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

OBJECTIVES: To investigate the mechanism mediating the regulatory effect of miR-155-5p on proliferation of human submandibular gland epithelial cells (HSGECs) in primary Sjogren's syndrome (pSS). METHODS: Dual luciferase reporter assay was used to verify the targeting relationship between miR-155-5p and the PI3K/AKT pathway. In a HSGEC model of pSS induced by simulation with TRAIL and INF-γ, the effects of miR-155-inhibitor-NC or miR-155 inhibitor on cell viability, cell cycle, apoptosis and proliferation were evaluated using CKK8 assay, flow cytometry and colony formation assay. ELISA and RT-PCR were used to detect the expressions of inflammatory cytokines and miR-155-5p mRNA in the cells; Western blotting was performed to detect the expressions of proteins in the PI3K/AKT signaling pathway. RESULTS: Dual luciferase assay showed that miR-155-5p targets the PI3K/AKT pathway via PIK3R1 mRNA. The HSGEC model of pSS showed significantly decreased cell viability, cell clone formation ability and expressions IL-10 and IL-4 and increased cell apoptosis, cell percentage in G2 phase, expressions of TNF‑α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-Akt/AKT ratio, and PIK3R1 protein expression. Treatment of the cell models with miR-155 inhibitor significantly increased the cell viability, G1 phase cell percentage, colony formation ability, and expressions of IL-10 and IL-4 levels, and obviously reduced cell apoptosis rate, G2 phase cell percentage, expressions of TNF-α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-AKT/AKT ratio, and PIK3R1 protein expression. CONCLUSIONS In HSGEC model of pSS, inhibition of miR-155-5p can promote cell proliferation and reduced cell apoptosis by targeting PI3K1 mRNA to negatively regulate the overexpression of PI3K/AKT signaling pathway.
Humans ; MicroRNAs/genetics* ; Cell Proliferation ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism* ; Sjogren's Syndrome/pathology* ; Epithelial Cells/cytology* ; Submandibular Gland/cytology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Apoptosis ; Class Ia Phosphatidylinositol 3-Kinase ; Cells, Cultured

OBJECTIVETo investigate the effect of CAL-101, a selective inhibitor of PI3Kδ, in combination with bortezomib on the proliferation and apoptosis in human mantle cell lymphoma cell lines Z138, HBL-2 and Jeko-1 in vitro, to explore its mechanisms and provide the foundation for effective treatment strategies against mantle cell lymphoma.

METHODSMTT assay was applied to detect the inhibitory effects of CAL-101 and bortezomib either alone or combined on Z138, HBL-2 and Jeko-1 cells. Calcusyn software was used to analyze the synergistic cytotoxicity. Western blot was used to detect the expression of PI3K-p110σ and p-Akt, Akt, p-ERK and ERK proteins after the cells were exposed to different concentrations of CAL-101. Flow cytometry was employed to assess the apoptosis rate. NF-κB kit was used to determine the changes of location of NF-κB P65, and Western blot was applied to detect the level of caswpase-3 and the phosphorylation of Akt in different groups.

RESULTSCAL-101 and BTZ inhibited the proliferation of Z138, HBL-2 and Jeko-1 cells in a dose- and time-dependent manner. CAL-101/BTZ combination induced significantly synergistic cytotoxicity in the MCL cells. The results of Western blot assay showed that CAL-101 significantly blocked the phosphorylation of Akt and ERK in the MCL cell lines. In addition, CAL-101 combined with BTZ induced pronounced apoptosis (P < 0.01). For example, after the Z138 cells exposed to the drugs for 48 h, the apoptosis rates of the control, CAL-101, BTZ and CAL-101 + BTZ groups were: (2.6 ± 1.8)%, (40.0 ± 3.0)%, (34.0 ± 1.0)%, and (67.4 ± 1.0)%, respectively; and when drug treatment was given to HBL-2 cells over 96 h, the apoptosis rates of these four cell groups were (7.4 ± 0.6)%, (30.7 ± 5.7)%, (12.0 ± 1.0)%, and (85.0 ± 4.0)%, respectively. The combination therapy contributed to the enhanced inactivity of nuclear factor-κB (NF-κB) and Akt inactivation in the MCL cell lines (P < 0.05), however, the casepase-3 activity was up-regulated.

CONCLUSIONSThe combination of CAL-101 and bortezomib is muchmore effective in inhibiting proliferation and promoting apoptosis of mantle cell lymphoma cell lines (Z138, HBL-2 and Jeko-1), which may be mediated through inhibiting PI3K/Akt signaling pathway and the transcription of NF-κB.

Antineoplastic Agents ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Boronic Acids ; Bortezomib ; pharmacology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Class Ia Phosphatidylinositol 3-Kinase ; antagonists & inhibitors ; Dose-Response Relationship, Drug ; Drug Synergism ; Formazans ; Humans ; Lymphoma, Mantle-Cell ; drug therapy ; pathology ; MAP Kinase Signaling System ; drug effects ; NF-kappa B ; metabolism ; Neoplasm Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Purines ; administration & dosage ; pharmacology ; Pyrazines ; Quinazolinones ; administration & dosage ; pharmacology ; Signal Transduction ; Software ; Tetrazolium Salts

OBJECTIVETo investigate the roles of ceruloplasmin (Cp) in PI3K/PTEN cell signaling pathway change in human embryonic lung fibroblasts (HELFs) induced by silica.

METHODSHELFs transfected with pGenesil1.1 plasmid and pGenesil1.1 with PTEN shRNA (PT) plasmid were successfully established. 100 µg/ml silica and different concentrations of Cp (10, 20, 30 µg/ml) were used in this experiment and Cp were treated cells after exposed to silica for 1h. Three different cell lines (including HELFs, PT and cells were transfected with p85 dominant negative mutant plasmid (DN-p85)) were divided into control groups, silica groups and silica+different concentrations of Cp groups. MTT assay was used to detect the effects of Cp on silica-induced cell proliferation after inhibiting PTEN and p85. When suppressing the expression of PTEN and p85, western blot assay was performed to detect the levels of p85, p110, AKT308, AKT473 and ERK, JNK and their phosphorylated levels.

RESULTSAfter inhibition of PTEN, the high levels of p85 induced by 100 µg/ml silica with 30 µg/ml Cp were markedly decreased (P<0.05). When suppressing p85, the increased cell proliferation was not observed. And the high levels of AKT308, AKT473, ERK and phosphorylated JNK and ERK stimulated by 100 µg/ml silica with 30 µg/ml Cp were decrease (P < 0.05).

CONCLUSIONCp could further strengthened silica-induced cell proliferation by PI3K/AKT/MAPK cell signaling pathway, of which the level of p85 was regulated by PTEN.

Cell Proliferation ; drug effects ; Cells, Cultured ; Ceruloplasmin ; pharmacology ; Class Ia Phosphatidylinositol 3-Kinase ; metabolism ; Fibroblasts ; drug effects ; metabolism ; Humans ; PTEN Phosphohydrolase ; metabolism ; Signal Transduction ; drug effects ; Silicon Dioxide ; toxicity

OBJECTIVETo investigate the effect of phosphatidylinositol- 3-kinases(PI3K) P85α silence on cell cycle and apoptosis of colorectal cancer cells.

METHODSFour shRNA vectors(shRNA/89, 324, 1073 and 1123) and one negative control vector were designed and stably transfected into SW480 cells. Western blotting was used to determine the expression level of P85α. Flow cytometry was used to determine the PI-labeled cycle after stable transfection. Annexin V-FITC kit was used to determine the apoptosis.

RESULTSWestern blotting analysis showed that the expression level of PI3K P85α protein was significantly decreased in cells transfected with shRNA/324 vector. The inhibition rate was 90%. The group was selected for the following experiments. G1 phase cells in the interference group and the control group were (62.4±2.7)% and (51.2±3.5)%, respectively. S phase cells in the interference group and the control group were (23.9±1.7)% and (34.1±3.4)%, respectively. Apoptosis cells induced by 5-FU of interference group and control group were(11.1±3.7)% and (1.4±0.6)%, respectively. The differences were all significant (P<0.05).

CONCLUSIONSDepletion of PI3K P85α can significantly induce SW480 cell cycle arrest and sensitize SW480 cells to 5-FU induced apoptosis. PI3K P85α may be a new therapeutic target for colorectal cancer cells.

Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Class Ia Phosphatidylinositol 3-Kinase ; genetics ; metabolism ; Colorectal Neoplasms ; metabolism ; pathology ; Humans ; RNA Interference

OBJECTIVETo research the effects and mechanism of Shenqi compound (SQC) on PTEN/ PI3K signal transducing path and angiogenesis in Goto-Kakizaki (GK) rats with diabetes mellitus type 2 (DM2) caused macroangiopathy.

METHODGK rats with blood sugar > or = 11.1 mmol/L were divided into 4 groups, the GK group, the model group, the Western medicine (WM) group treated by atorvastatin 1.5 mg/(kg x d) and the Chinese medicine (CM) group treated with SQC 1.44 g/(kg x d). All were fed 35 days with high fatty diet, but to the latter three groups, N omega-nitriyl-L-arginine methyl ester 0.1 mg/mL was added into their drinking water for macroangiopathy model establishing. Besides, a group of normal Wistar rats fed with ordinary forage was set for control. Rat's blood glucose and lipids were measured, morphology of abdominal aorta wall tissue was observed with HE staining, and mRNA expressions of PTEN and PI3Kp85 in aortic wall were detected by Real-time PCR.

RESULTSGeneral condition, gluco-lipid metabolism and aortic morphology in the CM group were significantly better than those in the model group. PTEN mRNA expression in the CM group (1.10 +/- 0.48) was significantly higher than that in the GK group (0.63 +/- 0.16) and the model group (0.17 +/- 0.07, both P < 0.01), but near to that in the WM group (1.11 +/- 0.46), while the PI3Kp85 mRNA expression in the TCM group (0.19 +/- 0.05) was lower than that in the GK group (1.38 +/- 0.43, P < 0.01), but near to that in the model group (0.33 +/- 0.09) and the WM group (0.11 +/- 0.06, both P > 0.05).

CONCLUSIONSQC could increase the PTEN mRNA expression and restrain the PI3Kp85 mRNA expression in aorta, which is possibly the partial mechanisms of action of the remedy in inhibiting angiogenesis and preventing diabetic macroangiopathy.

Angiogenesis Inhibitors ; pharmacology ; therapeutic use ; Animals ; Aorta ; metabolism ; Astragalus membranaceus ; chemistry ; Atherosclerosis ; prevention & control ; Class Ia Phosphatidylinositol 3-Kinase ; genetics ; metabolism ; Diabetes Mellitus, Type 2 ; complications ; drug therapy ; metabolism ; Diabetic Angiopathies ; drug therapy ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Male ; PTEN Phosphohydrolase ; genetics ; metabolism ; Panax ; chemistry ; Phytotherapy ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred Strains ; Rats, Wistar ; Signal Transduction ; drug effects

OBJECTIVETo explore the effect of PI3K p85alpha gene silencing on the 5-fluorouracil (5-FU)-induced apoptosis of colorectal cancer cells.

METHODSThe PI3K p85alpha/RNAi transfected cells (PI3K p85alpha/RNAi-LoVo) were cultured in RPMI 1640 supplemented with 10% fetal calf serum and 500 microg/ml G418. The 50% inhibitory concentration (IC50) values of 5-FU (0.000625, 0.00125, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32 micromol/ml) were evaluated by MTT assay. Mitochondrial membrane potential was detected by JC-1 fluorescence, and Western blotting was used to analyze the expression of apoptotic proteins Bcl-6 and Bim.

RESULTSCompared with the untransfected LoVo cells, PI3K p85alpha/RNAi-LoVo showed obviously decreased IC(50) of 5-FU (P=0.000). The mitochondrial membrane potential of PI3K p85alpha/RNAi-LoVo cells was significantly lower than that of LoVo cells, suggesting that silencing PI3K p85alpha expression increased the sensitivity of LoVo cells to 5-FU. The expression of apoptotic protein Bcl-6 and Bim were significantly higher in PI3K p85alpha/RNAi-LoVo cells treated with 5-FU than LoVo cells (P=0.000).

CONCLUSIONPI3Kp85alpha gene silencing can significantly promote 5-FU-induced apoptosis of colorectal LoVo cells.

Apoptosis ; genetics ; Cell Line, Tumor ; Class Ia Phosphatidylinositol 3-Kinase ; genetics ; metabolism ; Colorectal Neoplasms ; genetics ; pathology ; Fluorouracil ; pharmacology ; Genetic Therapy ; methods ; Humans ; RNA Interference