OBJECTIVE:To analyze associations between differentially expressed miRNAs and development of pancreatic cancers, and to screen biomarkers which might be related to prognosis of pancreatic cancers. METHODS:Data of GSE32678 and GSE71533 datasets in Gene Expression Omnibus (GEO) were downloaded to screen differentially expressed miRNAs by using R language and the intersection of the three data sets were taken. Bioinformatics analysis tool was used to predict target genes of overlapping miRNAs, to perform functional analysis on the target genes,and to construct a protein interaction network (PPI),a miRNA-mRNA network,and a miRNA-mRNA-pathway network. The markers which were related to prognosis of pancreatic cancers were screened by using the expression and prognosis data of miRNAs of pancreatic cancer samples in the Cancer Genome Atlas (TCGA). RESULTS:A total of 22 overlapping miRNAs were screened,and hsamiR-30a-5p,hsa-miR-551a,and hsa-miR-375 were closely related to the prognosis of pancreatic cancer. The core miRNAs in the miRNA-mRNA network were hsa-miR-125b-5p,hsa-miR-221-3p,hsa-miR-31-5p, hsa-miR-222-3p and hsa-miR-10a-5p. CONCLUSION:Analysis of data of pancreatic cancers in the GEO and TCGA databases reveal that hsa-miR-30a-5p,hsa-miR-551a,and hsa-miR-375 significantly affected the prognosis of pancreatic cancers and could be used as markers for prognosis.

OBJECTIVE: This study aimed to detect the expression of human fibroblast growth factor binding protein 2 (FGFBP2) mRNA and to explore its prognostic value in esophageal squamous cell carcinoma (ESCC). METHODS:Tissue microarrays and RNA in situ hybridization (RISH) were used to detect expression of FGFBP2 mRNA in 190 cases of ESCC tissues and 42 cases of adjacent normal tissues. Proliferation assays were utilized to explore the roles of FGFBP2 in ESCC cells in vitro. RESULTS:RISH results show positive expression of FGFBP2 mRNA in 49 out 190 (25.8%) ESCC tissues,whereas no FGFBP2 mRNA signal was detected in normal surgical margins tissues (P<0.01). High expression of FGFBP2 mRNA was correlated with shorter survival time of ESCC patients (P=0.002). Cox regression analyses indicate that FGFBP2 mRNA was an independent prognostic factor in ESCC patients (HR:2.291,95%CI:1.271-4.129,P=0.006). Knockdown of FGFBP2 inhibited the proliferation of ESCC cell lines KYSE150 and KYSE510. Western blotting results demonstrate that knockdown of FGFBP2 decreased the level of phosphorylated AKT (p-AKT) but did not affect the expression of phosphorylated ERK (p-ERK) protein. CONCLUSION: Our data suggest that FGFBP2 mRNA may be a prognosis biomarker of ESCC. With respect to the mechanism, our studies show that knockdown of FGFBP2 depressed proliferation of ESCC cells through activating the AKT pathway.

OBJECTIVE: To investigate the effect of 1,4-benzoquinone (1,4-BQ) exposure and G6PD deficiency on DNA methylation in K562 cells. METHODS:G6PD-deficient K562 cells and normal cells were treated with 0,10,20 μmol/L 1,4-BQ solution for 12,24 and 48 hours. Relative levels of DNA methylation in cell genome were detected by colorimetric analysis and expression levels of DNMT1,DNMT3a and DNMT3b were detected by real-time fluorescence quantitative PCR (qPCR). Expression levels of DNMT1 and DNMT3a proteins were detected by Western blot.RESULTS:The genomic DNA methylation levels in the normal K562 cells were increased in the 10 and 20 μmol/L dose groups (P<0.05) but not by the 20 μmol/L dose group at 24 hour. The methylation levels in the GPPD-deficient K562 cells were significantly higher than that in normal cells for all exposed groups (P<0.05). After 1,4-BQ exposure, the expression levels of DNMT1, DNMT3a, DNMT3b mRNA and DNMT1, DNMT3a proteins in G6PD-deficient cells were higher than those in normal cells (P<0.05). After the addition of GSH, the relative levels of whole genome DNA methylation, DNMTs mRNA and protein expression levels of G6PD-deficient cells and normal cells became non-statistically significant. CONCLUSION:G6PD deficiency might have increased the level of oxidative stress by inhibiting GSH synthesis,which ultimately led to an increase in the production of DNMTs and an increase in the DNAmethylation level of the whole genome.

OBJECTIVE: To construct a ROCK1 gene-knockout model using the CRISPR/Cas9 system and to investigate effects of the ROCK1 knockout on MCF-7 breast cancer cell migration and invasion. METHODS: Three pairs of 20 bp sgRNA targeting ROCK1 were chemically synthesized and inserted into CRISPR expression vectors. The ROCK1 knockout cells were selected with lentivirus infection of MCF-7 cells, and were identified by gene sequencing and Western blot. Effects of the knockout on MCF-7 cell migration and invasion were evaluated by wound-healing and Transwell assays. RESULTS:Western blot data show that ROCK1 knockout MCF-7 cells were successfully and stably established by usage of the CRISPR/Cas9 system. Wound-healing assays show that the wound closure of ROCK1 knockout cells was significantly lower than that of their parental cells[(60.600±0.047)% and (80.404±0.018)%,respectively;P=0.003]. Transwell assays without matrigel show that the migration capability of ROCK1 knockout cells was significantly decreased compared with the control group(271.3 ±5.0 and 448.3 ±5.5, respectively; P=0.000) by 24 h. Transwell assays with matrigel show the invasion capability of ROCK1 knockout cells at 48 h was deeply suppressed compared to that of the controls (1.7±2.9 and 298.3±5.7,respectively;P=0.000). CONCLUSION:Knockout of ROCK1 gene caused significant inhibition of the migration and invasion ability of breast cancer cells,suggesting that ROCK1 may play an important role in the invasion and metastasis of breast cancer.

OBJECTIVE:To investigate associations between expression of Forkhead box protein (FoxO1) and prognosis of prostate cancers (PCa). METHODS: FoxO1 expression data and the corresponding clinical data of PCa patients were downloaded from The Cancer Genome Atlas (TCGA). Expressions of FoxO1 mRNA in PCa tissues were measured by using the GEPIA database. Statistical analyses were performed for relationships between the expression and clinic-pathological factors and prognosis. Cox regression model was performed for the multivariate analysis. The Human Protein Atlas (HPA) was used to measure the UBE2C protein expression. RESULTS: Expression of FoxO1 mRNA was reduced in PCa compared to normal prostate tissues. The expression levels were correlated with the depth of tumor invasion, distant metastasis and degree of differentiation. The disease-free survival rates of patients with high FoxO1 expression were significantly higher than that of patients with low expression (P<0.05). Cox regression analyses show that the depth of tumor invasion and differentiation, and the expression of FoxO1 were independent prognostic factors for prostate cancers (P<0.05). Compared with the normal prostate tissues,immuno-histochemical staining show that FoxO1 expressions were significantly down-regulated in the PCa tissues (P<0.05). CONCLUSION:FoxO1 may be a tumor suppressor gene which can possibly provide the reference value for diagnosis and survival prognosis of patients with prostate cancer.

OBJECTIVE:This study aimed to investigate whether Fludarabine,at a low-concentration, would enhance induction of apoptosis in A549 cells by TRAIL. METHODS: A549 cultures were organized into different groups:control, Fludarabine, TRAIL, and Fludarabine plus TRAIL. The CCK-8 method was used to detect effect of Fludarabine and/or TRAIL on A549 and human umbilical vein endothelial cells (HUVEC). Flow cytometry was used to detect apoptosis rates. Western Blot was used to detect apoptosis-related proteins Survivin,Bcl-2,Bcl-xl,caspase-8,caspase-3,and the expression of JNK and p38. RESULTS: Fludarabine(25 or 50 μmol/L) or TRAIL alone had no significant effects on viability of A549 and HUVEC cells, or apoptosis in A540 cells. The inhibition rates from combined treatment of Fludarabine (25 or 50 μmol/L) and TRAIL were 30.9% and 44.9%, respectively, in A549 cells and the effect was concentration dependent. The combined treatment also caused apoptosis in 89.3% of A549 cells, as well as decreased expression of Survivin,Bcl-2 and Bcl-xl,promoted the activation of caspase-8 and caspase-3,and promoted the phosphorylation of JNK and p38. CONCLUSION:Fludarabine combined with TRAIL specifically induced apoptosis in A549 cells.

OBJECTIVE:To analyze the suptype and activity of spleen immune cells in apolipoprotein CⅢ (Apo C3) transgenic mice. METHODS:Using ICR Apo C3 transgenic mice as a research model,weights of transgenic and wild-type mice were measured and blood samples were collected from the posterior venous plexus of mice. The triglyceride assay kit was used to determine plasma triglyceride levels by enzyme reaction colorimetric method in a microplate reader. Mice with a plasma triglyceride level of ≥ 1 000 mg/dL (11.3 mmol/L) were selected for supsequent experiments. The spleens of these mice were prepared into single cell suspensions. The frequencies and suptypes of various immune cells in the spleen of the transgenic and wild-type mice were detected by flow cytometry using cell membrane surface molecules, intracellular molecules and intranuclear molecular staining. Then,the spleen cells of transgenic and wild-type mice were co-cultured with colon cancer cell MC38 in 1:1,3:1,3:1 ratios for 72 h,and expressions of CD107a in CD8⁺ T cells were determined. RESULTS: Plasma triglyceride levels were elevated in the transgenic mice but there was no significant difference in body weight compared to wild-type mice. Furthermore,the frequencies of CD3⁺CD8⁺ and CD3⁺CD8⁺ NKG2D⁺ cells in spleen of the transgenic mice were increased significantly;of myeloid-derived suppressor cells MDSC) were increased;of spleen NK cells was,however,significantly reduced; of CD3⁺CD4⁺NKG2D⁺,CD3⁺ CD4⁺IL^-17⁺ cells in spleens and of regulatory T cells,NKT cells,γδT cells,B cells and macrophages did not changed significantly. The frequencies of CD8⁺CD62L^-CD44⁺ and CD8⁺CD44⁺KLGR1⁺ in Apo C3 transgenic mice were increased, effector T cells mainly, and the secretions of IFN-γ by CD8 ⁺ T cells were enhanced. The expressions of CD107a in CD8⁺ T cells were increased in transgenic mice. CONCLUSION:Plasma triglyceride levels were significantly increased in Apo C3 transgenic mice. Compared with wild-type mice,the frequencies and activities of CD8 + T cells were up-regulated in transgenic mice, the frequencies of myeloid suppressor cells were increased,and that of NK cells were decreased. Moreover,the ability of CD8⁺T lymphocytes to kill target cells in transgenic mice was enhanced.

OBJECTIVE: To investigate the expression and clinical significance of miR-200b-3p and PAK2 mRNA in chemotherapy-resistant and sensitive ovarian cancers. METHODS: Bioinformatics method combined with comprehensive literature analyses were used to predict the target gene of miR-200b-3p as PAK2. From 2015 to 2018,25 tissue samples from patients with chemo-resistant ovarian cancers and 25 from chemo-sensitive ovarian cancers were collected from Shanxi Provincial Cancer Hospital. Relative expression of miR-200b-3p and PAK2 mRNA from the two groups of tissues were detected by using quantitative real-time PCR (qPCR). Their expression levels and clinic-pathological parameters of the two groups of cancer patients were compared. RESULTS:The expression level of miR-200b-3p was 15.78 times and significantly higher in the chemo-resistant ovarian cancer tissues than the sensitive ones. However, the expression level of PAK2 mRNA was 0.03 times and significantly lower in the resistant and in the sensitive tissues. The expression levels of miR-200b-3p and PAK2 mRNA were significantly correlated with the clinical stage and lymph node metastasis of ovarian cancer patients (P<0.05). CONCLUSION:Our data show that over-expression of miR-200b-3p and regulation of PAK2 mRNA were involved in the expression of chemoresistance and the promotion of ovarian cancer metastasis.

OBJECTIVE:To study the relationship between miR-146a rs2910164 G/C gene polymorphism and non-small cell lung cancer in the Han population. METHODS:rs2910164 genotypes in 198 patients with non-small cell lung cancers and 218 control subjects were detected by PCR-restriction fragment length polymorphism (PCR-RFLP). Logistic regression analysis was used. RESULTS: Compared with the control group,logistic regression analyses show that the genotype carrying the C allele was significantly associated with increased risk of non-small cell lung cancer[dominant model OR=5.04, 95% CI (4.72, 5.39), P<0.01; recessive model OR=2.75, 95% CI was (2.57, 2.94), P<0.01]. Further analyses show that there was no statistical relationship between the rs2910164 gene polymorphism and clinical features (gradation, stage, metastasis) (P>0.05) and there was no interaction between the rs2910164 gene polymorphism and smoking (P> 0.05). CONCLUSION:Our results show that the genotype carrying the miR-146a rs2910164 C allele may be associated with genetic susceptibility to non-small cell lung cancer.

OBJECTIVE:To investigate the significance of IL-24 and IL-6 concentrations in serum of patients with Diffuse large B-cell lymphoma (DLBCL). METHODS:Concentrations of serum IL-24 and IL-6 were detected using the ELISA assay. The relationship between the concentrations and the clinicopathological parameters in 43 cases of DLBCL patients was analyzed. RESULTS:Compared with the control patients,the concentrations of IL-24 and IL-6:(58.32 ±11.81) and (24.63 ±7.73) μg/mL, respectively, and the IL-24 concentration:(42.18 ±8.48) μg/mL in the DLBCL patients were significantly lower, whereas the IL-6 concentration:(45.29±12.71) μg/mL was significantly higher (P<0.01). Furthermore,the concentrations of IL-24 was significantly lower,while the IL-6 was higher in the serum of the patients who had bone marrow invasion than those without the invasion (P<0.01). The similar results were also shown in the patients with the high IPI scores (P<0.01). These findings suggest that the low IL-24 and high IL-6 concentrations may be predictors for poor clinical status and prognosis of DLBCL patients. CONCLUSION:Detection of the serum IL-24 and IL-6 concentrations may be useful as auxiliary diagnosis for improved clinical therapy in DLBCL.

OBJECTIVE:To investigate the association between JAK2 gene mutation and development of chronic myeloid leukemia (CML). METHODS: A systematic literature searches of CNKI, VIP, Wanfang, PubMed,Web of Science and CBM databases were conducted in September 2018. The identified publications were screened for inclusion and exclusion criteria by two researchers independently. Data were extracted from selected papers and evaluated for quality using a modified NOS score. Meta-analyses were performed by using the Stata 11.0 and RevMan 5.3 software. RESULTS:A total of 15 articles were selected which included 1 684 cases and 104 mutations (JAK2V617F mutation is characterized by a G to T transversion at nucleotide 1 849 in exon 12 or exon 14 of the gene). Meta-analyses show that the mutation rate of JAK2 in CML patients was 0.08[95%CI (0.05,0.11)]. Subgroup analyses show that the main reason for heterogeneity of whether Ph and JAK2 mutations involved different regions and exon mutation site. CONCLUSION:The JAK2 exon 12 and 14 mutations and mutant JAKV617F were associated with the development of CML.

OBJECTIVE:The purpose of this study was to determine the purification effect of modified biological slow filter, with activated alumina and activated carbon as defluorizers, on fluoride and related pollutants in high-fluorine water. METHODS: The chemical oxygen demand (COD_Mn), ammonia nitrogen (NH₄⁺-N),chromaticity and pH value were measured by fluoride ion-selective electrode method,acid potassium permanganate method, Nessler's reagent colorimetric method, platinum cobalt standard colorimetric method, and glass electrode method, respectively. The purification effects of the modified biological slow filter on fluoride and related pollutants in high-fluorine water were analyzed and evaluated. RESULTS: The average instantaneous fluoride removal rates,24 h-post-treatment fluoride removal rates,CODMn removal rates, NH₄⁺-N removal rates,standard-reaching rates of pH value and colority removal rates of the modified biological slow filters,using activated aluminium oxide/activated carbon as fluoride removal agents,were 73.17%/6.14%,87.57%/18.33%,41.77%/18.28%,37.66%/21.17%,100%/85.71%,and 65.71%/22.14%,respectively. CONCLUSION:The modified biological slow filter,with activated aluminium oxide as fluoride removal agent,has relatively good purification effects on fluoride and related pollutants and can be considered for application.

OBJECTIVE: To develop an in vitro PIG-A mutation assay in human lymphoblastoid TK6 cells and to evaluate its capacity in determining mutagenicity of Ethyl methanesulfonate (EMS) and Benzo(a) pyrene (B[a]P). METHODS:In order to reach a high sensitivity of the assay,it was necessary to eliminate the pre-existing GPI (-) cells (PIG-A mutant cells) in TK6 cells by means of immunomagnetic beads,and then determine the spontaneous mutation rate of PIG-A gene in TK6 cells up to 40 days. In this study,TK6 cells were divided into two groups,long time treatment without metabolic activation system (24 h -S9 group) and short time treatment with metabolic activation system (4 h +S9 group),and were then treated with ethyl EMS (50,75,100 μmol/L) and B[a]P (4,8,16 μmol/L) of 3 concentration gradients,then determined the mutation rate of PIG-A gene on day 11. Cells were stained with APC Mouse-Anti-Human CD19 antibody,PE Mouse Anti-Human CD55 antibody, PE Mouse Anti-Human CD59 antibody and nucleic acid dye 7-AAD, then analyzed the negative frequencies of CD55 and CD59(ratio of CD55^-CD59^-/CD19 ⁺ cells in CD19 ⁺ cells). RESULTS:The level of preexisting mutant cells were reduced to lower than 75×10^-6 after immunomagnetic beads separation. The spontaneous mutation rate for 40 days was 5.04×10^-7 cells/day. In the 24 h -S9 group, different concentrations of EMS exerted a dose-dependent increase in the mutation rate of PIG-A gene compared with the negative control. In the 4 h +S9 group with 2-fold increase,B[a]P different concentrations resulted in a similar dose-dependent effect. CONCLUSION: The flow cytometric in vitro PIG-A mutation assay was developed in human lymphoblastoid TK6 cells. This assay has the advantages of the low cost,easy and fast procedure,which may be useful for early genotoxicity testing of chemicals and pharmaceuticals.