Research Progress of Pseudogenes in Hepatocellular Carcinoma / 肿瘤防治研究

Pseudogenes were initially thought to have no function and were called by aliases, such as "junk genes." With the emergence of large-scale genomics projects and more and more experimental studies, pseudogenes have been shown to play an important role in the occurrence and development of solid tumors, especially playing an important regulatory role in the occurrence and develepment of liver cancer, such as regulating the proliferation, apoptosis, invasion, metastasis, and immunity of liver cancer cells. Recent studies showed that pseudogenes can act as regulators of oncogenes and tumor suppressors in hepatocellular carcinoma (HCC) and can thus serve as prognostic markers and even therapeutic targets for this cancer type. In this review, we systematically summarize the mechanisms and functions of different pseudogenes in HCC and present their future prospects as therapeutic targets.

Long non-coding RNA DUXAP9 promotes the proliferation and metastasis of head and neck squamous cell carcinoma / 口腔疾病防治

Objective@#To investigate the role of long non-coding RNA double homeobox A pseudogene 9 (DUXAP9) in head and neck squamous cell carcinoma (HNSCC) and to evaluate the expression level, molecular function and mechanism of DUXAP9 in HNSCC cells.@*Methods@#Differential expression of lncRNAs between normal and tumor tissues in HNSCC tissues were screened using lncRNA microarray, the expression level of DUXAP9 in HNSCC tissues and its relationship with prognosis were analyzed in the TCGA database. The expression levels of DUXAP9 in HNSCC tissues and cell lines were detected using qRT-PCR. The function in HNSCC cells after DUXAP9 silencing was evaluated using the CCK-8 assay, wound healing assay, Transwell migration assay and subcutaneous xenograft assay in nude mice. Changes in the transcription and translation of epithelial-mesenchymal transition (EMT)-related proteins in head and neck squamous cell carcinoma cells after DUXAP9 silencing were detected using qRT-PCR and Western blot.@*Results@#lncRNA microarray results showed that, compared to adjacent normal tissues, DUXAP9 was abnormally upregulated in HNSCC tissues. Analysis from TCGA database showed that, compared to HNSCC patients with low DUXAP9 expression, HNSCC patients with high DUXAP9 expression had poorer survival. The relative expression of DUXAP9 in HNSCC tissues and 4 HNSCC cell lines increased compared to paired adjacent normal tissues as detected using qRT-PCR. Silencing DUXAP9 significantly inhibited the proliferation, migration and expression of EMT-related genes in HNSCC cells. The silencing of DUXAP9 significantly inhibited subcutaneous tumorigenesis of the HNSCC cell line CAL27 in nude mice.@* Conclusion@#Silencing DUXAP9 significantly inhibited the proliferation of HNSCC cells and subcutaneous xenografts in nude mice. DUXAP9 may mediate the migration of head and neck squamous cell carcinoma cells via the EMT pathway.

Research progress of pseudogene gene PTENP1 in human tumors / 医学研究生学报

Phosphatase and tension homolog pseudogene 1 (PTENP1) is a pseudogene of phosphatase and tension homology deletion (PTEN), a well-known carcinoma suppressor gene.It belongs to processed pseudogenes which is unable to encode functional proteins. Its transcription product is a member of long noncoding RNA(lncRNA) family. PTENP1 primarily modulates the expression of PTEN at the levels of epigenetics and post-transcription, and then it plays a significant role in inhibiting oncogenesis via the signaling pathways of PI3/AKT, MAPK etc. The present paper is intended to summarized the existing researches of PTENP1 in human tumors and provide some help for future studies.

Identification and analysis of a processed cytochrome P450 pseudogene of the disease vector Aedes aegypti

OBJECTIVE@#To clone cytochrome P450 from Aedes aegypti (Ae. aegypti) and determine the characteristics using bioinformatics tools.@*METHODS@#Cytochrome P450 of Ae. aegypti was amplified using polymerase chain reaction, cloned and sequenced. Evolutionary relationship of the sequence was inferred and bioinformatics tools were used to predict subcellular localisation, signal peptide, transmembrane helix, phosphorylation, O-glycosylation, secondary and tertiary structures of the deduced protein.@*RESULTS@#Polymerase chain reaction rather amplified a cytochrome P450 pseudogene which was named CYP4H44P (GenBank accession number KF779932). The pseudogene has 1537 nucleotides and an open reading frame of 335 amino acids containing cytochrome P450 motifs except the WxxxR motif. It is highly homologous to CYP4H28 and CYP4H28v2. Phylogenetic analysis and evolutionary divergence showed strong clustering with CYP4H28 alleles and least divergence from the alleles respectively. The deduced protein was predicted to be found in the cytoplasm and likely to be phosphorylated but devoid of signal peptide, transmembrane helix and O-glycosylated sites. The secondary and tertiary structures were also generated.@*CONCLUSIONS@#A cytochrome P450 pseudogene, CYP4H44P was cloned from Ae. aegypti. The pseudogene is homologous with CYP4H28 alleles and seems to have recently diverged from this group. Isolating this pseudogene is an important step for evaluating its biological role in the mosquito and for the evolutionary analysis of Ae. aegypti CYPs.

Identification and analysis of a processed cytochrome P450 pseudogene of the disease vector Aedes aegypti

Objective To clone cytochrome P450 from Aedes aegypti (Ae. aegypti) and determine the characteristics using bioinformatics tools. Methods Cytochrome P450 of Ae. aegypti was amplified using polymerase chain reaction, cloned and sequenced. Evolutionary relationship of the sequence was inferred and bioinformatics tools were used to predict subcellular localisation, signal peptide, transmembrane helix, phosphorylation, O-glycosylation, secondary and tertiary structures of the deduced protein. Results Polymerase chain reaction rather amplified a cytochrome P450 pseudogene which was named CYP4H44P (GenBank accession number KF779932). The pseudogene has 1 537 nucleotides and an open reading frame of 335 amino acids containing cytochrome P450 motifs except the WxxxR motif. It is highly homologous to CYP4H28 and CYP4H28v2. Phylogenetic analysis and evolutionary divergence showed strong clustering with CYP4H28 alleles and least divergence from the alleles respectively. The deduced protein was predicted to be found in the cytoplasm and likely to be phosphorylated but devoid of signal peptide, transmembrane helix and O-glycosylated sites. The secondary and tertiary structures were also generated. Conclusions A cytochrome P450 pseudogene, CYP4H44P was cloned from Ae. aegypti. The pseudogene is homologous with CYP4H28 alleles and seems to have recently diverged from this group. Isolating this pseudogene is an important step for evaluating its biological role in the mosquito and for the evolutionary analysis of Ae. aegypti CYPs.

A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease

A pseudogene, designated as "ps(5.8S+ITS-2)", paralogous to the 5.8S gene and internal transcribed spacer (ITS)-2 of the nuclear ribosomal DNA (rDNA), has been recently found in many triatomine species distributed throughout North America, Central America and northern South America. Among characteristics used as criteria for pseudogene verification, secondary structures and free energy are highlighted, showing a lower fit between minimum free energy, partition function and centroid structures, although in given cases the fit only appeared to be slightly lower. The unique characteristics of "ps(5.8S+ITS-2)" as a processed or retrotransposed pseudogenic unit of the ghost type are reviewed, with emphasis on its potential functionality compared to the functionality of genes and spacers of the normal rDNA operon. Besides the technical problem of the risk for erroneous sequence results, the usefulness of "ps(5.8S+ITS-2)" for specimen classification, phylogenetic analyses and systematic/taxonomic studies should be highlighted, based on consistence and retention index values, which in pseudogenic sequence trees were higher than in functional sequence trees. Additionally, intraindividual, interpopulational and interspecific differences in pseudogene amount and the fact that it is a pseudogene in the nuclear rDNA suggests a potential relationships with fitness, behaviour and adaptability of triatomine vectors and consequently its potential utility in Chagas disease epidemiology and control.

CYP21A2 Mutation Analysis in Korean Patients With Congenital Adrenal Hyperplasia Using Complementary Methods: Sequencing After Long-Range PCR and Restriction Fragment Length Polymorphism Analysis With Multiple Ligation-Dependent Probe Amplification Assay

CYP21A2 mutation analysis of congenital adrenal hyperplasia (CAH) is challenging because of the genomic presence of a homologous CYP21A2 pseudogene and the significant incidence of pseudogene conversion and large deletions. The objective of this study was to accurately analyze the CYP21A2 genotype in Korean CAH patients using a combination of complementary methods. Long-range PCR and restriction fragment length polymorphism analyses were performed to confirm valid amplification of CYP21A2 and to detect large gene conversions and deletions before direct sequencing. Multiple ligation-dependent probe amplification (MLPA) analysis was conducted concurrently in 14 CAH-suspected patients and six family members of three patients. We identified 27 CYP21A2 mutant alleles in 14 CAH-suspected patients. The c.293-13A>G (or c.293-13C>G) was the most common mutation, and p.Ile173Asn was the second, identified in 25% and 17.9% of alleles, respectively. A novel frame-shift mutation of c.492delA (p.Glu 164Aspfs*24) was detected. Large deletions were detected by MLPA in 10.7% of the alleles. Mutation studies of the six familial members for three of the patients aided in the identification of haplotypes. In summary, we successfully identified CYP21A2 mutations using both long-range PCR and sequencing and dosage analyses. Our data correspond relatively well with the previously reported mutation spectrum analysis.

Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil

A cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC). A two-step approach was used to distinguish M. bovis from other members of MTC: (i) oxyR pseudogene amplification to detect MTC and, subsequently, (ii) allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5 percent) SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB) results and the source laboratory were associated (p < 0.05) with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.

Comparative analysis of human masculinity

To study rapidly evolving male specific Y (MSY) genes we retrieved and analyzed nine such genes. VCY, HSFY and RBMY were found to have functional X gametologs, but the rest did not. Using chimpanzee orthologs for XKRY, CDY, HSFY, PRY, and TSPY, the average silent substitution is estimated as 0.017 +/- 0.006/site and the substitution rate is 1.42 x 10(-9)/site/year. Except for VCY, all other loci possess two or more pseudogenes on the Y chromosome. Sequence differences from functional genes show that BPY2, DAZ, XKRY, and RBMY each have one pseudogene for each one that is human specific, while others were generated well before the human-chimpanzee split, by means of duplication, retro-transposition or translocation. Some functional MSY gene duplication of VCY, CDY and HSFY, as well as X-linked VCX and HSFX duplication, occurred in the lineage leading to humans; these duplicates have accumulated nucleotide substitutions that permit their identification.

High Correlation between Alu Elements and the Conversion of 3' UTR of mRNAs Processed Pseudogenes

Even though it represents 6 13% of human genomic DNA, Alu sequences are rarely found in coding regions. When in exon region, over 80 % of them are found in 3' untranslated region (UTR). Pseudogenes are an important component of human genome. Their functions are not clearly known and the mechanism of how they are generated is still debatable. Both the Alu and Pseudogenes are important research problems in molecular biology. mRNA is thought to be a prime source of pseudogene and active research is going on its molecular mechanism. We report, for the first time, that mRNAs containing Alu repeats at 3' UTR has a significantly high correlation with processed pseudogenes, suggesting a possibility that Alu containing mRNAs have a high tendency to become processed pseudogenes. It is known that about 10% of all human genes have been transposed. Transposed genes at 3' UTR without Alu repeat have about two processed pseudogenes per gene on average while we found with statistical significance that a transposed gene with Alu had over three processed Pseudogenes on average. Therefore, we propose Alu repeats as a new and important factor in the generation of pseudogenes.

Caracterización molecular en pacientes con enfermedad granulomatosa crónica por deficiencia en p47 phox / Molecular characterization in patients with chronic granulomatous disease due to p47phox deficiency

El sistema NAOPH oxidasa es un complejo enzimático transportador de electrones localizado en la membrana de las células fagocíticas. De este sistema hacen parte varias proteínas; un flavocitocromo b558' el cual está conformado por una cadena b (gp91-phox) y una cadena a (p22-phox) y poral menos 3 proteínas citosólicas (p47-phox, p67- phox, p40-phox). Una alteración gen ética en cualquiera de estas proteínas causa el síndrome de Enfermedad Granulomatosa Crónica (EGC). La caracterización de las mutaciones de los pacientes con EGC ha sido fundamental para dilucidar la estructura y función de los componentes del sistema NAOPH oxidasa. En el caso de la p47-phox, se han obtenido hallazgos importantes que la hacen un modelo interesante para estudiar el mecanismo molecular involucrado en regular la expresión y función bioquímica de este sistema. En los pacientes con defecto en la p47-phox investigados hasta ahora, se ha hallado una deleción del dinucleótido GT al comienzo del exón 2 , siendo la mayoría de ellos homocigóticos para esta deleción, la cual posiblemente se debe a eventos de recombinación entre el gen p47 -phox normal y un seudogen recientemente descrito. En el diagnóstico de pacientes no homocigóticos, cualquier mutación encontrada en el análisis del ONA (gONA o cONA) puede representar un cambio sufrido por el seudogen. Por lo tanto, para la identificación precisa del defecto genético es necesario separar el gen normal del seudogen y analizar las secuencias en forma individual. Los pacientes no homocigóticos posiblemente deben tener una segunda mutación en el alelo tipo silvestre diferente a la deleción GT. De otro lado, a través de mutagénesis sitio-dirigida se pueden modificar algunos de los aminoácidos o dominios de la p47-phox, los cuales pueden ser esenciales para su funcionamiento y su relación con la EGC. Con esta metodología, es posible introducir cambios en un gen cuya secuencia es totalmente conocida, el cual es amplificado; las mutantes así generadas pueden dar información acerca de la estructura y función de los genes analizados, observando su efecto sobre la función. De esta manera se puede determinar lo importante que puede ser un cambio estructural en la función de esta proteína.

NADPH oxidase system is an enzymatic electron transport complex localized in the membrane of phagocytic cells. Several proteins belong to this system: A flavocytochrome b558, formed by a b chain (gp91.phox) and an a chain (p22.phox) and, at least, 3 cytosolic proteins (p47.phox, p67.phox and p40 phox). Genetic alteration in any of these proteins causes the syndrome of Chronic Granulomatous Disease (CGD). Characterization of mutations in patients with CGD has been fundamental to elucidate the structure and function of NADPH oxidase system ComponentS. Several findings make p47.phoX an interesting model to study the molecular mechanism involved in regulating the expreSSion and bioChemical function ofthis system. So far, in patients with p47.phoX defect a deletion of dinucleotide GT has been foUnd at the beginning of exon 2; most of them are homocygotic for this deletion which is probably due to recombinant events between normal p47.phoX gen and a recently described pseudogen. Any mutation found when diagnosing non.homocygotic patients (gDNA or cDNA) may represent a pseudogen change. Therefore, for precise identification of the genetic defect it is necessary to separate the normal gen from the pseudogen and to analyze individual sequences. Non.homocygotic patients posibly have a second mutation in the wild type allele different fron GT deletion. On the other hand, through site. oriented mutagenesis it is posible to modify some of the aminoacids or domains of p47.phoX, which may be essential for its function and relationship with CGD. With this method010gy it is possible to introduce changes in a gen whoSe sequence is thoroughly known and which is amplified; mutants So generated can give information concerning the structure and function of the analyzed genes, observing their effect on function. In this way the importance of a structural change on the function of a protein can be determined.

Molecular biology of tubulin: Its interaction with drugs and genomic organization.

Microtubules are ubiquitous cellular structures found in eukaryotic organisms and responsible for a variety of functions. These functions include mitosis, motility, cytoskeletal architecture, intracellular transport and secretion. The major structural component of microtubules is tubulin, a dimeric protein molecule consisting of two similar but nonidentical subunits (α and β) each of about molecular weight 55,000. With the introduction of radioactive colchicine for the first time it has been reported that colchicine binds specifically to tubulin. At this point microtubule research stepped up to a new era linking microtubules with other spindle poisons which are structurally diverse as well as binding at different sites on to the tubulin heterodimer. These antimicrotubular agents have already provided valuable information regarding microtubule-mediated cellular functions and its association and dissociation phenomena. Tubulins appear to be conserved proteins based on in vitro copolymerization and comigration on polyacrylamide gel electrophoretic properties. Further, amino acid sequences of both α and β subunits from a variety of sources also appear to be mostly conserved. The evolutionary conservation of tubulin genes is highly reflected at the nucleic acid level as well. The estimation of the number of genes for tubulin and their organization in a variety of organisms have opened up a new dimension to microtubule and tubulin research. The multigene family for tubulins comprising also pseudogenes is suggestive that more than one gene for each α and β tubulin is functional in the cell. Therefore, it has been speculated that different tubulin gene products contribute to functionally different microtubules at specific stages in cell cycle and cell growth. Heterogeneity in both α and β tubulins has already been established during different stages of development of the cell. Obviously, it reflects that tubulin genes are highly regulated and this regulation might be at the transcriptional and/or translational level. Whatever is the actual control mechanism it appears that cells can detect an enhanced pool of depolymerized subunits and a rapid and specific control in tubulin gene expression at the transcriptional and/or post transcriptional level does occur.