BackgroundThe role of B7-H3 in acute monocytic leukemia U937 cells has not been thoroughly investigated.Materials and methodsB7-H3 knockdown in the U937 cell line was performed using small hairpin (sh)RNA lentivirus transduction. The effects on cell proliferation, cycle, migration, and invasion were investigated by Cell Counting Kit-8 assay, methyl cellulose colony-forming assay, propidium iodide staining, and Transwell assays in vitro. Changes in cell growth inhibition and apoptosis, when combined with chemotherapy drugs, were determined using the Cell Counting Kit-8 and Annexin V-FITC/PI assays. U937 xenograft models were used to assess the effects of B7-H3 on tumorigenicity and the therapeutic effect of B7-H3 knockdown in combination with chemotherapy drugs in vivo.ResultsDownregulation of B7-H3 significantly decreased U937 cell growth and colony-forming ability. The mean inhibition rate of tumor growth with B7-H3 knockdown was 59.4%, and the expression of both Ki-67 and PCNA in xenografts was significantly reduced. After B7-H3 silencing, the U937 cell cycle was arrested at the G0/G1 phase. The cell migration rate of B7-H3 knockdown cells was reduced more than fivefold, and invasion capacity decreased by 86.7%. B7-H3 RNAi profoundly increased the antitumor effect of chemotherapy in vitro and in vivo. On day 19, inhibition rates of tumor growth in B7-H3 shRNA combined with idarubicin, cytarabine, and idarubicin plus cytarabine were 70.5%, 80.0%, and 90.0%, respectively (P=0.006, P=0.004, and P=0.016, respectively).ConclusionB7-H3 may promote U937 cell progression, and shRNA targeting B7-H3 significantly enhances sensitivity to chemotherapeutic drugs. These results may provide new insight into the function of B7-H3 and a promising therapeutic approach targeting B7-H3 in acute monocytic leukemia.

Abstract. B7-H3 (CD276), known as a member of B7 immunoregulatory family, is a type I transmembrane glycoprotein aberrantly expressed in numerous types of cancer and associated with poor prognosis. However, the role of B7-H3 in oncogenesis and chemosensitivity of mantle cell lymphoma (MCL) remains unknown. We determined the effects of downregulating B7-H3 expression on tumor progression and the sensitivity of chemotherapeutic drug in mantle cell lymphoma. B7-H3 knockdown was performed using lentivirus transduction in the Maver and Z138 mantle cell lymphoma cell lines, respectively. The effects of B7-H3 on cell proliferation, cycle, migration and invasion were investigated by CCK-8 assay, methyl cellulose colony forming assay, PI staining, and Transwell assays in vitro. By establishing Maver and Z138 xenograft models, the effects of B7-H3 on tumorigenicity were observed, and Ki-67 and PCNA was detected by immunohistochemistry. The downregulation of B7-H3 significantly decreased tumor proliferation in MCL in vitro and in vivo. In the B7-H3 knockdown groups of Maver and Z138 xenograft models, the mean inhibition rate of tumor growth was 59.1 and 65.0% (p=0.010 and 0.003), and the expression of both Ki-67 and PCNA were significantly lower, respectively. After B7-H3 silencing, the cell cycles of Maver and Z138 were both arrested at G0/G1 phase, and the cell migration rates and invasion capacity were decreased as well. Moreover, the impacts of B7-H3 RNAi on the antitumor effect of chemotherapy drugs were determined with CCK-8 and Annexin V-FITC/PI assays in vitro and with xenograft models in vivo. The silencing of B7-H3 increased the sensitivity of Maver and Z138 cells to rituximab and bendamustine and enhanced the drug-induced apoptosis, respectively. Our study demonstrates for the first time that B7-H3 promotes mantle cell lymphoma progression and B7-H3 knockdown significantly enhances the chemosensitivity. This may provide a new therapeutic approach to mantle cell lymphoma. IntroductionMantle cell lymphoma (MCL), a heterogeneous subtype of B-cell non-Hodgkin lymphoma (NHL), accounts for ~7% of NHL cases in the USA and Europe and has one of the worst outcomes of all the lymphomas (1,2). It is characterized by the t(11;14) (q13;q32) translocation, which results in overexpression of cyclin D1 and deregulation of the cell cycle (2). At initial diagnosis, most patients with the median age ~68-70 years have advanced stage disease, and the median overall survival is 3-5 years (3). Although several novel agents have proven to be effective, MCL remains a largely incurable disease and the following relapse is still challenging. Therefore, understanding the molecular mechanisms of MCL pathogenesis and drug resistance will aid in the development of highly active targeted therapies for the disease.B7-H3, a new member of B7 immunoregulatory family with immunoglobulin-like structure (4), is induced in activated dendritic cells, monocytes and T cells (5). Aberrant expression of B7-H3 has been reported and associated with poor prog...

Background : Aberrant expression of the immunoregulatory protein B7-H3 in B7 family has been associated with more advanced disease and poor prognosis in a wide range of cancer. However, the role of B7-H3 in acute monocytic leukemia U937 cells has not been thoroughly investigated. In this study, we examined the gene expression and subcellular localization of B7-H3 in human peripheral blood cells and hematologic malignancy cell lines. Then, we determined the effects of down-regulating B7-H3 expression on U937 cells, which has the highest B7-H3 protein expression in the detected cell lines. Methods : B7-H3 expression in 12 healthy volunteer peripheral blood cell samples and 13 human hematologic malignancy cell lines was determined by RT-PCR, western blot and flow cytometry. B7-H3 knockdown in the U937 cell line was performed using shRNA lentivirus transduction. The effects on cell proliferation, cycle, migration and invasion were investigated by CCK-8 assay, methyl cellulose colony forming assay, PI staining, and transwell assays in vitro. U937 xenograft models were used to assess the effects of B7-H3 on tumorigenicity and Ki-67 and PCNA was detected through immunohistochemical. Changes in cell growth inhibition and apoptosis, when combined with chemotherapy drugs, were determined using CCK-8, Annexin V-FITC/PI and Hoechst 33342 staining assays in vitro. The therapeutic effect of B7-H3 knockdown in combination with chemotherapy drugs were also studied by U937 xenograft models in vivo. Results: B7-H3 mRNA was widely expressed in the 12 hematologic malignancy cell lines except for CZ1 and PB MNCs of volunteers. But the protein level of B7-H3 was only abnormally overexpressed in 12 hematologic malignancy cell lines except for CZ1, with subcellular localizations in nucleus and cytoplasm mostly determined. The down-regulation of B7-H3 in U937 cells significantly decreased cell growth and the rate of colony formation by 32.8% in 72 h and 70.3% in 14 d. Mean inhibition rate of tumor growth with B7-H3 knockdown was 59.4%, and expression of both Ki-67 and PCNA in xenografts was significantly reduced. After B7-H3 silencing, U937 cell cycle was arrested at G0/G1 phase, and the cell cycle-related proteins Cyclin D1 and CDK4 were lower. Cell migration rate of B7-H3 knockdown cells was reduced more than five-fold, and invasion capacity was decreased by 86.7%. The rates of distant metastasis in B7-H3 knockdown xenografts were significantly decreased. The invasion-related proteins MMP-2 and MMP-9 were lower in both B7-H3 knockdown cells and xenografts. B7-H3 RNAi profoundly increased the anti-tumor effect of chemotherapy and enhanced the activity of caspase-3 in vitro and in vivo. At the end of observation (on day 19 after inoculation), inhibition rates of tumor growth in B7-H3 shRNA combined with idarubicin, cytarabine, and idarubicin plus cytarabine groups were 70.5%, 80.0%, and 90.0%, respectively (P=0.006, 0.004 and 0.016). The TUNEL positive cells were significantly increased in the B7-H3 shRNA combined with chemotherapy drugs groups. Conclusions: B7-H3 protein was abnormally overexpressed in 12 hematologic malignancy cell lines except for CZ1, with subcellular localizations in nucleus and cytoplasm mostly determined. B7-H3 may promote U937 cell progression, and shRNA targeting B7-H3 significantly enhances sensitivity to chemotherapeutic drugs. These results may provide new insight into the function of B7-H3 and a promising therapeutic approach targeting B7-H3 in acute monocytic leukemia. Disclosures No relevant conflicts of interest to declare.

Ossification of the posterior longitudinal ligament (OPLL) is a disorder with multiple pathogenic mechanisms and leads to different degrees of neurological symptoms. Recent studies have revealed that non-coding RNA (ncRNA), including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), could influence the development of OPLL. Nevertheless, the molecular mechanisms linking circular RNAs (circRNAs) and the progression of OPLL is still unknown. The current research explored the expression profiles of OPLL-related circRNAs by microarray analysis, and applied qRT-PCR to validate the results. Subsequently, we confirmed the upregulation of hsa_circ_0007292 in OPLL cells by qRT-PCR and validated the circular characteristic of hsa_circ_0007292 by Sanger sequencing. Fluorescence in situ hybridization (FISH) unveiled that hsa_circ_0007292 was predominantly located in the cytoplasm. Functionally, gain-of-function and loss-of-function experiments showed that hsa_circ_0007292 promoted the osteogenic differentiation of OPLL cells. Mechanistically, the interaction of hsa_circ_0007292 and miR-508-3p was predicted and validated by bioinformatics analysis, dual-luciferase reporter assays, and Ago2 RNA immunoprecipitation (RIP). Similarly, we validated the correlation between miR-508-3p and SATB2. Furthermore, rescue experiments were performed to prove that hsa_circ_0007292 acted as a sponge for miR-508-3p, and SATB2 was revealed to be the target gene of miR-508-3p. In conclusion, our research shows that hsa_circ_0007292 regulates OPLL progression by the miR-508-3p/SATB2 pathway. Our results indicate that hsa_circ_0007292 can be used as a promising therapeutic target for patients with OPLL.

Aberrant expression of microRNAs (miRNAs) has been associated with spinal ossification of the posterior longitudinal ligament (OPLL). Our initial bioinformatic analysis identified differentially expressed ADORA2A in OPLL and its regulatory miRNAs miR-497 and miR-195. Hence, this study was conducted to clarify the functional relevance of miR-497-195 cluster in OPLL, which may implicate in Adenosine A2A (ADORA2A). PLL tissues were collected from OPLL and non-OPLL patients, followed by quantification of miR-497, miR-195 and ADORA2A expression. The expression of miR-497, miR-195 and/or ADORA2A was altered in posterior longitudinal ligament (PLL) cells, which then were stimulated with cyclic mechanical stress (CMS). We validated that ADORA2A was expressed highly, while miR-497 and miR-195 were down-regulated in PLL tissues of OPLL patients. miR-195 and miR-497 expression in CMS-treated PLL cells was restored by a demethylation reagent 5-aza-2′-deoxycytidine (AZA). Moreover, expression of miR-195 and miR-497 was decreased by promoting promoter CpG island methylation. ADORA2A was verified as the target of miR-195 and miR-497. Overexpression of miR-195 and miR-497 diminished expression of osteogenic factors in PLL cells by inactivating the cAMP/PKA signaling pathway via down-regulation of ADORA2A. Collectively, miR-497-195 cluster augments osteogenic differentiation of PLL cells by inhibiting ADORA2A-dependent cAMP/PKA signaling pathway.