MicroRNA 494 increases chemosensitivity to doxorubicin in gastric cancer cells by targeting phosphodiesterases 4D.

Acquired drug resistance is one of the main limitations in pharmacological therapy of malignancies including gastric cancer. MicroRNAs (miRNAs) are a class of small noncoding RNAs that suppress their targets by binding to the 3'UTR region of genes. In this study, we explored investigate the target gene of miR-494 and its roles in chemoresistance of gastric cancer. We found that miR-494 was significantly down-regulated in gastric cancer cells lines compared to the normal gastric epithelial cell line. Exogenous overexpression of miR-494 increased the chemosensitivity of gastric cancer cells to doxorubicin. Moreover, miR-494 expression was reduced in a doxorubicin-resistant gastric cancer cells (AGS/dox) compared with the parental cells. MTT assay showed that AGS/dox cells exhibited an elevated viability compared with the parental cells. Enforced expression of miR-494 inhibited AGS/dox cell viability and colony formation ability. In addition, we demonstrated that elevated expression of miR-494 inhibited the mRNA and protein expression of phosphodiesterases 4D (PDE4D) in gastric cancer cell. Luciferase assay showed that miR-494 directly targeted the 3'UTR region of PDE4D. Furthermore, restoration of PDE4D recovered the chemoresistance in miR-494-overexpressed gastric cancer cells. Taken together, this study demonstrated that miR-494 enhanced doxorubicin sensitivity via regulation of PDE4D expression, suggesting a novel therapeutic strategy for anti-chemoresistance in gastric cancer.

Prognostic value of neutrophil-to-lymphocyte ratio, lactate dehydrogenase, D-dimer, and computed tomography score in patients with coronavirus disease 2019.

BACKGROUND: This study aimed to explore the significance of neutrophil-to-lymphocyte ratio (NLR), lactate dehydrogenase (LDH), D-dimer, and CT score in evaluating the severity and prognosis of coronavirus disease 2019 (COVID-19). METHODS: Patients with laboratory-confirmed COVID-19 were retrospectively enrolled. The baseline data, laboratory findings, chest computed tomography (CT) results evaluated by CT score on admission, and clinical outcomes were collected and compared. Logistic regression was used to assess the independent relationship between the baseline level of the four indicators (NLR, LDH, D-dimer, and CT score) and the severity of COVID-19. RESULTS: Among the 432 patients, 125 (28.94%) and 307 (71.06%) were placed in the severe and non-severe groups, respectively. As per the multivariate logistic regression, high levels of NLR and LDH were independent predictors of severe COVID-19 (OR=2.163; 95% CI=1.162-4.026; p=0.015 for NLR>3.82; OR=2.298; 95% CI=1.327-3.979; p=0.003 for LDH>246 U/L). Combined NLR>3.82 and LDH>246 U/L increased the sensitivity of diagnosis in patients with severe disease (NLR>3.82 [50.40%] vs. combined diagnosis [72.80%]; p=0.0007; LDH>246 [59.2%] vs. combined diagnosis [72.80%]; p<0.0001). CONCLUSIONS: High levels of serum NLR and LDH have potential value in the early identification of patients with severe COVID-19. Moreover, the combination of LDH and NLR can improve the sensitivity of diagnosis.

Downregulation of the hexokinase II gene sensitizes human colon cancer cells to 5-fluorouracil.

BACKGROUND: Extensive trials have indicated that cancer cells with high glycolytic activity exhibit decreased sensitivity to anticancer agents. Moreover, recent research has proven that specific inhibitors of hexokinase (HK) II, a key glycolytic enzyme, may enhance the activity of anticancer drugs. The aim of this study was to investigate the effect and mechanisms of HK II on chemosensitivity of a colon cancer cell line (LoVo) to 5-fluorouracil (5-FU). METHODS: HK II gene expression was downregulated by RNA interference in the colon cancer cell line LoVo, which was detected by Western blot analysis. Then the IC(50) value of 5-FU was determined in LoVo cells via MTT assay. In addition, cell apoptosis and mitochondrial membrane potential (MMP) were assessed by flow cytometry and caspase-3 activity by its substrate color reaction. RESULTS: In LoVo cells, HK II downregulation resulted in a decreased IC(50) value of 5-FU and increased apoptosis. Furthermore, HK II downregulation resulted in a decreased MPP and activation of caspase-3. CONCLUSION: Our findings suggest that targeting HK II may be beneficial for patients with colon cancer treated with 5-FU.

[Expression of hexokinase-II gene in human colon cancer cells and the therapeutic significance of inhibition thereof].

OBJECTIVE: To investigate the expression of hexokinase (HK)-II gene in human colon cancer cells and the therapeutic significance of inhibition of HK-II gene. METHODS: Human colon cancer cells of the lines HCT-116, LOVO, HT-19, and SW480 were cultured. The mRNA expression and protein expression of HK-II in these cells were detected by RT-PCR and Western blotting respectively. 3-Bromopyruvic acid (3-BrPA), a HK-II specific inhibitor, of different concentrations was added into the culture fluid of the colon cancer cells for 48 h, then MTT method was used to examine the proliferation of the cells. 3-BrPA combined with adriamycin (ADM) of the concentrations of 0.05 and 0.1 microg/ml, or with oxaliplatin (L-OHP) of the concentration of 0.5 and 1.0 microg/ml was added into culture fluid for 48 h to observe the change of the cell proliferation rate. 3-BrPA of different concentrations was co-cultivated with LOVO cells for 24 h, and then enzyme labeling instrument was used to measure the activity of caspase-3, a cell apoptosis signal. 3-BrPA and CaCl2 were added into the culture fluid of LOVO cells and then ultra-violet spectrum was used to detect the mitochondrial permeability transition pore (PTP) opening degree. Flow cytometry (FCM), with addition of 10 microg/ml Rh123, was used to measure the mitochondrial membrane potential (DeltaPsim) of LOVO cells. RESULTS: Both HK-II mRNA and HK-II protein were expressed in the HCT-116, LOVO, HT-19, and SW480 cells. Treated by 3-BrPA combined with ADM of the concentrations of 0.05 and 0.1 microg/ml for 48 h, the cell proliferation inhibiting rates in the 4 lines were increased from 5.1% +/- 1.3% and 10.5% +/- 2.0% to 46.5% +/- 3.2% and 57.9% +/- 3.3% respectively (all P < 0.01). Treated by 3-BrPA combined with L-OHP of the concentration of 0.5 and 1.0 microg/ml for 48 h, the cell proliferation inhibiting rates were increased from 19.2% +/- 6.1% and 32.2% +/- 2.2% to 48.4% +/- 3.2% and 60.5% +/- 4.6% respectively (all P < 0.01). 24 h after the co-cultivation of 3-BrPA of different concentrations, the caspase-3 activity of the LOVO cells was increased along with the increase of the concentration of 3-BrPA (P = 0.000). The PTP opening degrees induced by 3-BrPA and CaCl(2) of the LOVO cells were 40.0% +/- 3.5% and 37.4% +/- 2.3% respectively (P = 0.348). After treated with 100 microml/L 3-BrPA for 1, 3, and 5 h, the DeltaPsim decrease rates of the LOVO cells were 12.7%, 15.4%, and 26.8% respectively. CONCLUSION: HK-II gene may be an effective therapeutic target for gene may be an effective therapeutic target for gene may be an effective therapeutic target for colon cancer.

A novel method for cryopreservation of individual human spermatozoa.

The purpose of this study is to develop a novel method for the cryopreservation and efficient post-thaw recovery of individual or small numbers of human spermatozoa. Spermatozoa equilibrated in cryoprotectant buffer were injected with an intracytoplasmic sperm injection (ICSI) needle into a droplet of cryoprotectant on a homemade cryoleaf. The droplet was of cryoprotectant and seminal plasma at a ratio of 1:1. The sperm-loaded cryoleaf was slowly lowered over and stored in liquid nitrogen. Spermatozoa were thawed in a 37°C oil bath without dilution and centrifugation. To test the fertilizing ability of these spermatozoa, the recovered spermatozoa were injected by ICSI into 1-d-old or in vitro-matured human oocytes. Fresh spermatozoa from the same semen samples served as controls. The trials were performed in two separate experiments. In the first set of experiments, 92 spermatozoa were thawed and carefully investigated. The spermatozoa from percutaneous epididymal sperm aspiration had a motility recovery of 92.9% (13/14); ejaculated spermatozoa had a motility recovery of 61.5% (48/78), and only 1.3% (1/78) was lost. Together in the first and second set of experiments, the fertilization rates for the fresh and frozen-thawed spermatozoa were 67.6% (25/37) and 60.6% (40/66), respectively (P = 0.052). The mean embryo cleavage rates in the fresh and frozen-thawed groups were 88% (22/25) and 85% (34/40), respectively (P = 0.990). This cryopreservation method for individual or small numbers of human spermatozoa was efficient and simple. These findings make this method a promising technique for the clinical application of ejaculated sperm from oligozoospermic patients.

Endogenous axon guiding chemorepulsant semaphorin-3F inhibits the growth and metastasis of colorectal carcinoma.

PURPOSE: To elucidate the role of Semaphorin-3F (SEMA3F), originally described as an axon guiding chemorepulsant implicated in nerve development, in the progression of colorectal carcinoma. EXPERIMENTAL DESIGN: SEMA3F and its receptor NRP2 were examined in 72 cases of human colorectal carcinoma specimens and cell lines LoVo, SW480, and SW620 with immunohistochemistry and Western blotting. SEMA3F mRNA expression in the frozen tissue specimens and cell lines was examined with quantitative reverse transcriptase-PCR. Confocal laser scanning microscopy was used for detection of cellular localization of the proteins by immunofluorescent staining. MTT assay, flow cytometry, cell adhesion and migration, and xenografts were used to evaluate biological significance of SEMA3F. RESULTS: SEMA3F was significantly reduced in colorectal carcinoma tissues and cell lines. Overexpression of SEMA3F resulted in reduced proliferation, adhesion to fibronectin, and migratory capability as well as reduced S-phase population and integrin αvß3 expression of SW480 colon cancer cells. In addition, SEMA3F-overexpressing cells exhibited diminished tumorigenesis when transplanted orthotopically in nude mice and reduced liver metastases. Moreover, transfection of siRNA targeting SEMA3F in colon cancer cells increased their tumorigenicity in vivo. CONCLUSIONS: Endogenous SEMA3F acts as a suppressor of the growth and metastasis of human colorectal cancer cells.