Finding key genes (UBE2T, KIF4A, CDCA3, and CDCA5) co-expressed in hepatitis, cirrhosis and hepatocellular carcinoma based on multiple bioinformatics techniques.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Hepatitis B virus (HBV) is one of the major causes of liver cirrhosis (LC) and HCC. Therefore, the discovery of common markers for hepatitis B or LC and HCC is crucial for the prevention of HCC. METHODS: Expressed genes for to chronic active hepaititis B (CAH-B), LC and HCC were obtained from the GEO and TCGA databases, and co-expressed genes were screened using Protein-protein interaction (PPI) networks, least absolute shrinkage and selection operator (LASSO), random forest (RF) and support vector machine - recursive feature elimination (SVM-RFE). The prognostic value of genes was assessed using Kaplan-Meier (KM) survival curves. Columnar line plots, calibration curves and receiver operating characteristic (ROC) curves of individual genes were used for evaluation. Validation was performed using GEO datasets. The association of these key genes with HCC clinical features was explored using the UALCAN database ( https://ualcan.path.uab.edu/index.html ). RESULTS: Based on WGCNA analysis and TCGA database, the co-expressed genes (565) were screened. Moreover, the five algorithms of MCODE (ClusteringCoefficient, MCC, Degree, MNC, and DMNC) was used to select one of the most important and most closely linked clusters (the top 50 genes ranked). Using, LASSO regression model, RF model and SVM-RFE model, four key genes (UBE2T, KIF4A, CDCA3, and CDCA5) were identified for subsequent research analysis. These 4 genes were highly expressed and associated with poor prognosis and clinical features in HCC patients. CONCLUSION: These four key genes (UBE2T, KIF4A, CDCA3, and CDCA5) may be common biomarkers for CAH-B and HCC or LC and HCC, promising to advance our understanding of the molecular basis of CAH-B/LC/HCC progression.

AURKB as a Promising Prognostic Biomarker in Hepatocellular Carcinoma.

The Aurora kinases form a family of 3 genes encoding serine/threonine kinases and are involved in the regulation of cell division during the mitosis. This study was designed to investigate the prognostic role of Aurora kinases in hepatocellular carcinoma (HCC). In this study, we analyzed the expression, overall survival (OS) data, promoter methylation level, and relationship with immunoinhibitors of Aurora kinases in patients with HCC from GEPIA2, UALCAN, OncoLnc, and TISIDB databases. Protein-protein interaction (PPI) network, gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analysis were performed using the STRING database and Cytoscape software. We found that the mRNA expression, stages of HCC, and OS of AURKA and AURKB in HCC tissues were significantly different from control tissues, but there were significant inconsistencies in promoter methylation level and relationship with immunoinhibitors for AURKA and AURKB. None of the above items were significantly different for AURKC. Furthermore, a hub module including AURKA, AURKB, and AURKC was identified within the PPI network constructed with the Molecular Complex Detection (MCODE) plug-in in Cytoscape software. Our results show that AURKB could be a potential biomarker for HCC prognosis.

Comparison of two methods for isolation and culture of human foreskin fibroblasts. / ä¸¤ç§äººåŒ çš®æˆçº¤ç»´ç»†èƒžåˆ†ç¦»åŸ¹å »æ–¹æ³•çš„æ¯”è¾ƒ.

OBJECTIVES: The efficient acquisition and purification of fibroblasts as ideal seed cells are very important. For optimization of the isolation and culture of human foreskin fibroblasts (HFF), we compared the improved tissue culture method (ITCM) and the enzyme digestion method (EDM). METHODS: In ITCM, the skin tissue was digested with 0.1% Type II collagenase overnight at 4 ℃, the epidermis was separated from the dermis and digested again with 0.25% trypsin at room temperature for 15 min, and then the tissue block was attached to the culture dish. In EDM, the skin tissue was digested with 0.25% trypsin overnight at 4 ℃, the epidermis was separated from the dermis and digested with 0.1% Type II collagenase overnight at 4 ℃, the tissue block was filtered and squeezed together with the enzyme mixture, the filter was rinsed with medium containing fetal bovine serum, and the cell suspension was cultured. Both ITCM and EDM used 2 digestion enzymes, but the order, digestion time, and temperature of the 2 enzymes were different. The final inoculations of ITCM and EDM in the dishes for subsequent culture were tissue blocks and cell suspensions, respectively. In this study, HFF cells were isolated and cultured with ITCM and EDM, and the cell morphology was observed from Passage 0 to Passage 3 in the ITCM and EDM groups. The cell purity was identified by staining for vimentin, CD68, and Pan-keratin. The growth curves of Passage 3 were plotted to compare the proliferation ability of the 2 groups. Passage 3 HFF cells in the ITCM and EDM groups were irradiated with medium-wave ultraviolet (UVB) at an energy value of 120 mJ/cm2 to establish a light damage model. The experiments were grouped into an UVB group and a control group (Control) according to the presence or absence of UVB irradiation. Platelet-poor plasma (PPP) was extracted by secondary centrifugation, and the HFF cells of ITCM and EDM groups were cultured in groups using complete medium containing different concentrations (0, 2.5%, 5.0%, and 10.0%) of PPP, and the proliferation of damaged cells was detected by cell counting kit-8 after 24 h of PPP incubation. RESULTS: A large number of HFF could be observed in the ITCM group up to day 3, which was less affected by impurities; the observation of HFF morphology in the EDM group was affected by more impurities. By day 9, cells in both ITCM and EDM groups could be passaged; HFF isolated and cultured in vitro by the 2 methods showed long spindle-shaped, swirling growth. The positive rates of vimentin in the ITCM and EDM groups when HFF cells were cultured up to Passage 2 were significantly different [(97.36±0.76)% vs (99.4±0.56)%, P<0.01)]. The positive rates of CD68 were also significantly different [(70.8±0.46)% vs (78.37±0.75)%, P<0.01]. The expressions of pan-keratin in the ITCM group and the EDM group were positive and negative, respectively. There was no difference in vimentin and pan-keratin staining results between the ITCM group and the EDM group when HFF were cultured to Passage 3. The positive rates of CD68 between the ITCM group and the EDM group were significantly different [(74.73±1.37)% vs (85.27±2.63)%, P<0.001]. The proliferative capacity of HFF cells in Passage 3 was significantly higher in the EDM group than that in the ITCM group (P<0.05). After UVB (120 mJ/cm2) irradiation, HFFs procured by the 2 isolation methods showed damage. The damage repair test demonstrated that the 2.5% PPP+UVB irradiation group showed significantly higher repair competence than the other groups (all P<0.05). CONCLUSIONS: In contrast with HFFs isolated via ITCM, HFF cells isolated by EDM have a faster purification rate and a stronger proliferative capacity. Therapy with PPP can moderately repair UVB-induced damage to HFFs. The results provide a theoretical basis for clinical treatment studies in the future.

Bioinformatics analysis for hepatocellular carcinoma genes based on the data of expression profile chip. / åŸºäºŽè¡¨è¾¾è°±èŠ¯ç‰‡æ•°æ®çš„è‚ç»†èƒžç™ŒåŸºå› çš„ç”Ÿç‰©ä¿¡æ¯å­¦åˆ†æž.

OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, especially in Asia and Africa. However, the underlying mechanism is still unclear. Consequently, it is important to explore its key genes and prognosis-related genes via bioinformatics. This study aimed to explore the molecular mechanism of HCC by using bioinformatics analysis for HCC gene chip data. METHODS: Microarray data of HCC genes were downloaded from public GEO database and screened for differentially expressed genes (DEGs) by GEO2R analysis. Then DAVID online tool was used for GO annotation and KEGG pathway enrichment analysis. STRING-DB online database and Cytoscape software were used for protein interaction network analysis.GEPIA and Ualcan were applied to evaluate prognosis and promoter methylation level. RESULTS: A total of 87 DEGs of HCC were screened, of which 15 genes were up-regulated and 72 genes were down-regulated. GO annotation indicated that most of the genes were involved in oxidation reduction,cellular amino acid derivative metabolic process, carboxylic acid catabolic process, and response to wounding. KEGG pathways were enriched in linoleic acid metabolism, retinol metabolism, complement and coagulation cascades,steroid hormone biosynthesis, drug metabolism, and other pathways. Two key modules and key genes AURKA and SPP2 were obtained by protein interaction network analysis. Prognostic analysis showed that the 2 genes were significantly correlated with the total survival time of patients with HCC. There was no significant difference in the methylation level of AURKA promoter between the primary tumor group and the normal group (P=0.296) and the methylation level of SPP2 promoter was significantly lower in the primary tumor group than that in the normal group (P<0.001). CONCLUSIONS: HCC-relevant AURKA and SPP2 are obtained via bioinformatics analysis, which are closely related to the prognosis of patients with HCC. Gene promoter methylation is not the main factor for AURKA and SPP2 expression levels.

Effects of different chemical groups on behaviors of bladder cancer cells.

Chemical groups of microenvironment play an important role in the adhesion, proliferation, and apoptosis of tumor cells. The different chemical groups (CH3 , OH, COOH) were grafted on the surfaces with the same density by self-assembly monolayer (SAM) technique to introduce the influence of different microenvironments of the human bladder cancer (5637) cells. The results indicated that the 5637 cells on COOH surface exhibited the lowest proliferation rate and the highest apoptosis rate on the first and fifth day because of negative charge and polarity of the COOH group, which might help optimize biomedicine materials and find new methods to treat bladder cancer.

Bioinformatics analysis to identify the key genes affecting the progression and prognosis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer, which has poor outcome. The present study aimed to investigate the key genes implicated in the progression and prognosis of HCC. The RNA-sequencing data of HCC was extracted from The Cancer Genome Atlas (TCGA) database. Using the R package (DESeq), the differentially expressed genes (DEGs) were analyzed. Based on the Cluepedia plug-in in Cytoscape software, enrichment analysis for the protein-coding genes amongst the DEGs was conducted. Subsequently, protein-protein interaction (PPI) network was built by Cytoscape software. Using survival package, the genes that could distinguish the survival differences of the HCC samples were explored. Moreover, quantitative real-time reverse transcription-PCR (qRT-PCR) experiments were used to detect the expression of key genes. There were 2193 DEGs in HCC samples. For the protein-coding genes amongst the DEGs, multiple functional terms and pathways were enriched. In the PPI network, cyclin-dependent kinase 1 (CDK1), polo-like kinase 1 (PLK1), Fos proto-oncogene, AP-1 transcription factor subunit (FOS), serum amyloid A1 (SAA1), and lysophosphatidic acid receptor 3 (LPAR3) were hub nodes. CDK1 interacting with PLK1 and FOS, and LPAR3 interacting with FOS and SAA1 were found in the PPI network. Amongst the 40 network modules, 4 modules were with scores not less than 10. Survival analysis showed that anterior gradient 2 (AGR2) and RLN3 could differentiate the high- and low-risk groups, which were confirmed by qRT-PCR. CDK1, PLK1, FOS, SAA1, and LPAR3 might be key genes affecting the progression of HCC. Besides, AGR2 and RLN3 might be implicated in the prognosis of HCC.

High expression of FAM13A was associated with increasing the liver cirrhosis risk.

AIM: Liver cirrhosis is a consequence of chronic liver disease, and it may be caused by multiple influences of both genetic and environmental factors. Family with sequence similarity 13 member A (FAM13A) has been previously associated with lung function in several lung diseases, including chronic obstructive pulmonary disease, asthma, lung cancer, and pulmonary fibrosis. The aim of this study was to explore whether FAM13A polymorphisms confer susceptibility to liver cirrhosis. METHODS: FAM13A expression was evaluated in liver cirrhosis tissues by immunohistochemistry staining. The relationship between FAM13A gene polymorphism and liver cirrhosis was determined by association analysis. The genotypes were assessed in the Agena MassARRAY platform. Statistical analysis was performed using chi-squared test/Fisher's exact test, genetic model analysis, and haplotype analysis. RESULTS: The results showed that the expression of FAM13A is obvious higher in the liver cirrhosis tissue cells than in the normal liver tissue cells. Moreover, association analysis results indicated that the minor allele "A" of rs3017895 was positively associated with high risk of liver cirrhosis in the allele model by the chi-squared test (OR = 1.32, 95%CI = 1.03-1.68, p = 0.028). Logistic regression analyses revealed that the risk of liver cirrhosis was significantly higher in subjects with the G/A-G/G genotype of rs3017895 than those with A/A genotype under the dominant model and log additive model, and the T/A-A/A genotype of rs1059122 was positively associated with higher liver cirrhosis than T/T genotype based on dominant model respectively. In addition, haplotype analysis showed that the G-A haplotype of rs3017895-rs1059122 of the FAM13A gene significantly increased the risk of liver cirrhosis. CONCLUSION: Our findings demonstrated that the high expression of FAM13A may be associated with an increased risk of liver cirrhosis.

Associations of TERT polymorphisms with hepatocellular carcinoma risk in a Han Chinese population.

Genetic association analysis and functional analysis have suggested that telomerase reverse transcriptase (TERT) gene affects the predisposition to various tumors. In this study, we wanted to explore the association between TERT variants and hepatocellular carcinoma (HCC) risk in a Han Chinese population via a case-control study of 473 HCC patients and 564 controls. Sequenom Mass-ARRAY platform was applied to determine the genotype of TERT polymorphisms in these subjects. Odds ratios and 95% confidence intervals that calculated by logistic regression analysis were used to assess the association under the genotype, dominant, recessive, and additive models. The "AA" genotype frequency of TERT rs2242652 in cases was significantly lower than in controls (1.69% versus 3.72%). We found two SNPs were associated with decreased risk of HCC with or without the adjustment for age and gender: rs10069690 under an additive model (adjusted OR = 0.77, 95% CI: 0.60-0.98, P = 0.038); rs2242652 under a dominant model (adjusted OR = 0.72, 95% CI: 0.54-0.95, P = 0.022) and an additive model (adjusted OR = 0.72, 95% CI: 0.56-0.92, P = 0.009). To our knowledge, the present study is the first to show the significant association between TERT polymorphisms and HCC susceptibility in a Han Chinese population from China, which may act as a potential prognostic biomarker in HCC patients.

Effects of ovariectomy and 17beta-estradiol treatment on the renin-angiotensin system, blood pressure, and endothelial ultrastructure.

The purpose of this study was to determine whether the renin-angiotensin system (RAS), nitric oxide (NO), atrial natriuretic peptide (ANP), blood pressure (BP), ultrastructural characteristics, and endothelium-dependent relaxation of thoracic aorta were modulated by the estrogen level. Rats were divided into 3 groups: ovariectomized (OVX); not ovariectomized (sham); and ovariectomized and treated with subcutaneous 17beta-estradiol (15 microg/kg/day, OVX+E(2)) (n=15-17 per group). For 13 weeks after surgery, blood pressure, serum estrogen, NO, plasma angiotensin II (Ang II), ANP, and renin activity levels were monitored. Thirteen weeks after surgery, the vasodilator responses of the aortic rings to acetylcholine and the ultrastructural characteristics of the thoracic aorta were determined. In the 9th and 13th week, OVX rats had a significantly higher blood pressure than the other two groups (p<0.05). Ovariectomy led to a significant decrease in plasma Ang II level and a significant increase in renin activity in OVX rats compared to sham rats; this effect could be reversed by estrogen treatment. In the 5th, 9th, and 13th weeks, the serum NO level was significantly lower in the OVX group than in the sham group (p<0.05); this effect could be reversed by estrogen treatment. Plasma ANP levels in the 9th and 13th weeks were significantly lower in the OVX group (p<0.05), and plasma ANP levels could be completely restored by estrogen treatment. Ovariectomy markedly reduced endothelium-dependent relaxation in response to acetylcholine in isolated rat thoracic aortic rings; chronic estrogen treatment significantly restored endothelium-dependent relaxation in response to acetylcholine. Under electron microscopy, the endothelial cells in OVX rats were swollen, even necrosed; estrogen treatment inhibited these changes. These results strongly suggest that estradiol protects rats from the development of hypertension and has a protective effect on the endothelium by increasing NO and ANP levels while decreasing renin activity. However, there was a discordance between the effects that estradiol had on angiotensin II and on blood pressure. This might be the result of negative feedback that ultimately results in the overall suppression of the RAS.

17Beta-oestradiol regulates the expression of Na+/K+-ATPase beta1-subunit, sarcoplasmic reticulum Ca2+-ATPase and carbonic anhydrase iv in H9C2 cells.

1. It is necessary to improve our understanding of the effect of 17beta-oestradiol (E2) on the heart at a molecular and cellular level. In the present study, the effects of E2 on Na(+)/K(+)-ATPase, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and carbonic anhydrase IV (CAIV) in H9C2 cells were investigated. To identify the mechanism of action of E2 on these proteins, the oestrogen receptor (ER) antagonist tamoxifen was used. 2. The results indicated that 1 and 100 nmol/L E2 can enhance the activity of Na(+)/K(+)-ATPase and SERCA and upregulate the expression of the Na(+)/K(+)-ATPase beta1-subunit, SERCA2a and CAIV at both the mRNA and protein level compared with 0 and 0.01 nmol/L E2. 17beta-Oestradiol had the greatest effect at 100 nmol/L; 1 micromol/L E2 did not further protein expression compared with 100 nmol/L E2. 3. Tamoxifen (10 nmol/L) significantly decreased the activity of SERCA, as well as the expression of the Na(+)/K(+)-ATPase beta1-subunit and SERCA at the mRNA and protein level, in H9C2 cells cultured with 1 nmol/L E2. Tamoxifen alone had no significant effect on these proteins in H9C2 cells. 4. It may be hypothesized that a suitable E2 concentration has a protective effect on the heart and that the actual dose of E2 used in hormone-replacement therapy is important in menopausal women.