Clinical significance of telomerase reverse transcriptase promoter mutation in radioactive iodine refractory papillary thyroid cancer / 中华核医学与分子影像杂志

Objective:To evaluate the influence of telomerase reverse transcriptase (TERT) promoter mutation on radioiodine uptake status of radioactive iodine refractory papillary thyroid cancer (RAIR-PTC) and radioiodine therapy response by analyzing the mutation frequency of TERT promoter in RAIR-PTC.Methods:A total of 37 patients with RAIR-PTC (15 males, 22 females, age (49.8±16.1) years) and 40 PTC patients with effective radioiodine therapy (13 males, 27 females, age (39.8±10.9) years) between January 2005 and June 2020 in JiangYuan Hospital Affiliated to Jiangsu Institute of Nuclear Medicine were retrospectively analyzed. TERT promoter mutation and B-Raf proto-oncogene, serine/threonine kinase (BRAF) V600E mutation of patients were observed. The differences across genotype patterns on radioiodine uptake status and therapy response were compared. The Fisher′s exact test and independent-sample t test were used for data analysis. Results:The incidence rate of TERT promoter mutation in the RAIR-PTC group was 40.54% (15/37, all C228T), which was significantly higher than that in the effective radioiodine therapy group (0, 0/40; P<0.001). No statistically significant difference was found for the mutation rate of BRAF V600E between the RAIR group (64.86%, 24/37) and the effective radioiodine therapy group (72.50%, 29/40; P=0.858). Patients with TERT promoter mutation were older ( t=3.76, P=0.001) and the non-intake rate of radioiodine in distant metastases of those patients was higher ( P=0.037). Furthermore, 2/3 of patients who received targeted therapies and 3/4 deaths had TERT promoter mutation. Among 35 patients with negative thyroglobulin antibody (TgAb), 11/14 of patients with TERT mutation had a rising stimulated thyroglobulin (sTg), while the percentage of the non-TERT mutation group was 57.1% (12/21; P=0.357). Conclusion:The TERT promoter mutation rate is significantly increased in RAIR-PTC patients and can serve as a prognostic predictor in RAIR.

Construction and transcriptional activity of human CREG promoter reporter gene plasmid / 解放军医学杂志

Objective To construct the promoter enhanced green fluorescent protein (pEGFP1) reporter gene vector of different truncated fragments of human cellular repressor of E1A-stimulated genes (hCREG), and compare the transcriptional activity of each promoter to determine the hCREG core promoter region. Methods The promoter fragment with length of 2003 (–1925/+78) bp was obtained by querying the hCREG sequence from US National Center of Biotechnology Information (NCBI) database and combining with the characteristics of the promoter. Five promoter fragments were truncated by PCR and double enzyme digestion and cloned into pEGFP1 to construct pEGFP1_hCREG_2003, pEGFP1_hCREG_945, pEGF P1_hCREG_586, pEGFP1_ hCREG_478 and pEG FP1_hCREG_358 reporter gene vector plasmid. The 293T cells were transiently co-transfected with the internal reference plasmid pGL4.73 [hRluc/SV40] for 48 hours. The green fluorescence expression of pEGFP1_hCREG promoter reporter gene was observed under fluorescence microscope, and the mRNA expression of each promoter was detected by real-time quantitative PCR, and the core promoter region was determined. Bioinformatics was used to predict the transcription factors that might bind to the core promoter region. Results Five hCREG promoter reporter gene vectors were successfully constructed by double enzyme digestion and gene sequencing. The results showed that the transcription activity of pEGFP1_ hCREG_586 was the highest (P0.05), implying that –867/ –509 bp is a negative regulatory region, and there existed enhancer sequences in –400/–281 bp and –508/–401 bp, so the core promoter region of hCREG gene is located in the upstream sequence of –508/–281 bp. Bioinformatics predicted that the possibly bound transcription factors in key promoter region –508/–281 bp were Pax5/P53, C/EBPβ, GR-β, GATA-1, GR-α, c-Jun, PRB/ PRA, YY1, RXR-α, AP-2, FOXP3, GR, TFIID, STAT4 and c-Ets-1. Conclusion The recombinant plasmid of hCREG gene promoter has been successfully constructed, the core promoter of which is located in –508/–281 bp, where several transcription factors might be bound.

Biological molecules found to serve as prostate cancer specific promoters during construction of oncolytic adenovirus / 肿瘤

Prostate cancer is a common malignant disease in elder men, and it is the second leading death cause in male cancer patients. The incidence rate of prostate cancer has been increasing year by year, and it is ranked the third in the incidence rate of urologic cancers in China. The oncolytic adenovirus carrying specific promoter has been used as a novel target of antitumor therapy. The adenovirus has specific effects on tumor cells and can express downstream therapeutic genes to inhibit the growth of tumor cells, but it can't influence the normal cells or only has weak influence. At present, oncolytic adenovirus carrying specific promoter which has been used widely in basic and clinical studies has showed unique advantages in great antitumor effect and medication safety. This paper reviews the several biological molecules, which have been found during the construction of oncolytic adenovirus, may potentially become to be prostate cancer-specific promoters.

Association of tumor necrosis factor-α gene promoter polymorphisms with generalized pustular psoriasis / 中华皮肤科杂志

Objective To investigate the association of tumor necrosis factor-ot (TNF-α) gene promoter polymorphisms with generalized pustular psoriasis.Methods Totally,91 patients of Han nationality with generalized pustular psoriasis (generalized pustular psoriasis group) and 102 health checkup examinees (healthy control group) were enrolled into this study.PCR and direct sequencing were performed to analyze the-238,-308 and-857 polymorphic sites of the TNF-α promoter.Results The frequency of the A allele at TNF-α-238 site was significantly higher in the generalized pustular psoriasis group than in the healthy control group (P =0.003,OR =4.819,95％ CI:1.581-14.694),so was the frequency of GA/AA genotype (P =0.006,OR =4.455,95％ CI:1.410-14.077).However,no significant differences were observed in the frequencies of G/A alleles (P =0.794) and GG/GA/AA genotypes (P =0.786) at TNF-o-308 site,or in the frequencies of C/T alleles (P =0.474) and CC/CT/TT genotypes (P =0.453) at TNF-α-857 site,between the generalized pustular psoriasis group and healthy control group.Conclusion TNF-α-238G ＞ A polymorphisms may be associated with the occur-rence of generalized pustular psoriasis.

Constructing the dual luciferase reporter vector containing human DRD1 promoter region / 中国组织工程研究

BACKGROUND:The polymorphisms of dopamine receptor in promoter region wil affect the expression of the receptor, thereby affecting the dopaminergic neurotransmitter, final y lead to related diseases. OBJECTIVE:To construct the dual luciferase reporter vector containing human DRD1 promoter region and determine its activity, which could provide the basic tool for studying the transcriptional regulation of DRD1 gene. METHODS:DRD1 promoter sequence was amplified by PCR using the human blood genomic DNA and cloned into pGM-T vector. After sequencing, the correctly constructed vectors were ligated to the firefly luciferase reporter plasmid pGL3-Basic. The cloned pGL3-Basic vectors were transfected into HEK293 using cationic liposome method. In the meanwhile, PGL3-Basic vector with no promoter was co-transfected with pGL3-TK plasmid as negative control group. The relative fluorescence intensity was measured by chemiluminescence. RESULTS AND CONCLUSION:(1) Recombinant luciferase reporter gene vectors were confirmed by restriction analysis and sequencing. (2) Compared with the negative control group, the HEK293 cel s transfected by recombinant vectors presented transcriptional activity. (3) In conclusion, luciferase reporter gene vectors containing DRD1 promoter region are successful y constructed and can provide the basic tool for further study on the transcriptional regulation of DRD1.

Location of DNase Ⅰ hypersensive sites exactly in the promoter region of CD133 in colonel cancer cell line SW480 cells by inverse-PCR / 肿瘤研究与临床

Objective To precious localize DNase Ⅰ hypersensive sites exactly in the promoter region of CD133 of cell line SW480 by inverse-PCR.Methods The colonel cancer cell SW480 nuclei were suspended in digested buffer,treated with DNase Ⅰ at the concentration of 10 U/ml for 10 min.The inversePCR was performed as follows.DNA treated by DNase Ⅰ was purified,fragmented with restricted enzyme EcoRI and Xmal Ⅰ.Then the ends were blunted,ligated by T4 ligase.PCR was performed,and production was sequenced.The restricted enzymes cut sites were near DNase Ⅰ cleavage sites.Results 9 DNase Ⅰ cut sites were identified in CD133 promoter region.The DNaseI hypersensitive sites all distributed in a region -300 bp--700 bp up to transcription start site.Conclusion The DNase Ⅰ cleavage sites could identified preciously by application of inverse-PCR.These sites locate in a region of-300 bp--700 bp up to transcription start site.

Methylation status of IGF -Ⅱgene P4 promoter in hepatocellular carcinoma / 中国基层医药

Objective To investigate the alternation of insulin -like growth factor -Ⅱ( IGF-Ⅱ) gene promoter P4 methylation status in hepatocellular carcinoma ( HCC) and explore its relationship with expression of P4 mRNA levels.Methods Liver specimens of 43 patients with HCC and normal liver specimens of 9 control patients were collected in operation .Tissue DNA and total RNA were extracted from these specimens .IGF-Ⅱ P4 methylation status and P4 mRNA expression levels were detected .Results (1)The incidence of IGF -ⅡP4 methyl-ation in HCC group was significantly lower than that in normal liver specimens (16.28%vs 88.89%,χ2 =19.12,P<0.01).(2)The expression level of IGF -ⅡP4 mRNA in HCC group was significantly higher than that that in normal liver specimens[(0.96 ±0.74) vs (0.25 ±0.19),t=5.48,P<0.01].(3)In HCC group,the IGF-Ⅱ P4 mRNA expression level with hypomethylation gene was significantly higher than that without hypomethylation gene [(1.18 ± 0.76) vs (0.32 ±0.27),t=5.28,P<0.01].Conclusion The hypomethylation alternation of IGF -Ⅱ P4 gene promoter which is accomplished by up -regulate P4 mRNA expression has a close relationship with HCC .

Construction and ldentification of Cell Division Cycle 2 Promoter Reporter Gene Vector / 天津医药

Objective To construct the luciferase reporter gene vector of cell division cycle 2 (Cdc2) gene promoter and determine its transcriptional activity. Methods Primers were designed based on human Cdc2 promoter sequence from UCSC software. Then Cdc2 promoter from human genome DNA was replicated. After pGL3-Basic vector and Cdc2 promoter were digested with restriction enzymes SacⅠand XhoⅠseparately, Cdc2 promoter was inserted into pGL3-Basic vector. The recombinant plasmid named pGL3-Cdc2-promoter was transiently co-transfected into U2OS cells with control vector pRL-SV40, and then the activity of dual luciferase was detected. Results pGL3-Cdc2-promoter was constructed successfully. The restriction analysis and sequencing proved the entirely correct sequencing results. The luciferase activity was higher in pGL3-Cdc2-promoter/pRL-SV40 group than that of pGL3-Basic/pRL-SV40 group (1.591 5±0.199 8 vs 0.049 9±0.010 4). Conclusion pGL3-Cdc2-promoter can be transcribed and activated in U2OS cells. This study provided an important basis for screening and evaluation of anticancer drugs.

Current Challenges in Bacterial Transcriptomics

Over the past decade or so, dramatic developments in our ability to experimentally determine the content and function of genomes have taken place. In particular, next-generation sequencing technologies are now inspiring a new understanding of bacterial transcriptomes on a global scale. In bacterial cells, whole-transcriptome studies have not received attention, owing to the general view that bacterial genomes are simple. However, several recent RNA sequencing results are revealing unexpected levels of complexity in bacterial transcriptomes, indicating that the transcribed regions of genomes are much larger and complex than previously anticipated. In particular, these data show a wide array of small RNAs, antisense RNAs, and alternative transcripts. Here, we review how current transcriptomics are now revolutionizing our understanding of the complexity and regulation of bacterial transcriptomes.

Treatment of human pancreatic cancer with adenovirus-mediated fusion gene system driven by KDR promoter in nude mice / 中华肝胆外科杂志

Objective To investigate the curative effect of the adenovirus-mediated fusion gene system driven by KDR promoter (AdKDR-CDglyTK) on a model of pancreatic cancer. Methods By using transplantation of the cultivated cells, human pancreatic cell line Capan-2 was injected subcutaneously on the back of nude mice to establish the animal model of the pancreatic cancer. Twenty nude mice were divided randomly and equally into four groups. The mice in group Ⅰ were injected with AdKDR-CDglyTK and 5-FC/GCV, those in group Ⅱ were injected with 5-FC/GCV, those in group Ⅲwere injected with AdKDR-CDglyTK and those in group Ⅳ received no any injection. AdKDR-CDglyTK was injected directly into the tumor and 5-FC/GCV was given by intraperitoneal injection. The observing parameters included common status, tumor bulk, tumor weight, inhibition rate of tumor growth, pathology, immunohistochemistry and treatment effect in each group. Electron microscopy was performed to observe the pathological changes of cells. The apoptotic cells in tumor were detected using the TUNEL assay. The expression of CDglyTK in tumors from each group was examined by RT-PCR. Results Tumor growth was dramatically inhibited in group Ⅰ. Tumor growth has no significant difference among groupⅡ , group Ⅲ and group Ⅳ. The apoptotic rate (34.20±4.60)% was significantly increased in group Ⅰ (F= 243. 22, P= 0. 00) and it had no significant difference among groupⅡ , group Ⅲ and group Ⅳ (P＞0.05). Conclusion AdKDR-CDglyTK with 5-FC/GCV can obviously inhibit the growth of human KDR-expressing pancreatic cell line Capan-2 and induce the cell apoptosis in vivo. The probable molecular mechanism lies in the facts that the system can cause a decline in the level of Bcl-2.

Tumor-specific promoters and their application in cancer gene targeted therapy / 国际肿瘤学杂志

Tumor-specific promoters can induce high-efficiency and specific expression of exogenous genes in tumor cells. At present, commonly used promoters include alpha-fetoprotein promoter, carcinoembryonic antigen promoter,prostate specific antigen promoter,human telomerase reverse transcriptase promoter and multidrug resistance gene promoter and etc. Dual promoters, enhancer, regulatory element and other physical or chem-ical factors could be used to reconstruct or modify promoters to increase the expression and location of exogenous genes in tumor cells. Tumor-specific promoters play an important role in cancer gene targeted therapy by cou-pling with suicide gene, anti- oncogene, antiseuse nucleic acid, apoptosis gene and RNA interference.

The cardioprotective effect of the inhibitor of JAK/STAT on simulating ischemia-reperfusion injury in cultured immature rat cardiacmyocytes / 中国医师杂志

Objective To investigate the cardioprotective effects of the inhibitor of JAK/STAT pathway on ischemia-reperfusion injury in cultured immature rat cardiacmyocytes.Methods The cultured immature rat cardiacmyocytes were randomly divided into normal control group,ischemia-reperfusion group,AG490 treatment group and RMP treatment group.The cell viability was measured by MTT method.The contents of lactate dehydrogenase(LDH) and malondialdehyde(MDA),the superoxide dismutase(SOD) activity and the rate of cell apoptosis were detected.Results Compared with the normal group,the cell viability decreased significantly,the contents of LDH and MDA in the supernatant of the cells increased significantly,the SOD activity decreased and the rate of apoptosis of cells increased significantly in ischemia-reperfusion group(all P