Correlation of microRNAs responding to high dose γ-irradiation with predicted target mRNAs in HeLa cells using microarray analyses

An increasing data indicates that altered microRNAs(miRNAs)participate in the radiation-induced DNA damage response.However,a correlation of mRNA and miRNA profiles across the entire genome and in response to irradiation has not been thoroughly assessed.We analyzed miRNA microarray data collected from HeLa cells after ionizing radiation(IR),quantified the expression profiles of mRNAs and performed comparative analysis of the data sets using target prediction algorithms,Gene Ontology(GO)analysis,pathway analysis,and gene network construction.The results showed that the altered miRNAs were involved in regulation of various cellular functions.miRNA-gene network analyses revealed that miR-186,miR-106b,miR-15a/b,CCND1and CDK6 played vital role in the cellular radiation response.Using qRT-PCR,we confirmed that twenty-two miRNAs showed differential expression in HeLa cells treated with IR and some of these miRNAs affected cell cycle progression.This study demonstrated that miRNAs influence gene expression in the entire genome during the cellular radiation response and suggested vital pathways for further research.     

Role of Ppt1 in multiple stress responses in Candida albicans

To study the function of CaPptl, we deleted PPT1 gene from the Candida albicans genome by sequentially replacing the entire coding region with the selectable markers ARG4 and HIS1. The results showed that the deletion of Pptl did not affect the hyphal formation of C. albicans under serum induction and caused enhanced sensitivity to DNA damage, Calcofluor white and salt- induced stress. We also found that Pptl was not required for the phenotypic response of cells treated with the genotoxins, methylmethane sulfonate and hydroxyurea. Flow cytometric analyses indicated that pptlA cells and wild-type cells showed similar G2/M arrest profiles when exposed to DNA damage stress. Pptl was not required for the activation of the DNA damage response pathway, as indicated by normal phosphorylation of Rad53 and Rfa2 in pptlA cells under DNA damage stress. We suggest that Pptl plays important roles in response to various stress conditions in C. albicans.     

Dynamic analysis of DNA damage induced miRNAs in colon cancer cells

It is known that microRNAs （miRNAs） expression profile shows substantial changes in cells under DNA damage. Here, we did miRNA microarray and quantitative real-time PCR to comprehensively identify the differentially expressed miRNAs in colon cancer cell lines HCT116 p53＋/＋ and HCT116 p53-/-. Cluster analysis revealed a panel of differentially expressed miRNAs which are regulated by p53 and/or UV-C induced DNA damage. These altered miRNAs tend to be located in chromosomes 13, X and 17. Moreover, pathways enrichment analysis estimated that MAPK pathway, focal adheren pathway, p53 pathway and Wnt pathway were mediated by these miRNAs to exert their functions in DNA damage response. Additionally, we found that miR- 320a, one of the UV-C induced miRNAs, play a role in protecting cells from DNA damage. Taken together, our results show that miRNAs are dynamic regulated in p53- dependent or -independent manners in different cell contexts and different situations following DNA damage.     

A novel element (NIRS) participates in the regulation ofinterleukin 2 receptorαgene expression

A novel element at -153/- 143 bp in the interleukin 2 receptor α(IL-2Rα) gene has been coined as NRE-inverse repeat sequence (NIRS) due to its inversely repeated to the known negative regulatory element (NRE) further upstream of the gene. In order to explore the role of NIRS in the expression of IL-2Rαgene,luciferase reporter plasmids driven by 4 individually deleted IL-2Rα genes promoter regions were constructed. Transfection of the reporter plasmids into Jurkat cells and HeLa cells respectively, we found that both NIRS and NRE were critical for repressing the constitutive expression of IL-2Rα gene and were also necessary for promoter activity induced by PHA. EMSA results showed that double-stranded NRE- and NIRS-binding proteins existed in both HeLa cells and Jurkat cells. However, single-stranded NIRS- and NRE-binding protein was only found in HeLa cells. Interestingly, the supershift band showed up in EMSA system with Jurkat cells (no matter whether activated or not) adding to the cell lysate of HeLa cells. UV-crosslinking showed a double stranded NRE- and NIRS-binding protein p83 in both Jurkat cells and HeLa cells. Our results suggest that trans-acting factors play a key role in regulating promoter activity of IL-2Rα gene by interacting with double or single stranded NRE and/or NIRS selectively in different cells.     

Blast fungus-induction and developmental and tissue-specific expression of a rice P450<Emphasis Type="Italic">CYP72A</Emphasis> gene cluster

Cytochrome P450 gene superfamily is widely involved in diverse processes of plant development and environmental responses including defense response to pathogens.We previously isolated a rice cDNA fragment in a DD-PCR screening for blast fungus-induced genes. In the current study, we isolated a CYP72A gene cluster consisting of 7 P450 CYP72A genes (CYP72A17-23) with the conserved cDNA sequence through the public rice genome data. There are total 14 putative CYP72A members in the rice genome, with high diversity at N-terminal sequences while high homology at C-terminal sequences of those 14 putative proteins. We analyzed expression profiles of the cloned 7 CYP72A genes during pathogen infection and development. The results showed that expression of CYP72A18, 19, 22 and 23 was differentially regulated in the incompatible and compatible interactions between rice and blast fungus. Except CYP72A20, a pseudogene, other 6 CYP72A genes also exhibited temporal and spatial expression patterns, respectively.These findings provide fundamental data for rice P450 gene function analysis.     

Clinical and pathological features of miR-10b and RHOC gene expression in hepatocellular carcinoma

Aberrant expression of microRNAs （miRNAs） was reported frequently in different human cancers. The major role of miRNA is targeting 31-UTR of coding gene and causing translational repression or mRNA degradation. miR-10b overexpression was reported to promote breast cancer metastasis by up-regulating RHOC expression. But its expression in hepatocellular carcinoma （HCC） remains unclear. Our study indicated that the expression of miR-10b was different in HCC and adjacent tissue samples, and reduced expression of miR-10b in HCC was related tovein invasion. High-level expression of RHOC was also related to vein invasion in HCC. But no correlation was found between miR-10b and RHOC expression. These results suggest that miR-10b and RHOC are independent predictors of HCC invasion and metastasis.     

Review： Deregulation of microRNA expression in thyroid tumors

MicroRNAs （miRNAs or miRs） are endogenous non-coding RNAs that negatively regulate gene expres- sion by binding to the 3＇ non-coding regions of target mRNAs, resulting in their cleavage or blocking their translation. miRNAs may have an impact on cell differentiation, proliferation, and survival, and their deregulation can be inclined to diseases and cancers, including thyroid tumors. The purpose of this review is to summarize the existing findings of deregulated miRNAs in different types of thyroid tumors and to exhibit their potential target genes, especially to demonstrate those involved in tumor invasion and metastasis. In addition, new findings of circulating miRNA expres- sion profiles, single nucleotide polymorphism （SNP） in thyroid tumors, and the correlation of somatic mutations with deregulated miRNA expression in thyroid tumors were all included in this review.     

MiR-205 inhibits the invasion and migration of esophageal squamous cell carcinoma by modulating SMAD1 expression

Esophageal squamous cell carcinoma （ESCC） is one of the most lethal cancers worldwide. In this study, we aimed to investigate the underlying mechanisms of metastasis inhibition by miR-205 in ESCC. In microRNA （miRNA） array and quantitative RT-PCR analyses, we found that the expression level of miR-205 was significantly lower in patients with lymph node metastasis compared with that in patients without lymph node metastasis. After transfection of miR-205 mimics or inhibitors into ESCC cell lines, a significant negative correlation was observed between the expression level of miR-205 and Smad 1. In luciferase reporter assays, we revealed that miR- 205 inhibited the expression of SMAD1 by targeting the 3＇ untranslated region （3＇-UTR） of SMAD1 mRNA in ESCC cells. Furthermore, our results showed that miR-205 sup- pressed the invasion and migration of ESCC cells, whereas Smadl increased their invasion and migration. Taken together, our study demonstrates that miR-205 functions as a suppressor of tumor metastasis by regulating SMAD1 expression through targeting the 3＇-UTR of SMAD1 mRNAin ESCC. Therefore, miR-205 may be a potential therapeutic target for miRNA-based therapy of ESCC.     

Analysis of genes differentially expressed during initial cellular dedifferentiation in cotton

The early phase of phytohormone induction is a vital stage of somatic embryogenesis. This phase includes a key process for acquiring cellular totipotency through cellular dedifferentiation. To unravel the molecular mechanism of cellular dedifferentiation in cotton, we constructed a cDNA library using the suppression subtractive hybridization method. A total of 286 differential cDNA clones were sequenced and identified. Among these clones, 112 unique ESTs were significantly up-regulated during the early phase of phytohormone induction, and 40.2% of the ESTs were first identified. GST was highly ex- pressed from 6 to 24 h after induction with phytohormone treatment. PRPs were predominantly ex- pressed and exhibited distinct expression patterns in different treatments, suggesting that they are closely related to cellular dedifferentiation in cotton. Putative GhSAMS, GhSAMDC, GhSAHH and GhAC03 involvement in SAM metabolism was identified in this library. The analysis of qRT-PCR showed that two remarkable increased expressions of the four SAM-related genes happened during the early phase of phytohormone induction, and that a highly positive correlation existed between GhSAMS and GhSAHHo The highest expression level of GhSAMS might be associated with its reentry into the cell cycle. The histological observations further showed that some cells accomplished cellular dedifferentiation and division within 72 h in 2,4-D treatment, and that cellular dedifferentiation might be regulated through two alterations in SAM-dependent transmethylation activity in cotton. In addition, the expression patterns of differential genes in different treatments disclosed the complicated interaction between 2, 4-D and kinetin.     

New feature extraction in gene expression data for tumor classification

Using gene expression data to discriminate tumor from the normal ones is a powerful method. However, it is sometimes difficult because the gene expression data are in high dimension and the object number of the data sets is very small. The key technique is to find a new gene expression profiling that can provide understanding and insight into tumor related cellular processes. In this paper, we propose a new feature extraction method based on variance to the center of the class and employ the support vector machine to recognize the gene data either normal or tumor. Two tumor data sets are used to demonstrate the effectiveness of our methods. The results show that the performance has been significantly improved.     

Interaction of hepatitis B virus with tumor suppressor gene p53: its significance and biological function

The mechanism of the interaction of hepatitis B virus (HBV) with tumor suppressor p53 and its role in the hepatocar-cinogenesis have been studied by PCR-directed sequencing, gel shift assays and in situ ultraviolet cross-linking assay. The biological function of the interaction of HBV with p53 gene was investigated by co-transfection of chloramphenicol acetyltransferase ( CAT) reporter gene. p53 and HBV DNA. and quantitative PCR. Among the 16 primary hepatocellular carcinoma (PHC) samples. 13 were HBV-DNA positive. 10 HBxAg positive and 9 p53 protein positive. The p53 gene point mutation was found in 5 samples, one of which had a G to T substitution located at codon 249. After analyzing the HBV genome by a computer program, a p53 response element binding sequence was found in HBV genome at upstream of enhancer I. from 1047 to 1059 nucleotides. This sequence could specifically bind to p53 protein, increase p53 protein accumulation in the PHC cells and stimulate the transactivating activity of p53 and HBV replication . The results also revealed that HBxAg could combine with p53 protein to form a complex in the cells and enhance CAT expression. Immunocytochemical staining showed that p53 protein complex was located in the cytoplasm and the process of p53 entry to nuclei was. in part, blocked. From our results, we conclude that the mutation of p53 gene at codon 249 is infrequent in HBV-associated PHC. the DNA-protein binding between HBV and p53. and the protein-protein binding between HBxAg and p53 might lead to the reduction or inactivation of p53 protein, which in turn resulting in HBV-associated hepatocarcinogenesis.     

Non-viral gene carrier mediated short hairpin RNA interference for inhibition of tumor cells growth

A tumor-targeting gene vector G250mAb-PEI-PEG has been prepared by modification of polyethylenimine （PEI） with polyethyleneglycol （PEG） and G250, a monoclonal antibody against the G250 antigen on tumor cell surface. The transfection efficiency was as high as 70% in G250 positive HeLa cells, whereas the transfection efficiency was relatively low （30%） in normal NIH3T3 cells. A plasmid encoding the short hairpin RNA （shRNA） specific for nucleostemin gene （NS） was efficiently transfected into the HeLa cells with this nonviral gene vector. RNA interference down-regulated the expression of NS gene in HeLa cells, inhibited cells proliferation and induced apoptosis. However, the growth and activity of the NIH3T3 cells were not affected under the same treatment. These results indicate that the reported nonviral gene vector, G250mAb-PEI-PEG, can target and efficiently deliver genes into HeLa cells, and has the potential for the cervical cancer treatment.     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize （Zea mays L.）

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutinl gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors （pUUOAH）. The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of CrylAh protein in the construct containing the ubil intron （pUUOAH） was 20% higher than that of the intronless construct （pUOAH）. Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

Rank sum method for related gene selection and its application to tumor diagnosis

Tumor diagnosis by analyzing gene expression profiles becomes an interesting topic in bioinformatics and the main problem is to identify the genes related to a tumor. This paper proposes a rank sum method to identify the related genes based on the rank sum test theory in statistics. The tumor diagnosis system is constructed by the support vector machine (SVM) trained on the set of the related gene expression profiles. The experiments demonstrate that the constructed tumor diagnosis system with the rank sum method and SVM can reach an accuracy level of 96.2% on the colon data and 100% on the leukemia data.     

Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.     

Discoveries and functions of virus-encoded MicroRNAs

Virus-encoded microRNAs （miRNAs） are a new kind of miRNAs that regulate the expression of target gene in host cells or viruses through inducing cleavage of mRNA, repressing translation, etc., and change the processes of host cells or replicate viruses to escape or resist immune surveillance of host and protect viruses themselves. It has become a hot topic to discover viral genes encoding miRNAs and their target genes, and to identify their functions. This review provides background information on the history of virally encoded miRNAs including their genomic distribution, functions and mechanisms. In addition, we discuss the similarities and differences between virus- and host-encoded miRNAs, the future directions of researches in viral miRNAs and their applications in diseases control and therapy.     

Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles ste- phensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I di- gestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively con- served in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.