[Effects of receptor interacting protein 140 on the biological activity of glia cells].

OBJECTIVE: To explore the effects of receptor interacting protein (RIP) 140 gene overexpression upon the in vitro proliferation, apoptosis, invasion and migration of microglioma cells. METHODS: The BV-2 RIP140 overexpression model (BV-2-1) was constructed by Lipofection and G418 selection, then validated by real-time PCR and Western blotting. The proliferation, apoptosis, invasion and migration potencies were compared between BV-2-1 and its parents by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, flow cytometry and Transwell chamber. RESULTS: The BV-2-1 model was successfully constructed. Compared to those of the BV-2 group, the RIP140 mRNA and protein expression levels of BV-2-1 were markedly higher than those of the BV-2 group (t = 49.794, P < 0.01). MTT assay showed that the absorbance values in the BV-2 group were 1.157 ± 0.013, 1.679 ± 0.005 and 2.609 ± 0.008 at 24, 48, and 72 hours respectively. And those were 0.929 ± 0.013, 1.188 ± 0.008 and 1.528 ± 0.012 in the BV-2-1 group respectively. The proliferation at the time points of 48 and 72 hours of the BV-2-1 group were significantly lower than that of the BV-2 group (t = 6.058 and 9.245, both P < 0.01). Annexin-V staining showed that there were significant differences in the apoptosis rates between the BV-2 and BV-2-1 cells [(5.35 ± 0.23)% vs (3.46 ± 0.45)%, t = 6.619, P = 0.003)]. Transwell assay showed that the invaded cell number of the BV-2-1 group was 166 ± 43. And it was obviously higher than that of the BV-2 group (93 ± 32, t = 3.403, P = 0.007). Transwell assay also showed that the migrated cell number of BV-2 cells was 101 ± 25. And the migration potency of the BV-2-1 group (202 ± 50) was significantly stronger than that of the BV-2 group (t = 4.104, P = 0.002). CONCLUSION: RIP140 effectively inhibits the proliferation and facilitates the apoptosis of microglioma cells. And it may effectively facilitate the in vitro invasion and migration of microglioma cells.

Abelson interactor 1 splice isoform-L plays an anti-oncogenic role in colorectal carcinoma through interactions with WAVE2 and full-length Abelson interactor 1.

BACKGROUND: Expression of the full-length isoform of Abelson interactor 1 (ABI1), ABI1-p65, is increased in colorectal carcinoma (CRC) and is thought to be involved in one or more steps leading to tumor progression or metastasis. The ABI1 splice isoform-L (ABI1-SiL) has conserved WAVE2-binding and SH3 domains, lacks the homeo-domain homologous region, and is missing the majority of PxxP- and Pro-rich domains found in full-length ABI1-p65. Thus, ABI1-SiL domain structure suggests that the protein may regulate CRC cell morphology, adhesion, migration, and metastasis via interactions with the WAVE2 complex pathway. AIM: To investigate the potential role and underlying mechanisms associated with ABI1-SiL-mediated regulation of CRC. METHODS: ABI1-SiL mRNA expression in CC tissue and cell lines was measured using both qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR. A stably ABI1-SiL overexpressing SW480 cell model was constructed using Lipofectamine 2000, and cells selected with G418. Image J software, CCK8, and transwell assays were used to investigate SW480 cell surface area, proliferation, migration, and invasion. Immunoprecipitation, Western blot, and co-localization assays were performed to explore intermolecular interactions between ABI1-SiL, WAVE2, and ABI1-p65 proteins. RESULTS: ABI1-SiL was expressed in normal colon tissue and was significantly decreased in CRC cell lines and tissues. Overexpression of ABI1-SiL in SW480 cells significantly increased the cell surface area and inhibited the adhesive and migration properties of the cells, but did not alter their invasive capacity. Similar to ABI1-p65, ABI1-SiL still binds WAVE2, and the ABI1-p65 isoform in SW480 cells. Furthermore, co-localization assays confirmed these intermolecular interactions. CONCLUSION: These results support a model in which ABI1-SiL plays an anti-oncogenic role by competitively binding to WAVE2 and directly interacting with phosphorylated and non-phosphorylated ABI1-p65, functioning as a dominant-negative form of ABI1-p65.

[Effects of receptor-interacting protein 140 gene knockdown on the proliferation of microglioma cells: experiment with rat microglioma cells].

OBJECTIVE: To investigate the effects of receptor-interacting protein (RIP)140 gene knockdown on the proliferation of microglioma cells. METHODS: Mouse microglioma cells of the line BV-2 were cultured and transfected with 2 kinds of recombinant RIP140-shRNA plasmids (V2MM-71674 and V2MM-71080) or blank plasmid MSCV-EGFP. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of RIP140; and the cell proliferation was detected by MTT assay. RESULTS: There were not significant differences in the RIP140 mRNA and protein expression between the BV-2 and BV-2-MGCV-EGFP groups. Compared to those of the BV-2 group, the RIP140 mRNA expression levels of the BV-2-71674 and BV-2-71080 groups were lower by 73% and 75% respectively. The protein expression levels of the BV-2-71674 and BV-2-71080 groups were remarkably lower than those of the BV-2 and BV-MSGV-EGFP groups. MTT assay showed that there were not significant differences in the proliferation rates at different time points between the BV-2 and BV-2-MSCV-EGFP groups, however, the proliferation rates at the time points of 24, 48, 72, and 96 h of the BV-2-71674 and BV-2-71080 groups were significantly lower than those of the BV-2 group (all P<0.01). CONCLUSION: RIP140 gene knockdown effectively inhibits the proliferation of microglioma cells.

Cystathionine beta synthase participates in murine oocyte maturation mediated by homocysteine.

Our previous study demonstrated that cystathionine beta synthase (CBS) is highly expressed in the cumulus-oocyte complex during ovulation. However, the role of CBS during oocyte maturation remains uncertain. In this study, a small-interfering (si) RNA interference (siRNA) approach was used to investigate the potential role of CBS during oocyte maturation. Accompanied with a gradual increase of homocysteine, the introduction of CBS-siRNA into murine granulosa cells selectively depleted the corresponding target mRNA and protein for CBS as assessed by semi-quantitative reverse-transcriptase PCR (RT-PCR) and immunofluorescence staining. When fully grown, germinal vesicle (GV) oocytes matured in vitro for 16 h using medium from transfected granulosa cells, the functional suppression of CBS resulted in a significant increase in the rate of GV-arrested oocytes. The results of this study provide evidence that CBS participates in the process of oocyte maturation. Furthermore, this effect may be fulfilled by conditioning the level of homocysteine in the microenvironment of the oocyte.

[Screening and identification of human chromosome 21 genes resulting in abnormal development of fetal cerebral cortex with Down syndrome].

OBJECTIVE: To screen the candidate human chromosome 21 (HC21) genes related to mental retardation (MR). METHODS: The expression of 127 known HC21 genes in the cerebral cortex specimen of a DS fetus and the specimen of a non-DS fetus induced due to maternal disease was detected with Affymetrix U133A gene chip. Semi-quantitative RT-PCR and reverse Northern blotting were used to identify the HC21 genes thus screened. RESULTS: Fifty-six of the 127 known HC21 genes were not expressed in both the specimens from the DS and control fetuses, while 71 of the 127 HC21 genes were expressed in both of them. The expression of 57 of these 71 genes was up-regulated and 14 of the 71 genes was down-regulated in the DS fetal cerebral cortex compared with the non-DS control. The expression of these 71 genes was upregulated by 1.5 times and the expression of 2 genes was downregulated by 1.5 times.21 candidate key genes were thus provided. CONCLUSION: Twenty-one HC21 genes are involved in the MD of DS.

[Expression profile of receptor-interacting protein 140 in the brain: experiment with prenatal and postnatal mice].

OBJECTIVE: To investigate the expression of receptor-interacting protein of 140,000 (RIP140) in the developmental brain. METHODS: The brain tissues of 15-day-old and 20-day-old fetal Balb/c mice and 1, 7, 14, 28, 42, and 56-day-old postnatal mice were collected. The expression of RIP140 was examined by immunohistochemistry. Real-time PCR and Western blotting were used to quantify the expression of the mRNA and protein levels of RIP140. RESULTS: (1) Immunochemistry showed that RIP140 was expressed extensively in the brain of embryonic and newborn mice, mainly present in the neurons in many different brain regions, such as the cerebral cortex, hippocampus and pituitary gland. The cellular location of RIP140 was confined to the nucleus. (2) Real-time PCR revealed that the RIP140 mRNA expression in brain was in an increment as the time went by, peaked on the 7th day after birth, then was in a instable level after that until the adulthood. (3) Western blotting indicated that the protein level of RIP140 was coincident with the mRNA level [r(s) = 0.767, P = 0.016 (bilateral)]. CONCLUSION: The expression and role of RIP140 may be involved in the whole neurodevelopment stages of brain, and may take part in the development and the function of the brain.

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embryonic stem cells into neural stem cells.

Overexpression of receptor-interacting protein 140 (RIP140) promotes neuronal differentiation of N2a cells via extracellular regulated kinase 1/2 (ERK1/2) signaling. However, involvement of RIP140 in human neural differentiation remains unclear. We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells. Moreover, RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation, and positively correlated with the neural stem cell marker Nestin during later stages. Thus, ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.

Localization of cystathionine beta synthase in mice ovaries and its expression profile during follicular development.

BACKGROUND: In vitro fertilization (IVF) researches have suggested that cystathionine beta synthase (CBS) is involved in oocyte development. However, little is known about the regional and cellular expression patterns of CBS in the ovary. The purpose of this study was to analyze the localization of CBS in mice ovaries and to investigate the expression profile during follicular development. METHODS: We used in situ hybridization and immunohistochemical analysis to determine CBS expression in the ovaries of female Balb/c mice. Then the follicles were collected from F1 (C57BL x Balb/c) mice and cultured in vitro. With the method of semi-quantitative RT-PCR, we also investigated the expression profile of CBS during follicular development. RESULTS: CBS was absent in the oocytes, although it was ubiquitously expressed in the ovary with the strongest expression in follicular cells at all stages. In late antral follicles, CBS expression was markedly higher in granulosa cells located close to the antrum and in cumulus cells around the oocyte. The semi-quantitative RT-PCR showed that CBS mRNA was detected in follicles at all stages in vitro. In cumulus-oocyte complexes superovulated, CBS expression also increased rapidly. CONCLUSIONS: CBS was located mainly in the follicular cells in the ovaries. The level of CBS expression is high in follicles during folliculogenesis in mice. Differences in the CBS expression profile between oocyte and follicular cells suggest a role for CBS as a mediator in interactions between oocyte and granulosa cells.

[Cloning, identification, and cellular localization of a down-regulated gene fragment related with Down Syndrome].

OBJECTIVE: To clone a novel gene and explore its expression patterns in tissues and cells, so as to find its role in the process of encephalopathy in DS. METHODS: On the base of our previous microarray's result together with the tissue type, we chose EST AI480014 to carry out RACE, then analyzed its expression profiles in liver, spleen, kidney, heart, brain by multi-tissues Northern blot, after that semi-quantitive RT-PCR was used to reexamine the expression profiles. Furthermore, we used ISH to find whether aim gene expressed in neuroglial cells cultured in vitro. Finally we performed semi-quantitive RT-PCR to explore whether it expressed differently between DS and normal. RESULTS: We gained a 682 bp new cDNA fragment (DQ275636) which expressed in all the tissues examined and had no alternative splices in them. It expressed highly in brain especially in frontal lobe and hippocampus. According to the ISH result we convinced that it expressed in neuroglial cells. Using bioinformatics we mapped DQ275636 to chromosome 5q14. CONCLUSION: We have obtained a new gene fragment based on the above results. According to its expression character and tissue type, it can be suggested that this gene has a probable role in the process of encephalopathy in DS.

[Preliminary exploration of the influence factors of degenerate oligonucleotide primered PCR of genome DNA].

OBJECTIVE: To explore the influence factors of the degenerate oligonucleotide primered PCR(DOP-PCR). METHODS: Genome DNA template from the mouse single oocyte or liver tissue were used to perform DOP-PCR. DOP-PCR was carried out with templates of different origin, different gradient dilution, with or without low melting point gel purified to wipe off the small fragment that might interfere with the following analysis, and then PCR of gene FTCD and CBS were carried out to evaluate the influence of these factors on the amplification efficiency and specificity. RESULTS: Compared with genome DNA template from mouse liver, the template from single oocyte had the same efficiency and specificity but a minor yield and different gradient dilution of DNA template had no effect on the efficiency and specificity. Furthermore, there was a higher specificity in the low melting point gel-purified DOP-PCR product than in untreated ones. CONCLUSION: We have got a satisfactory result and increased specificity from DOP-PCR product purified with the low melting point gel. Single oocyte of mice could be used for further investigation of special genes detection by DOP-PCR and of an optimization in the yield of the products.

[Cell-cycle negative regulatory gene ANA is over-expressed in the brain tissues of patients with Down syndrome].

OBJECTIVE: To investigate the expression level of genes located in chromosome 21 in the brain tissues of Down syndrome(DS). METHODS: An optimized semi-quantitative RT-PCR method was used to evaluate the expression levels of seven genes encoded in chromosome 21 in fetal cortex brain and cerebellum of DS and the control at the end of 20 weeks of gestation. B2M was used as internal reference to normalize cell loss. RESULTS: The expression levels of 6 genes in cortex and cerebellum, including DYRK1A, SYNJ1, PCP4, C21orf5, C21orf2 and C21orf106, were comparable between DS and the control. ANA, a cell-cycle negative regulatory gene, was over-expressed dramatically in the cortex but not in the cerebellum of DS. CONCLUSION: Over-expression of ANA may contribute to the reduction of neuronal density in DS brain.

[The expression profiles of human chromosome 21 orthologues in mouse M II oocyte].

OBJECTIVE: To analyse the expression profile of orthologues of human chromosome 21 (HC21) in mouse M II oocytes, and to discuss the relationship between this expression profile and early embryonic development and further to find the possible reasons of DS phenotypes genesis. METHODS: cDNA array and Global RT-PCR methods were used to analyse and identify the expression profile of 93 HC21 orthologues in mouse M II oocytes. RESULTS: 26 of 93 orthologues were proved to be expressed in mouse M II oocytes and these genes were involved in many biological procedure including transcriptional, metabolism, ionic channel, and ubiquitin pathway etc. CONCLUSION: To our knowledge, this is first report about the HC21 orthologues expression profile in M II oocytes and this expression profile indicates that 26 of 93 HC21 orthologues may directly play important role in early development, and the biological information obtained from this experiment would be beneficial to understand imbalance of HC21 genes expression and the molecular mechanism of DS phenotypes genesis.

Overexpression or knock-down of runt-related transcription factor 1 affects BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo in mice.

BACKGROUND: Runt-related transcription factor 1 (Runx1) plays a crucial role in hematogenesis and its dysfunction may contribute to leukemogenesis. However, it is not clear whether or not abnormal expression of Runx1 will induce leukemia and how the change of Runx1 expression level could affect BCR-ABL-induced leukemogenesis. In the present study, we aimed to analyze if abnormal expression of Runx1 in BaF3 cells alone would induce leukemogenesis. And we also wanted to know if abnormal expression of Runx1 in leukemic cells would affect leukemogenesis. Furthermore, we investigated whether overexpression or knock-down of Runx1 in BaF3 cells would induce leukemogenesis. METHODS: Plasmids containing full-length Runx1 cDNA were transduced into BaF3 cells and BaF3-P185wt cells (BCR-ABL transformed BaF3 cells) by electroporation. Plasmids containing a short hairpin RNA of Runx1 were transduced into BaF3 cells and BaF3-P185wt cells by electroporation. Runx1 expression level was quantified by Western blotting and quantitative real-time PCR. The effects of overexpression or knock-down of Runx1 on proliferation, apoptosis and migration of cells were detected in vitro. Then, using MSCV-P185wt-EGFP as a control, we transplanted MSCV-P185wt-Runx1 cells or MSCV-P185wt-shRNA cells into Balb/c mice through tail vein and observed tumorgenesis of the different phenotypes. RESULTS: In vitro analysis revealed that overexpression of Runx1 in P185wt cells could inhibit cell proliferation and slow down cell migration; while knock-down of Runx1 could promote cell proliferation and speed up cell migration. In vivo analysis indicated that mice transplanted with MSCV-P185wt-Runx1 survived longer than controls. In contrast, mice transplanted with MSCV-P185wt-shRNA survived shorter than the control group. Gross pathological analysis revealed that the MSCV-P185wt-Runx1 group had less severe splenomegaly and hepatomegaly compared to the control group, and the MSCV-P185wt-shRNA group had more severe splenomegaly and hepatomegaly. No splenomegaly or hepatomegaly was detected in mice transplanted with MSCV-BaF3-Runx1 cells or MSCV-BaF3-shRNA cells. Both the mice of MSCV-BaF3-Runx1 group and MSCV-BaF3-shRNA group were healthy with no sign of leukemia for up to three months. CONCLUSIONS: Overexpression or knock-down of Runx1 gene in BaF3 cells alone could not induce leukemogenesis. However, in BaF3-P185wt cells, alteration of Runx1 expression could affect BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo.