Expression Of Phosphorylated Protein Kinase Research Articles

SSRP1 affects the growth and apoptosis of gastric cancer cells through AKT pathway.

BackgroundWe aimed to determine the SSRP1's potential influence on the apoptosis and proliferation of gastric cancer (GC) cells and its regulatory mechanism.MethodsSSRP1 expression in GC cells and tissues was detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The interrelation between clinicopathological characteristics of GC patients and SSRP1 expression was analysed via x2 test, and the correlation between SSRP1 expression and overall survival rate was analysed using Kaplan-Meier survival analysis. After the knockdown of SSRP1 in AGS cells, the SSRP1 expression, colony formation ability, cell viability, cell cycle changes, apoptosis rate, and migration and invasion ability were detected through qRT-PCR, colony formation assay, CCK8 assay, flow cytometry and transwell test, respectively. Finally, the effects of down-regulation of SSRP1 on the expressions of phosphorylated-protein kinase B (p-AKT), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were explored using Western blotting.ResultsSSRP1 displayed a high expression in GC cells and tissues. SSRP1 expression was closely interrelated to the TNM stage, lymph node metastasis and tumour size. The survival rate of patients was markedly shorter in the high expression group than in the lower expression group. After the knockdown of SSRP1 in cells, the viability and colony formation ability of AGS cells were inhibited. In addition, the cell ratio in the G1 phase was increased, while that in the S phase declined, and the cell invasion and migration were obviously weakened. It was found from Western blotting that the knockdown of SSRP1 could evidently suppress the protein levels of Bcl-2 and p-AKT but promote the protein expression of Bax, indicating that silencing SSRP1 can inhibit the proliferative capacity and increase the number of GC cells through inactivating the AKT signalling pathway.ConclusionsSSRP1 rose up in GC tissues and cells. Reduction of SSRP1 can inhibit the proliferative capacity and increase the number of GC cells through inactivating the AKT signalling pathway.

MiR-19a suppress apoptosis of myocardial cells in rats with myocardial ischemia/reperfusion through PTEN/Akt/P-Akt signaling pathway.

To detect differentially expressed micro ribonucleic acids (miRNAs) in rats with myocardial ischemia/reperfusion (MIR), and to explore the influence of miR-19a on MIR rats and its mechanism. Firstly, the Sprague-Dawley (SD) rats were used to prepare MIR models, RNAs were extracted, and miRNA sequencing analysis was carried out to determine differentially expressed miRNAs related to MIR. Secondly, the predicted target genes of miR-19a were collected, and WebGestalt was applied to analyze gene ontology (GO) and pathway enrichment. Thirdly, the expression of the related proteins and the apoptosis of myocardial cells in MIR rats were detected via Western blotting. Fourthly, the interaction between miR-19a and the target gene phosphatase and tensin homolog (PTEN) was examined through Luciferase reporter assay. Compared with that in the Sham operation (Sham) group, the miR-19a expression in rat myocardial tissues in the MIR group was significantly increased (p<0.05). Compared with those in the miR-negative control (miR-NC) group, the messenger RNA (mRNA) and protein expressions of PTEN in the miR-19a group were notably decreased (p<0.05). In comparison with the miR-NC group, miR-19a group had elevated expression of phosphorylated protein kinase B (p-Akt) (p<0.05). The Luciferase reporter gene assay manifested the direct binding of miR-19a to PTEN mRNA. MiR-19a inhibits the PTEN expression by directly binding to the 3'-UTR of PTEN mRNA, thus activating the Akt/p-Akt signaling pathway to suppress the apoptosis of myocardial cells in MIR injury.

Expression and significance of phosphorylated protein kinase B and spleen tyrosine protein kinase in different TNM stages of gastric cancer patients

Objective To investigate the expression of phosphorylated protein kinase B (PKB/AKT) and spleen tyrosine protein kinase (Syk) in different tumor node metastasis (TNM) stages of gastric cancer patients. Methods From January 2015 to April 2018, 82 patients with gastric cancer confirmed by gastroscopy and surgical pathology were enrolled in this study. All patients were selected for cancer tissue, and 30 patients were randomly selected from normal gastric mucosa at 5 cm adjacent to the tumor. Immunohistochemistry was used to detect the expression of PKB/AKT and Syk protein. To compare the expression of PKB/AKT and Syk in gastric cancer and adjacent normal tissues, and to analyze the relationship between PKB/AKT and Syk expression and clinicopathological features in gastric cancer tissues, and the correlation between PKB/AKT and Syk and TNM staging of gastric cancer patients. Results The positive expression rate of PKB/AKT in gastric cancer tissues was higher than that in adjacent tissues (P<0.05). The positive expression rate of Syk was lower than that in adjacent tissues (P<0.05). PKB/Akt and Syk gene expression in gastric cancer were related to histological grade, tumor infiltration, TNM staging, lymph node metastasis and distant metastasis (P<0.05), and the expression of PKB/AKT was significantly increased with the increase of TNM staging in gastric cancer patients, and the positive expression of Syk was significantly decreased (P<0.05). Conclusions PKB/AKT is positively correlated with TNM staging of gastric cancer patients. Syk is negatively correlated with TNM staging of gastric cancer patients. The clinical expression of PKB/AKT and Syk can be used to determine the TNM staging of gastric cancer, which provides a strong basis for tumor treatment. It is of great significance in treatment. Key words: Stomach neoplasms; Neoplasm staging; Phosphorylation; Proto-oncogene proteins c-akt; Syk kinase

MiR-126 promotes endothelial cell apoptosis by targeting PI3K/Akt in rats with lower limb arteriosclerosis obliterans.

To investigate the influence of micro ribonucleic acid (miR)-126 on the rats with lower limb arteriosclerosis obliterans (ASO). Male Sprague- Dawley rats aged 3 months old were randomly divided into Sham operation group (Control group, n=10) and Model group (n=10), and the model of lower limb ASO was established. After modeling, the expression of miR-126 in arteries was detected using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and the change in the downstream signaling pathway was examined via Western blotting. The human umbilical vein endothelial cells (HUVECs) were induced by oxidized low-density lipoprotein (Ox-LDL) to establish the model of endothelial injury, followed by detection of miR-126 expression. Then, the Luciferase assay was performed to verify the downstream target gene of miR-126. After being cultured, HUVECs were set as Control group, Ox-LDL induction group, and Ox-LDL + miR-126 inhibitor group, and the expressions of phosphorylated-protein kinase B (p-Akt) and cleaved cysteine-aspartic protease-3 (Caspase-3) were detected in the above groups. After the establishment of the model, the expression level of miR-126 was raised in vessels, but the phosphatidylinositol 3-hydroxy kinase (PI3K)/Akt signals were weakened (p<0.01). Ox-LDL-induced endothelial cell apoptosis promoted the expression of miR-126, and the difference was statistically significant. The bioinformatics analysis results showed that PI3KR2 was a direct target of miR-126, which was also proven via the Luciferase assay. Moreover, the transfection with miR-126 inhibitor into endothelial cells suppressed Ox-LDL-induced cell apoptosis, thereby persistently activating the PI3K/Akt signaling pathway (p<0.01). In rats with lower limb arteriosclerosis obliterans (ASO), miR-126 represses the PI3K/Akt signaling pathway to accelerate endothelial cell apoptosis.

Role and mechanism of circular RNA-vimentin in the proliferation and apoptosis of colorectal cancer cells

Objective To investigate the role and mechanism of circular RNA-vimentin (circ-VIM) in the proliferation and apoptosis of colorectal cancer cells. Methods From December 2016 to December 2017, at Department of General Surgery of The First Affiliated Hospital of Zhengzhou University, the clinical data of 100 patients who underwent radical resection of colorectal cancer and were confirmed by pathological examination after operation were collected. The tumor tissues and corresponding paracancerous tissues (negative control) were also collected. The expression of circ-VIM in the colorectal cancer tissues was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation of HCT-116 and HT29 colorectal cancer cells was detected by cell counting kit-8 assay. The ratio of apoptosis of HCT-116 and HT29 cells was measured by annexin Ⅴ/propidium iodide double staining assay. The mitochondrial membrane potential of HCT-116 and HT29 cells was examined by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl-imidacarbocyanine iodide (JC-1) assay. The expression changes of protein kinase B and mammalian target of rapamycin were tested by Western blotting. The target miRNA of circ-VIM was predicted by miRDB software. T-test and chi-square test were performed for statistical analysis. Results The expression of circ-VIM in colorectal cancer tissues was 2.387±0.536, which was higher than that in corresponding paracancerous tissues (1.110±0.134), and the difference was statistically significant (t=23.096, P<0.01). And the expression levels of circ-VIM were significantly different in patients with different tumor size, TNM stage and lymph node metastasis (all P<0.05). The proliferation of HCT-116 cells and HT29 cells in lenti-circ-VIM group was 0.737±0.023 and 0.835±0.025, respectively, which were both higher than those in control group (0.449±0.020 and 0.531±0.019), and the differences were statistically significant (t=20.706 and -15.374, both P<0.01). The proliferation of HCT-116 cells and HT29 cells in lenti-circ-VIM-shRNA group was 0.236±0.027 and 0.243±0.019, which were lower than those in control group, and the differences were statistically significant (t=24.557 and -23.197, both P<0.01). The ratio of apoptosis of HCT-116 cells and HT29 cells in lenti-circ-VIM-shRNA group was (18.00±1.82)% and (20.80±0.61)%, which was higher than those in control group ((6.64±2.01)% and (7.35±1.36)%), and the differences were statistically significant (t=8.826 and 17.454, both P<0.01). The fluorescence intensity ratio of JC-1 aggregate and JC-1 monomer of HCT-116 cells and HT29 cells in lenti-circ-VIM-shRNA group was 2.21±0.12 and 1.40±0.11, which was lower than those in control group (14.54±1.00 and 9.24±1.18), and the differences were statistically significant (t=-19.558 and -15.685, both P<0.01), which indicated mitochondrial membrane potential decreased. After treated with lenti-circ-VIM-shRNA, the expression of phosphorylated protein kinase B, phosphorylated mammalian target of rapamycin, B-cell lymphoma-2 and mitochondrial cytochrome C at protein level were all down-regulated, however the expression of cytoplasmic cytochrome C, B-cell lymphoma-2 associated X protein and cleaved caspase-3 at protein level were all up-regulated. When the expression of circ-VIM was up-regulated, the expression of miRNA-147b, miRNA-4447 and miRNA-3656 was down-regulated. When the expression of circ-VIM was down-regulated, the expression of miRNA-147b, miRNA-4447 and miRNA-3656 was up-regulated. Conclusion The expression of circ-VIM in colorectal cancer is abnormally increased, which is involved in the proliferation and apoptosis of colorectal cancer cells. Key words: Colorectal neoplasms; Cell proliferation; Apoptosis; circ-VIM

The oxamate effect and mechanism in inhibiting the proliferation of anaplastic thyroid cancer

Objective To evaluate the antitumor effect of oxamate as a lactate dehydrogenase (LDH) inhibitor on Anaplastic human thyroid cancer, and to explore the therapeutic effect of inhibiting cancer metabolism on Anaplastic thyroid cancer (ATC). Methods Immunohistochemistry were used to detect the expression of LDHa in 12 cases of human ATC and adjacent tissues. Methyl thiazol tetrazolium (MTT) assay and plate cloning assay was used to detect the effect of Oxamate on the proliferation of ATC cell lines. The effects of Oxamate on the activity of LDH and the formation of lactate were detected by lactate dehydrogenase kit and lactate detection kit. Western blotting was used to detect changes in protein expression related to cell proliferation. Results Immunohistochemical staining showed that the positive rates of LDHa protein in cancer and adjacent tissues were 46.0% and 14.1% respectively, and the difference was statistically significant (P=0.004) LDH inhibitor oxamate was used to treat Hth83 and SW1736 cell lines in 0, 5, 10, 20, 40, 60, 80, 100 mmol/L, MTT assay showed that the 50% growth inhibitory concentration (IC50) of the Hth83 cell group was 64.3, 24.4, and 10.1 mmol/L, while in the SW1736 cell group, the IC50 values were 82.5 and 51.2, 25.3 mmol/L at 24, 48, and 72 h. The clone formation test indicated that two cell lines clone formation ability was also significantly declined, and the degree of decrease was positively correlated with the drug concentration (P=0.012). This inhibition was accompanied by a decrease in the activity of lactate dehydrogenase and the production of lactate. Western blotting analysis showed that the expression of phosphorylated protein kinase B (p-Akt), Cyclin D1, and phosphorylated glycogen synthase kinase 3β (p-GSK-3β) was down-regulated. Conclusion LDHa is highly expressed in human ATC tissues. Oxamate, as an inhibitor of LDH enzyme, can decrease the proliferation potential of Hth83 and SW1736 cell lines. The possible molecular mechanism is that the Akt-Cyclin D1 protein pathway is inhibited. Key words: Oxamate; Lactate dehydrogenase; Thyroid carcinoma; Proliferation

Effect of recombinant human annexin A5 on expression of p-PKCα and p120-catenin during endotoxin-induced damage to cardiomyocytes

Objective To evaluate the effect of recombinant human annexin A5 on the expression of phosphorylated protein kinase C alpha (p-PKCα) and p120-catenin during endotoxin-induced damage to cardiomyocytes. Methods H9c2 cells cultured in vitro were randomly divided into 3 groups (n=18 each) using a random number table: control group (group C), endotoxin group (group L), and recombinant human annexin A5 group (group A). Recombinant human annexin A5 (final concentration 5 ng/ml) was added, and the cells were incubated for 2 h in group A, and then lipopolysaccharide (final concentration 1 μg/ml) was added, and the cells were incubated for 4 h in L and A groups.At 4 h of incubation, cell apoptosis was detected using the cell apoptosis detection kit, the intercellular space was measured using the confocal microscopy, and the expression of p-PKCα and p120-catenin was determined by Western blot.The apoptosis rate was calculated. Results Compared with group C, the apoptosis rate was significantly increased, the intercellular space was significantly widened, the expression of p120-catenin was significantly down-regulated, and the expression of p-PKCα was significantly up-regulated in group L (P<0.05). Compared with group L, the apoptosis rate and intercellular space were significantly decreased, the expression of p120-catenin was significantly up-regulated, and the expression of p-PKCα was significantly down-regulated in group A (P<0.05). Conclusion Recombinant human annexin A5 can inhibit phosphorylation of PKCα and up-regulate the expression of p120-catenin, thus attenuating endotoxin-induced damage to cardiomyocytes. Key words: Annexin A5; Endotoxins; Myocytes, cardiac; Protein kinase C-alpha; Connexins

Expression and significance of p-AKT, p-GSK3β and β-catenin in epithelial carcinoma of ovary

To investigate the expressions of phosphorylated protein kinase B (p-AKT), phosphorylated glycogen synthase kinase 3β (p-GSK3β) and β-catenin proteins and to evaluate their relationship with the clinical pathological characteristics in epithelial tumors of the ovary. The expression of p-AKT, p-GSK3β, and β-catenin was detected with immunohistochemical staining (EnVision method) in 10 cases of benign epithelial neoplasia, 10 cases of borderline epithelial neoplasia and 70 cases of ovarian carcinoma. The relationship of the expression of p-AKT, p-GSK3β and β-catenin with the clinical pathological features was analyzed. The positive expression rates of p-AKT, p-GSK3β and β-catenin in epithelial ovarian carcinoma were 67.1% (47/70), 60.0% (42/70) and 71.4% (50/70), respectively. Compared to the results of benign and borderline epithelial neoplasia, the expression of the three proteins in carcinoma of the ovary was significantly different (all P < 0.05).Positive correlation was found between p-AKT and p-GSK3β, p-GSK3β and β-catenin, and p-AKT and β-catenin in epithelial ovarian carcinoma (r = 0.546, 0.581, 0.500, respectively; all P < 0.05). Compared to the results of benign and borderline epithelial neoplasia, the expression of p-AKT protein in epithelial ovarian carcinoma was significantly different (all P < 0.05). The expression of p-AKT was correlated with the differentiation of epithelial ovarian carcinoma (P < 0.05), but no relationship was found between its expression and histological classification and FIGO staging (P > 0.05). The expression of p-GSK3β and β-catenin in epithelial ovarian carcinoma were both higher than that in benign and borderline epithelial neoplasia (P < 0.05), and correlated with tumor differentiation and FIGO staging (P < 0.05), but no relationship were found between their expression with histological classification (P > 0.05). Positive correlations are found between p-AKT, p-GSK3β and β-catenin in epithelial ovarian carcinoma. The activation of β-catenin is possibly correlated with inactivation of p-GSK3β that binds to p-AKT.