1.Expressions of receptor tyrosine kinases mRNA and protein in carcinoma of bladder.
Jin WEN ; Han-zhong LI ; Zhi-gang JI ; Wei-gang YAN ; Bing-bing SHI
Acta Academiae Medicinae Sinicae 2011;33(4):393-396
OBJECTIVETo detect the expressions of receptor tyrosine kinases (RTKs) mRNA and protein and to explore potentially promising tumor markers and conceivable drug target in bladder cancer.
METHODSThe expressions of RTKs mRNA and protein in tissue from invasive urothelial carcinoma of the bladder were examined by real-time quantitative PCR array and cytokine antibody array, with normal bladder tissue as control. The Results were analyzed using bioinformatic approaches.
RESULTSThe expressions of TGFA, STAB1, SERPINE1, ANGPT2, SPINK5, ANGPTL1, PROK1, MDK, CXCL9, GRN, RUNX1, VEGFA, and TGFB1 were obviously upregulated in bladder cancer tissue, while those of EDIL3, PTN, CCL2, PDGFD, FGF13, KITLG, FGF2, SERPINF1, and TNF were downregulated. ALK, Btk, EphB2, ErbB4, PDGFR-α, ROS, Tie-2, Tyk2, and VEGFR3 were over-expressed in bladder cancer, while FRK, Fyn, IGF-IR, Insulin R, Itk, JAK1, JAK3, and LCK were low-expressed.
CONCLUSIONVascular endothelial growth factor/platelet-derived growth factor-targeted therapies may play an active role in treating carcinoma of bladder.
Carcinoma, Transitional Cell ; metabolism ; Humans ; RNA, Messenger ; genetics ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism ; Urinary Bladder Neoplasms ; metabolism
2.Advance of study on Mer function.
Journal of Experimental Hematology 2007;15(4):892-895
Mer is one member of Axl receptor tyrosine kinase family, and its ligand Gas6 can stimulate activity of Mer receptor tyrosine kinase after binding it, and then activate the downstream signal transduction pathway, Mer participates in cell inflammation, apoptosis, tumorigenesis, thrombosis and hemostasis. Rencet advances of study on Mer function were reviewed, and its potential prospects of antithrombosis and antitumor were discussed in this article.
Animals
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Fibrinolytic Agents
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Humans
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Intercellular Signaling Peptides and Proteins
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metabolism
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Proto-Oncogene Proteins
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genetics
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metabolism
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Receptor Protein-Tyrosine Kinases
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genetics
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metabolism
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Signal Transduction
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c-Mer Tyrosine Kinase
3.Effects of FMS-like tyrosine kinase 3 targeted RNA interference on proliferation and apoptosis of acute monocytic leukemia cell line THP-1.
Jie LU ; Guang-yao SHENG ; Xiang ZOU ; Xue-ju XU ; Xiao-ming ZHAO ; Song-ting BAI ; Pei-rong XU
Chinese Journal of Pediatrics 2007;45(8):615-619
OBJECTIVEFMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in (70-90)% pediatric patients with acute myeloid leukemia (AML) and appears to confer an adverse prognosis. Although several FLT3-selective small molecule inhibitors and antibodies were developed with varied degrees of success, to address the specificity and resistance, new approaches for specifically targeted FLT3 are needed and RNA interference is a promising choice. The aim of the present study was to investigate the efficacy of suppression of FLT3 induced by small hairpin interfering RNA (shRNA) on myeloproliferation and apoptosis in an acute monocytic leukemia (AMOL) cell line THP-1.
METHODSFLT3-targeted small hairpin interfering RNA (FLT3-shRNA) was designed and synthesized by transcription system in vitro was transfected into THP-1 cells. Firstly FLT3 mRNA level was detected by semi-quantitative RT-PCR and FLT3 protein level was detected by flow cytometry (FCM) to verify the efficacy on FLT3-shRNA interference at 48 h after transfection. Cell growth viability was measured at 24 h, 48 h and 72 h after treatment with CCK-8. The distribution of cell cycle was assayed by FCM, and apoptosis was analyzed by DNA Ladder and Annexin V-FITC Staining at 48 h.
RESULTSFLT3 targeted shRNAs was synthesized successfully and the concentration of 15 nmol/L for 48 h could obtain desirable downregulation of FLT3 expression, the inhibitory percentages of FLT3 mRNA and protein were (72.95 +/- 2.07)% and (65.39 +/- 5.57)%, respectively. The suppression of FLT3 induced by FLT3-shRNA resulted in marked inhibition of cell growth and the inhibitory percentages were (36.66 +/- 3.67)% at 48 h, (35.56 +/- 0.73)% at 72 h. FLT3-shRNA induced the inhibition of cell cycle from G(0)/G(1) phase to S phase, the percentage of sub-G(0)/G(1) phase (65.71 +/- 4.47)% was higher than those in the PBS-control group (52.23 +/- 2.98)%, NC-shRNA control group (51.81 +/- 1.44)%, P < 0.01; the percentage of S phase (25.11 +/- 2.70)% was lower than those in the PBS-control group (34.41 +/- 4.07)% and NC-shRNA control group (32.50 +/- 1.46)%, P < 0.05. Furthermore treatment with FLT3-shRNA for 48 h resulted in clear apoptosis ladder, the percentage of early apoptosis detected by Annexin V-FITC was (18.59 +/- 2.07)% which was significantly higher than that in the PBS-control group (4.00 +/- 0.50)% and the NC-shRNA control group (6.06 +/- 0.70)%, P < 0.001.
CONCLUSIONThe suppression of FLT3 induced by the shRNA can effectively inhibit cell proliferation, and apoptosis induction on THP-1 cells, which indicates that this approach may bear the therapeutic potential on childhood AMOL.
Apoptosis ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Child ; Humans ; Leukemia, Monocytic, Acute ; enzymology ; pathology ; Protein-Tyrosine Kinases ; metabolism ; RNA Interference ; physiology ; RNA, Small Interfering ; pharmacology ; Receptor Protein-Tyrosine Kinases ; metabolism ; fms-Like Tyrosine Kinase 3 ; metabolism
4.The role of FAK-ERK signal transduction pathway in apoptosis of hepatic stellate cell.
Jian-gang SHEN ; Xiao-lan ZHANG ; Xiao-xia HUO
Chinese Journal of Hepatology 2008;16(11):849-853
OBJECTIVESTo investigate the effects of FAK-related non-kinase (FRNK) on the apoptosis of hepatic stellate cells (HSC) in vitro and on the extracellular signal-regulated kinase (ERK) signal transduction pathway.
METHODSHSC were stimulated by fibronectin (FN), and then they were transfected with FAK-related non-kinase (FRNK) plasmids mediated by cationic liposome. The apoptosis of FRNK-induced HSC was examined by annexin-V/propidium iodide double-labeled flow cytometry (FCM), gel electrophoresis and transmission electron microscopy. Levels of FRNK, FAK, p-FAK (Tyr397), ERK1 and p-ERK in HSC were assayed by Western blot on the protein level, and by RT-PCR on the mRNA level.
RESULTSThe expression of FRNK was enhanced after FRNK plasmids were transiently transfected into the HSC. The apoptotic rate of the HSC exposed to FRNK plasmids for 48 h was higher than that in the non-FRNK plasmid group (25.37%+/-1.92% vs 9.28%+/-1.05%, P less than 0.01), and was accompanied by a significantly higher activity of caspase-3 both in the protein and in the mRNA levels [(264.17+/-12.60 vs 185.82+/-9.69), P less than 0.01; (4.19+/-0.48 vs 1.07+/-0.27), P less than 0.01]. After exposure of HSC to FRNK plasmids, compared with the non-FRNK plasmid group, the expressions of p-FAK, ERK1 and p-ERK in protein and mRNA levels were lower; on the contrary, compared with the control group, the expressions of p-FAK, ERK1 and p-ERK in the FN group were higher.
CONCLUSIONThe expression of FRNK was enhanced and the phosphorylation of FAK was inhibited after FRNK was transiently transfected into HSC in vitro. FRNK induces apoptosis of HSC. FAK-ERK signal transduction pathway perhaps is involved in the process.
Apoptosis ; Cell Line ; Cell Proliferation ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hepatic Stellate Cells ; metabolism ; pathology ; Humans ; Protein-Tyrosine Kinases ; genetics ; metabolism ; RNA, Messenger ; genetics ; Signal Transduction
5.Abnormality of anaplastic lymphoma kinase gene and its expression in pediatric neuroblastoma.
Shuo CHEN ; Chunju ZHOU ; Xiaoli MA ; Liping GONG
Chinese Journal of Pathology 2014;43(8):541-545
OBJECTIVETo correlate the abnormal expression of anapastic lymphoma kinase (ALK) protein with the genetic and epigenetic changes of ALK, and to analyze its clinical application in pediatric neuroblastoma.
METHODSThree neuroblastoma (NB) cell lines (two ALK positive: SH-SY5Y and SK-N-SH, one ALK negative: SK-N-AS) and 43 paraffin-embedded NB tissues were included in the study. In both cell lines and clinical cases, immunohistochemistry was used to detect ALK protein expression; PCR and Sanger sequencing were used to detect ALK point mutation; fluorescence in situ hybridization (FISH) was used to detect ALK abnormality and bisulfite sequencing PCR (BSP) was used to detect methylation of CpG island in the promoter area of ALK.
RESULTSThe cell lines SH-SY5Y and SK-N-SH were positive for ALK expression (cytoplasm), while the SK-N-AS was negative; among the 43 cases of NB, 26 (60.5%, 26/43) were positive for ALK protein (membrane and cytoplasm), and the rest were negative. Survival analysis showed ALK protein expression was related to survival time, with ALK positive cases having shorter survival time than ALK negative cases (P = 0.020). But ALK protein expression had no association with tumor differentiation (P = 0.503), tumor sites (P = 1.000) and age of patients (P = 0.063). FISH showed ALK amplification in two cases (4.6%, 2/43), ALK gain was found in 30 cases (69.7%, 30/43), and the remaining cases had normal ALK copy (25.6%, 11/43). The presence of extra copies (amplification and gain) of ALK was associated with ALK positive protein expression (P = 0.020), but there was no association with tumor differentiation (P = 1.000), tumor sites (P = 0.775) and age of patients (P = 0.328). No point mutation was found in all three cell lines. Of the 43 NB cases, only one case (2.3%, 1/43) showed point mutation in exon 23, and was a synonymous mutation [A1200A (G4552C)]. The case was ALK negative, but the patient died two months after diagnosis. BSP analysis showed that CpG island in ALK promoter region were all unmethylated in three cell lines and 6 NB cases (including 3 ALK positive, 3 ALK negative).
CONCLUSIONSALK protein is expressed in most NB, and the expression indicates poor outcome. ALK expression is associated with extra copies of ALK, but there is no association with the methylation status of CpG island of ALK; the presence of extra copies of ALK is the most common genetic aberration in NB. Point mutation of ALK is rare, and may predict poor prognosis in pediatric NB.
Adolescent ; Cell Line, Tumor ; Child ; Exons ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Neuroblastoma ; enzymology ; genetics ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism
6.Detection of ROS1 gene rearrangement by FISH and analysis of its clinical features in non-small cell lung cancer patients.
Hongxia CHENG ; Lun YE ; Liquan XUE
Chinese Journal of Oncology 2014;36(10):751-754
OBJECTIVETo detect the frequency of ROS1 gene rearrangement in non-small cell lung cancer ( NSCLC) patients by FISH, and to analyze the relationship between ROS1 gene rearrangement and clinical features (including age, sex, stage, histology, smoking history) with NSCLC.
METHODSThe ROS1 gene rearrangement in histological sections of 1 652 NSCLC tissues was detected by FISH. The extracted RNA was amplified and the sequences were analyzed by Sanger sequencing for ROS1-positive samples.
RESULTSROS1 rearrangement was identified in 53 specimens (3.2%) from the 1 652 NSCLC tissues. Among these positive cases, 15 were CD74-ROS1, 13 were SLC34A2-ROS1, 13 were SDC4-ROS1 and 12 were TPM3-ROS1. The frequency of ROS1 rearrangement was significantly higher in never-smoking patients (49 cases) than in smokers (4 cases) (P < 0.05). Patients with ROS1-positive NSCLC tended to be younger and there was no significant difference in sex (P > 0.05). All of the ROS1-positive samples were adenocarcinomas, with a tendency toward higher clinical stage (P < 0.05).
CONCLUSIONSROS1 rearrangement has diversity, and may be defined as a new molecular subtype of NSCLC. ROS1 rearrangement tends to occur in younger, and never-smoker lung adenocarcinoma patients.
Adenocarcinoma ; genetics ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; genetics ; Oncogenes ; Protein-Tyrosine Kinases ; genetics ; Proto-Oncogene Proteins ; genetics ; metabolism
8.Effects of antisense oligonucleotides on the expression of focal adhesion kinase gene and collagen synthesis in the cultured human fibroblasts of hypertrophic scar.
Rui CHEN ; Min-Gang FU ; Yi LU ; Lin WANG ; Ping PING ; Zhi-Hong FAN
Chinese Journal of Plastic Surgery 2008;24(6):475-477
OBJECTIVETo study the role of focal adhesion kinase (FAK) in the pathogenesis of human hypertrophic scar.
METHODSHuman hypertrophic scar fibroblasts (HSFB) were isolated from human hypertrophic scar and cultured in vitro. The cells were then divided into 3 groups as AT group (phosphorothioate FAK ASODN was transfected into the HSFB by liposome), LPC group (liposome only), and LC group (control group, without liposome or ASODN). The FAKmRNA index of HSFB was assessed by polymerase chain reaction method (FQ-PCR). The collagen synthesis of HSFB was assessed by 3H-proline incorporation method.
RESULTSThe FAK mRNA index of HSFB in AT group 48 hours after transfection was significantly lower than that in LPC and LC groups (0.043 +/- 0.030, 0.124 +/- 0.070, 0.127 +/- 0.0195, P < 0.05). The 3H-proline incorporation rate in AT group was lower than that in LPC and LC groups (257.0 +/- 15.14, 962.2 +/- 300.5, 930.8 +/- 28.97, P < 0.01).
CONCLUSIONThe expression of FAK gene and collagen synthesis of the cultured HSFB could be inhibited by FAK ASODN, indicating that FAK played a role in the development of excessive fibrosis of human hypertrophic scar.
Cells, Cultured ; Cicatrix, Hypertrophic ; genetics ; metabolism ; pathology ; Collagen ; biosynthesis ; Fibroblasts ; drug effects ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; genetics ; metabolism ; Humans ; Oligonucleotides, Antisense ; genetics ; pharmacology ; Transfection
9.The expression of Tec and the level of its phosphorylation in primary hepatic carcinomas.
Chuan CHEN ; Ge WANG ; Zhi-Min ZHANG ; Wen XU ; Qiong LI ; Qing HU ; Dong WANG ; Zeng-Peng LI ; Zhi-Xiang YANG ; Jin-You SUO ; Ji-Jun ZHENG ; Hong-Zhong WANG
Chinese Journal of Hepatology 2007;15(12):910-913
OBJECTIVESTo detect the expressions of Tec tyrosine kinase in hepatocellular carcinoma and the levels of phosphorylation of tyrosine kinase in liver cancer tissues, paracancerous tissues and normal liver tissues and to find the significance of their differences.
METHODS200 specimens of tissues, including liver cancer tissues, surrounding liver tissues not more than 1.5 cm from the cancers, and normal liver tissues were investigated for Tec protein expression and Tec phosphorylation by tissue microarrays and immunohistochemistry (SP method).
RESULTSThe positive immunohistochemical stainings of Tec in cancerous tissues and non-cancerous tissues showed no obvious differences, nevertheless, the immunostaining levels in liver cancer tissues were much higher than in non-cancerous tissues and they correlated with the grading of tumors (P < 0.05). The phosphorylation of Tec was significantly expressed in liver cancer tissues (73%) in comparison with other tissues (42%, 10% both P < 0.05), but it did not correlate with any clinicopathological characteristics.
CONCLUSIONOverexpression of Tec is associated with the tumorigenesis and development of liver cancer; inhibiting Tec or degrading Tec phosphorylation directly might affect the progression of liver cancer.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Hepatocellular ; metabolism ; Female ; Humans ; Liver Neoplasms ; metabolism ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; metabolism ; Phosphorylation ; Protein-Tyrosine Kinases ; genetics ; metabolism
10.Time course degeneration and expression of glial fibrillary acidic protein in mer-knockout mice.
Xiao-Ying LIANG ; Huai-Zhou WANG ; Ning-Li WANG
Chinese Medical Journal 2010;123(7):949-953
BACKGROUNDMüller cells in the mammalian retina normally express low levels of glial fibrillary acidic protein (GFAP); however, its expression is upregulated in response to the loss of retinal neurons. The change in expression of GFAP is one of the earliest indicators of retinal damage and is correlated with the time course of disease. The aim of this study was to investigate the time course of degeneration and the expression of GFAP in the retina of mer knockout mice.
METHODSA total of 30 mer knockout mice, aged from 15 - 20 days to 1 year and 32 age-matched wild type mice as controls were tested. Immunohistochemistry was used to show the expression of GFAP in the central and peripheral retina of mer knockout and control mice at postnatal age of 15 days (P15d), 20 days (P20d), 4 weeks (P4w), 6 weeks (P6w), 8 weeks (P8w), 3 months (P3m), 6 months (P6m) and 1 years (P1y).
RESULTSThe expression of GFAP in the central and peripheral retina of wild type mice was limited to the retinal ganglion cell and nerve fiber layers. In the central retina of mer knockout mice, GFAP expression was upregulated at P4w and GFAP immunolabelling penetrates across the entire thickness of the retina at P8w; whereas in the peripheral retina, the GFAP expression was upregulated at P20d and GFAP immunolabelling penetrates the entire retina after P4w.
CONCLUSIONSIncreased expression of GFAP in Müller cells of mer knockout mice occur at P20d in the peripheral retina and P4w in the central retina. GFAP expression in Müller cells appears to be a secondary response to the loss of retinal neurons. Increased expression of GFAP may occur prior to any detectable morphological changes in the retina. This study suggests that the loss of retinal neurons may begin in the early stages of retinitis pigmentosa, prior to the discovery of any morphological changes in the retina.
Animals ; Glial Fibrillary Acidic Protein ; metabolism ; Immunohistochemistry ; Mice ; Mice, Knockout ; Proto-Oncogene Proteins ; genetics ; Receptor Protein-Tyrosine Kinases ; genetics ; Retina ; metabolism ; pathology ; Retinitis Pigmentosa ; genetics ; metabolism ; c-Mer Tyrosine Kinase