OBJECTIVE: To construct a stably knockdown NF-YB gene in HeLa cells using the Lentivirus-mediated RNA interference (RNAi) technique. METHODS: shRNA-NF-YB primers were designed and the H1/GFP-Puro-shRNA-NF-YB plasmids were constructed by molecular cloning. The virus-infected HeLa cells were collected after transfection with 293T. Stably knock down cells were obtained by screening cells with puromycin. Cell lines were identified by Western blot and qPCR. RESULTS: The constructed plasmid was sequenced by the company and was consistent with the designed primer sequence. Western blot and qPCR results show that inhibition of NF-YB protein and mRNA expression was significant. CONCLUSION: In this experiment,NF-YB-shRNA-HeLa cell line was successfully constructed and HeLa cells with specific NF-YB gene silencing knockdown were obtained.

OBJECTIVE: To explore expression and prognostic values of the MYB family (MYB,MYBL1 and MYBL2) in breast cancer patients via data mining analyses. METHODS: The Oncomine database,GEPIA2,cBioPortal,bc-GenExMiner v4.4,Kaplan-Meier Plotter and DAVID were used to investigate expression distribution,genetic alteration,clinicopathological features,prognostic values and function enrichment of MYB family members in breast cancer patients. RESULTS: Expression of MYB family members were significantly higher in multiple breast cancer datasets than that in normal breast tissues (P < 0.05). Expression variation of these genes mainly involved gene amplification,and mRNA strength and weakness. Their expression levels were significantly associated with a number of clinicopathological characteristics of the patients,e.g. age,molecular subtypes,Scarff-Bloom-Richardson (SBR) grade (P < 0.01). Specifically,the mRNA expression levels of MYB were significantly downregulated,while those of MYBL1 and MYBL2 were significantly upregulated in triple negative breast cancers (TNBC),basal-like breast cancers(BLBC) and P53 mutated type breast cancers (P < 0.01).Breast cancer patients with the characteristics of low MYB and high MYBL2 mRNA expression levels showed worse relapse-free survival and post progression survival (P < 0.01). In addition,patients with metastatic recurrence of breast cancer,and low MYB and high MYBL2 expression levels had poor prognosis (Univariate Cox analyses;P < 0.01). Based on the multivariate Cox regression analyses,MYBL2 was an independent prognostic factor for metastatic recurrence survival of breast cancer patients (P < 0.05). Functional evaluation indicates that the MYBL2 co-expressed genes were mainly involved in the cell proliferation process and the cell cycle signaling pathway. CONCLUSION: Our integrative bioinformatics analyses indicate that the MYB gene family were significantly involved in metastatic relapse in breast cancer. Consequently,these genes can be used as biomarkers for clinical diagnosis and prognosis of breast cancer. Furthermore,our data provide a basis for molecular mechanism research and drug discovery for breast cancer.

OBJECTIVE: To mine the TCGA database and to identify regulatory network(s) which are related to lung squamous cell carcinoma (LUSC) and to drug actions. METHODS: Bioinformatics and network pharmacology methods were used on the following tasks:TCGA RNA-seq data,WGCNA analysis,differential expression analysis,GO analysis,PPI analysis and screening of core genes. Collected data were used to construct ceRNA network and molecular docking which were employed to analyze mechanisms of stigmasterol action on LUSC. RESULTS: A total of 801 genes with high reliability were identified by WGCNA combined with differential expression analysis. These genes were found to be involved with chromosome segregation,mitosis,chromosome centromere region,etc. Based on PPI analyses,the first 10 key genes (CDC20,BUB1,CCNB2,BUB1B,CDK1,CCNB1,KIF2C,NDC80,CDCA8,CENPF) were closely related to the survival rates. Finally,the ceRNA network of CDCA8 and its upstream miRNA hsa-let-7b-5p and associated 14 lncRNAs were constructed. Stigmasterol and CDCA8 docked well. CONCLUSION: Our data indicate that competitive regulation of CDCA8 by 14 lncRNAs and hsa-let-7b-5p played an important role in the occurrence and development of LUSC and that the mechanism involved stigmasterol intervention in LUSC.

OBJECTIVE: To access the TCGA database for tumor mutation burden (TMB) in muscle-invasive bladder cancers (MIBC) and to investigate their prognostic values. METHODS: MIBC sequencing data from the TCGA database and clinically significant TMB in the MIBC were used to determine their prognostic values that were related to differentially expressed immune-related genes. In addition,non-negative matrix decomposition CIBERSORT algorithm was used to determine correlations between immune cells and TMB subtypes. RESULTS: Our data indicate that single nucleotide polymorphisms (SNP) and C > T were the most common missense mutations in the 375 MIBC patients. In addition,mutation rates of TP53,TTN,KMT2D,MUC16 and ARID1A genes were elevated. Among the MIBC patients,those in the high TMB group had better prognosis (P < 0.01). In the COX regression model which was constructed by KIR2DL4,IL1RL1 and SSTR5,the low-risk group of MIBC patients compared with the higher risk group had a better prognosis,with the ROC of 0.71. In contrast with normal bladder tissues,expression of CD8⁺ T cells,activated CD4⁺ T cells and eosinophils was higher in the high TMB group,while expression of memory B cells and non-activated mast cells was higher in the low TMB group (P < 0.05). CONCLUSION: MIBC patients with higher TMB may have better prognosis in immunotherapy,and TMB has potential clinical application in predicting tumor immunotherapy. In addition,different components of immune cells were found to be differentially expressed in the TMB subgroup MIBC microenvironment.

OBJECTIVE: To investigate effects from regulation of expression of RBMS3 on proliferation,apoptosis and invasion of cervical cancer cells. METHODS: Immortalized human cervical cells,Ect1/E6E7,and cervical cancer cells:HeLa,Caski and SiHa were cultured,then Real-time quantitative PCR was used to detect expression of RBMS3-AS3 in cells,and Western blot was used to detect protein expression of RBMS3. HeLa cells were divided into control (cells were cultured normally),si RBMS3 AS3 (transfected with RBMS3 AS3 small interfering RNA),negative sequence (transfected with disordered and meaningless negative sequences),si RBMS3 AS3+si RBMS3 (co transfected with small interfering RNAs of RBMS3 AS3 and of RBMS3) and si RBMS3 AS3+negative sequence group (co transfected with RBMS3 AS3 small interfering RNA and disordered meaningless negative sequences). MTT assay was used to detect proliferation rates of HeLa cells in each group;flow cytometry to detect apoptosis rates;Transwell to detect HeLa cell invasion;Western blot to detect protein expression levels of Cyclin D1,Bcl 2,Bax and MMP 2. RESULTS: Compared with Ect1/E6E7 cells,expression levels of RBMS3 AS3 in cervical cancer cell lines HeLa,Caski and SiHa were significantly increased (P < 0.05),and expression levels of RBMS3 protein were decreased (P < 0.05). Compared with the negative sequence group,the number of invasive HeLa cells and their expression levels of Cyclin D1,Bcl 2 and MMP-2 proteins in the si RBMS3 AS3 group were decreased (P < 0.05),apoptosis rate and the expression levels of RBMS3 and Bax protein were increased (P < 0.05). Compared with the si RBMS3 AS3+negative sequence group,the number of invasive HeLa cells and their expression levels of Cyclin D1,Bcl-2 and MMP-2 were increased in the si-RBMS3-AS3+si-RBMS3 group (P < 0.05),apoptosis rate and Bax protein expression levels were decreased (P < 0.05). CONCLUSION: Inhibition of RBMS3 AS3 expression inhibited proliferation and invasion of cervical cancer cells and promoted apoptosis,which might be related to up-regulation of RBMS3 expression.

OBJECTIVE: To investigate the influence of electronic beams on protein expression of the insulin growth factor system and to provide evidence for radiation-induced early damage in livers of mice. METHODS: Mice were irradiated with electron beam at of 0,1,2 and 4 Gy. At 12,48 and 72 h after irradiation,their livers were removed and used for analyses. Western blot was used to detect changes of protein expressions in insulin growth factor 1 receptor (IGF-1R),insulin growth factor binding protein 1 (IGFBP1),insulin growth factor binding protein 4 (IGFBP4),and insulin growth factor 1 (IGF-1). RESULTS: When the irradiation dose was 1 Gy,IGF-1 protein was highly expressed at 48 h after irradiation,and that for IGFBP1,IGFBP4 and IGF1R protein were at 12,48 and 72 h. When the irradiation dose was 2 Gy,IGF-1 and IGFBP4 proteins were up regulated at 48 h after irradiation,and they were down regulated for the rest of the time. IGFBP1 protein was up regulated at 12 h after irradiation,and it was down regulated at 48 h and 72 h. IGF1R protein was down regulated at three time points after irradiation. When the irradiation dose was 4 Gy,the expressions of IGF-1,IGFBP1,and IGFBP4 proteins was down regulated. IGF1R protein was up regulated at 12 h after irradiation,and it was down regulated at 48 and 72 h. CONCLUSION: Our results show that electron beam irradiation caused down-regulation on protein expression of the insulin growth factor-1 system in the liver of mice. Our results are consistent with those performed using PCR. Based on the study,expression of the insulin growth factor-1 system may be useful as a biomarker for radiation-induced liver injury.

OBJECTIVE: To investigate the effect of mTOR inhibitor,AZD2014,on proliferation of HCC cells. METHODS: The CCK-8 method was used to detect proliferation of HCC cells after their treatment with different concentrations of AZD2014 (10,100,500,1 000 nmol/L). Expression levels of UHRF1 mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot,respectively. RESULTS: The CCK-8 results show that different concentrations of AZD2014 (10,100,500,1 000 nmol/L) significantly inhibited proliferation of hepatoma cells,and the degree of inhibition was positively dose-dependent. In addition,the RT-PCR and Western Blot data show that AZD2014 at concentrations of 500 nmol/L and 1 000 nmol/L reduced the expression of UHRF1 mRNA and protein in HCC cells. CONCLUSION: AZD2014 inhibited proliferation of HCC cells by repressing expression of UHRF1.

OBJECTIVE: To investigate the effect of microRNA-29a (miR-29a) on proliferation and migration of human gastric cancer cells in vitro. METHODS: Gastric cancer SGC-7901 cells were randomly divided into miR-29a transfection group,plasmid control group,blank control group,and signal transducers and activators of transcription 3 (STAT3) inhibition group. The miR-29a transfection and the plasmid control groups were transfected with the hsa miR-29a mimics plasmids and the siRNA control plasmids,respectively,by liposome transfection. The STAT3 inhibition group received the STAT3 inhibitor solution and the blank control group was not treated. Real-time fluorescent quantitative polymerase chain reaction (qPCR) and Western blot were used to detect expression of miR-29a,vascular endothelial growth factor (VEGF),STAT3,cyclin D1 mRNA and protein in the transfected cells. MTT and scratch tests were used to detect cell proliferation and migration. RESULTS: qPCR and Western blot results show that,compared with the blank control and the plasmid control groups,expression levels of miR-29a in the miR-29a transfection group was increased,and expression levels of VEGF,cyclin D1 mRNA and protein,p-STAT3/STAT3 were decreased (P < 0.01). MTT and scratch tests show that,compared with the blank control and the plasmid control groups,the miR-29a transfection group had lower cell proliferation and migration rates at 24,48 and 72 h (P < 0.01). After application of the STAT3 inhibitor,compared with the blank control group,expression levels of cyclin D1 mRNA and protein,p-STAT3/STAT3,and MTT test cell relative proliferation rates at 24,48,72 h were lower in the STAT3 inhibition group (P < 0.01). CONCLUSION: Our data show that MiR-29a reduced the expression of VEGF mRNA and protein and inhibited the proliferation and migration of human gastric cancer cells. A mechanism for these effects may be related to down-regulation of p-STAT3,and cyclin D1 mRNA and protein expression.

OBJECTIVE: Based on the cancer genome map (TCGA) database,associations between expression of abnormal spindle head malformation associated gene (ASPM) in lung adenocarcinoma and clinicopathological parameters were investigated. METHODS: The TCGA data on mRNA expression,and clinical characteristics of lung adenocarcinoma tissues and adjacent tissues were retrieved. The differences of ASPM mRNA expression in cancer and adjacent tissues were compared,and the relationship between ASPM expression level and clinicopathological parameters and prognosis of lung adenocarcinoma patients was statistically analyzed. Through R language ballgown and ggplot package,the differentially expressed genes between ASPM groups and drew volcano maps were screened. The David tools were used to analyze the differential genes,and GSEA was used to predict possible signal pathways which could be regulated by ASPM. String and Cytoscape were used to analyze key genes and interactions among differentially expressed genes. RESULTS: The expression level of ASPM mRNA in lung adenocarcinoma was significantly higher than that in adjacent tissues. In addition,the expression level of ASPM was correlated with survival time. The prognosis of patients with lung adenocarcinoma having high expression of ASPM was poor. This was also an independent risk factor for prognosis of lung adenocarcinoma patients. There were 183 differentially expressed genes between the ASPM high and low expression groups. The differentially expressed genes were enriched in biological process (BP),cell component (CC) and molecular function (MF) in three categories,including 19 subclasses and 18 KEGG pathways. Four key proteins were found by the Cytoscape software. CONCLUSION: ASPM mRNA was highly expressed in patients with lung adenocarcinoma and prognosis for the high expression group was poor. Our observations can be used as prognostic markers of lung adenocarcinoma.

OBJECTIVE: To determine immunophenotype characteristics of plasma cells in bone marrow of multiple myeloma (MM) patients and to correlate with clinical prognosis. METHODS: Bone marrow smears and sections were stained with Wright Giemsa and HE,respectively,and their morphological and pathological characteristics were analyzed. In addition,bone marrow or peripheral blood samples were collected for flow cytometry immunophenotyping and related laboratory tests. RESULTS: There was a significantly increased number of bone marrow plasma cells in MM patients and marked morphological abnormality. Their distributions included plug solid type in 16 cases (45.71%),nodular type in 14 cases (40.00%) and interstitial type in 5 cases (14.29%). Proportion of increased serum LDH,β2-MG,Cr and BUN in MM patients was 80%,85.71%,62.86% and 60%,respectively. There was positive expression of CD38 and CD138 in the plasma cells. The absence of expression of Igκor Igλ chain was monoclonal hyperplasia. The positive rates of CD56,CD200,CD117 and CD28 were 65.71%,94.29%,60% and 54.29%,respectively,and those of CD20,CD81,CD33,CD27 and CD13 were 37.14%,42.86%,28.57%,57.14% and 34.29%,respectively,as opposed to 22.86% and 31.42% in CD19 and CD45,respectively. The expressions of CD27 and CD28 were closely related to prognosis of MM patients,and the 5-year overall survival rate was lower in patients with the loss of CD27 expression in plasma cells and the positive expression of CD28 (P < 0.05). CONCLUSION: Abnormal immunophenotype was observed in plasma cells from bone marrow of MM patients,and expressions of CD27 and CD28 were significantly correlated with disease progression and prognosis. Therefore,immunophenotype detection can be of great significance in clinical diagnosis and treatment monitoring of MM patients.

OBJECTIVE: To determine efficiency in using two assays,the human cell line activation test (h-CLAT) and the occluded patch test of Buchler (BT),in detecting skin sensitization of two preservatives commonly used in cosmetics. METHODS: BT was carried out according to "cosmetics safety technical specification (The 2015 Edition)". Skin sensitizations to ethylparaben,butylparaben andglycerin were used as negative controls and to 2,4-dinitrochlorobenzene (DNCB) as positive control. In test animals,skin sensitization was evaluated by score. The h-CLAT was set up,according to the OECD No 442 (E) toxicology test method,to evaluate the ethylparaben,butylparaben,glycerin and the DNCB groups. The CCK-8 (cell counting kit-8) method was used to test evaluate cytotoxicity and to calculate the CV₇₅ value. The RFI values of CD86 and CD54 were detected by flow cytometry. RESULTS: The results from the BT test show that glycerin was a non-sensitizer while DNCB was strong sensitizer,ethylparaben was a nonsensitizer and butylparaben was a moderate sensitizer. The results from h-CLAT show that the CV₇₅ value of ethylparaben was 126 μg/mL,RFI_CD86 was 110%-140%,RFI CD54 was 140%-260%;the CV₇₅ value of butylparaben was 18 μg/mL,RFI_CD86 was 75%-200%,RFI_CD54 was 90%-210%,the CV₇₅ value of glycerol as more than 5 000 μg/mL,RFI_CD86 was 75%-100%,RFI_CD54 was 60%-140%,the CV₇₅ value of DNCB was 2.5 μg/mL,RFI_CD86 was 185%-640% and RFIcd54 was 100%-460%. The test results indicate that ethylparaben and butylparaben were weak and strong sensitizers,respectively,DNCB was a very strong sensitizer and glycerol was a non-sensitizer. CONCLUSION: In this comparative test,the sensitivity of h-CLAT was higher than BT. In addition,this cell culture method can be used as a screening method for some allergenicity evaluations.

OBJECTIVE: To determine acute toxicity and mutagenicity of Aronia melanocarpa extract. METHODS: The Aronia melanocarpa extract solution was given orally to mice with a dose of 20 g/kg to determine its acute oral toxicity. In addition,mutagenicity was assessed by a bacterial reverse mutation assay,micronucleus test and sperm shape abnormality test. RESULTS: The oral maximum tolerated dose (MTD) of Aronia melanocarpa extract in mice was more than 20 g/kg. No increase in the number of revertant colonies was found in the bacterial reverse mutation assay with or without S9 mixture in 8,40,200,1 000,5 000 μg/plate of Aronia melanocarpa extract. The micronucleus rates and sperm abnormality rates in 2.5,5,10 g/kg dose were not significantly different from the negative control. CONCLUSION: This Aronia melanocarpa extract showed no acute oral toxicity and nor mutagenicity in mice.