1.Rapamycin inhibits the proliferation of prostate cancer cell line 22RV1 and activity of S6K1.
Kai LIANG ; De-Xiang LU ; Ning CHU ; Jie CHENG
National Journal of Andrology 2012;18(4):327-330
OBJECTIVETo explore the effects of rapamycin on the proliferation of prostate cancer cell line 22RV1 and the activity of S6K1.
METHODSProstate cancer 22RV1 cells cultured in vitro were treated with rapamycin at the concentrations of 0, 50, 100, 200 and 400 nmol/L. The inhibition rate of the cells'proliferation was detected by MTT, and the activity of S6K1 was determined by liquid scintillation counting.
RESULTSRapamycin significantly inhibited the proliferation of the prostate cancer 22RV1 cells and the activity of S6K1 in a dose- dependent manner, most obviously at 400 nmol/L (P<0.01).
CONCLUSIONRapamycin can effectively suppress the proliferation of prostate cancer 22RV1 cells by regulating the expression of S6K1, the downstream protein of mammalian target of rapamycin (mTOR).
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Male ; Prostate ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Sirolimus ; pharmacology
2.Effect of rapamycin on proliferation of rat heart valve interstitial cells in vitro.
Yan TAN ; Ji-Ye WANG ; Ren-Liang YI ; Jian QIU
Journal of Southern Medical University 2016;36(4):572-576
OBJECTIVETo investigate the effect of rapamycin on the proliferation of rat valvular interstitial cells in primary culture.
METHODSThe interstitial cells isolated from rat aortic valves were cultured and treated with rapamycin, and the cell growth and cell cycle changes were analyzed using MTT assay and flow cytometry, respectively. RT-PCR was used to detect mRNA expression levels of S6 and P70S6K in cells, and the protein expressions level of S6, P70S6K, P-S6, and P-P70S6K were detected using Western blotting.
RESULTSRat aortic valvular interstitial cells was isolated successfully. The rapamycin-treated cells showed a suppressed proliferative activity (P<0.05), but the cell cycle distribution remained unaffected. Rapamycin treatment resulted in significantly decreased S6 and P70S6K protein phosphorylation level in the cells (P<0.05).
CONCLUSIONThe mechanism by which rapamycin inhibits the proliferation of valvular interstitial cells probably involves suppression of mTOR to lower S6 and P70S6K phosphorylation level but not direct regulation of the cell cycle.
Animals ; Blotting, Western ; Cell Cycle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Heart Valves ; cytology ; Phosphorylation ; Rats ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Sirolimus ; pharmacology
3.Effect of moxibustion on autophagy in mice with Alzheimer's disease based on mTOR/p70S6K signaling pathway.
Yang-Yang WU ; Xiao-Ge SONG ; Cai-Feng ZHU ; Sheng-Chao CAI ; Xia GE ; Ling WANG ; Yu-Mei JIA
Chinese Acupuncture & Moxibustion 2022;42(9):1011-1016
OBJECTIVE:
To investigate the effect of moxibustion on autophagy and amyloid β-peptide1-42 (Aβ1-42) protein expression in amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD).
METHODS:
After 2-month adaptive feeding, fifty-six 6-month-old APP/PS1 double transgenic AD mice were randomly divided into a model group, a moxibustion group, a rapamycin group and an inhibitor group, 14 mice in each group. Another 14 C57BL/6J mice with the same age were used as a normal group. The mice in the moxibustion group were treated with monkshood cake-separated moxibustion at "Baihui"(GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min; the mice in the rapamycin group were intraperitoneally injected with rapamycin (2 mg/kg); the mice in the inhibitor group were treated with moxibustion and injection of 1.5 mg/kg 3-methyladenine (3-MA). All the treatments were given once a day for consecutive 2 weeks. The morphology of hippocampal tissue was observed by HE staining; the ultrastructure of hippocampal tissue was observed by transmission electron microscopy; the expression of Aβ1-42 protein in frontal cortex and hippocampal tissue was detected by immunohistochemistry; the expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), p70 ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6K (p-p70S6K) protein in hippocampus were detected by Western blot method.
RESULTS:
Compared with the normal group, the number of neuron cells was decreased, cells were necrotic and deformed, and autophagy vesicle and lysosome were decreased in the model group. Compared with the model group, the number of neuron cells was increased, cell necrosis was decreased, and autophagy vesicle and lysosome were increased in the moxibustion group and the rapamycin group. Compared with the normal group, the protein expressions of Aβ1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the model group were increased (P<0.05); compared with the model group, the protein expressions of Aβ1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group, rapamycin group and inhibitor group were decreased (P<0.05); compared with the inhibitor group, the protein expressions of Aβ1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group and rapamycin group were decreased (P<0.05); compared with the rapamycin group, the protein expressions of mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group were decreased (P<0.05).
CONCLUSION
Moxibustion could enhance autophagy in hippocampal tissue of APP/PS1 double transgenic AD mice and reduce abnormal Aβ aggregation in brain tissue, the mechanism may be related to the inhibition of mTOR/p70S6K signaling pathway.
Alzheimer Disease/therapy*
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Amyloid beta-Peptides/genetics*
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Animals
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Autophagy
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Disease Models, Animal
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Hippocampus/metabolism*
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Mammals/metabolism*
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Mice
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Mice, Inbred C57BL
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Mice, Transgenic
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Moxibustion
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Ribosomal Protein S6 Kinases, 70-kDa/pharmacology*
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Signal Transduction
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Sirolimus/pharmacology*
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TOR Serine-Threonine Kinases/metabolism*
4.P70S6K is involved in the inhibition of testosterone production in TM3 mouse Leydig cells overexpressing Cox7a2.
Liang CHEN ; Jin-Ming JIA ; Wei ZHONG ; Yan-Lei ZHANG ; Jian LÜ ; Hong-Wu WANG ; Zheng ZHOU ; Ming-Xiao WANG ; Zhong-Cheng XIN ; Ying-Lu GUO
National Journal of Andrology 2011;17(4):291-295
OBJECTIVETo investigate the effects of Cox7a2 on the LH-induced testosterone production and the involved autophagy regulating signals in TM3 mouse Leydig cells.
METHODSThe Cox7a2-pEYFP-N1 fluorescent protein vector was constructed and transfected into TM3 mouse Leydig cells. The level of testosterone was determined by ELISA, and the effects of Cox7a2 on the expression of the steroidogenic acute regulatory protein (StAR) and the phosphorylation of the autophagy regulatory factor P70S6K were detected by Western blot.
RESULTSLH stimulation increased the StAR protein expression and testosterone production, while Cox7a2 decreased P70S6K phosphorylation, reduced StAR expression and consequently inhibited LH-induced testosterone biosynthesis in the TM3 Leydig cells.
CONCLUSIONCox7a2 inhibits testosterone production by decreasing the StAR protein expression, which might be at least in part related with the activation of autophagy in TM3 mouse Leydig cells.
Animals ; Autophagy ; Cells, Cultured ; Electron Transport Complex IV ; genetics ; Leydig Cells ; metabolism ; Luteinizing Hormone ; pharmacology ; Male ; Mice ; Phosphoproteins ; metabolism ; Phosphorylation ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Testosterone ; biosynthesis
5.Blockage of mTOR signaling pathway by rapamycin contributes to inhibition of tumor cell proliferation in ALK-positive lymphoid cell strains.
Ling GU ; Jin-Fan LI ; Ju GAO ; Yi-Ping ZHU ; Qiang LI ; Cang-Song JIA ; Cheng-Yan ZHOU ; Zhi-Gui MA
Chinese Journal of Hematology 2008;29(10):662-666
OBJECTIVETo investigate the relationship between mTOR signaling pathway and ALK-positive lymphoid cell lines.
METHODSThe expression of the downstream effector proteins of mTOR were analyzed by Western blot before and after Karpas299, BaF3/NPM-ALK and BaF3 cell lines treated with rapamycin. Effect of rapamycin on cell proliferation was detected by MTT assay. FACS was used to analyze apoptosis and cell cycles.
RESULTSmTOR signaling phosphoproteins, p-p70S6K and p-4E-BP1 were highly expressed in ALK(+) Karpas299, BaF3/NPM-ALK and parental BaF3 cell lines, and they were dephosphorylated after 1 h withdrawal of IL-3 in BaF3 cells. After 48 h exposure to 10 nmol/L rapamycin, p-p70S6K and p-4E-BP1 proteins expression were decreased, and mainly for the former. The relative inhibitory rate to its control cells was 24.4% in Karpas299, 37.8% in BaF3/NPM-ALK and 61.6% in BaF3. The apoptotic ratio was increased from (11.97 +/- 0.11)% to (15.87 +/- 0.62)% in Karpas299 (P < 0.05), from (3.23 +/- 0.11)% to (7.67 +/- 0.49)% in BaF3 (P < 0.05) and from (1.90 +/- 0.47)% to (2.80 +/- 0.27)% in BaF3/NPM-ALK (P > 0.05). The fraction of G(1) phase cells increased from (37.63 +/- 1.91)% to (69.77 +/- 5.44)% in BaF3/NPM-ALK, from (31.13 +/- 2.51)% to (40.70 +/- 1.47)% in Karpas299 and (53.57 +/- 2.22)% to (63.70 +/- 1.20)% in BaF3 (P < 0.05).
CONCLUSIONNPM-ALK kinase can activate mTOR signaling pathway. Rapamycin can inhibit the proliferation of ALK(+) lymphoid cells by blocking mTOR signaling pathway and inducing cell cycling arrest at G(1) phase.
Animals ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Lymphoma ; metabolism ; pathology ; Mice ; Protein-Serine-Threonine Kinases ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Receptor Protein-Tyrosine Kinases ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; drug effects ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases
6.Down-regulation of mTOR activity and survivin expression during tamoxifen-induced apoptosis in hepatoblastoma cells.
Ren-Hua GUO ; Tong-Shan WANG ; Xiao-Feng CHEN ; Zu-Hu HUANG ; Yong-Qian SHU
Chinese Journal of Oncology 2010;32(12):903-906
OBJECTIVEThe aim of this study was to investigate the changes in mTOR activity and survivin expression in liver cancer cell line HepG2 cells treated with tamoxifen.
METHODSSurvivin transcription level and p70S6K was demonstrated by PCR, dual-luciferase reporter assay and Western blot analysis, respectively, and the apoptosis in the HepG2 cells was detected by flow cytometry.
RESULTSTamoxifen leads to apoptosis of the cells and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6K. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduced the survivin protein level but not affected the survivin transcription, indicating that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells.
CONCLUSIONSOur results suggest that tamoxifen down-regulates survivin expression in HepG2 cells and it is mediated by transcriptional and post-transcriptional level via PI3K/Akt/mTOR pathway to induce apoptosis.
Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents, Hormonal ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Drug Synergism ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; metabolism ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Tamoxifen ; pharmacology
7.The effect of relgulation of PPAR-α on cardiac hypertrophy and the relationship between the effect of PPAR-α with PI3K/Akt/mTOR pathway.
Yang WU ; Bao-xia WANG ; Yuan-yuan GUO ; Yu-qin WANG
Chinese Journal of Applied Physiology 2015;31(3):284-288
OBJECTIVETo investigate the effect of peroxisiome proliferator activated receptor-α (PPAR-α) on the regulation of cardiomyocyte hypertrophy and the relationship between the effect of PPAR-α with PI3K/Akt//mTOR signal pathway.
METHODSCardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (Leica). The expressions of atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC) and PPAR-α mRNA were detected by qRT-PCR. The protein expressions of Akt, mTOR and P70S6K were detected by Western blot. The expression of PPAR-α was suppressed by RNAi.
RESULTS(1) The expression of PPAR-α was significantly reduced in cardiomyocyte hypertrophy. PPAR-α activator Fenofibrate (Feno) increased the expression of PPAR-α and suppressed cardiomyocyte hypertrophy. The inhibitory effect of Feno on cardiomyocyte hypertrophy was reversed by PPAR-α RNAi. (2) Feno significantly inhibited the increase of the protein expressions of p-Akt, p-mTOR and p-p70S6K in ISO induced cardiomyocyte hypertrophy, which could be blocked by PPAR-α RNAi. (3) PI3K antagonist LY294002 (LY) or mTOR antagonist rapamycin (RAPA) markedly-inhibited cardiomyocyte hypertrophy. The inhibitory effects of LY or RAPA on cardiomyocyte hypertrophy were reversed by PPAR-α RNAi.
CONCLUSIONPPAR-α can negatively regulate cardiomyocyte hypertrophy. The effect might be associated with PPAR-α inhiting PI3K/ Akt/mTOR signal pathway.
Atrial Natriuretic Factor ; metabolism ; Cardiomegaly ; metabolism ; Cells, Cultured ; Fenofibrate ; pharmacology ; Humans ; Isoproterenol ; adverse effects ; Myocytes, Cardiac ; drug effects ; metabolism ; Myosin Heavy Chains ; metabolism ; PPAR alpha ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism
8.Effects of rosuvastatin in homocysteine induced mouse vascular smooth muscle cell dedifferentiation and endoplasmic reticulum stress and its mechanisms.
Chang-Zuan ZHOU ; Sun-Lei PAN ; Hui LIN ; Li-Ping MENG ; Zheng JI ; Ju-Fang CHI ; Hang-Yuan GUO
Chinese Journal of Applied Physiology 2018;34(1):43-48
OBJECTIVE:
To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS).
METHODS:
VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected.
RESULTS:
Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01).
CONCLUSIONS
Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.
Actins
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metabolism
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Animals
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Calcium-Binding Proteins
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metabolism
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Cell Dedifferentiation
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drug effects
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Cells, Cultured
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Endoplasmic Reticulum Stress
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drug effects
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Heat-Shock Proteins
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metabolism
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Homocysteine
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Membrane Proteins
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metabolism
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Mice
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Microfilament Proteins
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metabolism
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Muscle, Smooth, Vascular
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cytology
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Myocytes, Smooth Muscle
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cytology
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drug effects
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Ribosomal Protein S6 Kinases, 70-kDa
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metabolism
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Rosuvastatin Calcium
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pharmacology
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TOR Serine-Threonine Kinases
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metabolism
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X-Box Binding Protein 1
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metabolism
9.Effects of mTOR siRNA on mTOR/p70S6K signaling pathway in esophageal squamous cell carcinoma cells and the growth of transplanted tumor in nude mice.
Ming-yue LIU ; Gui-qin HOU ; Yan ZHANG ; Wei-juan BEI ; Ai-hua YAN
Chinese Journal of Oncology 2011;33(5):334-339
OBJECTIVETo investigate the effects of mTOR siRNA on mTOR-p70S6K signaling pathway in esophageal squamous cell carcinoma (ESCC) cells in vitro,and growth and apoptosis in transplanted tumor in nude mice.
METHODSmTOR siRNA was transfected into ESCC cell line EC9706 cells. The expressions of factors of the mTOR/p70S6K signaling pathway were detected by RT-PCR and Western blot. DNA contents and cell apoptosis were determined by flow cytometry, and cell proliferation was measured by CCK-8 assay. The effects of mTOR siRNA on the transplanted tumor growth were assessed in nude mice.
RESULTSThe levels of mTOR and p-p70S6K were significantly decreased (P < 0.05) while the level of p70S6K was increased (P < 0.05) in the cells transfected with mTOR siRNA, compared with that in untransfected cells and cells transfected with control siRNA. After being interfered by mTOR siRNA, the number of apoptotic cells was increased, cell proliferation became slower and cell cycle was arrested in G(1) phase compared with that in control cells. Also, mTOR siRNA inhibited the growth of transplanted tumor in vivo.
CONCLUSIONSmTOR siRNA can effectively interfere in mTOR-p70S6K signaling pathway, induce cell apoptosis and inhibit cell proliferation and tumor growth, suggesting that mTOR-p70S6K signaling pathway plays an important role in the carcinogenesis and development of esophageal squamous cell carcinoma.
Animals ; Apoptosis ; Carcinoma, Squamous Cell ; enzymology ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; enzymology ; pathology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; pharmacology ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases ; genetics ; metabolism ; Transfection ; Tumor Burden
10.MicroRNA-223-3p inhibits the angiogenesis of ischemic myocardial microvascular endothelial cells via modulating Rps6kb1/HIF-1α signal pathway.
Guohua DAI ; Xianbo SONG ; Peize MA ; Ning LIU ; Jing YAO
Chinese Journal of Cardiology 2014;42(12):1039-1047
OBJECTIVETo explore the role of microRNA on the myocardial microvascular endothelial cells (CMECs) of ischemic heart rats in the process of angiogenesis and related regulation mechanism.
METHODSMyocardial ischemic rats model was established by coronary ligation.Seven days after operation, the ischemic CMECs were cultured by the method of planting myocardium tissue and identified by immunocytochemistry to observe the biological characteristics of ischemic CMECs angiogenesis, to determine the window period of migration, proliferation, tube formation in the process of its angiogenesis. Dynamic expression changes of microRNA in the process of ischemic CMECs angiogenesis was detected using microRNA chip and further verified by real-time PCR, the core microRNA of the ischemic CMECs was defined and the predicted target genes of core microRNA were determined by bioinformatics methods and real-time PCR. At the same time, the protein expression of target gene and angiogenesis related genes of p38MAPK, PI3K,Akt,VEGF were measured by Western blot.
RESULTSThe CMECs of rats presented typical characteristics of microvascular endothelial cells, and factor VIII, CD31 related antigens were all positively stained by immunocytochemical analysis. The migration window period was on the first day, and the tube formation window period was on the second day of both control and ischemic groups, while the proliferation window period was on the third day for the normal group, and the sixth day for ischemic group. According to the expressional difference and their relationship with angiogenesis, miRNA-223-3p was ultimately determined as the core microRNA in the process of ischemic CMECs angiogenesis, real-time PCR verified this hypothesis. Bioinformatics methods predicted that Rps6kb1 is the target genes of miRNA-223-3p, the pathway analysis showed that Rps6kb1 could regulate angiogenesis via HIF-1α signal pathway. Moreover, the mRNA and protein expression of VEGF, p38MAPK, PI3K,Akt, which were the downstream molecules of Rps6kb1/HIF-1α signal pathway, were also significantly downregulated in ischemic CMECs from migration and proliferation stage.
CONCLUSIONOur results show that the miRNA-223-3p is the core microRNA of ischemic CMECs angiogenesis. MiRNA-223-3p could regulate Rps6kb1/HIF-1α signal pathway, inhibit the process of migration and proliferation of ischemic CMECs angiogenesis. MiRNA-223-3p is thus likely to be a core target for enhancing angiogenesis of ischemic heart disease.
Animals ; Blotting, Western ; Endothelial Cells ; drug effects ; physiology ; Endothelium, Vascular ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; MicroRNAs ; pharmacology ; Myocardial Ischemia ; Myocardium ; Myocytes, Cardiac ; Neovascularization, Pathologic ; Phosphatidylinositol 3-Kinases ; Platelet Endothelial Cell Adhesion Molecule-1 ; RNA, Messenger ; Rats ; Ribosomal Protein S6 Kinases, 70-kDa ; biosynthesis ; Signal Transduction