BACKGROUND: The transcription factor POU2F1 regulates the expression levels of microRNAs in neoplasia. However, the miR-29b1/a cluster modulated by POU2F1 in gastric cancer (GC) remains unknown. METHODS: Gene expression in GC cells was evaluated using reverse-transcription polymerase chain reaction (PCR), western blotting, immunohistochemistry, and RNA in situ hybridization. Co-immunoprecipitation was performed to evaluate protein interactions. Transwell migration and invasion assays were performed to investigate the biological behavior of GC cells. MiR-29b1/a cluster promoter analysis and luciferase activity assay for the 3'-UTR study were performed in GC cells. In vivo tumor metastasis was evaluated in nude mice. RESULTS: POU2F1 is overexpressed in GC cell lines and binds to the miR-29b1/a cluster promoter. POU2F1 is upregulated, whereas mature miR-29b-3p and miR-29a-3p are downregulated in GC tissues. POU2F1 promotes GC metastasis by inhibiting miR-29b-3p or miR-29a-3p expression in vitro and in vivo . Furthermore, PIK3R1 and/or PIK3R3 are direct targets of miR-29b-3p and/or miR-29a-3p , and the ectopic expression of PIK3R1 or PIK3R3 reverses the suppressive effect of mature miR-29b-3p and/or miR-29a-3p on GC cell metastasis and invasion. Additionally, the interaction of PIK3R1 with PIK3R3 promotes migration and invasion, and miR-29b-3p , miR-29a-3p , PIK3R1 , and PIK3R3 regulate migration and invasion via the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway in GC cells. In addition, POU2F1 , PIK3R1 , and PIK3R3 expression levels negatively correlated with miR-29b-3p and miR-29a-3p expression levels in GC tissue samples. CONCLUSIONS The POU2F1 - miR-29b-3p / miR-29a-3p-PIK3R1 / PIK3R1 signaling axis regulates tumor progression and may be a promising therapeutic target for GC.
MicroRNAs/metabolism* ; Humans ; Stomach Neoplasms/pathology* ; Cell Line, Tumor ; Cell Movement/physiology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Animals ; Mice ; Octamer Transcription Factor-1/metabolism* ; Mice, Nude ; Class Ia Phosphatidylinositol 3-Kinase/metabolism* ; Neoplasm Invasiveness ; Gene Expression Regulation, Neoplastic/genetics* ; Male ; Immunohistochemistry ; Female

Objective To investigate the effect of the HtrA serine peptidase 3(HTRA3)gene on choroidal neovascu-larization(CNV)and M2 macrophage polarization.Methods Fasting venous blood was collected from 30 patients with wet age-related macular degeneration(wAMD group)and 30 healthy subjects(normal group).The serum HTRA3 messen-ger ribonucleic acid(mRNA)level was detected by quantitative reverse transcription polymerase chain reaction(qRT-PCR).RF/6A cells were randomly divided into the control group,NC-sh group and HTRA3-sh group.Lentiviral vectors of NC-shRNA and HTRA3-shRNA were transfected into RF/6A cells in the NC-sh group and HTRA3-sh group by Lipo-fectamine2000.HTRA3 transfection was detected by qRT-PCR and Western blot.Then,the RF/6A cells were randomly di-vided into the N group,H group,H+NC-sh group and H+HTRA3-sh group.After cell transfection,RF/6A cells in the N group were cultured in a RPMI 1640 complete medium at a normoxia state,and cells in other groups were cultured in a RP-MI 1640 medium with 200 mmol·L-1 CoCl2 at a hypoxia state.Tubule formation was measured by Matrigel.The C57BL/6J mice were divided into the control group,CNV group,CNV+NC-sh group and CNV+HTRA3-sh group,with 12 mice in each group.Mice in the control group were unmodeled mice,and mice in the other groups were laser-induced CNV model mice.NC-shRNA and HTRA3-shRNA lentiviral vectors with a titer of 1 × 1011 TU·mL-1 were administered to mice in the CNV+NC-sh group and CNV+HTRA3-sh group via intravitreal injection.Mice in the control group and CNV group were in-jected with phosphate buffered saline.After 7 days of treatment,the mice were examined by fundus fluorescein angiogra-phy,and the eyeballs received hematoxylin & eosin staining.The mRNA levels of HTRA3,chitinase-like protein 3(Ym-1),arginase 1(Arg-1),inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2)and vascular endothelial growth factor(VEGF)in RF/6A cells or choroidal tissues were detected by qRT-PCR.The protein expression levels of HTRA3,VEGF and nuclear factor kappa B(NF-κB)p65 in RF/6A cells or choroidal tissues were detected by Western blot.Re-sults Compared with the normal group,serum HTRA3 mRNA level of patients in the wAMD group increased(t=11.804,P＜0.001).Compared with the control group and NC-sh group,the expressions of HTRA3 mRNA and protein in RF/6A cells in the HTRA3-sh group decreased(all P＜0.05).Compared with the N group,the number of closed lumen and the mRNA and protein expressions of HTRA3 and VEGF in RF/6A cells in the H group increased(all P＜0.05).Compared with the H+NC-sh group,the number of closed lumen and the mRNA and protein expressions of HTRA3 and VEGF decreased in RF/6A cells in the H+HTRA3-sh group(all P＜0.05).Compared with the control group,the mRNA and protein expression levels of HTRA3 increased,the relative fluorescence intensity of CNV increased,the mRNA levels of Ym-1 and Arg-1 in-creased,the iNOS and COX-2 mRNA levels decreased,and the NF-κB p65 protein expression level increased in mice of the CNV group(all P＜0.05).Compared with the CNV+NC-sh group,the mRNA and protein expression levels of HTRA3 de-creased,the relative fluorescence intensity of CNV decreased,the mRNA levels of Ym-1 and Arg-1 decreased,the mRNA levels of iNOS and COX-2 increased,and the NF-κB p65 protein expression level decreased in mice of the CNV+HTRA3-sh group(all P＜0.05).Conclusion Down-regulation of HTRA3 can inhibit the formation of CNV and the polarization of M2 macrophages.HTRA3 may be an important potential target for the prevention and treatment of wAMD.

Objective To detect the effects of siRNA targeting CDX2 gene expression on of BCR-ABL, caspase and Bax expressions, and the mechanisms thereof. Methods According to the earlier experiments, siRNA specifically targeting CDX2 gene (CDX2-siRNA) and the negative control sequence (CDX2-siRNA-NC) were selected, and then were transfected into K562 cells by Roche X-tremeGENE HP DNA Transfection Reagent. The flow cytometry analysis was used to detect the effects of siRNA on cell apoptosis. The expressions of BCR-ABL, caspase-9, Bax mRNA and protein were tested by RT-PCR and Western blot assay. Results MTT and flow cytometry analysis showed that after the silence of CDX2 gene expression, the proliferation of K562 cells was prohibited and the apoptotic rate of K562 cells was distinctly increased compared with that of normal cell group, but the negative control group had no significant change. According to the RT-PCR and Western blot assay, in comparison with the normal cell group and the negative control group, the expression levels of BCR-ABL mRNA and protein were obviously decreased, and the difference was statistic significance. On the other hand, the expressions of caspase-9 and Bax mRNA and protein were significantly higher than those of other two groups (P<0.05). Conclusion CDX2-siRNA can promote apoptosis of K562 cells obviously, and the mechanism is related with the down-regulation of BCR-ABL and the up-regulation of caspase-9 and Bax.

Acute myeloid leukemia(AML) is a heterogeneous kind of disease,which is accounting for 15％ ～ 20％ of all tumors diagnosed in children under 15 years of age.In the past few decades,remarkable improvements have been yielded in long-term outcomes for children with AML.A better risk-group stratification of disease,which is based on clinical and biological features,a more effective use of anti-leukemia agents and enormous improvements in supportive care have increased the rate of cure by approximately 60％.This paper review the new improvements in the chemotherapeutic regimens and drugs.