Using differential display PCR, we have identified a gene [NOEY2, ARHI (designation by the Human Gene Nomenclature Committee)] with high homology to ras and rap that is expressed consistently in normal ovarian and breast epithelial cells but not in ovarian and breast cancers. Reexpression of NOEY2 through transfection suppresses clonogenic growth of breast and ovarian cancer cells. Growth suppression was associated with down-regulation of the cyclin D1 promoter activity and induction of p21 WAF1͞CIP1 . In an effort to identify mechanisms leading to NOEY2 silencing in cancer, we found that the gene is expressed monoallelically and is imprinted maternally. Loss of heterozygosity of the gene was detected in 41% of ovarian and breast cancers. In most of cancer samples with loss of heterozygosity, the nonimprinted functional allele was deleted. Thus, NOEY2 appears to be a putative imprinted tumor suppressor gene whose function is abrogated in ovarian and breast cancers.
ARHI is a maternally imprinted tumor suppressor gene whose expression is markedly downregulated in breast cancer. Reactivation of ARHI expression in breast cancer cells is associated with increased histone H3 acetylation and decreased lysine 9 methylation of histone H3. An ARHI promoter segment that spanned bases À420 to þ 58 (designated the P2 region) exhibits significantly higher promoter activity in normal cells than in cancer cells. To better understand the molecular mechanisms contributing to this differential transcriptional activity, we sought to identify transcription factors that bind to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies can supershift the A2-protein complexes, whereas anti-E2F2 and 6 antibodies cannot, demonstrating that the A2 fragment interacts with specific members of the E2F family proteins. When compared with normal breast epithelial cells, breast cancer cells have significantly elevated expression of E2F1, 4 and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor -trichostatin A (TSA). When SKBr3 cells were cotransfected with an ARHI/ luciferase reporter and E2F-expression vectors, E2F1 and 4 reduced ARHI promoter activity 2-3-fold, and this reduction could be reversed by TSA treatment. The negative regulation by E2F-HDAC complexes could also be reduced by small interfering RNA of E2F1 and 4. While the retinoblastoma protein, pRB, alone had no effect on ARHI promoter activity, repression by E2F1, but not E2F4, was enhanced by the coexpression of pRB. Taken together, our results suggest that E2F1, 4 and their complexes with HDAC play an important role in downregulating the expression of the tumor suppressor gene ARHI in breast cancer cells.
Acute respiratory distress syndrome virus-2 (SARS-CoV-2) responsible for coronavirus disease 2019 (COVID-19) infection, which causes global public health emergencies, has sped widely for more than 5 months and has the risk of long-term transmission. No effective treatment has been discovered to date. In the cases we report, the patient continued to deteriorate even after administration of antiviral drugs such as lopinavir/ritonavir, interferon-α, and ribavirin, as well as intravenous injection of meropenem, methylprednisolone, and immunoglobulin. So, we infused the patient with convalescent plasma (CP), and the absolute lymphocyte count increased the next day and returned to normal on the fourth day. Followed by intravenous infusion of mesenchymal stem cells (MSCs), bilateral infiltrates were absorbed and the pulmonary function was significantly improved. We note that the intravenous infusion of CP and MSCs for the treatment of severe COVID-19 patients may have synergistic characteristics in inhibiting cytokine storm, promoting the repair of lung injury, and recovering pulmonary function. We hope to provide a reference for the research direction of COVID-19 clinical strategies.
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