Identification of CDK6 and RHOU in Serum Exosome as Biomarkers for the Invasiveness of Non-functioning Pituitary Adenoma / 中国医学科学杂志(英文版)

Objective To explore circulating biomarkers for screening the invasiveness of non-functioning pituitary adenomas (NF-PAs). Methods The exosomal RNAs were extracted from serum of patients with invasive NF-PA (INF-PA) or noninvasive NF-PA (NNF-PA). Droplet digital PCR was adapted to detect the mRNA expression of candidate genes related to tumor progression or invasion, such as cyclin dependent kinase 6 (CDK6), ras homolog family member U (RHOU), and spire type actin nucleation factor 2 (SPIRE2). Student's -test was used to analyze the statistical difference in the mRNA expression of candidate genes between the two groups. Receiver operating characteristic (ROC) curve was used to establish a model for predicting the invasiveness of NF-PAs. The accuracy, sensitivity, specificity and precision of the model were then obtained to evaluate the diagnostic performance. Results CDK6 (0.2600±0.0912 . 0.1789±0.0628, =3.431, =0.0013) and RHOU mRNA expressions (0.2696±0.1118 . 0.1788±0.0857, =2.946, =0.0052) were upregulated in INF-PAs patients' serum exosomes as compared to NNF-PAs. For CDK6, the area under the ROC curve (AUC) was 0.772 (95% : 0.600-0.943, =0.005), the accuracy, sensitivity, specificity and precision were 77.27%, 83.33%, 75.00% and 55.56% to predict the invasiveness of NF-PAs. For RHOU, the AUC was 0.757 (95% : 0.599-0.915, =0.007), the accuracy, sensitivity, specificity and precision were 72.73%, 83.33%, 68.75% and 50.00%. In addition, the mRNA levels of CDK6 and RHOU in serum exosomes were significantly positively correlated (=0.935, <0.001). After combination of the cut-off scores of the two genes, the accuracy, sensitivity, specificity and precision were 81.82%, 83.33%, 81.25% and 62.50%. Conclusions CDK6 and RHOU mRNA in serum exosomes can be used as markers for predicting invasiveness of NF-PAs. Combination of the two genes performs better in distinguishing INF-PAs from NNF-PAs. These results indicate CDK6 and RHOU play important roles in the invasiveness of NF-PAs, and the established diagnostic method is valuable for directing the clinical screening and postoperative treatment.

Methyltransferase-like 3 Promotes the Proliferation of Acute Myeloid Leukemia Cells by Regulating N6-methyladenosine Levels of MYC / 中国医学科学院学报

Objective To investigate the role of methyltransferase-like 3(METTL3) in the proliferation of acute myeloid leukemia (AML) cells and its mechanism. Methods METTL3 expression in AML patients was analyzed in Gene Expression Omnibus data files. METTL3 expression was inhibited by lentivirus-mediated gene transduction in MOLM13 cells,after which cell proliferation was analyzed by cell counting kit-8,N6-methyladenosine (m6A) levels of total mRNA was analyzed by ELISA,specific m6A on MYC was analyzed by gene-specific m6A RNA immunoprecipitation,and MYC expression was analyzed by RT-qPCR and Western blot analysis. Results METTL3 level was slightly increased in AML-M5 patients,and its expression was significantly higher in immature cells than in mature monocytes (t=4.504,P=0.0098). METTL3 knock-down significantly suppressed cell proliferation (P<0.001),reduced m6A level of total mRNA (t=3.606,P=0.042) and specific m6A level on MYC mRNA (P<0.01),and suppressed MYC expression (P<0.01). Conclusion METTL3 acts as an oncogene in MOLM13 cells by upregulating MYC expression.

Function of RNA binding protein KSRP in type M5 acute myeloid leukemia cells / 基础医学与临床

Objective To investigate the expression of RNA binding protein KSRP in multiple acute myeloid leuke-mia(AML) patients. Methods KSRP expression among AML patients and normal controls was analyzed in gene expression omnibus (GEO) datasets and the cancer genome atlas(TCGA) database. KSRP expression was inhibi-ted by lentivirus-mediated gene transduction in THP-1 cells,cell proliferation and apoptosis were analyzed. Results KSRP has lower expression in t(15;17) acute promyelocytic leukemia while has higher expression in mixed lineage leukemia(MLL) translocated acute monocytic leukemia or acute myelomonocytic leukemia. KSRP knock-down sig-nificantly promoted apoptosis (P<0.01),as well as suppressed cell proliferation (P<0.01). In addition, overex-pression of miR-129 increased cell proliferation(P<0.05). Conclusions KSRP may regulate AML-M5 cell growth through promoting miR-129 processing.

Inhibitory effects of vina-ginsenoside R7 on activation of rat C6 astrocytes induced by LPS and TNF-α combination / 中国中药杂志

To investigate the inhibitory effect and mechanism of vina-ginsenoside R7 (R7) on the activation of rat C6 astrocytes cells induced by LPS/TNF-α, cells in logarithmic growth phase were cultured in DMEM medium without FBS for 24 h. After dissociated using 0.25% EDTA-trypsin, the cells were seeded into respective plates at the density of 1.5×10⁶ cells per mL and cultured overnight. The cells were divided into the following groups： control group (no treatment), model group (treated with LPS 1 μg•mL⁻¹ and TNF-α 10 μg•L⁻¹ treated for 24 h), R7 groups (pre-treated with 6.25, 12.5, 25, 50, and 75 μmol•L⁻¹ R7, 4 μmol•L⁻¹ L-NMMA for 2 h and then stimulated with LPS 1 mg•L⁻¹ and TNF-α 10 μg•L⁻¹ for 24 h). Cell viability was analyzed by CCK-8 kit. Secretion of nitric oxide (NO) in the medium was measured by Greiss method. Concentrations of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) were assayed by ELISA kits. Gene expressions of inflammatory factors were examined by quantitative-PCR analysis. Activation of NF-κB was detected by dual luciferase reporter gene assay kit. The results showed that R7 could significantly inhibit the secretion of NO from C6 cells in a dose-effect manner, with an IC₅₀ of 34 μmol•L⁻¹. And it could reduce cell proliferation induced by LPS/TNF-α stimulation. Furthermore, R7 at 50 μmol•L⁻¹ significantly down-regulated gene expressions of iNOS (P<0.001), TNF-α (P<0.001), IL-1β(P<0.05), and COX-2 (P<0.001), but could not change gene expression of IL-6. However, R7 reduced the secretion of TNF-α (P<0.001) and IL-6 (P<0.001). Further studies disclosed that, different concentrations of R7 (25, 50, 100 μmol•L⁻¹) could significantly inhibit the transcription activity of NF-κB(P<0.05, P<0.01, and P<0.001). In conclusion, R7 could inhibit inflammatory responses in C6 cells induced by LPS/TNF-α probably by inhibiting the transcription activity of NF-κB, which indicates its possible therapeutic effect in neurological diseases related to neuroinflammation.

Role of spleen tyrosine kinase in phenotypic modulation of vascular smooth muscle cell induced by platelet-derived growth factor-BB / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the role of spleen tyrosine kinase (syk) in the phenotypic modulation induced by platelet-derived growth factor (PDGF-BB) in rat pulmonary vascular smooth muscle cells (VSMC).</p><p><b>METHODS</b>Vascular smooth muscles were isolated from pulmonary media of SD rats, cultured, adopted, and divided into 3 groups: blank control group, control group and medicine intervention group. The changes of proliferation and ultrastructure of vascular smooth muscle cells by using [(3)H] thymidine incorporation and electron microscopy. The mRNA and protein expression level of syk, alpha-smooth muscle-actin (α-SM-actin) and smooth muscle protein 22alpha (SM22α) were detected by RT-PCR and Western blotting. The change of fluorescence intensity was detected by laser scanning confocal microscope.</p><p><b>RESULTS</b>Treatment with PDGF-BB for 24 h resulted in a significant increase in [(3)H] thymidine incorporation (2429.25 ± 253.36 vs. 242.75 ± 14.33,P < 0.01) and marked change in phenotype and cytoskeleton, the level of average optical density decreased significantly (263.75 ± 19.21 vs.1146.23 ± 62.61, P < 0.01). Meanwhile, the mRNA (1.70 ± 0.25 vs. 1.01 ± 0.12, P < 0.05) and protein level of syk significantly increased, the mRNA and protein expression of α-SM-actin (0.10 ± 0.00 vs. 1.00 ± 0.00, P < 0.01) and SM22α (0.18 ± 0.00 vs. 1.00 ± 0.01, P < 0.01) significantly decreased in VSMC induced by PDGF-BB. Piceatannol could inhibit significantly these biological effects. Compared with control group, the level of [(3)H] thymidine incorporation (527.00 ± 27.76 vs. 2429.25 ± 253.36,P < 0.01) was significantly down-regulated and the VSMC presented an apoptotic status in medicine intervention group, the level of average optical density increased significantly (810.65 ± 37.94 vs. 263.75 ± 19.21,P < 0.01) in medicine intervention group. Meanwhile, the mRNA (0.36 ± 0.07 vs. 1.70 ± 0.25, P < 0.01) and protein level of syk significantly decreased. The mRNA and protein levels of α-SM-actin (0.22 ± 0.00 vs. 0.10 ± 0.00, P < 0.01) and SM22α (0.31 ± 0.00 vs. 0.18 ± 0.00, P < 0.01) were significantly higher in medicine intervention group than in control group. The level of average optical density increased significantly (810.65 ± 37.94 vs. 263.75 ± 19.21, P < 0.01).</p><p><b>CONCLUSION</b>Syk plays an important role in vascular remodeling by changing the phenotypes and cytoskeleton of VSMC stimulated by PDGF-BB.</p>