Effects of circular RNA-SEC31A on the invasion and migration of pancreatic cancer cells and molecular mechanism / 中华胰腺病杂志

Objective:To determine the expression of circular RNA-SEC31A(circSEC31A) in pancreatic cancer and investigate the effects on the invasion and migration of pancreatic cancer cells and the underlying molecular mechanism.Methods:Differentially expressed circRNAs between pancreatic cancer cells (BXPC-3, PANC1, CaPan-2, SW1990) and human normal pancreatic cells (HPDE) were identified by qRT-PCR. Then, two cell lines with high circSEC31A expression were selected to conduct next experiments. According to the sequence of the back-splicing site in circSEC31A, siRNAs for downregulation of circSEC31A were designed and transfected by liposome to silence circSEC31A in pancreatic cancer cells, and grouped as followed siR-circSEC31A#1 and siR-circSEC31A#2. Meanwhile, siR-NC group transfected with non-specific siRNA served as control. Transwell assays and wound healing assays were operated to evaluate the functional role of circSEC31A on the invasion and migration of pancreatic cancer cells. RNA Pull-down assay with circSEC31A probe and oligo control probe was used to screen the miRNA combining with circSEC31A and the effects of miRNA on cell invasion and migration of pancreatic cancer cells were validated. The effect of miR-200c-3p and circSEC31A silencing on the expression of PDK1 mRNA was identified by qRT-PCR. The protein expression of PDK1, downstream Akt and p-Akt after circSEC31A silencing was verified by Western blotting assays.Results:The relative expression level of circSEC31A in HPDE (1.000±0.120) was obviously lower than that in BXPC-3 (1.920±0.130), SW1990 (2.93±0.528), PANC1 (4.557±0.692) and CaPan-2 (5.247±0.194), and all the differences were statistically significant ( P<0.001). Compared with the PANC1 siR-NC group (1301.3±94.6) and CaPan-2 siR-NC group (1835.0±70.1) per 100 high power field, transwell assays showed that the numbers of invasive pancreatic cancer cells was highly decreased in PANC1 siR-circSEC31A#1 group (727.3±92.9), siR-circSEC31A#2 group (792.0±18.1), CaPan-2 siR-circSEC31A#1 group (718.0±90.6), siR-circSEC31A#2 group (692.7±84.8). Wound healing assays showed that silencing circSEC31A decreased the wound healing rate of pancreatic cancer cells in PANC1 siR-circSEC31A#1 group (20.667±3.215)%, siR-circSEC31A#2 group (20.000±4.583)%, CaPan-2 siR-circSEC31A#1 group (28.000±8.185)%, siR-circSEC31A#2 group (29.667±5.686)%, compared with the PANC1 siR-NC group (55.000±4.359)% and CaPan-2 siR-NC group (69.000±3.606)%. RNA Pull-down assays showed that compared with PANC1 oligo probe group (1.000±0.091) and CaPan-2 oligo probe group (1.000±0.153), miR-200c-3p was significantly enriched in the PANC1 circSEC31A probe group (2.237±0.175) and CaPan-2 circSEC31A probe group (2.166±0.156). Compared with PANC1 siR-NC group (939.3±57.0) and CaPan-2 siR-NC group (786.7±51.5) per 100 high power field, the numbers of invasive pancreatic cancer cells were up-regulated in PANC1 siR-miR-200c-3p group (1206.0±99.1) and CaPan-2 siR-miR-200c-3p group (1838.0±105.7), while the low numbers of invasive pancreatic cancer cells were observed in PANC1 siR-miR-200c-3p+ siR-circSEC31A group (932.7±116.4) and CaPan-2 siR-miR-200c-3p+ siR-circSEC31A group (785.3±58.8). Compared with PANC1 siR-NC group (1.000±0.103) and CaPan-2 siR-NC group (1.000±0.107), the relative expression of PDK1 mRNA in PANC1 siR-miR-200c-3p group (1.898±0.159) and CaPan-2 siR-miR-200c-3p group (2.102±0.337) was upregulated. Furthermore, the expression of PDK1 mRNA was decreased in the siR-miR-200c-3p+ siR-circSEC31A group (0.980±0.070, 1.015±0.079). Western blot assays showed that the expression of PDK1 protein in PANC1 siR-NC group, siR-circSEC31A#1 group, siR-circSEC31A#2 group was 0.767±0.086, 0.281±0.191, 0.333±0.062 and in CaPan-2 siR-NC group, siR-circSEC31A#1 group, siR-circSEC31A#2 group was 0.712±0.038, 0.353±0.061, 0.308±0.018. The expression of p-Akt protein in PANC1 siR-NC group and siR-circSEC31A group was 0.741±0.050, 0.114±0.027, 0.139±0.041. In addition, p-Akt protein expression in CaPan-2 siR-NC group and siR-circSEC31A group was 0.823±0.052, 0.141±0.045, 0.280±0.089. PDK1 and p Akt expression in siR circSEC31A group was obviously lower than those in sir NC group. All the differences between either groups above were statistically significant ( P<0.05). Conclusions:circSEC31A is upregulated in pancreatic cancer cells, which facilitates the invasion and metastasis of pancreatic cancer cells via miR-200c-3p/PDK1/Akt signaling pathway, supporting that circSEC31A may function as a new diagnostic and therapeutic target for pancreatic cancer patients.

AAZ2 induces mitochondrial-dependent apoptosis by targeting PDK1 in gastric cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Drastic surges in intracellular reactive oxygen species (ROS) induce cell apoptosis, while most chemotherapy drugs lead to the accumulation of ROS. Here, we constructed an organic compound, arsenical N-‍(4-(1,3,2-dithiarsinan-2-yl)phenyl)acrylamide (AAZ2), which could prompt the ROS to trigger mitochondrial-dependent apoptosis in gastric cancer (GC). Mechanistically, by targeting pyruvate dehydrogenase kinase 1 (PDK1), AAZ2 caused metabolism alteration and the imbalance of redox homeostasis, followed by the inhibition of phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway and leading to the activation of B-cell lymphoma 2 (Bcl2)/Bcl2-associated X (Bax)/caspase-9 (Cas9)/Cas3 cascades. Importantly, our in vivo data demonstrated that AAZ2 could inhibit the growth of GC xenograft. Overall, our data suggested that AAZ2 could contribute to metabolic abnormalities, leading to mitochondrial-dependent apoptosis by targeting PDK1 in GC.

Effect of Liangfang Wenjing Decoction on expression of key glycolytic enzymes in uterus and ovaries of rats with coagulating cold and blood stasis syndrome / 中国中药杂志

This study aimed to investigate the relationship between coagulating cold and blood stasis syndrome and glycolysis, and observe the intervention effect of Liangfang Wenjing Decoction(LFWJD) on the expression of key glycolytic enzymes in the uterus and ovaries of rats with coagulating cold and blood stasis. The rat model of coagulating cold and blood stasis syndrome was established by ice-water bath. After modeling, the quantitative scoring of symptoms were performed, and according to the scoring results, the rats were randomly divided into a model group and LFWJD low-, medium-and high-dose groups(4.7, 9.4, 18.8 g·kg~(-1)·d~(-1)), with 10 in each group. Another 10 rats were selected as the blank group. After 4 weeks of continuous administration by gavage, the quantitative scoring of symptoms was repeated. Laser speckle flowgraphy was used to detect the changes of microcirculation in the ears and uterus of rats in each group. Hematoxylin-eosin(HE) staining was used to observe the pathological morphology of uterus and ovaries of rats in each group. The mRNA and protein expressions of pyruvate dehydrogenase kinase 1(PDK1), hexokinase 2(HK2) and lactate dehydrogenase A(LDHA) in the uterus and ovaries of rats were examined by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. The rats in the model group showed signs of coagulating cold and blood stasis syndrome, such as curl-up, less movement, thickened veins under the tongue, and reduced blood perfusion in the microcirculation of the ears and uterus, and HE staining revealed a thinning of the endometrium with disorganized arrangement of epithelial cells and a decrease in the number of ovarian follicles. Compared with the model group, the treatment groups had alleviated coagulating cold and blood stasis, which was manifested as red tongue, reduced nail swelling, no blood stasis at the tail end as well as increased blood perfusion of the microcirculation in the ears and uterus(P<0.05 or P<0.01). Among the groups, the LFWJD medium-and high-dose groups had the most significant improvement in coagulating cold and blood stasis, with neatly arranged columnar epithelial cells in uterus, and the number of ovarian follicles was higher than that in the model group, especially mature follicles. The mRNA and protein expressions of PDK1, HK2, LDHA in uterus and ovaries were up-regulated in the model group(P<0.05 or P<0.01), while down-regulated in LFWJD medium-and high-dose groups(P<0.05 or P<0.01). The LFWJD low-dose group presented a decrease in the mRNA expressions of PDK1, HK2 and LDHA in uterus and ovaries as well as in the protein expressions of HK2 and LDHA in uterus and HK2 and PDK1 in ovaries(P<0.05 or P<0.01). The therapeutic mechanism of LFWJD against coagulating cold and blood stasis syndrome is related to the down-regulation of key glycolytic enzymes PDK1, HK2 and LDHA, and the inhibition of glycolytic activities in uterus and ovaries.

Apigenin inhibits liver fibrosis through PDK1/AKT signaling pathway in mice / 中国药理学通报

Aim To evaluate the therapeutic effect of apigenin on liver fibrosis in mice anrl the pharmacologi¬cal mechanism.Methods Carbon tetrachloride ( CC14) -induced liver fibrosis mouse model was estab¬lished.The mice were divided into six groups of con¬trol, model, silibinin(55 mg • kg 1 • d 1 ) , apigenin in high dosage (60 mg • kg 1 • d 1 ) , apigenin in mid¬dle dosage( 30 mg • kg 1 • d 1 ) and apigenin in low dosage( 15 mg • kg 1 • d 1 ).The general life status, body weight and liver coefficient of the mice in every group were recorded.HE staining, Masson staining, immunohistochemistry and Western blot were used to e- valuate the effect of apigenin on the pathological chan¬ges, the markers related to epithelial-mesenchymal transition and signaling pathways of liver tissues.Re¬sults In CCI4-induced liver fibrosis mice, middle and high-dosage of apigenin could improve the general life status, increase body weight, decrease liver coeffi¬ cient, and significantly improve liver lesions.Middle and high-dosage of apigenin significantly increased the expression of the epithelial marker protein E-cadherin and significantly decreased the expression of the mes¬enchymal marker protein Vimentin in liver tissues of mice with the disease.The further results showed that middle and high-dosage apigenin could significantly in¬hibit the expression of phosphorvlated PDK1 and phos- phorvlated AKT protein in liver tissues of model mice.Conclusions Apigenin can inhibit EMT by inhibiting PDK1/AKT signaling pathway, which plays an anti-fi- brosis role.The apigenin has the potential to be further developed as a drug to protect the liver and treat liver fibrosis.

The effects of PDK1-Akt signaling pathway intervention on cardiomyocyte HCN4 ion channels / 中华心血管病杂志

Objective: To explore the effects of 3-phosphate dependent protein kinase 1-protein kinase B (PDK1-Akt) signaling pathway on the transcription, expression and function of cardiac hyperpolarized activated cyclic nucleotide gated 4 (HCN4) ion channels. Methods: Atrial myocytes were obtained from healthy male wild-type C57 mice and heart-specific PDK1 knockout mice (PDK1-KO) by enzymolysis. Then the atrial myocytes were divided into blank control group and PDK1-KO group. In further studies, the isolated atrial myocytes were cultured and further divided into drug control group (treated with dimethyl sulfoxide (DMSO)) and PDK1 knockdown group (treated with 1 μg/ml PDK1 short hairpin RNA (shRNA) interference plasmid), SC79 group (treated with 8 μmol/ml SC79), GSK2334470 group (treated with 10 nmol/L GSK2334470) and PDK1 knockdown+SC79 group (8 μmol/ml SC79 and 1 μg/ml PDK1 shRNA interference plasmid). Real time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of PDK1 and HCN4, Western blot was used to detect the protein expression levels of PDK1, Akt and HCN4, the whole cell patch clamp was used to detecte the current density of HCN, and immunofluorescence was used to detecte the expression of HCN4 protein on atrial cells. Results: (1) the expression levels of HCN4 mRNA (1.46±0.03 vs. 0.99±0.01, P<0.001) and protein (1.14±0.02 vs. 1.00±0.06, P=0.017) in PDK1-KO group were higher than those in blank control group. The HCN current density in PDK1-KO group was higher than that in blank control group((-17.47±2.00) pA/pF vs. (-12.15±2.25) pA/pF, P=0.038). (2) The functions of PDK1 shRNA and specific Akt agonist SC79 were verified by comparing the PDK1 knockdown group and SC79 group with the drug control group. The results showed that the expression levels of PDK1 mRNA and protein in PDK1 knockdown group were lower than those in drug control group, and the expression level of phosphorylated Akt (Thr 308) protein in SC79 group was higher than that in drug control group. (3) The expression levels of HCN4 mRNA (3.61±0.46 vs. 1.00±0.08, P<0.001) and protein (2.33±0.11 vs. 1.00±0.05, P<0.001) in GSK2334470 group were higher than those in drug control group. (4) To reduce the effect of drug-miss target, the cultured atrial myocytes were transfected with shRNA plasmid of PDK1 and intervened with SC79. The results showed that the expression of HCN4 mRNA in PDK1 knockdown group was higher than that in the drug control group (1.76±0.11 vs. 1.00±0.06, P<0.001), and PDK1 knockdown+SC79 group (1.76±0.11 vs. 1.33±0.07, P=0.003). In PDK1 knockdown+SC79 group, the mRNA expression level was also higher than that in the drug control group (1.33±0.07 vs. 1.00±0.06, P<0.001). The expression level of HCN4 protein in PDK1 knockdown group was higher than that in drug control group (1.15±0.04 vs. 1.00±0.05, P=0.003). As for the The expression level of HCN4 protein, there was no significantly statistical difference between the PDK1 knockdown+SC79 group and the drug control group (P>0.05), but PDK1 knockdown+SC79 group was lower than PDK1 knockdown group (0.95±0.01 vs. 1.15±0.04, P<0.001). In patch clamp experiments, the results showed that the HCN current density was (-13.27±1.28) pA/pF in the drug control group, (-18.76±2.03) pA/pF in the PDK1 knockdown group, (-13.50±2.58) pA/pF in the PDK1 knockdown+SC79 group; the HCN current density of PDK1 knockdown group was higher than that of drug control group (P<0.001), but there was no significant difference between PDK1 knockdown+SC79 group and drug control group (P>0.05). (5) The results of immunofluorescence showed that the brightness of green fluorescence of PDK1 knockdown group was higher than that of drug control group, indicating that the expression of HCN4 localized on cell membrane was increased. However, the green fluorescence of PDK1 knockdown+SC79 group was lighter than that of PDK1 knockdown group, suggesting that the expression of HCN4 in PDK1-knockdown cell membrane decreased after further activating Akt. Conclusion: PDK1-Akt signaling pathway is involved in the regulation of HCN4 ion channel transcription, expression and function.

MicroRNA-4290 suppresses PDK1-mediated glycolysis to enhance the sensitivity of gastric cancer cell to cisplatin

The development of chemotherapy resistance significantly impairs the efficiency of chemotherapy, but the underlying mechanisms of chemotherapy resistance in gastric cancer (GC) are complicated and still need to be further explored. Here, we aimed to reveal the effects of miR-4290/PDK1 (pyruvate dehydrogenase kinase 1) axis on chemotherapy resistance of GC in vitro. The expression patterns of miR-4290 in GC tissues and cell lines were determined by real-time quantitative PCR. Kaplan-Meier was used to assess the relationship between miR-4290 expression levels and patients' overall survival. CCK-8 and flow cytometry technologies were applied to detect cell proliferation and apoptosis. The luciferase gene reporter assay was used to evaluate the interaction between miR-4290 and PDK1. miR-4290 was lowly expressed in GC tissues and cell lines, which was closely associated with the shorter overall survival of GC patients. miR-4290 mimics significantly inhibited cell proliferation and induced cell apoptosis, as well as induced a significant reduction in the expression of PDK1. Moreover, miR-4290 significantly inhibited glycolysis and decreased the IC50 value to cisplatin in SGC7901 cells, whereas these effects were abolished and cell apoptosis was promoted when PDK1 was overexpressed. In conclusion, this study revealed that miR-4290 suppressed PDK1-mediated glycolysis to enhance the sensitivity of GC cells to cisplatin.

A new double MEK/PDK1 inhibitor 9za retards cell cycle progression at G0/G1 phase and induces mitochondrial apoptosis in non-small cell lung cancer cells

Objective: To identify the molecule mechanism of potential cytotoxicity and proapoptosis of 9za in non-small cell lung cancer (NSCLC) cells. Methods: MTT assay, cell cycle detection kit, JC-1 staining assay were adopted to detect the cell viability, the cell cycle distribution and the mitochondrial membrane potential, respectively. Cell apoptosis was measured by Annexin V-FITC/propidium iodide apoptosis detection and the morphology was observed under a light microscope and the colorimetric TUNEL assay. Western blot was used to monitor the cell cycle-, apoptosisrelated proteins and some proteins involved in the pathways. Results: MTT assay showed that 9za significantly weakened the viability of NSCLC cells. Cell cycle analysis demonstrated that the low concentrations of 9za arrested the cell cycle at G0/G1 phase, which was further confirmed by the down-regulated levels of Cyclin D1, cyclin-dependent kinase 4 and cyclin-dependent kinase 6. In addition, Annexin V-FITC/propidium iodide apoptosis analysis, morphological observations and TUNEL assays revealed that the high concentrations of 9za could induce cell apoptosis. Furthermore, the JC-1 staining assay indicated that the mitochondrial membrane potential was reduced after 9za treatment. Western blot also manifested that 9za dramatically decreased the protein levels of the total Bcl-2, Cytochrome C in the mitochondria and BCL2 associated X in the cytoplasm. However, the levels of BCL2 associated X in the mitochondria, Cytochrome C in the cytoplasm, cleaved caspase-9, cleaved caspase-3 and the ratio of cleaved- PARP1/PARP1 showed the opposite trends. Moreover, the dose-dependently decreased phosphorylation levels of PDK1, protein kinase B, MEK and extracellular signal regulated kinase 1/2 following 9za treatment confirmed that 9za was indeed a double MEK/PDK1 inhibitor as we expected. Coadministration with PD0325901 or BX517 strengthened the cytotoxic and pro-apoptotic effect of 9za, verifying that 9za inhibited cell proliferation and induced cell apoptosis through the double MEK/PDK1 signaling pathways in NSCLC cells. Conclusions: This study demonstrates that 9za could play the effect of cytotoxicity and pro-apoptosis via the dual MEK/PDK1 signaling pathways in NSCLC cells and provides certain experimental foundation for 9za as a new type of drug candidate against non-small cell lung cancer.

The role of PDK1 in the transition of endothelial to hematopoietic cells / 中华血液学杂志

Objective: To explore the role of PDK1 in the transition of endothelial to hematopoietic cells and its effect on the generation and normal function of HSC. Methods: PDK1 was deleted specifically in endothelial cells expressing VEC (Vascular Endothelial Cadherin). CFU-C was performed to detect the effect of PDK1 on the function of hematopoietic progenitor cells using the cells from PDK1(fl/fl), PDK1(fl/+) and Vec-Cre; PDK1(fl/fl) AGM region. Hematopoietic stem cell transplantation assay was conducted to determine the effect of PDK1 on hematopoietic stem cells. Flow cytometry was performed to analyze the influence of PDK1 on percentage, cell cycle and apoptosis of CD31(+)c-Kit(high) cell population. Real-time PCR was conducted to measure the expression of transcription factors involved in process of transition from endothelial to hematopoietic cells. Results: In contrast to the wild type group, the CFU from PDK1-deficient hematopoietic progenitor cells showed smaller in morphology and fewer in quantity. CFU-GM was (24±5)/ee in knockout group, and the control group was (62±1)/ee (P=0.001). PDK1 deletion severely impaired the ability to repopulate hematopoietic cells and differentiate into committed cells. hematopoietic progenitor cells from knockout group was transplanted into 5 recipients without any recipients reconstructed. However, 5 of 7 recipients were reconstructed in control group (P=0.001). The proportion of intra-vascular clusters in the AGM was decreased (the frequency of CD31(+)c-Kit(high) in the knockout group was (0.145±0.017)%, and the control group ratio was (0.385±0.040)% (P=0.001), but not due to the inhibition of cell proliferation and/or increase of apoptosis. Further study found that the absence of endothelial PDK1 causes a decreased expression of RUNX1, P2-RUNX1, GATA2 and other important hematopoietic-related transcription factors in hemogenic cluster. Conclusion: PDK1 deletion impairs the transition of endothelial cells to hematopoietic cells as well as the generation and function of HSC.

The role of PDK1 in the transition of endothelial to hematopoietic cells / 中华血液学杂志

Objective@#To explore the role of PDK1 in the transition of endothelial to hematopoietic cells and its effect on the generation and normal function of HSC.@*Methods@#PDK1 was deleted specifically in endothelial cells expressing VEC (Vascular Endothelial Cadherin). CFU-C was performed to detect the effect of PDK1 on the function of hematopoietic progenitor cells using the cells from PDK1fl/fl, PDK1fl/+ and Vec-Cre; PDK1fl/fl AGM region. Hematopoietic stem cell transplantation assay was conducted to determine the effect of PDK1 on hematopoietic stem cells. Flow cytometry was performed to analyze the influence of PDK1 on percentage, cell cycle and apoptosis of CD31+c-Kithigh cell population. Real-time PCR was conducted to measure the expression of transcription factors involved in process of transition from endothelial to hematopoietic cells.@*Results@#In contrast to the wild type group, the CFU from PDK1-deficient hematopoietic progenitor cells showed smaller in morphology and fewer in quantity. CFU-GM was (24±5)/ee in knockout group, and the control group was (62±1)/ee (P=0.001). PDK1 deletion severely impaired the ability to repopulate hematopoietic cells and differentiate into committed cells. hematopoietic progenitor cells from knockout group was transplanted into 5 recipients without any recipients reconstructed. However, 5 of 7 recipients were reconstructed in control group (P=0.001). The proportion of intra-vascular clusters in the AGM was decreased (the frequency of CD31+c-Kithigh in the knockout group was (0.145±0.017)%, and the control group ratio was (0.385±0.040)% (P=0.001), but not due to the inhibition of cell proliferation and/or increase of apoptosis. Further study found that the absence of endothelial PDK1 causes a decreased expression of RUNX1, P2-RUNX1, GATA2 and other important hematopoietic-related transcription factors in hemogenic cluster.@*Conclusion@#PDK1 deletion impairs the transition of endothelial cells to hematopoietic cells as well as the generation and function of HSC.

Mechanism of HIF-1α-PDK1 signaling system involved in glucose metabolism and drug resistance in acute monocytic leukemia / 上海交通大学学报（医学版）

Objective • To investigate the mechanism of HIF-1α-PDK1 signaling system mediated glucose metabolism and drug resistance in acute monocytic leukemia cells. Methods • The expression of pyruvate dehydrogenase kinase 1 (PDK1) mRNA in U937, U937/DNR and acute monocytic leukemia cells was detected by quantitative polymerase chain reaction (qPCR). siRNA HIF-1α plasmid was constructed and transferred to U937 and U937/ DNR cells for 24 h by gene silencing. Cell proliferation inhibition was examined by MTT assay. The level of PDK1 mRNA was detected by qPCR, and the expression of PDK1 and multi-drug resistance gene 1 (MDR1) proteins was detected by Western blotting. Cell membrane potential was measured by flow cytometry using JC-1. Lactic acid level in the culture fluid was determined by blood gas analyzer. Dichloroacetate (DCA) and daunorubicin (DNR) were added to treat U937/DNR and acute monocytic leukemia cells for 24 h, MTT was used to calculate cell proliferation inhibition and Western blotting was used to estimate the expression of PDK1 and MDR1 proteins. Results • PDK1 mRNA was highly expressed in U937, U937/DNR and acute monocytic leukemia cells. Silencing hypoxia-inducible factor-1α (HIF-1α) significantly inhibited the proliferation activity, PDK1 and MDR1 expression and lactic acid production in U937 and U937/DNR cells. DCA could reverse the resistance to DNR in U937/DNR and relapsed acute monocytic leukemia cells. Conclusion • HIF-1α-PDK1 signaling system may regulate glucose metabolism and participate in the drug resistance of acute monocytic leukemia.

Ubiquitin ligase Smurf1 promotes poly-ubiquitination of PDK1 / 军事医学

Objective To investigate the mechanism of 3-phosphoinositide-dependent protein kinase 1(PDK1)poly-ubiquitination.Methods Co-immunoprecipitation(Co-IP)and Western blot(WB)were used to analyze poly-ubiquitination of PDK1.It was confirmed that ubiquitin ligase smad ubiquitylation regulatory factor 1(Smurf1)inprove PDK1 poly-ubiquitination within MEF cells,site-directed mutagenesis and WB before PDK1 poly-ubiquitination sites were determined.Results We found that PDK1 could undergoes poly-ubiquitination,ubiquitin ligase Smurf1 was found to be a direct E3 ligase for PDK1 poly-ubiquitination and might rely on the ubiquitin ligase Smurf 1 K699 site activity.K304 was PDK1 poly-ubiquitination modification site point.Conclusion The ubiquitin ligase Smurf1 can promate poly-ubiquitination of PDK1.

MiR-128b is down-regulated in gastric cancer and negatively regulates tumour cell viability by targeting PDK1/Akt/NF-κ B axis.

Gastric cancer (GC) is the fourth most prevalent type of cancer worldwide, which is usually caused by the interaction between environmental and genetic factors, or epigenetic aspects. Referring to the non-coding RNAs, miR-128b has been reported to be associated with many tumour cases, and exerts distinct functions in different types of cancers. However, the function of miR-128b in GC onset and progression largely remains unknown. In the present study, we found that miR-128b expression was down-regulated in tissues from 18 GC patients and 3 carcinoma cell lines. In turn, over-expression of miR-128b suppressed GC cell proliferation, invasion and promoted apoptosis. Moreover, miR-128b was predicted to bind the 3 UTR of PDK1 gene using bioinformatic target-screening tools. Accordingly, ectopic expression of miR-128b inhibited the PDK1 expression at both transcriptional and post-transcriptional levels, and furthermore, the expression of gene tailed by the 3 UTR of PDK1 gene was significantly decreased in a dualluciferase reporter assay, suggesting that PDK1 was a direct target of miR-128b in GC cells. In the conditon of miR- 128b over-expression, we also observed spontaneous inactivation of the Akt/NF-κB signalling, implying PDK1 was a potential regulator of this pathway. In conclusion, our study shed some novel light on miR-128b-PDK1/Akt/NF-κB axis on GC progression.

Therapy with radio-attenuated vaccine in experimental murine visceral leishmaniasis showed enhanced T cell and inducible nitric oxide synthase levels, suppressed tumor growth factor-beta production with higher expression of some signaling molecules

Background: Visceral leishmaniasis (VL) or Kala-Azar (KA) is one of the most deadly forms of disease among all neglected tropical diseases. There are no satisfactory drugs or vaccine candidates available for this dreaded disease. Our previous studies showed promising therapeutic and prophylactic efficacy of the live, radio-attenuated parasites through intramuscular (I.M.) and intraperitoneal (I.P.) route in BALB/c mice model. Methods: The T-cell proliferation level, the mRNA expression level of inducible nitric oxide synthase (iNOS) and tumor growth factor-beta (TGF-β) genes and finally the phosphorylation levels of phosphoinositide dependent kinase 1 (PDK1), phosphoinositide 3 kinase (PI3K) and p38 mitogen activated protein kinase (p38MAPK) molecules were checked in BALB/c mice model immunized with radio-attenuated Leishmania donovani parasites through I.M. route. Results: Higher T-cell proliferation, increased iNOS level, and suppressed TGF-β level were found in treated infected animal groups (100 and 150 Gy) in relation to untreated infected animals. Likewise, phosphorylation levels of PDK1, PI3K and p38MAPK of these two groups were increased when compared to untreated infected controls. Conclusion: The clearance of the parasites from treated infected groups of animals may be mediated by the restoration of T-cell due to therapy with radio-attenuated L. donovani parasites. The killing of parasites was mediated by increase in nitric oxide release through PDK1, PI3K and p38MAPK signaling pathways. A lower TGF-β expression has augmented the restored Th1 ambience in the 100 and 150 Gy treated animal groups proving further the efficacy of the candidate vaccine. .

Abnormal vascular endothelial cell junctions caused by PDK1 deficiency / 实用医学杂志

Objective To reveal the role of PDK1 in vascular endothelial cells. Methods PDK1 was knocked out in endothelial cells by recombinase-mediated Cre to observe it′s effects on vascular endothelial cells. Results Abnormality of vascular development in rats could be found as a result of endothelial PDK1 deletion. Meanwhile, vascular leakage and bleeding phenotype were observed, and tissue analysis showed vascular endothelial cells abnormalities. Conclusions PDK1 plays an important role in the functioning and integrity of vascular endothelial cells, which made tentative explanation for PDK1-Akt signal path and laid basis for further research.

Effect of Gymnema Sylvestre on Protein Kinase B in Adipose Tissues of Insulin Resistance Mice / 世界科学技术-中医药现代化

This article was aimed to study effects and mechanisms of Gymnema sylvestre on protein kinase B (PKB) and its phosphorylation in adipose tissues of KKAy mice which were mainly characterized by insulin resistance (IR). A total of 18 KKAy mice were randomly divided into the diabetes model (DM) group and Gymnema sylvestre (GS) group according to body weight levels. And 9 normal C57BL/6J mice were used as the normal control (NC) group. Intragastric administration of medication was given to mice for 8 weeks. At the end of the experiment, all animals were tested for fasting plasma glucose (FPG) and fasting insulin level (Fins) for evaluation of insulin sensitivity index (ISI). Expressions of phosphoinositide-dependent kinase-1 (PDK1), PKB, P-PKB (Ser473), P-PKB (Thr 308) in adi-pose tissues of epididymis were determined. The expression of phosphatase and tensin homolog (PTEN) mRNA was also determined. The results showed that compared with the DM group, the GS group showed lower FPG and Fins, higher ISI. The expression of P-PKB (Ser473) phosphorylation and P-PKB (Thr 308) were increased, and the PDK1 and PTEN mRNA were decreased. It was concluded that GS can improve insulin sensitivity of KKAy mice through activating PKB by up-regulate the expression of P- PKB (Ser473) and its phosphorylation ratio and P- PKB (Thr 308) in adipose tissues.