Long Non-Coding RNA FAM157C Contributed to Clonal Proliferation in Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disease of hematopoietic stem cells. However, the mechanism of proliferative advantage of PNH clone is unclear. Long noncoding RNAs (LncRNAs) have a wide range of biological functions, including regulation of gene expression, cell differentiation, and proliferation, while its role in PNH remains unclear. Methods: In our study, CD59－and CD59+ granulocytes and monocytes from 5 PNH patients were sorted, and LncRNAs and mRNAs were detected by RNA sequencing. The proliferation-related NF-κB pathway was focused on. A total of 8 mRNAs and 5 LncRNAs were verified by qRT-PCR, and analyzed the correlation with clinical data. Meanwhile, the function of LncRNA was studied.Results: LncRNA FAM157C were verified to be upregulated in PNH clone cells, which were positively correlated with LDH level and CD59- granulated and monocytes cells ratio. After knockdown of FAM157C gene in PIGA-KO-THP-1 cell line, we found that the cells were blocked in G0/G1 phase and S phase, and the apoptosis rate increased, while the proliferation ability decreased. Conclusions: LncRNA FAM157C was proved to promote PNH clone proliferation, which is the first time to explore the role of LncRNAs in PNH.

Jet-printing microfluidic devices on demand

ABSTRACTThere is an unmet demand for microfluidics in biomedicine. We describe contactless fabrication of microfluidic circuits on standard Petri dishes using just a dispensing needle, syringe pump, 3-way traverse, cell-culture media, and an immiscible fluorocarbon (FC40). A submerged micro-jet of FC40 is projected through FC40 and media on to the bottom of a dish, where it washes media away to leave liquid fluorocarbon walls pinned to the substrate by interfacial forces. Such fluid walls can be built into almost any imaginable 2D circuit in minutes, which we exploit to clone cells using limiting dilution in a way that beats the Poisson limit, sub-culture adherent cells, and feed arrays of cells continuously for a week. This general method should have wide application in biomedicine.One sentence summaryIn the everyday world, we cannot build complex structures out of liquids as they collapse into puddles; in the microworld we can.

Intratumoral cycling hypoxia inhibits expression level of estrogen receptor to promote the formation of heterogeneous sub-clones in luminal A breast cancer

Background The expression level of estrogen receptor (ER) is positively correlated with chemoresistance in patients with Luminal A tumor (LAT). ER expression level alters frequently after neoadjuvant chemotherapy which may be related with hypoxia. The spatial and temporal heterogeneity of tumor sub-clones is one of the major contributors to treatment failure. It is essential to investigate how the intratumoral heterogeneous sub-clones survive hypoxic microenvironment in LAT. Material and Methods LAT with largest cross section was divided into micro-sections, the expressions of hypoxia inducible factor-1 (HIF-1) and ER were then detected by immunohistochemistry. The intratumoral distribution of micro-vessels was assessed by 3D reconstruction and stained CD34; the cycling hypoxia model of MCF-7 was established in a step fashion to investigate the changes in HIF-1 and ER expressions. All statistical analyses were performed using SPSS software (version 17.0 for Windows). Results There was a negative correlation between the expressions of ER-alpha and HIF-1alpha (c=-2.40; p=0.044) as assessed by mean optical density values in the maximal cross section of tumor. As shown by 3D ultrasound image, the center of tumor had less functional micro-vessels compared with the periphery (P<0.05). From the periphery to the center of tumors in the large sections from 8 patients with LAT, the expressions of ER, progesterone receptor and CD34 were gradually decreased with HIF-1alpha expression showing an opposite direction. There was a negative correlation between expressions of ER-alpha and HIF-1alpha, and between expressions of CD34 and HIF-1alpha. No significant difference was noted in the outcomes of cytometry between the hypoxia and control groups. ER-alpha expression gradually decreased with the time course of cycling hypoxia with HIF-1alpha showing an opposite direction. Conclusion LAT is composed of heterogeneous sub-clone cells which can be distinguished by ER-alpha and HIF-1alpha. The distribution of heterogeneous sub-clone cells was closely related with heterogeneous microenvironment of hypoxia / reoxygenation. Keywords: Luminal A breast cancer, cycling hypoxia, estrogen receptor, heterogeneous sub-clones.

The Expressions of CD114, CD117 and STAT5 in CD34+CD59− and CD34+CD59+ Bone Marrow Cells of the Patients with Paroxysmal Nocturnal Hemoglobinuria

Abstract 4404 Objective: To measure the expressions of granulocyte colony-stimulating factor receptor (G-CSFR, CD114) and stem cell factor receptor (C-KIT, CD117) on the membrane of CD34+CD59− and CD34+CD59+ bone marrow cells of the patients with paroxysmal nocturnal hemoglobinuria(PNH), and the signaling pathway protein STAT5 within the cytoplasm of those cells. Methods: The expressions of CD114 and CD117 on the cell membrane and STAT5 protein within the cytoplasm of bone marrow CD34+CD59+ and CD34+CD59− cells from 26 PNH patients and 14 normal controls were examined by flow cytometry (FCM). Results: The percentage of CD114 positive cells in CD34+CD59− cells of PNH patients was (43.23±19.77)%,which was significantly lower than that in CD34+CD59+ cells of PNH patients (73.72±17.42) (P<0.01) or normal controls (65.91±13.70)% (P<0.01). There was no statistic difference between the two latter (P>0.05). The percentage of CD117 positive cells in CD34+CD59− cells of PNH patients was (49.20±26.80)%,which was significantly lower than that in CD34+CD59+ cells of PNH patients (67.62±17.41) (P<0.01) or normal controls (70.21±12.68)% (P<0.01). There was no statistic difference between the two latter (P>0.05). The STAT5 MFI in CD34+CD59− and CD34+CD59+ cells of PNH patients and CD34+CD59+ cells of normal controls was (270.01±181.26), (205.05±146.16), (227.39±156.65) respectively. There was no statistic difference among the three groups (P>0.05). Conclusions: In PNH, CD114 and CD117 expressed lower on bone marrow PNH clone cells than normal clone cells, but the expressions of signaling pathway protein STAT5 within the cytoplasm was normal. Disclosures: No relevant conflicts of interest to declare.

Curdione Plays an Important Role in the Inhibitory Effect ofCurcuma aromaticaon CYP3A4 in Caco-2 Cells

Curcuma aromaticais a plant belonging to genusCurcumaof familyZingiberaceaeand is widely used as supplements in Japan. Rhizomes ofC. aromaticahave curcumin as a major yellow pigment and curdione as a main ingredient of essential oils. In this study, we investigated the affect ofC. aromaticaon CYP3A4 using 1α,25-(OH)2-D3-treated Caco-2 clone cells. Caco-2 cells were treated with methanol extract (0.1 mg ml−1), its hexane soluble fraction (0.1 mg ml−1), curcumin (4 μM) and curdione (20 μM) for 72 hours. Nifedipine was used as a substrate of CYP3A4. Methanol extract, hexane fraction and curdione inhibited the formation of oxidized nifedipine by 50–70%, and curcumin showed no effect. The IC50s of methanol extract, hexane fraction and curdione to oxidized nifedipine formation were 21, 14 and 3.9 μg ml−1(16.9 μM), respectively. The content of curdione in methanol extract was 11.4%. Moreover, all of methanol extract, hexane fraction and curdione decreased CYP3A4 protein expression but had no affect on CYP3A4 mRNA expression. Our results showed that these drugs further decreased the CYP3A4 protein expression level after the protein synthesis was inhibited by cychroheximide. These findings suggest that curdione plays an important role in the CYP3A4 inhibitory activity ofC. aromaticaand curdione might inhibit the activity by accelerating the degradation of CYP3A4.

Over-Expression of IGF-IR in Malignant Clonal Cells in Bone Marrow of Myelodysplastic Syndromes.

Abstract 4832 To explore the expression level of insulin-like growth factor-1 receptor (IGF-IR) in malignant clone cells of myelodysplastic syndromes (MDS). Method Fluorescence in situ hybridization (FISH) and immunochemistry (APAAP, alkaline phosphatase anti-alkaline phosphatase) were used together to detect the expression of IGF-IR in the bone marrow cells of 26 MDS patients with known abnormal karyotypes. Result The average IGF-IR expression level on the surface of clone cells from the 26 MDS cases was markedly elevated compared to the corresponding level in normal cells (78.2±13.7% vs 14.1±14.0%,P <0.0001). The percentages of malignant clone cells in all 26 MDS cases were significantly correlated with the respective percentages of IGF-IR positive nucleated cells (r = 0.909; P <0.0001). No significant difference in the IGF-IR expression level on the clone cells were observed either between high- and low-risk MDS patients or among MDS patients with different abnormal karyotypes. Conclusion IGF-IR might be taken as a marker of clone cells in MDS because of its propensity to cause malignant proliferation. Disclosures No relevant conflicts of interest to declare.

HEPATITIS B VIRUS

Dried leaves of Marchantia convoluta are largely used to protect livers,and to treat tumefaction of skins in China. Flavonoids from Marchantia Convoluta (MCF) was one of the mostpotentially effective anti-inflammatory. MCF was studied here for its ability to inhibit the proliferation of 2,2,15 cells(clone cells derived from HepG2 cells that were transected with a plasmid containing HBV, DNA). All concentrations(5,10,20 and 40 :g/ml) of MCF inhibit hepatitis B surface antigen (HbsAg) and hepatitis B E antigen (HbeAg) in thecultured medium released from 2.2.15 cells. Analysis of morphological changes of MCF-treated phase- contrastmicroscope revealed a possible model of action for MCF to inhibit Proliferation of 2.2.15 cells by inducing apoptosis.

Expression of Major Histocompatibility Complex Class II and CD80 by Gingival Epithelial Cells Induces Activation of CD4+ T Cells in Response to Bacterial Challenge

ABSTRACT HLA-DR (major histocompatibility complex [MHC] class II) is often expressed by epithelial cells in gingival tissues with periodontal disease but not by cells in healthy gingival tissues. Confocal microscopic analyses revealed that gingival epithelial cells (GEC) from tissue with periodontal disease express both HLA-DR and B7-1 (CD80) costimulatory molecules. Rat GEC lines were established to elucidate the possible role of MHC class II and B7-1 expression by GEC. Stimulation of a rat GEC line with gamma interferon (IFN-γ) induced the expression of MHC class II, whereas the cell line constitutively expressed B7-1 costimulatory molecules as determined by reverse transcription-PCR and flow cytometry. Actinobacillus actinomycetemcomitans Omp29-specific CD4+ Th1 clone cells proliferated in response to pretreatment of GEC with fixed A. actinomycetemcomitans and IFN-γ. However, the Th1 cells did not respond to pretreatment of GEC with the bacteria alone or IFN-γ alone. The activation of Th1 clone cells induced by the GEC was inhibited by antibody to MHC class II or by CTLA4 immunoglobulin (CTLA4-Ig). Lymph node T cells did not demonstrate superantigen activity to A. actinomycetemcomitans, although both lymph node T cells and Th1 clone cells were sensitive to superantigen activity of staphylococcal enterotoxin A as cultured in the presence of IFN-γ-treated GEC. These results suggested that GEC can take up bacterial antigen and consequently process and present the bacterial antigen to CD4+ T cells by MHC class II in conjunction with B7 costimulation. GEC appeared to play a role in the adaptive immune response by stimulating antigen-specific CD4+ T cells.