UHRF1/DNMT1-MZF1 axis-modulated intragenic site-specific CpGI methylation confers divergent expression and opposing functions of PRSS3 isoforms in lung cancer

As confusion mounts over RNA isoforms involved in phenotypic plasticity, aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity (ITH). Protease serine 3 (PRSS3), possessing four splice variants (PRSS3-SVs; PRSS3-V1-V4), is an indispensable trypsin that shows paradoxical effects on cancer development. Here, we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer, exhibiting opposing functions and clinical outcomes, namely, oncogenic PRSS3-V1 and PRSS3-V2 versus tumor-suppressive PRSS3-V3, by targeting different downstream genes. We identified an intragenic CpG island (iCpGI) in PRSS3. Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1 (MZF1) to regulate PRSS3 transcription. The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression. Epigenetic silencing of PRSS3-V3 via iCpGI methylation (iCpGIm) in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease. Thus, UHRF1/DNMT1-MZF1 axis-modulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs, conferring nongenetic functional ITH, with implications for early detection of lung cancer and targeted therapies.

Hematopoietic regulation of transcription factor GATA1 and mutations causing hereditary erythroid and megakaryocytic disorders / 中国输血杂志

GATA-binding protein 1 (GATA1), an important hematopoietic transcription factor, specifically regulates the proliferation and differentiation of erythroid and megakaryoid cells at the transcription level, which maintains the normal development and maturation of these two lineages. The functional structure of GATA1 is composed of one N-terminal transactivation domain (N-TAD) and two zinc fingers (NF and CF). GATA1 is highly conserved in different species. Alteration of GATA1 expression or function will lead to transcriptional disorder of erythrocyte and megakaryocyte related genes, resulting in various clinical phenotypes. This article reviews the molecular structure of GATA1, its transcriptional regulation in erythrocyte and megakaryocyte, and the hereditary hematopoietic regulatory disorders of these two lineages caused by GATA1 mutations.

Effect of zinc finger protein 22 gene on proliferation, apoptosis, invasion and metastasis of hepatocellular carcinoma cells / 中华肝胆外科杂志

Objective:To investigate the expression of zinc finger protein 22 (ZNF22) gene in hepatocellular carcinoma (HCC) and its effect on tumor proliferation, apoptosis, invasion and metastasis of HCC.Methods:The expression of ZNF22 in 32 HCC specimens, and 371 HCC samples from the cancer genome atlas database were analyzed. ZNF22 knockdown and negative control SNU-449 and JHH-7 HCC cell lines were constructed. The effects of ZNF22 on HCC cells were observed by cell proliferation assay, plate clone formation assay, apoptosis assay, scratch healing assay, Transwell invasion assay, subcutaneous tumor formation, tail vein injection transfer, and small animal live imaging assay in nude mice.Results:The expression of ZNF22 gene is higher in HCC tissues than in paracellular carcinoma tissues, and the difference was statistically significant ( P<0.001). The growth rate of SNU-449 and JHH-7 cells in ZNF22 knockdown group was lower than that in control group, and the difference was statistically significant ( P<0.001). Compared with negative control group, the clone number formed by SNU-449 cells in ZNF22 knockdown group decreased (26±8 vs. 59±5, P<0.01), the level of apoptosis increased (6.60%±0.22% vs. 2.38%±0.30%, P<0.001), the migration rate decreased (14.47%±6.42% vs. 68.84%±8.01%, P<0.001), and the number of invasive cells decreased (48.00±2.23 vs. 179.00±4.81, P<0.001). There was no obvious tumor growth after subcutaneous injection of JHH-7 cells into nude mice in ZNF22 knockdown group, and the systemic fluorescence expression was lower than that of the negative control group, and the difference was statistically significant ( P<0.05). No metastases were observed on autopsy in knockdown group nude mice. Conclusion:ZNF22 is highly expressed in HCC while knockdowing ZNF22 gene inhibited the growth, proliferation, invasion, metastasis of HCC cells, and induced apoptosis of HCC cells.

The efficacy comparison of sakubatril valsartan and valsartan in the treatment of patients with chronic cardiac insufficiency and the influence on zinc finger protein A20 and nuclear factor-κB in peripheral blood mononuclear cells / 中国医师进修杂志

Objective:To compare the efficacy of sakubatril valsartan and valsartan in the treatment of patients with chronic cardiac insufficiency and the influence on zinc finger protein A20 and nuclear factor-κB (NF-κB) in peripheral bloodmononuclear cells (PBMCs).Methods:Ninety-senven patients with chronic cardiac insufficiency admitted to the Affiliated Hospital of Jining Medical College from February 2019 to January 2020 were continuously selected and randomly divided into the control group (48 cases) and the observation group (49 cases). Both groups received routine anti-heart failure according to the guidelines. The control group added with valsartan and the observation group added with sakubatril valsartan treatment. Before the treatment and after 3 months of treatment, the changes of cardiac function indexes and the changes of inflammatory markers such as hypersensitive C-reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α), matrix metalloproteinase 9 (MMP-9), and N-terminal pro B-type natriuretic peptide (NT-proBNP) were compared. PBMCs was extracted to detect zinc finger protein A20 and NF-κB levels. The incidence of adverse reactions in the two groups was recorded, and the relationship between zinc finger proteins A20, NF-κB and the myocardial injury marker NT-proBNP were analyzed.Results:After 3 months of treatment, the changes of cardiac function indexes in the observation group were better than those in the control group and the levels of hs-CRP, TNF-α, MMP-9, NT-proBNP in the observation group were lower than those in the control group: (1.96 ± 0.57) mg/L vs. (2.87 ± 0.79) mg/L, (7.11 ± 1.46) μg/L vs. (8.24 ± 1.57) μg/L, (110.14 ± 10.63) μg/L vs. (129.52 ± 17.96) μg/L, (716.91 ± 105.78) ng/L vs. (965.25 ± 97.41) ng/L, there were statistical differences ( P<0.05). After 3 months of treatment, the levels of finger protein A20, NF-κB in the observation group were lower than those in the control group: (3.57 ± 1.13) % vs. (4.41 ± 1.32) %, (29.87 ± 6.58) ng/L vs. (35.71 ± 10.02) ng/L, there were statistical differences ( P<0.05). Finger protein A20 and NF-κB in patients with chronic cardiac insufficiency were positively correlated with NT-proBNP ( r = 0.487, 0.738, P<0.01). Conclusions:On the basis of conventional treatment, compared with valsartan, the addition of sakubatril valsartan, can improve the cardiac function of patients with chronic cardiac insufficiency, reduce the body′s inflammatory response, reduce the expression of myocardial injury marker NT-proBNP, inhibit the activation of PBMCs NF-κB, and reduce the level offinger protein A20.

Regulatory effect of Ac-SDKP on phosphorylated heat shock protein 27/SNAI1 pathway in silicotic rats / 中华劳动卫生职业病杂志

Objective: To study the effect of anti-fibrotic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on phosphorylated heat shock protein 27 (P-HSP27) and zinc finger family transcriptional repressor 1 (SNAI1) expression to explore the anti-silicosis fibrosis effect of Ac-SDKP. Methods: In December 2014, the rat silicosis animal model was prepared by one-time bronchial infusion of silicon dioxide (SiO(2)) dust. 80 SPF healthy adult Wistar rats were selected, and the rats were divided into 8 groups according to the random number table method, 10 in each group. Model control group for 4 weeks (feeding for 4 weeks) , model control group for 8 weeks (feeding for 8 weeks) : bronchial perfusion with normal saline 1.0 ml per animal. Silicosis model group for 4 weeks (feeding for 4 weeks) and silicosis model group for 8 weeks (feeding for 8 weeks) : bronchial perfusion of 50 mg/ml SiO(2) suspension 1.0 ml per animal. Ac-SDKP administration group for 4 weeks (feeding for 4 weeks) , Ac-SDKP administration group for 8 weeks (feeding for 8 weeks) : Ac-SDKP 800 μg·kg(-1)·d(-1) was administered by intraperitoneal pump. Ac-SDKP preventive treatment group: 48 h after Ac-SDKP 800 μg·kg(-1)·d(-1) administration, bronchial perfusion of SiO(2) suspension 1.0 ml per animal, raised for 8 weeks. Ac-SDKP anti-fibrosis treatment group: after bronchial perfusion of 1.0 ml of SiO(2) suspension for 4 weeks, Ac-SDKP 800 μg·kg(-1)·d(-1) was administered for 4 weeks. Western blotting was used to detect the expression of P-HSP27, SNAI1, α-smooth muscle actin (α-SMA) , and collage typeⅠ and Ⅲ in each group. The expression of P-HSP27 and SNAI1 was detected by immunohistochemistry, and the co-localized expression of P-HSP27 and α-SMA was detected by laser confocal microscopy. Results: Compared with the model control group, the expressions of P-HSP27, SNAI1, α-SMA, and collage typeⅠ and Ⅲ in the silicosis fibrosis area of the rats in the silicosis model group were enhanced, and the differences were statistically significant (P<0.05) . After Ac-SDKP intervention, compared with silicosis model group for 8 weeks, the expressions of P-HSP27, SNAI1 α-SMA, and collage typeⅠ and Ⅲ in the Ac-SDKP preventive and anti-fibrosis treatment groups were significantly decreased, and the differences were statistically significant (P<0.05) . However, the expressions of P-HSP27 SNAI1, and collage typeⅠ and Ⅲ between the Ac-SDKP administration group and the model control group did not change significantly, and the differences were not statistically significant (P>0.05) . Laser confocal results showed that the positive cells expressing P-HSP27 and α-SMA in the lung tissue of the silicosis model group were more than those in the model control group. Compared with the silicosis model group, the Ac-SDKP prevention and anti-fibrosis treatment groups expressing the positive cells of P-HSP27 and α-SMA decreased. Compared with the model control group for 8 weeks, there were some double-positive cells expressing P-HSP27 and α-SMA in the nodules of the silicosis model group for 8 weeks. Conclusion: Ac-SDKP may play an anti-silicic fibrosis effect by regulating the P-HSP27/SNAI1 pathway.

Expression and significance of GLI1 and Shh in the malignant transformation of ovarian endometriosis / 中华妇产科杂志

Objective:To investigate the expression levels and clinical significance of glioma-associated oncogene homolog 1 (GLI1) and sonic hedgehog signaling molecule (Shh) in the malignant transformation of ovarian endometriosis (EM).Methods:The expressions of GLI1 and Shh were detected by real-time reverse transcription (RT)-polymerase chain reaction (PCR) and EnVision method in 50 cases of ovarian EM tissues, 35 cases of atypical endometriosis (aEM) and 50 cases of endometriosis-associated ovarian cancer (EAOC). The expression differences of two molecular markers in the malignant transformation of ovarian EM were compared, and the relationships between two molecular markers and the clinicopathological features and prognosis of EAOC were analyzed.Results:(1) RT-PCR showed that the expression levels of GLI1 mRNA in EM, aEM and EAOC group were 1.77±0.40, 3.54±0.44, and 7.80±0.24, respectively. The expression levels of Shh mRNA were 0.95±0.21, 3.14±0.35, and 5.41±0.31, respectively. GLI1 and Shh mRNA in EAOC group were significantly higher than those in EM and aEM group (all P<0.01), and there were statistically significant differences between EM and aEM group (all P<0.01). The percentages of GLI1 in ovarian EM, aEM and EAOC were 32% (16/50), 57% (20/35), and 66% (33/50), respectively, meanwhile, the positive expression rates of Shh were 20% (10/50), 49% (17/35), and 54% (27/50), respectively (all P<0.01). GLI1 mRNA expression was positively correlated with Shh mRNA expression in EAOC tissues ( r=0.721, P<0.01). The expressions of GLI1 protein were proportionated to Shh protein in EAOC tissues ( r=0.608, P=0.001). (2) The expression of GLI1 was significantly related to the International Federation of Gynecology and Obstetrics (FIGO) stage, cancer antigen 125 (CA 125) levels, lymph node metastasis, and Platinum resistance in EAOC patients (all P<0.05). The expression of Shh were related to FIGO stage and lymph node metastasis in EAOC patients (all P<0.05). Logistic regression analysis showed that GLI1 expression was an independent risk factor for poor prognosis in EAOC patients ( P<0.05). Kaplan-meier survival analysis showed that the overall survival rate of EAOC patients with high GLI1 expression and low GLI1 expression was 12.1% and 35.3%, respectively, with statistical significance ( χ2=10.73, P<0.01). The overall survival rate of EAOC patients with high and low expression of Shh protein was 11.1% and 30.4%, in which there was statistically significant difference ( χ2=3.96, P=0.047). Conclusion:GLI1 and Shh are highly associated with the malignant transformation of ovarian EM, which may play a role in promoting malignant degeneration of ovarian EM, and the high expression of GLI1 and Shh indicates a poor prognosis in EAOC patients.

Expression and clinical significance of zinc finger protein 18 in pancreatic ductal adenocarcinoma / 中华胰腺病杂志

Objective:To investigate the expression of the zinc finger protein 18 (ZNF18) in the pancreatic ductal adenocarcinoma (PDAC) tissue and analyze its relationship with the clinicopathological features and prognosis.Methods:Cancer tissue specimens from 131 patients with PDAC who were surgically resected and pathologically confirmed at First Affiliated Hospital of Naval Medical University from March 2017 to December 2019 were collected. ZNF18 protein expression in cancer tissue was detected by immunohistochemical staining, and high and low ZNF18 expression groups were divided based on the expression level of ZNF18. . Kaplan-Meier method and Log-Rank test were used to analyze the relationship between ZNF18 expression level and overall survival rate of PDAC patients. Furthermore, the correlation between ZNF18 protein expression level and clinicopathological features was analyzed, and the Cox regression hazards model was applied for the univariate and multivariate analysis of the factors affecting the prognosis of PDAC patients.Results:82 of 131(62.60%) PDAC tissues had high ZNF18 expression, and 49(37.40%) PDAC tissues had low ZNF18 expression; the survival time in the high ZNF18 expression group was significantly higher than that in the low ZNF18 expression group (21.53±0.69 months vs 12.17±1.57 months), and the difference was statistically significant( P<0.001). Low ZNF18 expression in PDAC was associated with lymph node metastasis( P<0.05), but was not correlated with gender, age, tumor location, tumor volume, differentiation degree, TNM stage, nerve invasion, and vascular cancer thrombus. The results of the univariate analysis showed that tumor differentiation degree, TNM stage, lymph node metastasis, and ZNF18 expression were all associated with the prognosis of PDAC patients. The results of the multivariate analysis showed that tumor differentiation degree( HR=0.463, 95% CI 0.279-0.769, P=0.003), lymph node metastasis( HR=2.062, 95% CI 1.247-3.409, P=0.005), and ZNF18 expression( HR=0.416, 95% CI 0.255-0.676, P<0.001) were independent risk factors affecting the prognosis of patients with PDAC. Conclusions:Low ZNF18 expression in PDAC tissues was closely associated with lymph node metastasis and poor prognosis. ZNF18 can be used as an important molecular marker to evaluate the prognostic survival of PDAC patients.

Comparative study of the SBP-box gene family in rice siblings

SBP-box genes are a class of plant-specific transcription factors which have a common DNA-binding domain(SBP-domain) with an unusual zinc-finger architecture. Many of the genes in this class are thought to play adevelopmental role and a few are involved in the determination of plant architecture. We have made acomparative study of these genes in the genomes of rice (Oryza sativa japonica and Oryza sativa indica) andits nine siblings using a recently proposed hybrid method for orthology and paralogy detection (HyPPO).According to HyPPO, the SBP-box proteins of rice siblings could be divided into twenty primary orthologousgroups on the basis of their overall sequence features. This contrasts with a much less number of groups foundin earlier work with other plant genomes using phylogenetic analysis of the SBP-domains only. The orthologous groups reported by HyPPO showed close correspondence in exon–intron structure and motif conservation. Comparison between different Oryza species revealed disparity in the maintenance of orthologousgenes which may result in their different developmental characteristics. Inclusion of the SBP-box proteins fromA. thaliana did not result in any change in the orthologous groups except for the A. thaliana proteins beingadded to some of the existing groups. The closer correspondence between the proteins in the primaryorthologous clusters is expected to help in a more reliable prediction of their functions. It is also expected toprovide better insight into the evolutionary history of this class of plant-specific proteins.

Enhance of Cellulase Production and Biomass Degradation by Transformation of the Trichoderma reesei RUT-C30∆ zface1 Strain

Abstract The second-generation bioethanol employs lignocellulosic materials degraded by microbial cellulases in their production. The fungus Trichoderma reesei is one of the main microorganisms producing cellulases, and its genetic modification can lead to the optimization in obtaining hydrolytic enzymes. This work carried out the deletion of the sequence that encodes the zinc finger motif of the transcription factor ACE1 (cellulase expression repressor I) of the fungus T. reesei RUT-C30. The transformation of the RUT-C30 lineage was confirmed by amplification of the 989 bp fragment relative to the selection marker, and by the absence of the zinc finger region amplification in mutants, named T. reesei RUT-C30Δzface1. The production of cellulases by mutants was compared to RUT-C30 and measured with substrates carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel®) and Whatman filter paper (PF). The results demonstrated that RUT-C30Δzface1 has cellulolytic activity increased 3.2-fold in Avicel and 2.1-fold in CMC and PF. The mutants presented 1.4-fold higher sugar released in the hydrolysis of the biomass assays. These results suggest that the partial deletion of ace1 gene is an important strategy in achieving bioethanol production on an industrial scale at a competitive price in the fuel market.

miR-9 regulates the malignant biological behaviors of small cell lung cancer by targeting zinc finger E-box binding homeobox 2 and its possible mechanism / 中国肿瘤生物治疗杂志

@#[Abstract] Objective：To explore the regulatory effect of miR-9 on biological behaviors of small cell lung cancer (SCLC) cells by targeting zinc finger E-box binding homeobox 2 (ZEB2), and to analyze the role of miR-9 in SCLC and its possible mechanism. Methods: qPCR, WB and immunohistochemistry methods were used to detect the mRNA and protein expressions of ZEB2 in cancer tissues and corresponding adjacent tissues of 67 SCLC patients who received surgical treatment at the Department of Oncology, Fourth Hospital of Hebei Medical University from February 2018 to November 2019. TargetScan was used to predict the potential target gene of miR-9, which was later verified by Dual luciferase reporter gene assay, qPCR and WB methods. CCK-8 method, Flow cytometry and Transwell experiment were used to detect the effect of miR-9 and ZEB2 over-expression on the biological behaviors of NCI-H446 cells, and WB was used to detect the protein expressions of E-cadherin, N-cadherin and Vimentin in cells. NCI-H446 cells overexpressing miR-9 were used to construct SCLC nude mouse xenograft model, and the effect of miR-9 on the growth of xenografts was observed. Results: The mRNA and protein expression levels of ZEB2 in SCLC tissues were significantly higher than those in adjacent tissues (P<0.01). There is a potential binding site on the 3' UTR of ZEB2 to bind with miR-9. Compared with the control group, the mRNA and protein expression levels of ZEB2 in NCI-H446 cells of the miR-9 over-expression group were significantly reduced (P<0.01); the proliferation, migration and invasion abilities of NCI-H446 cells were significantly suppressed (P<0.05 or P<0.01), and the expression of EMT protein was reduced; However, simultaneous over-expression of ZEB2 could reverse above effects. In in vivo experiments, the size and weight of transplanted tumors in the miR-9 over-expression group were significantly lower than those in the control group (P<0.05 or P<0.01). The expression of ZEB2 protein in the tumor tissues of nude mice in the miR-9 overexpression group was significantly lower than that in the control group (P<0.01). Conclusion: miR-9 can inhibit the biological behaviors of SCLC cells and the growth of NCI-H446 transplanted tumors in nude mice by targeting and regulating ZEB2.

Role of zinc finger proteins in the development and progression of hepatocellular carcinoma / 临床肝胆病杂志

There are complex mechanisms in the development and progression of hepatocellular carcinoma, which have not been fully clarified at present. Zinc finger protein family is the largest transcription factor family in human genome, and more and more evidence has shown that zinc finger proteins play an important role in the development and progression of hepatocellular carcinoma and are expected to become new tumor biomarkers and therapeutic targets for hepatocellular carcinoma. This article reviews the structure and biological functions of zinc finger proteins and their role and regulatory mechanisms in hepatocellular carcinoma.

High expression of ZNF652 promotes carcinogenesis and progression of breast cancer / 南方医科大学学报

OBJECTIVE@#To investigate the expression of ZNF652 in breast cancer tissues and cells and explore its role in breast cancer cell proliferation, invasion and migration.@*METHODS@#We exploited the data from the TCGA database to analyze the differential expression of ZNF652 in breast cancer tissues and adjacent tissues and the correlations of ZNF652 expression with the clinicopathological characteristics of breast cancer patients including molecular subtypes, pathological types, TNM stages and clinical stages. RT-qPCR and Western blotting were used to detect the expression of ZNF652 in 5 breast cancer cell lines including MCF-7, MDA-MB-231, SK-BR-3, UACC-812 and BT-474. Using a lentivirus system and siRNA technique, we assessed the effects of ZNF652 over-expression and knockdown on proliferation, colony forming ability, migration and invasion of breast cancer cells with CCK-8 assay, clonogenic assay, Transwell assay and wound healing assay. The subcellular localization of ZNF652 in 293T cells was determined using immunofluorescence assay.@*RESULTS@#ZNF652 was significantly up-regulated in breast cancer tissues (@*CONCLUSIONS@#ZNF652 is highly expressed in breast cancer tissues and cells to promote the development and progression of breast cancer and may serve as a potential molecular target for diagnosis and treatment of the malignancy.

SNHG6 promotes invasion and metastasis of esophageal squamous cell carcinoma TE1 cells via upregulating ZEB1 / 中国肿瘤生物治疗杂志

@# Objective: To explore the roles and mechanisms of long non-coding RNA (lncRNA) small nucleolar RNA host gene 6 (SNHG6) in promoting invasion and metastasis of esophageal squamous carcinoma (ESCC). Methods: Real time quantitative polymerase chain reaction (qPCR) was used to detect the expression of SNHG6 in ESCC and matched para-carcinoma tissues. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of SNHG6 in ESCC cell lines (TE1, Yes-2, Eca9706 and Kyse150). Then, TE1 cell line which harbored highest expression of SNHG6 was used in following experiments. siRNAs were used to knock down the expression of SNHG6. Clone formation, wound-healing and transwell assay were used to detect the abilities of proliferation, migration andinvasionofTE1cells,respectively.Westernblottingwasusedtodetecttheexpressions of MMP-2, MMP-9andZEB1 protein before and after knockdownofSNHG6inTE1cells.Results:SNHG6washighlyexpressedinESCC tissues, compared to para-carcinoma tissues (P<0.01). The expression of SNHG6 was significantly decreased after transfection of SNHG6siRNA (all P<0.01). The abilities of proliferation, migration and invasion of TE1 cells in si-SNHG6-1 and si-SNHG6-2 group were significantly lower than those in the control group (all P<0.01). The expressions of ZEB1, MMP-2and MMP-9 in si-SNHG6-1 and si-SNHG6-2 group were significantly lower than those in the control group (all P<0.05). Conclusion: SNHG6 is highly expressed in ESCC tissues and promotes the malignant biological behavior of ESCC cells. Its mechanism of promoting the occurrence and development of ESCC may be related to the upregulation of ZEB1 expression.

Progress on TMPRSS2-ERG fusion gene product transcription regulation mechanism in prostate cancer cells / 中国临床药理学与治疗学

Prostate cancer is the most common malignant tumor in men in Europe and the United States, but the mechanism of prostate cancer occurrence and development is not completely clear. In prostate cancer, the TMPRSS2-ERG fusion gene has a high incidence, which promotes ERG overexpression and causes changes in target genes and signaling pathways, such as androgen receptors, spotted zinc finger structural proteins, Notch pathways. In-depth understanding of the transcriptional regulation mechanism of TMPRSS2-ERG fusion gene products in prostate cancer cells can provide new targets for drug action in the treatment of prostate cancer.

Expression and purification of the transcription factor StMsn2 from Setosphaeria turcica in Escherichia coli

Background: In Saccharomyces cerevisiae, Msn2, which acts as a key transcription factor downstream the MAPKHOG cascade pathway, also regulates the expression of genes related to stress responses. However, little is known about the regulation mechanisms of the transcription factor in Setosphaeria turcica. Results: In this study, a zinc finger DNA-binding protein, designated as StMSN2, was cloned from S. turcica. Sequencing results showed that StMSN2 had a 1752 bp open reading frame (ORF), which was interrupted by an intron (135 bp) and encoded a putative 538-amino acid protein. Phylogenetic analysis further revealed that StMsn2 was more closely related to Msn2 of Aspergillus parasiticus. StMSN2 was cloned into the pET-28a vector with His (Histidine) tags and induced with 1 mM IPTG (isopropyl-ß-D-thiogalactoside) at 37°C. The recombinant His-tagged StMsn2 was purified, and a band of size approximately 58.8 kDa was obtained. The high specificity of the polyclonal antibody Msn2-2 was detected with the StMsn2 protein from S. turcica and prokaryotic expression system, respectively. Conclusions: A new gene, named StMSN2, with 1617 bp ORF was cloned from S. turcica and characterized using bioinformatics methods. StMsn2 was expressed and purified in a prokaryotic system. A polyclonal antibody, named Msn2-2, against StMsn2 with high specificity was identified.

Effect of silencing ZEB1 gene on epithelial to mesenchymal transition in glioma U87 cells / 吉林大学学报(医学版)

Objective: To investigate the effects of silencing zinc finger E-box binding homeoboxl (ZEB1) gene on the expressions of mesenchymal markers and cell migration in the glioma U87 cells, and to clarify the effect of ZEB1 on the epithelial to mesenchymal transition (EMT) in the glioma cells. Methods: The constructed ZEB1 shRNA interfering plasmid and control plasmid (shCtrl) were transfected into the glioma U87 cells and the interfering effects were detected by Western blotting method. The glioma U87 cells were divided into control group (the glioma U87 cells were transfected with shCtrl), EMT group (EMT was induced by TGF-fil in the glioma U87 cells transfected with shCtrl) and ZEB1 silence group (EMT was induced by TGF-fil in the glioma U87 cells transfected with ZEB1 shRNAs plasmid). The protein expression levels of mesenchymal markers (N-cadherin, Vimentin), and matrix metalloproteinase-9 (MMP-9) in the glioma U87 cells were measured by Western blotting method. The scratch-healing assay was performed to examine the migration ability of glioma cells. Results: The Western blotting results showed that the expression levels of ZEB1 in the glioma U87 cells transfected with shZEBl # 1 and shZEBl # 2 were significantly lower than that in the cells transfected with shCtrl (P<0. 05 or P< 0. 01), and the inhibitory effect of shZEBl #2 on the ZEB1 expression was more obvious, indicating that ZEB1 was stably transfected into the U87 cells. Compared with control group, the expression levels of mesenchymal markers N-cadherin, Vimentin, and MMP-9 in EMT group were significantly increased (P<0. 05 or P<0. 01). Compared with EMT group, the expression levels of the above proteins in ZEB1 silencing group were markedly reduced (P< 0. 05 or P<0. 01). The cell migration rate in EMT group was obviously elevated compared with control group (P< 0. 01), and the cell migration rate of the glioma U87 cells in ZEB1 silence group was significantly lower than that in EMT group (P<0. 01). Conclusion: Silencing ZEB1 gene expression can inhibit the EMT in the glioma U87 cells and reduce the cell migration abilities, suggesting ZEB1 as an important therapeutic target of invasive glioma.

Downregulation of miR-4772-3p promotes enhanced regulatory T cell capacity in malignant pleural effusion by elevating Helios levels / 中华医学杂志(英文版)

Background@#Malignant pleural effusion (MPE) is a complicated condition of patients with advanced tumors. Further dissecting the microenvironment of infiltrated immune cells and malignant cells are warranted to understand the immune-evasion mechanisms of tumor development and progression.@*Methods@#The possible involvement of microRNAs (miRNAs) in malignant pleural fluid was investigated using small RNA sequencing. Regulatory T cell (Treg) markers (CD4, CD25, forkhead box P3), and Helios (also known as IKAROS Family Zinc Finger 2 [IKZF2]) were detected using flow cytometry. The expression levels of IKZF2 and miR-4772-3p were measured using quantitative real-time reverse transcription polymerase chain reaction. The interaction between miR-4772-3p and Helios was determined using dual-luciferase reporter assays. The effects of miR-4772-3p on Helios expression were evaluated using an in vitro system. Correlation assays between miR-4772-3p and functional molecules of Tregs were performed.@*Results@#Compared with non-malignant controls, patients with non-small cell lung cancer had an increased Tregs frequency with Helios expression in the MPE and peripheral blood mononuclear cells. The verified downregulation of miR-4772-3p was inversely related to the Helios+ Tregs frequency and Helios expression in the MPE. Overexpression of miR-4772-3p could inhibit Helios expression in in vitro experiments. However, ectopic expression of Helios in induced Tregs reversed the effects induced by miR-4772-3p overexpression. Additionally, miR-4772-3p could regulate Helios expression by directly targeting IKZF2 mRNA.@*Conclusion@#Downregulation of miR-4772-3p, by targeting Helios, contributes to enhanced Tregs activities in the MPE microenvironment.

Inhibition Effect of Weichang'an Granule and Decoction on Tumor Growth and Lymph Node Metastasis by RUFY3, SNAI1, VEGF, EMT Related Proteins in Nude Mice with Subcutaneous Xenograft Tumor of Human Gastric Carcinoma MKN45 / 中国实验方剂学杂志

Objective: To observe the effect of Weichang'an on the tumor growth and lymph node metastasis, and RUN and FYVE domain-containing protein 3(RUFY3),Zinc finger protein 1(SNAI1),vascular endothelial growth factor(VEGF),epithelial-mesenchymal transition(EMT)-related proteins in nude mice with subcutaneous xenograft tumor human gastric carcinoma MKN45, so as to discuss the mechanism of Weichang'an on MKN45 human gastric metastasis. Method: The nude mice model of human gastric carcinoma MKN45 cells was established and randomly divided into normal saline group (0.5 mL/mouse), Weichang'an granule group (3.54 g·kg-1) and Weichang'an decoction group (35.49 g·kg-1). The tumor weight and volume of axillary lymph nodes in each group were observed. The morphology of lymph nodes in each group was detected by hematoxylin-eosin (HE) staining. The expressions of related proteins were detected by immunohistochemistry, including RUFY3,SNAI1,VEGF,E-cadherin,N-cadherin,Vimentin. Result: Compared with the normal saline group, the tumor weight and volume of axillary lymph node were decreased (PPPConclusion: Both Weichang'an granule and Weichang'an decoction can inhibit the tumor growth and metastasis of axillary lymph nodes, obviously down-regulate the expressions of RUFY3,SNAI1,VEGF,N-cadherin,Vimentin and up-regulate the expression of E-cadherin in human gastric MKN45 subcutaneous transplanted tumor in nude mice. This suggested that Weichang'an may inhibit the tumor metastasis through regulation expressions of RUFY3,SNAI1,VEGF and EMT-related proteins.

Depletion of PRDM9 inhibited the osteogenic differentiation potential of periodontal ligament stem cells / 中华口腔医学杂志

Objective@#To investigate the effect of PR domain zinc finger protein 9 (PRDM9), one of the histone methylated transferases, on osteogenic differentiation ability of periodontal ligament mesenchymal stem cells (PDLSC).@*Methods@#PDLSC with PRDM9 gene knocked down by PRDM9 shRNA using recombinant lentiviral vector were allocated into the PRDM9sh group, and the transfected shRNA was as the control group. The gene expression efficiency was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Alkaline phosphatase activity (ALP), alizarin red staining, mineralization and osteocalcin, which belongs to osteogenic differentiation markers detected by RT-PCR and Western blotting to detect the osteogenic differentiation ability of stem cells from periodontal ligaments in vitro. In vivo, PRDM9sh and control group cells was transplanted into the dorsal dermal to explore the osteogenesis. The area percentage of new osteogenic tissue was calculated by image pro software and statistically analyzed.@*Results@#RT-PCR results showed that the relative expression of PRDM9 gene in PRDM9sh (0.460±0.017) was significantly lower than that in control group (1.000±0.107) (P<0.05). The results of ALP activity determined at 5 days postinduction in a significant decrease in PRDM9sh cells (0.762±0.063) compared with control group (1.225±0.058) (P<0.01). Alizarin red staining induced by osteogenesis at 2 weeks and 3 weeks showed that the staining of PRDM9sh was significantly lighter than that in control group. Quantitative calcium analysis results showed that the calcium ion concentration induced by osteogenesis at 2 weeks and 3 weeks [(0.071±0.004), (0.075±0.001)] in PRDM9sh was significantly lower than that in control group at 2 weeks and 3 weeks [(0.282±0.006), (0.485+0.004)] (P<0.01). RT-PCR results showed that the relative expression of osteocalcin mRNA in PRDM9sh (1.059±0.148) was significantly lower than that in control group at 2 weeks (2.542±0.190) (P<0.01). Western blotting results showed that osteocalcin expression in PRDM9sh was significantly lower than that in control group at 1 and 2 weeks after osteogenesis induction. Animal transplantation experiments results indicated that PRDM9 significantly inhibited the osteogenesis of PDLSC in vivo, and the proportion of osteogenic area calculated showed that the osteogenic capacity of PRDM9sh [(3.8±2.41)%] was significantly lower than that in control group [(24.54±7.06)%](P<0.05).@*Conclusions@#Depletion of PRDM9 repressed the osteogenic differentiation of stem cells from periodontal ligament in vitro and in vivo.

Effect of hepatitis B virus X gene integration on expression of zinc finger protein ZBTB20 in chronic hepatitis B patients complicated with hepatocellular carcinoma / 中华传染病杂志

Objective@#To investigate the effect of hepatitis B virus (HBV) X gene integration on expression of zinc finger protein ZBTB20 in chronic hepatitis B (CHB) patients complicated with hepatocellular carcinoma (HCC).@*Methods@#Eighteen CHB patients complicated with HCC who underwent surgical treatment in Taizhou Enze Medical Center Enze Hospital and Taizhou Central Hospital from July 2015 to June 2017 were enrolled. Samples of carcinoma tissue, para-carcinoma tissue and corresponding normal liver tissue were collected from each case. DNA was extracted from three kinds of tissue samples. HBV-Alu-polymerase chain reaction (PCR) was used to amplify the integrated HBVX fragments and their bilateral flanking sequences in human genomic DNA. The integrated HBV fragments were determined by PCR products sequencing. Protein was extracted from three kinds of tissue samples.The level of expression of ZBTB20 was detected by protein imprinting. Statistical analysis was performed using t test, analysis of variance, and χ2 test.@*Results@#Among the 18 CHB patients complicated with HCC, integration of HBVX gene was found in 13 carcinoma tissue samples, 16 para-carcinoma tissue samples and 9 corresponding normal liver tissue samples. The difference was statistically significant (χ2=6.353, P=0.037). Of the 18 patients, the protein expressions of ZBTB20 in carcinoma tissue, para-carcinoma tissue and corresponding normal tissue were (50.14±11.25)%, (40.71±7.17)% and (39.06±5.17)%, respectively, which was statistically different (F=9.420, P<0.01). HBVX gene integration was detected at ZBTB20 locus in five patients. The expression levels of ZBTB20 in patients with HBVX gene integration at this locus in carcinoma tissue, para-carcinoma tissue and corresponding, normal liver tissue were all significantly lower than those in patients without HBVX gene integration (carcinoma tissue (37.37±10.30)% vs (55.06±7.06)%, para-carcinoma tissue (32.06±2.61)% vs (44.04±5.24)%, corresponding normal tissue (34.66±5.59)% vs (40.76±4.04)%, t=4.205, 4.821 and 2.589, respectively, all P<0.05).@*Conclusions@#Incidence of HBVX integration in para-carcinoma tissue is higher than that in carcinoma tissue in CHB patients complicated with HCC.The expression level of ZBTB20 in carcinoma tissue is higher than that in para-carcinoma tissue. Integration of HBVX gene at ZBTB20 locus may decreases the expression of ZBTB20.