Up-regulation of GINS1 highlighted a good diagnostic and prognostic potential of survival in three different subtypes of human cancer / A regulação positiva de GINS1 destacou um bom potencial diagnóstico e prognóstico de sobrevivência em três subtipos diferentes de câncer humano

Abstract Cancer is a fatal malignancy and its increasing worldwide prevalence demands the discovery of more sensitive and reliable molecular biomarkers. To investigate the GINS1 expression level and its prognostic value in distinct human cancers using a series of multi-layered in silico approach may help to establish it as a potential shared diagnostic and prognostic biomarker of different cancer subtypes. The GINS1 mRNA, protein expression, and promoter methylation were analyzed using UALCAN and Human Protein Atlas (HPA), while mRNA expression was further validated via GENT2. The potential prognostic values of GINS1 were evaluated through KM plotter. Then, cBioPortal was utilized to examine the GINS1-related genetic mutations and copy number variations (CNVs), while pathway enrichment analysis was performed using DAVID. Moreover, a correlational analysis between GINS1 expression and CD8+ T immune cells and a the construction of gene-drug interaction network was performed using TIMER, CDT, and Cytoscape. The GINS1 was found down-regulated in a single subtypes of human cancer while commonly up-regulated in 23 different other subtypes. The up-regulation of GINS1 was significantly correlated with the poor overall survival (OS) of Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), and Kidney renal clear cell carcinoma (KIRC). The GINS1 was also found up-regulated in LIHC, LUAD, and KIRC patients of different clinicopathological features. Pathways enrichment analysis revealed the involvement of GINS1 in two diverse pathways, while few interesting correlations were also documented between GINS1 expression and its promoter methylation level, CD8+ T immune cells level, and CNVs. Moreover, we also predicted few drugs that could be used in the treatment of LIHC, LUAD, and KIRC by regulating the GINS1 expression. The expression profiling of GINS1 in the current study has suggested it a novel shared diagnostic and prognostic biomarker of LIHC, LUAD, and KIRC.

Resumo O câncer é uma doença maligna fatal e sua crescente prevalência mundial exige a descoberta de biomarcadores moleculares mais sensíveis e confiáveis. Investigar o nível de expressão de GINS1 e seu valor prognóstico em cânceres humanos distintos, usando uma série de abordagens in silico em várias camadas, pode ajudar a estabelecê-lo como um potencial biomarcador de diagnóstico e prognóstico compartilhado de diferentes subtipos de câncer. O mRNA de GINS1, a expressão da proteína e a metilação do promotor foram analisados ​​usando UALCAN e Human Protein Atlas (HPA), enquanto a expressão de mRNA foi posteriormente validada via GENT2. Os valores prognósticos potenciais de GINS1 foram avaliados por meio do plotter KM. Em seguida, o cBioPortal foi utilizado para examinar as mutações genéticas relacionadas ao GINS1 e as variações do número de cópias (CNVs), enquanto a análise de enriquecimento da via foi realizada usando DAVID. Além disso, uma análise correlacional entre a expressão de GINS1 e células imunes T CD8 + e a construção de uma rede de interação gene-droga foi realizada usando TIMER, CDT e Cytoscape. O GINS1 foi encontrado regulado negativamente em um único subtipo de câncer humano, enquanto comumente regulado positivamente em 23 outros subtipos diferentes. A regulação positiva de GINS1 foi significativamente correlacionada com a sobrevida global pobre (OS) de Carcinoma Hepatocelular de Fígado (LIHC), Adenocarcinoma de Pulmão (LUAD) e Carcinoma de Células Claras Renais de Rim (KIRC). O GINS1 também foi encontrado regulado positivamente em pacientes LIHC, LUAD e KIRC de diferentes características clínico-patológicas. A análise de enriquecimento de vias revelou o envolvimento de GINS1 em duas vias diversas, enquanto poucas correlações interessantes também foram documentadas entre a expressão de GINS1 e seu nível de metilação do promotor, nível de células imunes T CD8 + e CNVs. Além disso, também previmos poucos medicamentos que poderiam ser usados ​​no tratamento de LIHC, LUAD e KIRC, regulando a expressão de GINS1. O perfil de expressão de GINS1 no estudo atual sugeriu que é um novo biomarcador de diagnóstico e prognóstico compartilhado de LIHC, LUAD e KIRC.

Diagnostic and prognostic significance of long noncoding RNA LINC00173 in patients with melanoma

SUMMARY OBJECTIVE: A growing volume of literature has suggested long noncoding RNAs (lncRNAs) as important players in tumor progression. In this study, we aimed to investigate the expression and prognostic value of lncRNA LINC00173 (LINC00173) in melanoma. METHODS: LINC00173 expression was measured in 163 paired cancerous and noncancerous specimen samples by real-time polymerase chain reaction. The correlations between LINC00173 expression with clinicopathological characteristics and prognosis were analyzed by chi-square test, log-rank test, and multivariate survival analysis. Receiver-operating characteristic curves were used for the assessment of the diagnostic value of LINC00173 for melanoma patients. RESULTS: The expression level of LINC00173 in melanoma specimens was distinctly higher than that in adjacent non-neoplasm specimens (p<0.01). Besides, LINC00173 was expressed more frequently in patients with advanced melanoma than in patients with early melanoma. Multivariate assays confirmed that LINC00173 expression level was an independent prognostic predictor of melanoma patients (p<0.05). CONCLUSION: Our data indicated that LINC00173 expression could serve as an unfavorable prognostic biomarker for melanoma patients.

Construction and clinical evaluation of N6-methyladenosine risk signature of YTHDC2, IGF2BP2, and HNRNPC in head and neck squamous cell carcinoma / 华西口腔医学杂志

OBJECTIVES@#This work aimed to construct N6-methyladenosine (m6A) regulator-based prognostic signature and evaluate the prognostic value and the intervention on tumor immune microenvironment of this m6A risk signature.@*METHODS@#Using transcriptome and clinical data of head and neck squamous cell carcinoma (HNSCC) from The Cancer Genome Atlas (TCGA), we profiled m6A regulators and constructed an m6A risk signature. The relationship between m6A modulation and immune function was studied by differential gene expression, cell type enrichment, and correlation analyses.@*RESULTS@#Fifteen m6A regulators had aberrant expression in HNSCC. A three-gene m6A prognostic signature (i.e., YTHDC2, IGF2BP2, and HNRNPC) was constructed and identified as an independent prognostic indicator for HNSCC. The m6A regulator signature-based high-risk group revealed pro-tumoral immune microenvironment due to the dysregulation of immune-related gene expression, abnormal enrichment of multiple immunocytes, and production of immunoregulatory factors.@*CONCLUSIONS@#This comprehensive analysis of m6A regulators and tumor immune landscape in HNSCC revealed that the m6A signature of YTHDC2, IGF2BP2, and HNRNPC could serve as a promising biomarker for monitoring HNSCC development and may be a potential target for tumor therapy in the future.

Exosomal miR-485-3p derived from pancreatic ductal epithelial cells inhibits pancreatic cancer metastasis through targeting PAK1 / 中华医学杂志(英文版)

BACKGROUND@#Cell competition is an important feature in pancreatic cancer (PC) progression, but the underlying mechanism remains elusive. This study aims to explore the role of exosomes derived from normal pancreatic ductal epithelial cells involved in PC progression.@*METHODS@#PC cells and pancreatic stellate cells (PSCs) were treated with exosomes isolated from pancreatic ductal epithelial cells. Cell proliferation was assessed by CCK8 assays. Cell migration and invasion were assessed by Transwell assays. PC and matched adjacent non-tumor tissue specimens were obtained from 46 patients pathologically diagnosed with PC at Peking University First Hospital from 2013 to 2017. Tissue miR-485-3p and p21-activated kinase-1 (PAK1) expression was examined by real-time polymerase chain reaction (RT-PCR), and the relationship of the two was analyzed using Pearman's product-moment correlation. The clinical significance of miR-485-3p was analyzed using the Chi-square test, Wilcoxon rank-sum test, and Fisher exact probability, respectively. The binding of miR-485-3p to PAK1 5'-untranslated region (5'-UTR) was examined by luciferase assay. PC cells were xenografted into nude mice as a PC metastasis model.@*RESULTS@#Exosomes from pancreatic ductal epithelial cells suppressed PC cell migration and invasion as well as the secretion and migration of PSCs. MiR-485-3p was enriched in the exosomes of pancreatic ductal epithelial cells but deficient in those of PC cells and PSCs, in accordance with the lower level in PSCs and PC cells than that in pancreatic ductal cells. And the mature miR-485-3p could be delivered into these cells by the exosomes secreted by normal pancreatic duct cells, to inhibit PC cell migration and invasion. Clinical data analysis showed that miR-485-3p was significantly decreased in PC tissues (P < 0.05) and was negatively associated with lymphovascular invasion (P = 0.044). As a direct target of miR-485-3p, PAK1 was found to exert an inhibitory effect on PC cells, and there was a significantly negative correlation between the expression levels of miR-485-3p and PAK1 (r = -0.6525, P < 0.0001) in PC tissues. Moreover, miR-485-3p could suppress PC metastasis in vivo by targeting p21-activated kinase-1.@*CONCLUSIONS@#Exosomal miR-485-3p delivered by normal pancreatic ductal epithelial cells into PC cells inhibits PC metastasis by directly targeting PAK1. The restoration of miR-485-3p by exosomes or some other vehicle might be a novel approach for PC treatment.

MicroRNA-20a-5p regulates the epithelial-mesenchymal transition of human hepatocellular carcinoma by targeting RUNX3 / 中华医学杂志(英文版)

BACKGROUND@#MicroRNA-20a (miR-20a) is dysregulated in many types of malignancies, including human hepatocellular carcinoma (HCC), but its expression level and functional significance in HCC are still disputed. We aimed to study the role of miR-20a-5p in HCC and its downstream molecular mechanisms.@*METHODS@#We used real-time polymerase chain reaction to detect the expression of miR-20a-5p and runt-related transcription factor 3 ( RUNX3 ) in HCC and paraneoplastic tissue, transfected Huh7 and highly metastatic human hepatocellular carcinoma (MHCC97H) cells. A live cell workstation was used to observe the proliferation and migration of transfected cells. The invasiveness of transfected cells was verified by Transwell assay. Cell apoptosis was detected by flow cytometry. The expression levels of proteins after transfection were measured using simple western immunoblot measurements. Gene expression profiles between HCC and normal samples were obtained from The Cancer Genome Atlas. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results were processed by the database for annotation, visualization and integrated discovery. Potential target genes of miR-20a-5p were predicted to further investigate how miR-20a-5p regulates epithelial-mesenchymal transition (EMT) in HCC.@*RESULTS@#MiR-20a-5p was significantly highly expressed in HCC tissues, and overexpression of miR-20a-5p significantly promoted HCC cell proliferation, migration, and invasion and inhibited apoptosis in vitro. The protein expression of E-cadherin was decreased and that of vimentin was increased after overexpression of miR-20a-5p in HCC cells. We discovered the intersection of genes from miRDB, miR TarBase, and TargetScan, obtained 397 target genes and finally focused on RUNX3. RUNX3 was not only reduced in HCC specimens but also drastically reduced in HCC cells overexpressing miR-20a-5p. RUNX3 expression decreased with elevated miR-20a-5p, which activated downstream EMT signaling and promoted cell proliferation, migration, and invasion.@*CONCLUSIONS@#Since RUNX3 is involved in EMT in HCC, as proven by previous research, our findings provide further evidence for a novel regulatory pathway comprising the miR-20a/RUNX3/EMT axis that upregulates EMT signaling and enhances the migration of HCC cells.

Down-regulation of lncRNA TTTY15 targeting miR-4500 to inhibit the biological characteristics of A172 glioma cells / 中华医学遗传学杂志

OBJECTIVE@#To explore the effect and mechanism of down-regulating lncRNA TTTY15 targeting miR-4500 on the proliferation, apoptosis, migration and invasion of A172 glioma cells.@*METHODS@#The difference in TTTY15 expression between the glioma cells and tissue was determined with a qRT-PCR method. Complementary binding sites of TTTY15 and miR-4500 were predicted with Starbase software, and the targeting relationship was validated with a luciferase reporter system. A172 glioma cells were divided into Control, si-NC (transfected with control siRNA), si-TTTY15 (transfected with TTTY15 siRNA), si-TTTY15+Anti-miR-NC (co-transfected with TTTY15 siRNA and inhibitor control) and si-TTTY15+Anti-miR-4500 (co-transfected with TTTY15 siRNA and miR-4500 inhibitor) groups. Proliferation, apoptosis, migration and invasion, and the expression of Bax, Bcl-2, MMP-2 and MMP-9 proteins of the A172 glioma cells were respectively detected with CCK-8, flow cytometry, Transwell chamber and Western blotting assays.@*RESULTS@#The expression of TTTY15 in glioma cells and glioma tissues have both increased. The expression levels of TTTY15 and miR-4500 in glioma tissues were inversely correlated. TTTY15 and miR-4500 are mutually targeted. Compared with those of the Control and si-NC groups, the glioma cells in the si-TTTY15 group showed increased level of miR-4500, decreased survival rate, increased apoptosis rate, enhanced cell migration and invasion, increased expression of Bax protein, and decreased expression of Bcl-2, MMP-2 and MMP-9 proteins (P<0.05). Compared with those of the si-TTTY15+Anti-miR-NC group, the A172 glioma cells in the si-TTTY15+Anti-miR-4500 group showed decreased level of miR-4500, increased cell survival rate, decreased apoptosis rate, enhanced cell migration and invasion, decreased expression of Bax protein, and increased expression of Bcl-2, MMP-2, and MMP-9 proteins (P<0.05).@*CONCLUSION@#Down-regulating TTTY15 targeting miR-4500 can inhibit the proliferation, migration, invasion and induce apoptosis of the A172 glioma cells.

Effect of Circular RNA hsa_circ_0067582 on the Proliferation and Invasion Ability of Gastric Cancer Cells / 中国医学科学院学报

Objective To investigate the effects on cell proliferation and invasion of the circular RNA hsa_circ_0067582 in gastric cancer(GC). Methods After hsa_circ_0067582 overexpression (Oe-circ_0067582) plasmid was transfected into AGS and SGC-7901 cells,the cell viability,proliferation,invasion ability,and apoptosis were detected by CCK-8,colony formation and EdU assays,Transwell assay,and flow cytometry,respectively.Western blotting was employed to detect the expression levels of proteins related to the cell apoptosis and epithelial-mesenchymal transition(EMT).The effect of Oe-circ_0067582 on the growth of SGC-7901 cells in nude mice was observed.Bioinformatics tools were used to predict the binding target miRNA of hsa_circ_0067582,and the competing endogenous RNA(ceRNA)regulatory network was established.Finally,functional enrichment was performed to analyze the biological functions of the target genes of the predicted miRNA. Results Compared with the pLO-ciR(empty plasmid)group,the Oe-circ_0067582 group in AGS and SGC-7901 cells attenuated the cell viability(t=7.883,P=0.001;t=5.679,P=0.005),proliferation(t=6.709,P=0.003;t=5.857,P=0.003),and invasion ability(t=7.782,P=0.002;t=6.342,P=0.003)and induced cell apoptosis(t=7.225,P=0.002;t=11.509,P=0.001).Western blotting showed that the Oe-circ_0067582 group in AGS and SGC-7901 cells up-regulated the protein levels of cysteinyl aspartate specific proteinase (Caspase) 3(t=6.863,P=0.002;t=7.024,P=0.001),Caspase 7(t=3.295,P=0.04;t=6.008,P=0.004),Caspase 9(t=4.408,P=0.012;t=6.278,P=0.004),and E-cadherin(t=12.453,P=0.002;t=10.867,P=0.001),while down-regulated those of Vimentin(t=7.242,P=0.002;t=5.694,P=0.004)and N-cadherin(t=6.480,P=0.003;t=7.446,P=0.001).Furthermore,Oe-circ_0067582 significantly inhibited the growth of tumor in the SGC-7901 tumor-bearing nude mice(t=3.526,P=0.017).The prediction based on TargetScan and miRnada suggested that hsa_circ_0067582 can competitively bind to hsa-miR-181b-3p,hsa-miR-337-3p,hsa-miR-421,and hsa-miR-548d-3p.The functional enrichment indicated that the target genes of miRNA were involved in multiple cancer-related biological processes including negative regulation of apoptotic process,gene expression,transcriptional misregulation in cancer,transforming growth factor-β,and p53 signaling pathways. Conclusion Oe-circ_0067582 can inhibit the proliferation and attenuate EMT process to reduce the invasion ability of AGS and SGC-7901 cells,which provides a new target for the treatment of GC.

Differential Expression of Long Non-coding RNA Cancer Susceptibility Candidate 2 and Imprinted Gene H19 in Extrahepatic Cholangiocarcinoma / 中国医学科学院学报

Objective To investigate the expression and the potential roles of long non-coding RNA(lncRNA)cancer susceptibility candidate 2(CASC2)and imprinted gene H19 in extrahepatic cholangiocarcinoma(ECC). Methods Four samples from patients with ECC were collected for high-throughput sequencing which was conducted to reveal the transcriptomic profiles of lncRNA CASC2 and H19.Bioinformatics tools were employed to predict the potential roles of the two genes.Another 22 ECC tissue samples and the cholangiocarcinoma cell lines(RBE,QBC939,HuH-28,and HuCCT1)with different degrees of differentiation were selected for validation.The para-carcinoma tissue and normal human intrahepatic biliary epithelial cell(HIBEC)were used as the control groups.The expression levels of lncRNA CASC2 and H19 in carcinoma tissue,para-carcinoma tissue,and cell lines were determined by real-time quantitative polymerase chain reaction(qRT-PCR).The correlation analysis was carried out for the clinical indicators of patients with the expression levels of the target genes. Results The two target genes showed significantly different expression between carcinoma tissue and para-carcinoma tissue(all P<0.05).Specifically,CASC2 had higher expression level in the carcinoma tissue than in the para-carcinoma tissue(t=1.262,P=0.025),whereas the expression of H19 showed an opposite trend(t=1.285,P=0.005).The expression levels of CASC2 in QBC939(t=8.114,P=0.015)and HuH-28(t=9.202,P=0.012)cells were significantly higher than that in the control group.The expression levels of H19 were significantly lower in RBE(t=-10.244,P<0.001),QBC939(t=-10.476,P<0.001),HuH-28(t=-19.798,P<0.001),and HuCCT1(t=-16.193,P=0.004)cells than in the control group.Bioinformatics analysis showed that CASC2 was mainly involved in the metabolic process and H19 in the development of multicellular organisms.Both CASC2 and H19 were related to catalytic activity.The expression level of lncRNA CASC2 was correlated with pathological differentiation(χ 2=6.222,P=0.022)and lymph node metastasis(χ2=5.455,P=0.020),and that of lncRNA H19 with pathological differentiation(χ2=1.174,P=0.029)and tumor size(χ2=-0.507,P=0.037). Conclusions In the case of ECC,lncRNA CASC2 and H19 have transcription disorders.lncRNA CASC2 is generally up-regulated in the carcinoma tissue,while H19 is down-regulated.Both genes have the potential to become new molecular markers for ECC.

circBIRC6 contributes to the development of non-small cell lung cancer via regulating microRNA-217/amyloid beta precursor protein binding protein 2 axis / 中华医学杂志(英文版)

BACKGROUND@#Circular RNAs (circRNAs) are considered to be important regulators in cancer biology. In this study, we focused on the effect of circRNA baculoviral inhibitor of apoptosis protein (IAP) repeat containing 6 (circBIRC6) on non-small cell lung cancer (NSCLC) progression.@*METHODS@#The NSCLC and adjacent non-tumor tissues were collected at Shanghai Ninth People's Hospital. Quantitative real-time polymerase chain reaction was conducted for assessing the levels of circBIRC6, amyloid beta precursor protein binding protein 2 (APPBP2) messenger RNA (mRNA), baculoviral IAP repeat containing 6 mRNA (BIRC6), and microRNA-217 (miR-217). Western blot assay was adopted for measuring the protein levels of APPBP2, E-cadherin, N-cadherin, and vimentin. Colony formation assay, transwell assay, and flow cytometry analysis were utilized for evaluating cell colony formation, metastasis, and apoptosis. Dualluciferase reporter assay and RNA immunoprecipitation assay were carried out to determine the interaction between miR-217 and circBIRC6 and APPBP2 in NSCLC tissues. The murine xenograft model assay was used to investigate the function of circBIRC6 in tumor formation in vivo. Differences were analyzed via Student's t test or one-way analysis of variance. Pearson's correlation coefficient analysis was used to analyze linear correlation.@*RESULTS@#CircBIRC6 was overexpressed in NSCLC tissues and cells. Knockdown of circBIRC6 repressed the colony formation and metastasis and facilitated apoptosis of NSCLC cells in vitro and restrained tumorigenesis in vivo. Mechanically, circBIRC6 functioned as miR-217 sponge to promote APPBP2 expression in NSCLC cells. MiR-217 inhibition rescued circBIRC6 knockdown-mediated effects on NSCLC cell colony formation, metastasis, and apoptosis. Overexpression of miR-217 inhibited the malignant phenotypes of NSCLC cells, while the effects were abrogated by elevating APPBP2.@*CONCLUSIONS@#CircBIRC6 aggravated NSCLC cell progression by elevating APPBP2 via sponging miR-217, which might provide a fresh perspective on NSCLC therapy.

Paired box 5 increases the chemosensitivity of esophageal squamous cell cancer cells by promoting p53 signaling activity / 中华医学杂志(英文版)

BACKGROUND@#Gene promoter methylation is a major epigenetic change in cancers, which plays critical roles in carcinogenesis. As a crucial regulator in the early stages of B-cell differentiation and embryonic neurodevelopment, the paired box 5 (PAX5) gene is downregulated by methylation in several kinds of tumors and the role of this downregulation in esophageal squamous cell carcinoma (ESCC) pathogenesis remains unclear.@*METHODS@#To elucidate the role of PAX5 in ESCC, eight ESCC cell lines, 51 primary ESCC tissue samples, and eight normal esophageal mucosa samples were studied and The Cancer Genome Atlas (TCGA) was queried. PAX5 expression was examined by reverse transcription-polymerase chain reaction and western blotting. Cell apoptosis, proliferation, and chemosensitivity were detected by flow cytometry, colony formation assays, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays in ESCC cell lines with PAX5 overexpression or silencing. Tumor xenograft models were established for in vivo verification.@*RESULTS@#PAX5 methylation was found in 37.3% (19/51) of primary ESCC samples, which was significantly associated with age (P = 0.007) and tumor-node-metastasis stage (P = 0.014). TCGA data analysis indicated that PAX5 expression was inversely correlated with promoter region methylation (r = -0.189, P = 0.011 for cg00464519 and r = -0.228, P = 0.002 for cg02538199). Restoration of PAX5 expression suppressed cell proliferation, promoted apoptosis, and inhibited tumor growth of ESCC cell lines, which was verified in xenografted mice. Ectopic PAX5 expression significantly increased p53 reporter luciferase activity and increased p53 messenger RNA and protein levels. A direct interaction of PAX5 with the p53 promoter region was confirmed by chromatin immunoprecipitation assays. Re-expression of PAX5 sensitized ESCC cell lines KYSE150 and KYSE30 to fluorouracil and docetaxel. Silencing of PAX5 induced resistance of KYSE450 cells to these drugs.@*CONCLUSIONS@#As a tumor suppressor gene regulated by promoter region methylation in human ESCC, PAX5 inhibits proliferation, promotes apoptosis, and induces activation of p53 signaling. PAX5 may serve as a chemosensitive marker of ESCC.

PRKCDBP Methylation is a Potential and Promising Candidate Biomarker for Non-small Cell Lung Cancer / 中国肺癌杂志

BACKGROUND@#The occurrence and development of lung cancer are closely linked to epigenetic modification. Abnormal DNA methylation in the CpG island region of genes has been found in many cancers. Protein kinase C delta binding protein (PRKCDBP) is a potential tumor suppressor and its epigenetic changes are found in many human malignancies. This study investigated the possibility of PRKCDBP methylation as a potential biomarker for non-small cell lung cancer (NSCLC).@*METHODS@#We measured the methylation levels of PRKCDBP in the three groups of NSCLC tissues. Promoter activity was measured by the dual luciferase assay, with 5'-aza-deoxycytidine to examine the effect of demethylation on the expression level of PRKCDBP.@*RESULTS@#The methylation levels of PRKCDBP in tumor tissues and 3 cm para-tumor were higher than those of distant (>10 cm) non-tumor tissues. Receiver operating characteristic (ROC) curve analysis between tumor tissues and distant non-tumor tissues showed that the area under the line (AUC) was 0.717. Dual luciferase experiment confirmed that the promoter region was able to promote gene expression. Meanwhile, in vitro methylation of the fragment (PRKCDBP_Me) could significantly reduce the promoter activity of the fragment. Demethylation of 5'-aza-deoxycytidine in lung cancer cell lines A549 and H1299 showed a significant up-regulation of PRKCDBP mRNA levels.@*CONCLUSIONS@#PRKCDBP methylation is a potential and promising candidate biomarker for non-small cell lung cancer.

Mechanism of miR-155 Promoting Drug Resistance in Childhood Acute Lymphoblastic Leukemia by Regulating Wnt/β-Catenin Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the mechanism of miR-155 promoting drug resistance of children B-ALL to Ara-C by regulating Wnt/β-Catenin signaling pathway.@*METHODS@#The expression of miR-155 in bone marrow tissue and cell line of B-ALL was detected by PCR. The chemotherapy resistant strain REH/ Ara-C was constructed by using REH cells. REH/ Ara-C cells were transfected with miR-155 inhibitor. The proliferation of REH/Ara-C cells was detected by EdU. The apoptosis of REH/ Ara-C cells was detected by flow cytometry. The drug resistance of REH/Ara-C cells were analyzed by CCK-8 method and colony formation assay. The expression of Wnt/β-Catenin signaling pathway related proteins were determined by Western blot. MiR-155 inhibitor and Wnt activator agonist were used to transfect REH/Ara-C cells, and their effects on cell proliferation, apoptosis and drug resistance were determined.@*RESULTS@#Compared with normal tissues and cells, the expression level of miR-155 in B-ALL bone marrow tissue/cell line was increased (P<0.05); Compared with drug sensitive B-ALL tissues/cell lines, the expression level of miR-155 in drug resistant B-ALL tissues and cell lines was increased (P<0.05); Inhibition of miR-155 expression decreased the proliferation of REH/Ara-C cells (P<0.05), promoted apoptosis (P<0.05), enhanced the cytotoxicity of Ara-C (P<0.05), and inhibited Wnt/β-Catenin signaling pathway related protein and MDR1 gene expression (P<0.05), which could be reversed by activating Wnt expression (P<0.05).@*CONCLUSION@#The expression of miR-155 is up-regulated in bone marrow of children with B-ALL, which may be related to the activation of Wnt/β-Catenin signaling pathway promotes the proliferation of B-ALL cells and inhibits apoptosis, which leads to chemotherapy resistance.

Glucose-6 phosphatase catalytic subunit inhibits the proliferation of liver cancer cells by inducing cell cycle arrest / 中华肝脏病杂志

Objective: To investigate the effects of glucose-6-phosphatase catalytic subunit (G6PC) recombinant adenovirus on proliferation and cell cycle regulation of liver cancer cells. Methods: Recombinant adenovirus AdG6PC was constructed. Huh7 cells and SK-Hep1 cells were set as Mock, AdGFP and AdG6PC group. Cell proliferation and clone formation assay were used to observe the proliferation of liver cancer cells. Transwell and scratch assay were used to observe the invasion and migration of liver cancer cells. Cell cycle flow cytometry assay was used to analyze the effect of G6PC overexpression on the proliferation cycle of liver cancer cells. Western blot was used to detect the effect of G6PC overexpression on the cell-cycle protein expression in liver cancer cells. Results: The recombinant adenovirus AdG6PC was successfully constructed. Huh7 and SK-Hep1 cells proliferation assay showed that the number of proliferating cells in the AdG6PC group was significantly lower than the other two groups (P < 0.05). Clone formation assay showed that the number of clones was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression could significantly inhibit the proliferation of liver cancer cells. Transwell assay showed that the number of cell migration was significantly lower in AdG6PC than the other two groups (P < 0.05). Scratch repair rate was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression can significantly inhibit the invasion and migration of liver cancer cells. Cell cycle flow cytometry showed that G6PC overexpression had significantly inhibited the Huh7 cells G(1)/S phase transition. Western blot result showed that G6PC overexpression had down-regulated the proliferation in cell-cycle related proteins expression. Conclusion: G6PC inhibits the proliferation, cell-cycle related expression, and migration of liver cancer cells by inhibiting the G(1)/S phase transition.

Regulatory effect of small nuclear ribonucleoprotein-associated protein B on proliferation and metastasis of liver cancer cells / 中华肝脏病杂志

Objective: To study the expression and effect of small nuclear ribonucleoprotein-associated protein B (SNRPB) on proliferation and metastasis of liver cancer tissues and cells. Methods: The bioinformatics database starBase v3.0 and GEPIA were used to analyze the expression of SNRPB in liver cancer tissue and normal liver tissue, as well as the survival and prognosis of liver cancer patients. The expression of SNRPB mRNA and protein in liver cancer cell lines were analyzed by qRT-PCR and Western blot. RNA interference technique (siRNA) was used to determine SNRPB protein expression down-regulation. The proliferation effect on hepatocellular carcinoma cells was observed by MTT assay. Transwell invasion and migration assay was used to detect the changes in the metastatic ability of liver cancer cells after SNRPB down-regulation. Western blot was used to detect the changes of epithelial mesenchymal transition (EMT) markers in liver cancer cells after down-regulation of SNRPB expression. Data were compared between two groups and multiple groups using t-test and analysis of variance. Results: The expression of SNRPB was significantly higher in liver cancer tissue than normal liver tissue, and its expression level was correlated with the prognosis of liver cancer patients. Compared with the immortalized hepatocyte LO(2), the expression of SNRPB was significantly increased in the liver cancer cells (P < 0.01). siRNA-SNRPB had significantly inhibited the expression of SNRPB mRNA and protein in liver cancer cells. MTT results showed that the absorbance value was lower in SNRPB knockdown group than negative control group, and the difference at 96 h after transfection was most significant (P < 0.01). Transwell assay results showed that compared with the negative control group, the SNRPB knockdown group (MHCC-97H: 121.27 ± 8.12 vs. 46.38 ± 7.54; Huh7: 126.50 ± 6.98 vs. 41.10 ± 8.01) invasion and migration (MHCC-97H: 125.20 ± 4.77 vs. 43.18 ± 7.32; Huh7: 132.22 ± 8.21 vs. 38.00 ± 6.78) ability was significantly reduced (P < 0.01) in liver cancer cells. Western blot showed that the expression level of epithelial phenotype marker E-cadherin was decreased after down-regulation of SNRPB, while the expression levels of mesenchymal phenotype markers N-cadherin and vimentin was increased, suggesting that down-regulation of SNRPB inhibited EMT in liver cancer cells. Conclusion: SNRPB expression is significantly increased in liver cancer tissues and cells, and it is involved in regulating the proliferation, metastasis and EMT of liver cancer cells.

Mechanism of circZNF609 targeting miR-153 to regulate the proliferation and apoptosis of diffuse large B-cell lymphoma / 中华肿瘤杂志

Objective: To investigate the molecular mechanism of circZNF609 targeting miR-153 to regulate the proliferation and apoptosis of diffuse large B-cell lymphoma. Methods: Fifty cases of lymphoma tissue from patients with diffuse large B-cell lymphoma who were diagnosed and treated in the First Affiliated Hospital of Zhengzhou University from July 2018 to December 2019 were collected. Thirty cases of normal lymph node tissues that were confirmed to be reactive hyperplasia by pathological diagnosis during the same period were selected as controls. Real time quantitative polymerase chain reaction (PCR) was used to detect the expression of circZNF609 in diffuse large B-cell lymphoma tissues and control hyperplasia lymph nodes. Diffuse large B-cell lymphoma OCI-LY19 cells were divided into control group (blank control), si-con group (transfected with siRNA control), si-ZNF609 group (transfected with circZNF609 siRNA), and si-ZNF609+ Anti-NC group (co-transfected with circZNF609 siRNA and inhibitor control) and si-ZNF609+ Anti-miR-153 group (co-transfected with circZNF609 siRNA and miR-153 inhibitor). Cell counting kit-8 (CCK-8) was used to detected proliferation, flow cytometry was used to detect cell cycle and apoptosis. Western blot was used to detect the protein expressions of C-caspase-3, cyclin D1, p21. The luciferase reporter system was used to identifie the relationship between circZNF609 and miR-153. Results: The expression level of circZNF609 in diffuse large B-cell lymphoma tissue was (1.44±0.22), higher than (0.37±0.14) in the control tissues (P<0.001). The cell survival rate of the si-ZNF609 group was (51.74±6.39)%, lower than (100.00±10.23)% of the control group and the (99.64±11.67)% of the si-con group (P<0.001). The proportion of cells in the G(0)/G(1) phase was (63.25±4.11)%, higher than (48.62±4.32)% of the control group and (47.12±3.20)% of the si-con group (P<0.001), the apoptosis rate was (13.36±1.42)%, higher than (3.65±0.47)% of the control group and (3.84±0.62)% of the si-con group (P<0.05). The expression levels of C-caspase-3 and p21 protein were (0.85±0.09) and (0.90±0.08), higher than (0.38±0.04) and (0.65±0.07) in the control group and (0.39±0.05) and (0.66±0.05) in the si-con group (P<0.001). The expression level of cyclin D1 protein was (0.40±0.03), lower than (0.52±0.06) of the control group and (0.53±0.04) of the si-con group (all P<0.001). CircZNF609 and miR-153 are mutually targeted. The cell survival rate of the si-ZNF609+ Anti-miR-153 group was (169.92±13.25)%, higher than (100.00±9.68)% of the si-ZNF609+ Anti-NC group (P<0.001), the ratio of cells in G(0)/G(1) phase and apoptosis rate were (52.01±3.62)% and (8.20±0.87)%, respectively, lower than (64.51±5.17)% and (14.03±1.17)% in the si-ZNF609+ Anti-NC group (P<0.001). The protein expression levels of C-caspase-3 and p21 were (0.42±0.06) and (0.52±0.06), lower than (0.80±0.07) and (0.92±0.10) of the si-ZNF609+ Anti-NC group (P<0.001). The protein expression level of cyclin D1 was (0.68±0.07), higher than (0.39±0.04) in the si-ZNF609+ Anti-NC group (P<0.001). Conclusion: Down-regulation of circZNF609 inhibits the proliferation of diffuse large B-cell lymphoma OCI-LY19 cells and induces apoptosis by targeting miR-153.

The effect of HOXC10 gene on biological behaviors of glioma cells and mechanism in tumor microenvironment / 中华肿瘤杂志

Objective: To study the effects of Homeobox C10 (HOXC10) on biological characteristics such as migration, invasion and proliferation of glioma cancer cells and to explore the role of HOXC10 gene in glioma microenvironment. Methods: The expression level of HOXC10 in high grade glioma (glioblastoma) and low grade glioma and its effect on patient survival were analyzed by using The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) database. Hoxc10-siRNA-1, HOXC10-siRNA-2 and siRNA negative control (NC) were transfected into U251 cells according to the operation instructions of HOXC10-siRNA transfection. 100 ng/ mL recombinant protein chemokine ligand 2 (reCCL2) was added into the transfection group, and was labeled as HOXC10-siRNA-1+ reCCL2 and HOXC10-siRNA-2+ reCCL2 groups. The expressions of HOXC10 mRNA and target protein in each group was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot. The proliferation ability of cells in each group was detected by cell counting kit 8 (CCK8) method. The migration ability of cells was detected by Transwell assay and Nick assay, and cell apoptosis was detected by flow cytometry. The expression of chemokines in each group was detected by multiple factors. Co-incubation assays were performed to determine the role of HOXC10 and chemokine ligand 2 (CCL2) in recruiting and polarizing tumor-associated macrophages (M2-type macrophages). Results: The median expression level of HOXC10 in high grade gliomas was 8.51, higher than 1.00 in low grade gliomas (P<0.001) in TCGA database. The median expression level of HOXC10 in high grade gliomas was 0.83, higher than 0.00 in low grade gliomas (P=0.002) in CGGA database. The 5-year survival rate of patients with high HOXC10 expression in TCGA database was 28.2%, lower than 78.7% of those with low HOXC10 expression (P<0.001), and the 5-year survival rate of patients with high HOXC10 expression in CGGA database was 20.3%, lower than 58.0% of those with low HOXC10 expression (P<0.001). The numbers of cell migration in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (45±3) and (69±4) respectively, lower than (159±3) in NC group (P<0.05). The cell mobility of HOXC10-siRNA-1 group and HOXC10-siRNA-2 group at 48 hours were (15±2)% and (28±4)% respectively, lower than (80±5)% of NC group (P<0.05). The expressions of vimentin in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (141 740.00±34 024.56) and (94 655.00±5 687.97), N-cadherin were (76 810.00±14.14) and (94 254.00±701.45), β-catenin were (75 786.50±789.84) and (107 296.50±9 614.53), lower than (233 768.50±34 114.37), (237 154.50±24 715.50) and (192 449.50±24 178.10) of NC group (P<0.05). The A value of HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (0.44±0.05) and (0.32±0.02) at 96 hours, lower than 0.92±0.12 of NC group (P<0.05). The apoptosis rates of HOXC10-siRNA-1 group and HOXC10 siRNA-2 group were (10.23±1.24)% and (13.81±2.16)%, higher than (4.60±0.07)% of NC group (P<0.05). The expression levels of CCL2 in U251 cells in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were (271.63±44.27) and (371.66±50.21), lower than (933.93±29.84) in NC group (P<0.05). The expression levels of CCL5 (234.81±5.95 and 232.62±5.72), CXCL10 (544.13±48.14 and 500.87±15.65) and CXCL11 (215.75±15.30 and 176.18±16.49) in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were higher than those in NC group (9.98±0.71, 470.54±18.84 and 13.55±0.73, respectively, P<0.05). The recruited numbers of CD14(+) THP1 in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were (159.33±1.15) and (170.67±1.15), respectively, lower than (360.00±7.81) in NC group (P<0.05), while addition of reCCL2 promoted the recruitment of CD14(+) THP1 cells (287.00±3.61 and 280.67±2.31 in HOXC10-siRNA-1+ reCCL2 group and HOXC10-siRNA-2+ reCCL2 group, respectively, P<0.05). The expressions level of M2-type macrophage-related gene TGF-β in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (0.30±0.02) and (0.28±0.02), respectively, lower than (1.06±0.10) in NC group (P<0.05). The expressions level of M1-related gene NOS2 in HOXC10-siRNA-1 and HOXC10-siRNA-2 were (11 413.95±1 911.85) and (5 894.00±945.21), respectively, higher than (13.39±4.32) in NC group (P<0.05). Conclusions: The expression of HOXC10 in glioma is high and positively correlated with the poor prognosis of glioma patients. Knockdown of HOXC10 can inhibit the proliferation, migration and metastasis of human glioma U251 cells. HOXC10 may play an immunosuppressive role in glioma microenvironment by promoting the expression of CCL2 and recruiting and polarizing tumor-associated macrophages (M2 macrophages).

Bioinformatics analysis of differently expressed genes in osteoblastic sarcoma and screening of key genes / 中华肿瘤杂志

Objective: To screen the different expressed genes between osteosarcoma and normal osteoblasts, and find the key genes for the occurrence and development of osteosarcoma. Methods: The gene expression dataset GSE33382 of normal osteoblasts and osteosarcoma was obtained from Gene Expression Omnibus (GEO) database. The different expressed genes between normal osteoblasts and osteosarcoma were screened by limma package of R language, and the different expressed genes were analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. The protein interaction network was constructed by the String database, and the network modules in the interaction network were screened by the molecular complex detection (MCODE) plug-in of Cytoscape software. The different expressed genes contained in the first three main modules screened by MCODE were analyzed by gene ontology (GO) using the BiNGO module of Cytoscape software. The MCC algorithm was used to screen the top 10 key genes in the protein interaction network. The gene expression and survival dataset GSE39055 of osteosarcoma was obtained from GEO database, and the survival analysis was performed by Kaplan-Meier method. The data of 48 patients with osteosarcoma treated in the First Affiliated Hospital of Fujian Medical University from January 2005 to December 2015 were selected for verification. The expression of STC2 protein in osteosarcoma was detected by immunohistochemical method, and the survival analysis was carried out combined with the clinical data of the patients. Results: A total of 874 different expressed genes were identified from GSE33382 dataset, including 402 down-regulated genes and 472 up-regulated genes. KEGG enrichment analysis showed that different expressed genes were mainly related to p53 signal pathway, glutathione metabolism, extracellular matrix receptor interaction, cell adhesion molecules, folate tolerance, and cell senescence. The top 10 key genes in the interaction network were GAS6, IL6, RCN1, MXRA8, STC2, EVA1A, PNPLA2, CYR61, SPARCL1 and FSTL3. STC2 was related to the survival rate of patients with osteosarcoma (P<0.05). The results showed that the expression of STC2 protein was related to tumor size and Enneking stage in 48 cases of osteosarcoma. The median survival time of 25 cases with STC2 high expression was 21.4 months, and that of 23 cases with STC2 low expression was 65.4 months. The survival rate of patients with high expression of STC2 was lower than that of patients with low expression of STC2 (P<0.05). Conclusions: Bioinformatics analysis can effectively screen the different expressed genes between osteosarcoma and normal osteoblasts. STC2 is one of the important predictors for the prognosis of osteosarcoma.

The enhanced genomic 6 mA metabolism contributes to the proliferation and migration of TSCC cells / 国际口腔科学杂志·英文版

In contrast to the well-established genomic 5-methylcytosine (5mC), the existence of N6-methyladenine (6 mA) in eukaryotic genomes was discovered only recently. Initial studies found that it was actively regulated in cancer cells, suggesting its involvement in the process of carcinogenesis. However, the contribution of 6 mA in tongue squamous cell carcinoma (TSCC) still remains uncharacterized. In this study, a pan-cancer type analysis was first performed, which revealed enhanced 6 mA metabolism in diverse cancer types. The study was then focused on the regulation of 6 mA metabolism, as well as its effects on TSCC cells. To these aspects, genome 6 mA level was found greatly increased in TSCC tissues and cultured cells. By knocking down 6 mA methylases N6AMT1 and METTL4, the level of genomic 6 mA was decreased in TSCC cells. This led to suppressed colony formation and cell migration. By contrast, knockdown of 6 mA demethylase ALKBH1 resulted in an increased 6 mA level, enhanced colony formation, and cell migration. Further study suggested that regulation of the NF-κB pathway might contribute to the enhanced migration of TSCC cells. Therefore, in the case of TSCC, we have shown that genomic 6 mA modification is involved in the proliferation and migration of cancer cells.

Genetic variation of YWHAE gene-"Switch" of disease control / 中南大学学报(医学版)

YWHAE gene is located on chromosome 17p13.3, and its product 14-3-3epsilon protein belongs to 14-3-3 protein family. As a molecular scaffold, YWHAE participates in biological processes such as cell adhesion, cell cycle regulation, signal transduction and malignant transformation, and is closely related to many diseases. Overexpression of YWHAE in breast cancer can increase the ability of proliferation, migration and invasion of breast cancer cells. In gastric cancer, YWHAE acts as a negative regulator of MYC and CDC25B, which reduces their expression and inhibits the proliferation, migration, and invasion of gastric cancer cells, and enhances YWHAE-mediated transactivation of NF-κB through CagA. In colorectal cancer, YWHAE lncRNA, as a sponge molecule of miR-323a-3p and miR-532-5p, can compete for endogenous RNA through direct interaction with miR-323a-3p and miR-532-5p, thus up-regulating K-RAS/ERK/1/2 and PI3K-AKT signaling pathways and promoting the cell cycle progression of the colorectal cancer. YWHAE not only mediates tumorigenesis as a competitive endogenous RNA, but also affects gene expression through chromosome variation. For example, the FAM22B-YWHAE fusion gene caused by t(10; 17) (q22; p13) may be associated with the development of endometrial stromal sarcoma. At the same time, the fusion transcript of YWHAE and NUTM2B/E may also lead to the occurrence of endometrial stromal sarcoma. To understand the relationship between YWHAE, NUTM2A, and NUTM2B gene rearrangement/fusion and malignant tumor, YWHAE-FAM22 fusion gene/translocation and tumor, YWHAE gene polymorphism and mental illness, as well as the relationship between 17p13.3 region change and disease occurrence. It provides new idea and basis for understanding the effect of YWHAE gene molecular mechanism and genetic variation on the disease progression, and for the targeted for the diseases.

MiR-133b regulates the proliferation, colony formation, and invasion of bladder cancer cells via inhibiting SOX4 / 中南大学学报(医学版)

OBJECTIVES@#Bladder cancer is one of the most common urothelial tumors with high incidence and mortality rates. Although it has been reported that microRNA (miR)-133b can regulate tumorigenesis of bladder cancer, the mechanism remains unclear. Sex-determining region Y-box transcription factor 4 (SOX4) exhibits an important role in tumorigenesis, but it is unclear whether SOX4 and miR-133b are associated with regulation of pathogenesis of bladder cancer. This study aims to determine the expressions of SOX4 and miR-133b in bladder cancer tissues and cells, investigate their effects on the proliferation, colony formation, and invasion of bladder cancer cells, and to explore the association between miR-133b and SOX4 in regulating biological featurss of bladder cancer cells.@*METHODS@#The bladder cancer and adjacent tissue samples of 10 patients who underwent surgical resection in the Second Xiangya Hospital of Central South Universty from Januray to June 2015 were obtained. The levels of miR-133b were tested by real-time PCR, and the protein levels of SOX4 were evaluated using Western blotting in bladder cancer tissues, matched adjacent tissues, and cell lines. The correlation between miR-133b expression and SOX4 expression in bladder cancer tissues was analyzed. Using the online database TargetScan, the relationship between SOX4 and miR-133b was predicted. MiR-133b mimics, miR-133b inhibitor, and short hairpin RNA (shRNA)-SOX4 were transfected into T24 cells by Lipofectamine 2000. The relationship between miR-133b and SOX4 was also verified by a dual-luciferase reporter assay. The proliferation of T24 cells cultured for 0, 12, 48, 72, and 96 h was evaluated by cell counting kit-8 (CCK-8) assay. The colony formation capacity of bladder cancer cells was tested after 14-day culture, and cell invasion capacity was evaluated with Transwell invasion assay.@*RESULTS@#Bladder cancer tissue and bladder cancer cells had low level of miR-133b but high level of SOX4, compared with matched adjacent tissues and normal bladder epithelial cells. A negative correlation between miR-133b mRNA and SOX4 protein levels in bladder cancer tissues was also found (r=-0.84). The results of online database TargetScan showed that miR-133b targets at SOX4, and overexpression of miR-133b significantly attenuated the expression of SOX4 in T24 cells. Both overexpression of miR-133b and knockdown of SOX4 significantly inhibited the proliferation, colony formation, and invasion capacity of bladder cancer cells in vitro. SOX4 down-regulation restored the effects of miR-133b inhibitor on the proliferation, colony formation, and invasion capacity of T24 cells.@*CONCLUSIONS@#The up-regulation of SOX4 contributes to the progression of bladder cancer, and miR-133b can regulate the proliferation, colony formation, and invasion of bladder cancer cells via inhibiting SOX4.