Interferon regulatory factor-3 and its alternative splicing isoforms in tumor / 国际肿瘤学杂志

As a member of interferon regulatory factor family (IRF),IRF3 plays an important role in triggering the expression of type Ⅰ interferons and downstream interferon-stimulated genes,contributing to many biological process.Researches have found that it plays an anti-oncogene role in inhibiting tumor proliferation and migration,inducing cell apoptosis.The mechanism involves in tumor immunity and inflammatory reaction,apoptosis and epithelial mesenchymal transition.The alternative splicing isoforms of IRF3 act as negative modulators of IRF3 and affect tumor development progress.The recent signaling pathways and pathogenesis researches provide new ideas for early diagnosis and treatment of cancer.

Inhibición parcial de dos genes que codifican para proteínas del empalmosoma en Giardia intestinalis / Partial inhibition of two genes that encode spliceosomal proteins in Giardia intestinalis

Introducción. Giardia intestinalis es un organismo tempranamente divergente en el que recientemente se demostró la presencia de intrones. La maquinaria responsable de la remoción de intrones en organismos eucariotas superiores es el empalmosoma, el cual está conformado por cinco ribonucleoproteínas, cada una de las cuales tiene un ARN pequeño nuclear, un set de siete proteínas Sm (B, D1, D2, D3, E, F y G) y varias proteínas específicas. En G. intestinalis se han identificado los genes de algunas proteínas del empalmosoma por bioinformática. Aunque se asume que este es el responsable del empalme en el parásito, su caracterización bioquímica no se ha hecho. Objetivo. Inhibir dos genes que codifican para proteínas del empalmosoma de G. intestinalis con el fin de determinar si esta inhibición afecta el crecimiento o el enquistamiento del parásito. Materiales y métodos. En un vector específico para G. intestinalis se clonaron secuencias antisentido de los genes que codifican para las proteínas SmB y SmD3 del empalmosoma del parásito. Posteriormente, se transfectó G. intestinalis con los vectores recombinantes y se seleccionaron aquellos parásitos que lo incorporaron. Se confirmó la disminución del mensajero mediante reacción en cadena de la polimerasa (PCR) en tiempo real, y se evaluaron el crecimiento y el enquistamiento en parásitos silvestres y transfectados. Resultados. Se observó una disminución de 40 y 70 % en el ARNm de SmB y SmD3, respectivamente. El crecimiento y el enquistamiento no se vieron afectados en estos parásitos. Conclusión. La disminución de SmB y SmD3 no afectó al parásito, lo que indica que el empalmosoma sigue siendo funcional, o que el empalme no es una función vital del parásito.

Introduction. Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.

A novel spliceosome encoding isoform of suppresser of fused in human pancreatic cancer / 中华胰腺病杂志

Objective To investigate spliceosome of suppresser of fused(SUFU),a major member of hedgehog signaling pathway in human pancreatic cancer.Methods SUFU fragment was amplified by using reverse transcription and 3' RACE.After sequencing,a new exon was discovered,and then nSUFU was amplified by RT-PCR.nSUFU and SUFU were transfected into SW1990 by liposomes,and then the expressions of SUFU protein encoded by new spliceosome in SW1990 cells and pancreatic cancer tissues were detected by Western blot.Results PCR products by 3'RACE were of 600 bp,after sequencing and comparison with Blast data of NCBI,it was detected that a new exon was inserted between SUFU mRNA isoforml (NM_016169.3) exon 10 and exon 11.After verification with SW1990,it was noted the entire new spliceosome containing new exon was of 1400 bp.SW1990 with nSUFU transfection strongly expressed nSUFU protein,and pancreatic cancer tissues expressed both SUFU and nSUFU protein.Conclusions A new spliceosome of SUFU,which can encode SUFU protein,is present in pancreatic cancer tissue and cell.

Effects of IL-24 delE5 on human leukemia cell line K562 / 白血病·淋巴瘤

Objective To investigate the antitumor activity of IL-24 delE5 in human leukemia cell line K562. Methods The expression of mda-7/IL-24 and its splice variant induced by TPA in leukemic cell lines, U937 and HL-60, was evaluated. The effects of IL-24 delE5 in K562 on cell proliferation, colony-forming ability, cell cycle, apoptosis, and tumor growth in vivo by using MTr assay, colony forming assay, flow cytometry, Annexin-V/PI and tumor xenograft models in nude mice were assessed. Meantime, the effects of IL-24 delE-5 and mda-7/IL-2A were compared. Results The expression of IL-24 dciE5 was detected in differentiated U937 and HL-60 cells. Transfection with IL-24 delE5 significantly reduced tumor cell viability, inhibited colony formation. Comparing with the control, G_0/G_1 stage add from (24.46±3.99) % to (42.69±3.04) %, caused cell cycle arrest in G_0/G_1 stage and significantly inhibited the growth of K562 transplantation tumor. No significant differences in the aforementioned antileukemia characteristics between IL-24 delE5 and mda-7/IL-24 was found. Conclusion Similar with mda-7/IL-24, IL-24 delE5 can efficiently inhibit the proliferation of K562 in vitro and in vivo, probably through induction of G_0/G_1 cell cycle arrest.

Expression of interleukin-7 mRNA and identification of its splice variant in human hepatic cancer / 中国病理生理杂志

AIM: To investigate the expression of interleukin-7(IL-7) in human liver cancer and identify the splice variant of IL-7. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used for investigating the expression of IL-7. According to IL-7 gene cDNA, the primers were designed and synthesized, then the splice variant of IL-7 in human liver cancer cells was identified, cloned into vector and sequenced. RESULTS: The expression of IL-7 mRNA was equal between normal liver tissue and hepatic cancer cells.A new band of IL-7 was obtained from hepatic cancer cells, which lacks exon 4. CONCLUSION: IL-7 gene is expressed highly in both normal liver tissue and hepatic cancer cells. In addition, hepatic cancer cells can produce splicing variant by alternative splicing.

Structure and function of a truncated spliced isoform of interferon regulatory factor IRF-3c / 中华传染病杂志

Objective Interferon regulatory factors 3 (IRF-3) is a key transcription factor to regulate gene expression of interferon after virus infection. This study aims to look for new spliced isoforms of IRF-3 and to investigate their structures and functions. Methods RNA extracts from human embryonic kidney 293 cells were amplified by RACE and RT-PCR. New sequences were compared with published sequences of IRF-3 and murine EST database using bioinformatics method. A new sequence, IRF-3c, was subcloned into pcDNA3.1-flag. The IRF-3c/pcDNA3.1-flag plasmid was transfected in HEK 293 cells. Whole cell extract was analysed by Western blot and then probed with monoclonal Flag antibody. Luciferase assay was carried out by co-transfection with reporter plasmid, pGL2B with interferon ? promoter, and IRF-3c cDNA expression plasmid. At 16 hours posttransfection, cells were infected with Sendai virus for 8 hours. Cells were collected and assayed for luciferase activity. Results A novel spliced isoform of IRF-3, named IRF-3c was discovered. The new isoform is almost the same as IRF-3, except for the utilization of the 180 bp bases in intron 6 adjacent to exon 6. The first 2,3 and 4 bases are a stop codon, which may produce a protein with a truncated C-terminal stoped at amino acids 327. Western blot analysis confirmed an expected 44 kDa strong band. The new inserted bases can be found in murine EST database, suggesting a conservative function in evolution. The functional luciferase assay showed that IRF-3c inhibited the IFN? promoter activity to (around) 40%～50% as that of control after Sendai virus infection. Conclusions The discovery of a new isoform of IRF-3 provides a new insight into the functional regulation of IRF-3 family. It is a dominant-negative inhibitor for interferon ? promoter activity in the virus infection pathway, provides a mechanism for the fine-tuning of the virus-induced activation of the interferon response, and prevents interferon ? from its overexpression and its toxic effects. It is worthwhile to explore the role of IRF-3c in the pathogenesis of human diseases using IRF-3c’s specific sequence.