[Detection of JAK2V617F and CALR Gene Mutations by Multiplex of Patients with Myeloproliferative Neoplasms PCR-Capillary Electrophoresis].

OBJECTIVE: To evaluate the proformance of multiplex PCR and capillary electrophoresis(MPCE) in the detection of JAK2V617F and CALR mutation in myeloproliferative neoplasms(MPN). METHODS: The specificity primers of JAK2617F gene mutation and the primers of CALR gene were designed at the same time. The JAK2V617F and CALR gene primers were labeled with Cy5 fluorescence, all the primers were mixed in one tube for multiplex PCR and the PCR prodcuts were analysised by capillary electrophoresis. Then detection limit and sensitivity of MPCE were evaluated, and compared with comercial diagnostic kit. RESULTS: JAK2V617F and CALR gene mutations could be detect by MPCE in one PCR test. JAK2V617F mutation could be detected at 0.01 ng genomic DNA, double positive JAK2V617F and CLAR gene mutations could be detected at 0.1 ng genomic DNA, at least 0.1% JAK2V617F positive mutation could be detected. The consistency between MPCE and commercial diagnostic gene mutation kit was 100%. CONCLUSION: It is developed that a new gene mutation detection method of JAK2 V617F and CLAR gene based on MPCE in our experiment and it can be used as a new reagent for molecular diagnosis of MPN patients.

LRTM1 promotes the differentiation of myoblast cells by negatively regulating the FGFR1 signaling pathway.

The proliferation and differentiation of myoblast cells are regulated by the fibroblast growth factor receptor (FGFR) signaling pathway. Although the regulation of FGFR signaling cascades has been widely investigated, the inhibitory mechanism that particularly function in skeletal muscle myogenesis remains obscure. In this study, we determined that LRTM1, an inhibitory regulator of the FGFR signaling pathway, negatively modulates the activation of ERK and promotes the differentiation of myoblast cells. LRTM1 is dynamically expressed during myoblast differentiation and skeletal muscle regeneration after injury. In mouse myoblast C2C12 cells, knockout (KO) of Lrtm1 significantly prevents the differentiation of myoblast cells; this effect is associated with the reduction of MyoD transcriptional activity and the overactivation of ERK kinase. Notably, further studies demonstrated that LRTM1 associates with p52Shc and inhibits the recruitment of p52Shc to FGFR1. Taken together, our findings identify a novel negative regulator of FGFR1, which plays an important role in regulating the differentiation of myoblast cells.

LSD1 negatively regulates autophagy in myoblast cells by driving PTEN degradation.

Lysine-specific demethylase 1 (LSD1) is a well characterized transcriptional regulator functioning on the chromatin to remove mono- and di-methyl groups from lysine 4 or lysine 9 of histone 3 (H3K4 or H3K9). LSD1 also has non-transcriptional activities via targeting non-histone substrates that participate in diverse biological processes. In this report, we determined that LSD1 negatively regulates autophagy in skeletal muscle cells by promoting PTEN degradation in a transcription-independent mechanism. In C2C12 cells, LSD1 inhibition or depletion significantly induced the initiation of autophagy; and autophagy resulted from LSD1 inhibition is associated with AKT/mTORC1 inactivation. Notably, the proteins of PTEN, a prominent repressive AKT modulator, are stabilized by LSD1 inhibition despite a decrease of its mRNA levels. Further data demonstrated that LSD1 interacts with PTEN protein and enhances its ubiquitination and degradation. Together, our findings identify a novel biological function of LSD1 in autophagy, mediated by regulating the stability of PTEN and the activity of AKT/mTORC1.

[Efficacy of Quercetin-sensitized Adriamycin for Treatment of Refractory Acute Leukemia].

OBJECTIVE: To investigate the chemotherapeutic efficency of quercetin sensitized adriamycin. METHOD: CCK-8 was used to detect the inhibitory effect of different doses of adriamycin, quercetin and quercetin combined with adriamycin on the proliferation of primary leukemia cells from patients with clinically refractory acute leukemia. Quercetin, adriamycin and their combination were used to treat non-irradiated T-ALL leukemia mice to observe the changes of survival curve and myocardial injury. RESULT: There was no significant difference in the inhibition rate of primary leukemia cell proliferation between the adriamycin concentration group (6, 0.6 and 0.06 µg/ml) and the adriamycin half-dose (3, 0.3 and 0.03 µg/ml) plus quercetin (0.25 mmol/L) group at three different time points (24, 48 and 72 hours). There was a significant difference in the inhibition rate of primary leukemia cell proliferation among the drug concentration groups, and the inhibition rate of primary leukemia cell proliferation was time-and concentration-dependent (r24h，a\c\e=0.995、r48h，a\c\e=1.000、r72h，a\c\e=0.984、r24h，b\d\f=0.993、r48h，b\d\f=0.999、r72h，b\d\f=0.960). In vivo experiments showed that the survival time of non-irradiated T-ALL leukemia mice treated with low-dose adriamycin combined with quercetin was not significantly prolonged compared with the high-dose adriamycin treatment group. The survival time of non-irradiated T-ALL leukemia mice treated with high dose of adriamycin and quercetin was significantly prolonged (P＜0.05). Compared with adriamycin group, the SOD activity in adriamycin combined with quercetin group increased significantly and the MDA content decreased. The results of transcriptome sequencing analysis showed that the expression of Ighv1-84 and Igkv6-14 in adriamycin combined quercetin group and quercetin group was lower than that in adriamycin group. The Ms4a1, Podx1, Mecom, Sh3bgr12, Bex4 and Tdrp expression in adriamycin combined quercetin group and adriamycin group were higher than that in quercetin group, while Crabp1 expression was lower. CONCLUSION: Quercetin can inhibit the proliferation of primary leukemia cells in a time-dependent manner. Quercetin combined with adriamycin inhibit the proliferation of primary leukemia cells significantly, and had synergistic and additive effects on the proliferation of primary leukemia cells, and the inhibiting effect of quercetin combined with adriamycin is concentration-and time-dependent. Quercetin combined with high-dose adriamycin can significantly prolong the survival time of non-irradiated T-ALL leukemia mice and reduce the myocardial damage caused by adriamycin.

High glucose downregulates the effects of autophagy on osteoclastogenesis via the AMPK/mTOR/ULK1 pathway.

Diabetes is a chronic disease that disrupts the balance between bone formation and bone desorption, which can lead to osteoporosis, increasing the risk of fracture. However, compared with osteoblasts, the biological effects of hyperglycemia on osteoclastogenesis remain to be elucidated. Therefore, we investigated the impact of glucose at different concentrations (5.5, 10.5, 15.5, 20.5, 25.5, and 30.5 mM) on osteoclastogenesis using RAW264.7 cells. Cell proliferation was measured with the cell counting kit-8 assay, and osteoclastogenesis was detected with tartrate-resistant acid phosphatase staining and bone resorption assays, as well as protein cathepsin K expression. Compound C, the AMP-activated protein kinase (AMPK) pathway inhibitor, was used to examine the relationship between the AMPK/mTOR/ULK1 signaling pathway and autophagy in osteoclasts. Autophagy was evaluated with transmission electron microscopy and immunofluorescence microscopy and associated proteins were detected with western blotting. The pharmacological autophagic reagents bafilomycin A1, 3-methyladenine, and rapamycin were used to determine the effect of autophagy on osteoclastogenesis. Our results showed that glucose negatively affected osteoclast formation and function but did not affect the proliferation of RAW264.7 cells. Suppression of the AMPK/mTOR/ULK1 signaling axis decreased autophagy in glucose-mediated osteoclast. Furthermore, High levels of glucose decreased autophagy level in osteoclasts. Additionally, interfering with autophagy affected osteoclast formation and function. These findings clarify the mechanisms underlying the effects of glucose-mediated osteoclastogenesis and will help identify novel therapeutic strategies for the protection of skeletal health in diabetic osteoporosis.

[Influence of Mouse Spleen Microenviroment on T-ALL Progression].

OBJECTIVE: To investigate the influence of spleen on disease status of mouse T-ALL. METHODS: The leukemia cells were transplanted into the mice, then the development levels of leukemia cells in different organs of transplanted mice were monitored at different time points after transplantation; the transplanted leukemia cell level in different organs was detected by flow cytometry at different time points after transplantation; the survival of transplanted mice was analyzed by means of splenectomy. RESULTS: The spleen change displayed most severely in process of T-ALL, the number of T-ALL cells in the spleen obviously increased at initial period. The detection of organs showed that along with the progression of leukemia, spleen weight change was the most significant, following by the lever change. The splenectomy test showed that the spleen played a promotive role in progession of T-ALL, and the spleneetomy could difinitely postpone the progression of T-ALL in mice, there was significant difference between splenectomy and non-splenectomy. CONCLUSIONS: In early stage after transplantation of T-ALL cells, the spleen has the promotive effect on function of T-ALL cells, which suggests that the spleen may be a important microenvironment for T-ALL cell migrating into body.

Anticancer bioactive peptides suppress human colorectal tumor cell growth and induce apoptosis via modulating the PARP-p53-Mcl-1 signaling pathway.

AIM: We have reported novel anticancer bioactive peptides (ACBPs) that show tumor-suppressive activities in human gastric cancer, leukemia, nasopharyngeal cancer, and gallbladder cancer. In this study, we investigated the effects of ACBPs on human colorectal cancer and the underlying mechanisms. METHODS: Cell growth and apoptosis of human colorectal tumor cell line HCT116 were measured using cell proliferation assay and flow cytometry, respectively. The expression levels of PARP, p53 and Mcl1A were assessed with Western blotting and immunohistochemistry. For evaluation of the in vivo antitumor activity of ACBPs, HCT116 xenograft nude mice were treated with ACBPs (35 µg/mL, ip) for 10 days. RESULTS: Treatment of HCT116 cells with ACBPs (35 µg/mL) for 4-6 days significantly inhibited the cell growth. Furthermore, treatment of HCT116 cells with ACBPs (35 µg/mL) for 6-12 h significantly enhanced UV-induced apoptosis, increased the expression of PARP and p53, and decreased the expression of Mcl-1. Administration of ACBPs did not change the body weight of HCT116 xenograft nude mice, but decreased the tumor growth by approximately 43%, and increased the expression of PARP and p53, and decreased the expression of Mcl-1 in xenograft mouse tumor tissues. CONCLUSION: Administration of ACBPs inhibits human colorectal tumor cell growth and induces apoptosis in vitro and in vivo through modulating the PARP-p53-Mcl-1 signaling pathway.

[Expression of ADAR1 isoforms in murine acute T-ALL leukemia model].

This study was purposed to investigate the expression of ADAR1 isoforms of P110 and P150 during the development of murine leukemia. A Notch1 over-expressing murine T cell acute lymphoblastic leukemia model was used to study the expression of ADAR1. BMMNC were isolated at different stages of disease and CD45.2(+)GFP(+) leukemia cells were sorted by flow cytometry at late stage. The expression of ADAR1 was detected by real time quantitative PCR. The results showed that mouse bone marrow cells from both leukemia and control groups expressed P110 and P150. Difference of P110 and P150 mRNA expression were observed during the development of leukemia. The expression of P110 dramatically increased and was significantly higher than that in control group. However, the expression level of P150 in leukemia group decreased stably and reached one-fourth of that in control group at 14 day. Furthermore, similar expression patterns could be detected in sorted CD45.2(+)GFP(+) leukemia cells. It is concluded that the mRNA expressions of P110 and P150 show diverse patterns in the development of leukemia, suggesting that RNA editing mediated by ADAR1 isoforms may play different roles in leukemia.

[Effects of catalase on human umbilical cord mesenchymal stem cells].

This study was aimed to investigate the growth and multiple differentiation potential of human umbilical cord tissue derived mesenchymal stem cells (UC-MSCs) transfected by a retroviral vector with catalase (CAT) gene. The UC-MSCs cultured in vitro were transfected by using pMSCV carrying GFP (pMSCV-GFP) and pMSCV carrying CAT (pMSCV-GFP-CAT) respectively, then the MSC-GFP cell line and MSC-GFP-CAT cell line were obtained by sorting of flow cytometry. The GFP expression was observed by a fluorescent microscopy at 48 hours after CAT gene transfection. The GFP+ cells were sorted by flow cytometry. The activity of CAT in GFP+ cells was detected by catalase assay kit. The proliferative capacity of transfected UC-MSCs was determined by cell counting kit-8. The differentiation ability of gene-transfected GFP+ cells into osteogenesis and adipogenesis was observed by von Kossa and oil red O staining. The results indicated that green fluorescence in UC-MSCs was observed at 48 hours after transfection, and the fluorescence gradually enhanced to a steady level on day 3. The percentage of MSCs-GFP was (25.54+/-8.65)%, while the percentage of MSCs-GFP-CAT was (35.4+/-18.57)%. The activity of catalase in UC-MSCs, MSCs-GFP, MSCs-GFP-CAT cells were 19.5, 20.3, 67.2 U, respectively. The transfected MSCs-GFP-CAT could be induced into osteoblasts and adipocytes. After 21 days, von Kossa staining showed induced osteoblasts. Many lipid droplets with high refractivity occurred in cytoplasm of the transfected UC-MSCs, and showed red fat granules in oil red O staining cells. There were no significant differences between transfected and non-transfected UC-MSCs cells (p>0.05). It is concluded that UC-MSCs are successfully transfected by retrovirus carrying GFP or CAT gene, the activity of catalase increased by 3.4-fold. The transfected UC-MSCs maintain proliferation potential and ability of differentiation into osteoblasts and adipocytes.