliferation and tumor formation through suppression of the PTEN/AKT pathway in nasopharyngeal carcinoma.

Bromodomain containing 7 (BRD7) was identified as a nuclear transcriptional regulatory factor. BRD7 functions as a tumor suppressor in multiple cancers, including nasopharyngeal carcinoma (NPC). In this study, we reported a novel mechanism of BRD7 in NPC progression. We demonstrated that the expression of miR-141 was remarkably increased in NPC tissues and was negatively correlated with the expression of BRD7 and the survival rate of NPC patients. Decreased expression levels of miR-141, including the primary, the precursor and the mature forms of miR-141, were found in BRD7-overexpressing HEK293, 5-8F and HNE1 cells compared the control cells, while there was no obvious effect on the expression levels of the two critical enzymes Drosha and Dicer. BRD7 can negatively regulate the promoter activity of miR-141, while no obvious binding site of BRD7 was found in the potential promoter region of miR-141. Moreover, ectopic expression of miR-141 can significantly promote cell proliferation and inhibit apoptosis in NPC, and rescuing the expression of miR-141 in BRD7-overexpressing NPC cells could partially reverse the tumor suppressive effect of BRD7 on cell proliferation and tumor growth in vitro and in vivo. Furthermore, the activation of the PTEN/AKT pathway mediated by the overexpression of BRD7 could be inhibited by rescuing the expression of miR-141, which accordingly results in the partial restoration of cell proliferation and tumor growth. Our findings demonstrate that the BRD7/miR-141/PTEN/AKT axis has critical roles in the progression of NPC and provide some promising targets for the diagnosis and treatment of NPC.

Negative feedback loop between p66Shc and ZEB1 regulates fibrotic EMT response in lung cancer cells

The epithelial-to-mesenchymal transition (EMT) program is crucial for the epithelial cancer progression and fibrotic diseases. Our previous work has demonstrated that p66Shc, a focal adhesion-associated adaptor protein, is frequently downregulated in lung cancers and its depletion promotes metastasis behavior through anoikis resistance. However, mechanism underlying loss of p66Shc and EMT response is not fully understood. Here, we showed that p66Shc deficiency enhanced the expression of ZEB1, the known mesenchymal transcription factor and consequently increased Vimentin, and decreased epithelial markers of E-cadherin and β-catenin. p66Shc depletion also increased cell invasion and migration. In addition, ChIP and luciferase assays showed that these effects were directly mediated by ZEB1 repression of p66Shc promoter. Thus, our findings define a critical role of p66Shc in the suppression of fibrotic EMT response with a negative feedback loop between p66Shc and ZEB1 in lung epithelial cancer cells.

Integrating ChIP-sequencing and digital gene expression profiling to identify BRD7 downstream genes and construct their regulating network

BRD7 is a single bromodomain-containing protein that functions as a subunit of the SWI/SNF chromatin-remodeling complex to regulate transcription. It also interacts with the well-known tumor suppressor protein p53 to trans-activate genes involved in cell cycle arrest. In this paper, we report an integrative analysis of genome-wide chromatin occupancy of BRD7 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and digital gene expression (DGE) profiling by RNA-sequencing upon the overexpression of BRD7 in human cells. We localized 156 BRD7-binding peaks representing 184 genes by ChIP-sequencing, and most of these peaks were co-localized with histone modification sites. Four novel motifs were significantly represented in these BRD7-enriched regions. Ingenuity pathway analysis revealed that 22 of these BRD7 target genes were involved in a network regulating cell death and survival. DGE profiling identified 560 up-regulated genes and 1088 down-regulated genes regulated by BRD7. Using Gene Ontology and pathway analysis, we found significant enrichment of the cell cycle and apoptosis pathway genes. For the integrative analysis of the ChIP-seq and DEG data, we constructed a regulating network of BRD7 downstream genes, and this network suggests multiple feedback regulations of the pathways. Furthermore, we validated BIRC2, BIRC3, TXN2, and NOTCH1 genes as direct, functional BRD7 targets, which were involved in the cell cycle and apoptosis pathways. These results provide a genome-wide view of chromatin occupancy and the gene regulation network of the BRD7 signaling pathway.

Rac1 overexpression is correlated with epithelial mesenchymal transition and predicts poor prognosis in non-small cell lung cancer

Objective: Ras-related C3 botulinum toxin substrate1(Rac1) and epithelial mesenchymal transition (EMT) are key therapeutic targets in cancer. We investigated the clinical significance of Rac1 and markers of EMT expression in non-small cell lung cancer (NSCLC), and their possible correlation with EMT phenotype. Methods: Immunohistochemistry was used to assess the expression of Rac1, Snail1, Twist1, N-cadherin (N-cad), Vimentin (Vim), and E-cadherin (E-cad) in 153 NSCLC paraffin-embedded specimens and 45 normal specimens adjacent to tumors. The correlation of Rac1 and EMT markers with clinicopathological characteristics and the relationship between the protein levels and progression-free survival (PFS) and overall survival (OS) were analyzed. Results: Compared with non-tumor tissues, the NSCLC tissues showed marked elevation in the levels of Rac1, Snail1, Twist1, N-cad, and Vim levels, whereas the E-cad levels were significantly decreased (P < 0.05). The aberrant expression of Rac1 and EMT markers was significantly associated with TNM stage and metastasis (P < 0.05). Increased expression of Rac1 may be associated with poor OS and PFS compared with low expression (P<0.001 and P=0.004). Significant correlations were observed between the EMT markers expressed and OS or PFS(P<0.01). In addition, multivariate analysis indicated that the expression of Rac1, Snail1, Twist1, N-cad, Vim, and E-cad was an independent prognostic factor in NSCLC. Interestingly, Rac1 expression was positively correlated with Snail1, Twist1, N-cad, and Vim levels (r=0.563, r=0.440, r=0.247 r=0.536, P<0.01, respectively) and negatively correlated with E-cad levels (r=-0.464, P<0.001) in NSCLC tissues. Rac1, Twist, Snail1, Vim and N-cad were highly expressed in lung cancer patients resistant to radiotherapy, while E-cad was poorly expressed. Conclusion: Rac1 may promote NSCLC progression and metastasis via EMT, which may be considered as a potential therapeutic target.

Knockdown of c-Myc inhibits cell proliferation by negatively regulating the Cdk/Rb/E2F pathway in nasopharyngeal carcinoma cells

The proto-oncogene c-Myc encodes a transcription factor that is involved in the regulation of cellular proliferation, differentiation, and apoptosis. Several studies indicate that the over-expression of c-Myc is a frequent genetic abnormality in nasopharyngeal carcinoma (NPC). Therefore, specifically reducing its level by genetic means in established NPC cell lines helps to better understand its role in the pathogenesis of NPC. In this study, for the first time, we successfully established and characterized NPC 5-8F cell line with stably suppressed c-Myc expression by employing a DNA-based RNA interference approach. The suppression of c-Myc resulted in reduced cell growth, colony formation, and cell cycle progression in 5-8F cells. In vivo tumor formation assays revealed that the knockdown of c-Myc reduced the tumorigenic potential of 5-8F cells in nude mice. At the molecular level, we found that the knockdown of c-Myc could decrease the expression of several critical molecules involved in the Cdk/Rb/E2F pathway, including CDK4, cyclin D1, CDK2, pRb, E2F3, and DP2, and significantly reduced the promoter activity of cyclin D1. Taken together, these findings provide valuable mechanistic insights into the role of c-Myc in nasopharyngeal carcinogenesis and suggest that the knockdown of c-Myc may be a potential therapeutic approach for the treatment of NPC.

BRD7 plays an anti-inflammatory role during early acute inflammation by inhibiting activation of the NF-кB signaling pathway

Increasing evidence has shown a strong association between tumor-suppressor genes and inflammation. However, the role of BRD7 as a novel tumor suppressor in inflammation remains unknown. In this study, by observing BRD7 knockout mice for 6–12 months, we discovered that compared with BRD7+/+ mice, BRD7−/− mice were more prone to inflammation, such as external inflammation and abdominal abscess. By using mouse embryo fibroblast (MEF) cells from the BRD7 knockout mouse, an in vitro lipopolysaccharide (LPS)-stimulated MEF cell line was established. The mRNA levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), chemokine (C-X-C motif) ligand 1 (CXCL-1) and inducible nitric oxide synthase (iNOS) were significantly increased in BRD7−/− MEF cells compared with BRD7+/+ MEF cells after LPS stimulation for 1 or 6 h. In addition, the cytoplasm-to-nucleus translocation of nuclear factor kappa-B (NF-κB; p65) and an increased NF-κB reporter activity were observed in BRD7−/− MEF cells at the 1 h time point but not at the 6 h time point. Furthermore, an in vivo dextran sodium sulfate (DSS)-induced acute colitis model was created. As expected, the disease activity index (DAI) value was significantly increased in the BRD7−/− mice after DSS treatment for 1–5 days, which was demonstrated by the presence of a significantly shorter colon, splenomegaly and tissue damage. Moreover, higher expression levels of IL-6, TNF-α, p65, CXCL-1 and iNOS, and an increased level of NF-κB (p65) nuclear translocation were also found in the DSS-treated BRD7−/− mice. These findings suggest that BRD7 has an anti-inflammatory role during early acute inflammation by inhibiting activation of the NF-кB signaling pathway, which provides evidence to aid in understanding the therapeutic effects of BRD7 on inflammatory diseases.

Elevated microRNA-125b levels predict a worse prognosis in HER2-positive breast cancer patients

Breast cancer, the second most common cancer worldwide, is the leading cause of cancer-associated mortality in women, accounting for ~15% of all cancer-associated mortalities in women. The development, local invasion and metastasis of breast cancer are associated with the dysregulation and mutation of numerous genes and epigenetic mechanisms, including coding RNA and non-coding RNA, such as microRNAs (miRs/miRNAs). Previous studies have shown a dual-faced role of miR-125b in breast cancer. In the present study, a total of 221 paraffin-embedded breast cancer and 49 paraffin-embedded non-cancerous breast tissue samples were collected. In situ hybridization was used to analyze the expression of miR-125b in the breast cancer tissues. Spearmans rank correlation analysis was used to analyze the expression correlation between miR-125b and human epidermal growth factor 2 (HER2). The overall survival estimates over time were calculated using the Kaplan-Meier method with log-rank test. It was found that miR-125b expression was significantly increased in the breast cancer tissues compared with that in the non-cancerous tissues, and high miR-125b expression indicated a poor prognosis in the breast cancer patients. In addition, miR-125b expression was positively correlated with HER2, but not with progesterone receptor and estrogen receptor. Notably, high miR-125b expression was significantly correlated with tumor size and Tumor-Node-Metastasis stage in the HER2-positive breast cancer patients, along with a poor prognosis. The present study provides clinical data to confirm the oncogenic potential of miR-125b, particularly in HER2-positive human breast cancer. Thus, identification of miR-125b may be a potential molecular biomarker for the prediction of clinical outcomes in breast cancer patients, particularly HER2-positive cases that will receive paclitaxel-based neoadjuvant chemotherapy.

Inactivation of BRD7 results in impaired cognitive behavior and reduced synaptic plasticity of the medial prefrontal cortex

BRD7 is a bromodomain-containing protein (BCP), and recent evidence implicates the role of BCPs in the initiation and development of neurodevelopmental disorders. However, few studies have investigated the biological functions of BRD7 in the central nervous system. In our study, BRD7 was found to be widely expressed in various regions of the mouse brain, including the medial prefrontal cortex (mPFC), caudate putamen (CPu), hippocampus (Hip), midbrain (Mb), cerebellum (Cb), and mainly co-localized with neuron but not with glia. Using a BRD7 knockout mouse model and a battery of behavioral tests, we report that disruption of BRD7 results in impaired cognitive behavior leaving the emotional behavior unaffected. Moreover, a series of proteins involved in synaptic plasticity were decreased in the medial prefrontal cortex and there was a concomitant decrease in neuronal spine density and dendritic branching in the medial prefrontal cortex. However, no significant difference was found in the hippocampus compared to the wild-type mice. Thus, BRD7 might play a critical role in the regulation of synaptic plasticity and affect cognitive behavior.

High Bak Expression Is Associated with a Favorable Prognosis in Breast Cancer and Sensitizes Breast Cancer Cells to Paclitaxel.

Breast cancer has become the leading cause of cancer-related death among women. A large number of patients become resistant to drug chemotherapy. Paclitaxel (Taxol) is an effective chemotherapeutic agent used to treat cancer patients. Taxol has been widely used in human malignancies including breast cancer because it can stabilize microtubules resulting in cell death by causing an arrest during the G2/M phase of the cell cycle. Pro-apoptotic Bcl-2 antagonist killer 1 (Bak) plays an important role in Taxol-induced apoptosis in breast cancer. In our present study, we investigated the expression of the Bak protein and clinicopathological correlations in a large sample of breast cancer tissues by immunohistochemistry. We found that the percentage of high scores of Bak expression in breast cancer was significantly lower than that of the non-cancerous breast control tissue. In addition, lower Bak expression was positively associated with the clinical TNM stage of breast cancer with a significant decrease in overall survival compared with those with higher Bak expression especially in the Luminal and HER2 subtypes. Importantly, higher Bak expression predicted a favorable clinical outcome in the cases treated with Taxol indicated by a higher overall survival than that of patients with lower Bak expression especially in Luminal and HER2 subtypes. Furthermore, these results were confirmed in vitro since overexpression of Bak sensitized breast cancer cells to Taxol by inhibiting proliferation and promoting apoptosis; in contrast, downregulation of Bak through siRNA transfection inhibited Taxol induced-apoptosis. Therefore, our results demonstrate that Bak acts as a sensitive biomarker and favorable prognostic factor for Taxol treatment in breast cancer. The restoration of Bak expression would be therapeutically beneficial for Taxol resistant breast cancer patients.