Hypermethylation and expression regulation of secreted frizzled-related protein genes in colorectal tumor

AIM： To investigate the functions of promoter hypermethylation of secreted frizzled-related proteins （sFRPs） genes in colorectal tumorigenesis and progression. METHODS： The promoter hypermethylation and expression of sFRP genes in 72 sporadic colorectal carcinomas, 33 adenomas, 18 aberrant crypt foci （ACF） and colorectal cancer cell lines RKO, HCT116 and SW480 were detected by methylation-specific PCR and reverse transcription PCR, respectively. RESULTS： None of the normal colorectal mucosa tissues showed methylated bands of any of four sFRP genes, sFRP1, 2, 4 and 5 were frequently methylated in colorectal carcinoma, adenoma and ACF （sFRP1 〉 85%, sFRP2 〉75%, sFRP5 〉 50%）, and the differences between three colorectal tissues were not significant （P 〉 0.05）. IVlethylation in colorectal tumors was more frequent than in normal mucosa and adjacent normal mucosa. The mRNA of sFRP1-5 genes was expressed in all normal colorectal mucosa samples. Expression of sFRP1, 2, 4 and 5 and sFRP1, 2 and 5 was downregulated in carcinoma and adenoma, respectively. The downregulation of sFRP2, 4 and 5 was more frequent in carcinoma than in adenoma. Expression of sFRP3 which promoter has no CpG island was downregulated in only a few of colorectal tumor samples （7/105）. The downregulation ofsFRP1, 2, 4 and 5 expression was significantly associated with promoter hypermethylation in colorectal tumor. After cells were treated by DAC/TSA combination, the silenced sFRP mRNA expression could be effectively re-expressed in colorectal cancer cell lines. CONCLUSION： Hypermethylation of sFRP genes is a common early event in the evolution of colorectal tumor, occurring frequently in ACF, which is regarded as the earliest lesion of multistage colorectal carcinogenesis. It appears to functionally silence sFRP genes expression. Methylation of sFRP1, 2 and 5 genes might serve as indicators for colorectal tumor.

Clonality and allelotype analyses of focal nodular hyperplasia compared with hepatocellular adenoma and carcinoma

AIM： To identify clonality and genetic alterations in focal nodular hyperplasia （FNH） and the nodules derived from it. METHODS： Twelve FNH lesions were examined. Twelve hepatocellular adenomas （HCAs） and 22 hepatocellular carcinomas （HCCs） were used as references. Nodules of different types were identified and isolated from FNH by microdissection. An X-chromosome inactivation assay was employed to describe their clonality status. Loss of heterozygosity （LOH） was detected, using 57 markers, for genetic alterations.RESULTS： Nodules of altered hepatocytes （NAH）, the putative precursors of HCA and HCC, were found in all the FNH lesions. Polyclonality was revealed in 10 FNH lesions from female patients, and LOH was not detected in any of the six FNH lesions examined, the results apparently showing their polyclonal nature. In contrast, monoclonality was demonstrated in all the eight HCAs and in four of the HCCs from females, and allelic imbalances were found in the HCAs （9/9） and HCCs （15/18）, with chromosomal arms 11p, 13q and 17p affected in the former, and 6q, 8p, 11p, 16q and 17p affected in the latter lesions in high frequencies （≥ 30%）. Monodonality was revealed in 21 （40%） of the 52 microdissected NAH, but was not found in any of the five ordinary nodules. LOH was found in all of the 13 NAH tested, being highly frequent at six loci on 8p, 11p, 13q and 17p. CONCLUSION： FNH, as a whole, is polyclonal, but some of the NAH lesions derived from it are already neoplastic and harbor similar allelic imbalances as HCAs.

Genomic structure analysis of SNC6, a progesterone-receptor associated protein gene, and cloning and characterization of its 5＇-flanking region

Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (accession number: Z98048) covering the whole SNC6 gene was used to analyze the genomic structure of SNC6 and design primers for PCR amplification of its 5'\|flanking region. A 1894 bp fragment of the 5'\|flanking region \{(-1814\} to +75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments (1423 bp, 632 bp and 416 bp, which correspond to -1344 to +75, -552 to +75 and -337 to +75 respectively), were subcloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luciferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All transfected SW620 cells with the above 5\|flanking region\|containing constructs showed luciferase activities. The highest luciferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luciferase activities were measured in transfected cells with vectors containing 416 bp fragments. Luciferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in transfected cells with vectors containing 1423 bp fragments. Conclusion: The basic transcription\|promoting element (promoter) for SNC6 expression resides between 0 to -337, and two transcription\|enhancing elements (enhancer) resides between -337 to -552 and -1344 to -1814, whereas one transcription\|inhibiting element (silencer) exists between -552 to -1344.

Analysis of the action mechanism of Fang Ji Huang Qi decoction in treating rheumatoid arthritis by network pharmacology

Objective:To explore the pharmacological action mechanism of Fang Ji Huang Qi decoction(FHD)in the treatment of rheumatoid arthritis(RA)by network pharmacology.Methods:The chemical compositions and functional targets of the TCM were retrieved using the systematic pharmacological analysis platform TCMSP,and the gene name of each target protein was obtained from the UniProtKB network platform.The targets of RA were queried through the CTD database.The protein–protein interaction network was constructed in the STRING database,and the network visualization analysis was performed in Cytoscape.The Gene Ontology and Kyoto Gene and Genomic Encyclopedia pathways enrichment analyses of key target proteins were performed using the DAVID data platform.Results:A total of 472 drug active ingredients were screened from the TCMSP database.Seventy-five disease targets from the CTD database were screened.The compound-target network map contained further screened out 98 components and corresponding 75 targets.The key compounds included quercetin and kaempferol.The key targets were prostaglandin G/H synthase 2 and nitric oxide synthase 2.The protein-protein interaction network consisted of 75 proteins,of which 37 were key proteins,including tumor protein 53,JUN and interleukin-6.There were 260 Gene Ontology entries,of which 246 were biological processes.Fifty-five Kyoto Gene and Genomic Encyclopedia pathways were enriched,mainly the cancer pathway,NOD-like receptor signaling pathway,and Toll-like receptor signaling pathway,which are involved in the action mechanism of FHD.Conclusion:The results of this study preliminarily verified the basic pharmacological action mechanism of FHD in the treatment of RA,laying a foundation for elucidating its mechanism of action.

The 5'-flanking cis-acting elements of the human ε-globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII, -446bp-419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.

KLFs与肿瘤发生发展的关系

Krüpple是果蝇分节基因"间隙基因"类的一个成员,它编码一个包含一个DNA结合序列的蛋白质,该序列由几个Cys2/His2型的锌指构成,一群与Krüpple的锌指结构高度同源的蛋白质被发现,命名为Krüpple样因子(Krüpple-like factor,KLFs),它们通过调控多种富含GC或CACCC的启动子的基因的表达,参与细胞增殖、分化、凋亡和肿瘤发生、发展等多种生理、病理过程。本文综述KLFs的结构、表达调控功能、主要家族成员及其在肿瘤研究中的现状,探讨其与肿瘤发生发展的关系。

Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin

Artemisinin, the key ingredient of first-line antimalarial drugs, has large demand every year. The native plant, which produces small quantities of artemisinin, remains as its main source and thus results in a short supply of artemisinin. Intensified efforts have been carried out to elevate artemisinin production. However, the routine metabolic engineering strategy, via overexpressing or down-regulating genes in artemisinin biosynthesis branch pathways, was not very effective as desired. Glandular secretory trichomes, sites of artemisinin biosynthesis on the surface of Artemisia annua L.(A. annua), are the new target for increasing artemisinin yield. In general, the population and morphology of glandular secretory trichomes in A. annua(Aa GSTs) are often positively correlated with artemisinin content. Improved understanding of Aa GSTs will shed light on the opportunities for increasing plant-derived artemisinin. This review article will refresh classification of trichomes in A. annua and provide an overview of the recent achievements regarding Aa GSTs and artemisinin. To have a full understanding of Aa GSTs,factors that are associated with trichome morphology and density will have to be further investigated, such as genes,micro RNAs and phytohormones. The purpose of thisreview was to(1) update the knowledge of the relation between Aa GSTs and artemisinin, and(2) propose new avenues to increase artemisinin yield by harnessing the potential biofactories, Aa GSTs.

Analysis of genotypes and phenotypes in Chinese children with tuberous sclerosis complex

Tuberous sclerosis complex(TSC) is a neurocutaneous syndrome with serious clinical presentations, an autosomal dominant genetic disorder involving multiple organs and systems. We retrospectively investigated the clinical manifestations and genotypes of 20 Chinese children with TSC to enable informed diagnostic and surveillance recommendations in China. A retrospective analysis of clinical manifestations in 20 children(7.00±5.30 years old) with TSC was conducted. A genetic testing of the genes TSC1 and TSC2 was performed in 14 children.The earliest manifestations of TSC were skin lesions(80% of patients) and seizures(75%). Fourteen of the children presented with retinal hamartomas, and 2 of these underwent eye enucleation at other hospitals through misdiagnosis. On magnetic resonance imaging, 18 children exhibited subependymal nodules, and 16 ones showed cortical nodules. 5 cases of non-renal hamartomas, 5 cases of multiple renal cysts, and 5 cases of cardiac rhabdomyomas were observed.The genotyping of TSC1 and TSC2 in 14 children revealed 11 with mutations in TSC2, 2 with mutations in TSC1, and no mutations of either gene in one patient. Eight of these observed mutations are reported here in for the first time. The illness presentations of the TSC2-mutated patients were more severe than that of patients carrying TSC1 mutations.There were differences in the mutations of TSC genes in Chinese children from those reported in other countries. The described clinical characteristics and genotyping will help pediatric neurologists to understand, diagnosis, and treat TSC.