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 N2 a 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.

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

Cytotoxic effects of mono-（2-ethylhexyl） phthalate on human embryonic stem cells

Background Mono-（2-ethylhexyl） phthalate （MEHP）, the metabolite of di-（2-ethylhexyl） phthalate （DEHP）, was suspected to be toxic to human embryos. This study contributes to investigating its toxic effects by an embryonic stem cell test （EST） based on two human embryonic stem cell （hESCs） lines. Methods CH1 established in our own lab and H1, a federally registered cell line were two human embryonic stem cell lines used in this test. Four endpoint measurements were performed consisting of cell viability, proliferation ability, apoptosis as well as changes of gene expression patterns after spontaneous differentiation were determined. For measuring effects on the first three endpoints, the cells were treated with various concentrations of MEHP dissolved in dimethyl sulfoxide （DMSO） and only with DMSO which served as control and harvested after 5 days. For measuring effects during spontaneous differentiation, the RNA of embryoid bodies （EBs） formed after 8 days＇ MEHP exposure was collected and changes in differentiation specific gene expression patterns were analyzed by quantitative real time RT-PCR. Results As a result the viability and proliferation ability of both cell lines decreased significantly at 1000 μmol/L MEHP, while there was no effect on apoptosis or cell morphology. In addition MEHP also changed the gene expression pattern in the EBs of both cell lines. Conclusion MEHP in a high dose was cytotoxic and affected the development of hESCs, which indicates its embryo toxicitv in human embrvos.

G-protein Coupled Receptor 34 Knockdown Impairs the Proliferation and Migration of HGC-27 Gastric Cancer Cells In Vitro

Background：Overexpression of G-protein coupled receptor 34 （GPR34） affects the progression and prognosis of human gastric adenocarcinoma,however,the role of GPR34 in gastric cancer development and progression has not been well-determined.The current study aimed to investigate the effect of GPR34 knockdown on the proliferation,migration,and apoptosis of HGC-27 gastric cancer cells and the underlying mechanisms.Methods：The expression of GPR34 in gastric cancer cell line HGC-27 was detected by quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting.HGC-27 cells were employed to construct the stable GPR34 knockdown cell model in this study.Real-time RT-PCR and Western blotting were applied to validate the effect of short hairpin RNA （ShRNA） on the expression of GPR34 in HGC-27 gastric cells.The proliferation,migration of these cells were examined by Cell Counting Kit-8 and transwell.We also measured expression profile of PI3K/PDK1/AKT and ERK using Western blotting.Results：The ShRNA directed against GPR34 effectively inhibited both endogenous mRNA and protein expression levels of GPR34,and significantly down-regulated the expression of PIK3CB （P ＜ 0.01）,PIK3CD （P ＜ 0.01）,PDK1 （P ＜ 0.01）,phosphorylation of PDK1 （P ＜ 0.01）,Akt （P ＜ 0.01）,and ERK （P ＜ 0.01）.Furthermore,GPR34 knockdown resulted in an obvious reduction in HGC-27 cancer cell proliferation and migration activity （P ＜ 0.01）.Conclusions：GPR34 knockdown impairs the proliferation and migration of HGC-27 gastric cancer cells in vitro and provides a potential implication for therapy of gastric cancer.

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 （Runxl） plays a crucial role in hematogenesis and its dysfunction may contribute to leukemogenesis. However, it is not clear whether or not abnormal expression of Runxl will induce leukemia and how the change of Runxl expression level could affect BCR-ABL-induced leukemogenesis. In the present study, we aimed to analyze if abnormal expression of Runxl in BaF3 cells alone would induce teukemogenesis. And we also wanted to know if abnormal expression of Runxl in leukemic cells would affect leukemogenesis. Furthermore, we investigated whether overexpression or knock-down of Runxl in BaF3 cells would induce leukemogenesis. Methods Plasmids containing full-length Runxl cDNA were transduced into BaF3 cells and BaF3-P185wt cells （BCR-ABL transformed BaF3 cells） by electroporation. Plasmids containing a short hairpin RNA of Runxl were transduced into BaF3 cells and BaF3-P185wt cells by electroporation. Runxl expression level was quantified by Western blotting and quantitative real-time PCR. The effects of overexpression or knock-down of Runxl 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 Runxl in P185wt cells could inhibit cell proliferation and slow down cell migration; while knock-down of Runxl 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-Runxl cells or MSCV-BaF3-shRNA cells. Both the mice of MSCV-BaF3-Runxl group and MSCV-BaF3-shRNA group were healthy with no sign of leukemia for up to three months. Conclusions Overexpression or knock-down of Runxl gene in BaF3 cells alone could not induce leukemogenesis. However, in BaF3-P185wt cells, alteration of Runxl expression could affect BCR-ABL-induced proliferation and migration in vitro and leukemoaenesis in vivo.

Aberrant Alternative Polyadenylation is Responsible for Survivin Up-regulation in Ovarian Cancer

Background： Survivin is an oncoprotein silenced in normal mature tissues but reactivated in serous ovarian cancer （SOC）. Although transcriptional activation is assumed for its overexpression, the long 3＇-untranslated region （3＇-UTR） in survivin gene, which contains many alternate polyadenylation （APA） sites, implies a propensity for posttranscriptional control and therefore was the aim of our study. Methods： The abundance of the coding region, the proximal and the distal region of survivin mRNA 3＇-UTR, was evaluated by real-time polymerase chain reaction （PCR） in SOC samples, cell lines, and normal fallopian tube （NFT） tissues. The APA sites were confirmed by rapid amplification ofcDNA 3＇ ends and DNA sequencing. Real-time PCR were used to screen survivin-targeting microRNAs （miRNAs） that were inversely correlated with survivin. The expression of an inversely correlated miRNA was restored by pre-miRNA transfection or induction with a genotoxic agent to test its inhibitory effect on survivin overexpression. Results： Varying degrees of APA were observed in SOC by comparing the abundance of the proximal and the distal region of survivin 3＇-UTR, and changes of 3＇-UTR correlated significantly with survivin expression （r = 0.708, P 〈 0.01）. The main APA sites are proved at 1197 and 1673 of survivin 3＇-UTR by DNA sequencing. Higher level of 3＇-UTR proximal region than coding region was observed in NFT, as well as in SOC and cell lines. Among the survivin-targeting miRNAs, only a few highly expressed miRNAs were inversely correlated with survivin levels, and they mainly targeted the distal part of the 3＇-UTR. However, in ovarian cancer cells, restoration of an inversely correlated miRNA （miR-34c） showed little effect on survivin expression. Conclusions： In NFT tissues, survivin is not transcriptionally silenced but regulate posttranscriptionally. In SOC, aberrant APA leads to the shortening of survivin 3＇-UTR which enables it to escape the negative regulation of miRNAs and is responsible for survivin up-regulation.

Xingshentongqiao Decoction Mediates Proliferation, Apoptosis, Orexin-A Receptor and Orexin-B Receptor Messenger Ribonucleic Acid Expression and Represses Mitogen-activated Protein Kinase Signaling

Background：Hypocretin （HCRT） signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy.Our previous study showed that xingshentongqiao decoction （XSTQ） is clinically effective for the treatment of narcolepsy.To determine whether XSTQ improves narcolepsy by modulating HCRT signaling,we investigated its effects on SH-SY5Y cell proliferation,apoptosis,and HCRT receptor 1/2 （orexin receptor 1 [OXl R] and orexin receptor 2 [OX2R]） expression.The signaling pathways involved in these processes were also assessed.Methods：The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays.OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis.Western blotting for mitogen-activated protein kinase （MAPK） pathway activation was performed to further assess the signaling mechanism of XSTQ.Results：XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells.This effect was accompanied by the upregulation of OX 1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase （Erk） 1/2,p38 MAPK and c-Jun N-terminal kinase （JNK）.Conclusions：XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells.XSTQ also promotes OX1R and OX2R expression.These effects are associated with the repression of the Erkl/2,p38 MAPK,and JNK signaling pathways.These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation,which may explain its ability to treat narcolepsy.