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ObjectiveTo investigate the effects of maltol aluminum exposure on miR-193a-3p, demethylase AlkB homolog 5 (ALKBH5), phosphatase and tensin homolog deleted on chromosome ten (PTEN) and protein kinase B (AKT), and whether miR-193a-3p is involved in aluminum-induced cognitive impairment by regulating ALKBH5/PTEN/AKT signaling pathway. Methods Specific pathogen-free male SD rats were randomly divided into control group and low-, medium- and high- dose groups according to their body weight, with eight rats in each group. Rats in the low-, medium-, and high- dose groups were intraperitoneally injected with maltol aluminum solution at concentrations of 10.00, 20.00, and 40.00 μmol/kg body weight, respectively, while the rats in control group were given an equal volume of 0.9% sodium chloride solution. Rats were injected for five days every week for three months. After injection, the novel object recognized test was used to assess the learning and memory ability of the rats. The relative expression of miR-193a-3p and B-cell lymphocytoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cysteine aspartate protease-3 (Caspase-3) mRNA in rat hippocampus was detected using the real-time quantitative polymerase chain reaction. The relative protein expression of ALKBH5, PTEN, and AKT2 in the rat hippocampus was detected using Western blot. Results The discrimination index and the preference index of the new object recognition test of the rats in high-dose group were lower than those in control group and low-dose group (all P<0.05). The relative expression of miR-193a-3p and Bcl-2 mRNA in the hippocampus of the rats in high-dose group was lower than those in control group and low-dose group (all P<0.05). The relative mRNA expression of Bax in the high-dose group was higher than those in the control group and low-dose group (both P<0.05). The relative mRNA expression of Caspase-3 of the rats in the high-dose group was higher than that in the other three groups (both P<0.05). The relative protein expression of ALKBH5 in the hippocampus of the rats in the high-dose group was lower than that in the control group (P<0.05). The relative expression of PTEN protein was higher than those in the control group and low-dose group (both P<0.05). The relative protein expression of AKT2 was lower than those in the control group and low-dose group (both P<0.05). Conclusion Sub-chronic aluminum exposure can inhibit the expression of miR-193a-3p in the hippocampus of rats, which may disrupt the ALKBH5/PTEN/AKT pathway and affect normal neuronal homeostasis and cellular function. This pathway may play an important role in aluminum-induced cognitive impairment.
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Background Aluminum can cause synaptic plasticity damage in the hippocampus, probably due to blocked interneuronal signal transmission. MicroRNA-29a (miR-29a) can target phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and participate in the generation of neuronal networks, and may be involved in the effect of aluminum on the electrical activity of neuronal networks. Objective To study the role and mechanism of miR-29a-targeted PTEN in aluminum-induced neuronal network injury in primary hippocampal neurons of ICR mice treated with maltol aluminum [Al(mal)3] in vitro. Methods Primary hippocampal neurons of ICR mice born within 24 h were cultured in vitro. The purity of neurons was determined by labeling neuron-specific microtubule-associated protein 2 (MAP2) by immunofluorescence staining on day six of the culture; neurons were treated with different concentrations of Al(mal)3, and divided into a control group, and 10, 20, and 40 μmol·L−1 Al(mal)3 groups, and neuronal cell viability was detected by CCK-8 method. Al(mal)3 at 20 μmol·L−1 was selected for subsequent experiments to establish a neuronal network injury model for intervention. The lentivirus infection method was used to transfect miR-29a into neurons, which were divided into mNG, mNG+20 μmol·L−1 Al(mal)3, miR-29a, and miR-29a+20 μmol·L−1 Al(mal)3 groups, and micro-electrode array (MEA) was used to analyze the firing of neuronal network. The expressions of miR-29a and PTEN mRNA in each group were detected by real-time PCR (RT-PCR), and the expression of PTEN protein in each group was detected by Western blotting. Results The purity of primary mouse hippocampal neurons was greater than 90%, and the viability of the neurons was above 80% in all groups. At 48 h of the designed Al(mal)3 treatments, the changes in spike frequency, burst frequency, network burst frequency, and synchrony index of neurons cultivated on MEA plates in the control group were 207.56%±38.70%, 73.19%±46.43%, 75.42%±33.04%, and 117.13%±15.54%, respectively; the Al(mal)3 groups’ neuronal network electrical activity showed a decreasing trend. Compared with the control group, the spike frequency, burst frequency, network burst frequency, and synchrony index of the 20 and 40 μmol·L−1 Al(mal)3 groups significantly decreased (The changes were 171.70%±28.08%, 49.20%±23.23%, 50.20%±18.18%, and 85.45%±20.30%; 150.68%±26.15%, 43.43%±15.54%, 52.05%±26.31%, and 26.80%±8.29%, respectively, P < 0.05). Compared with the control group (1.00), the miR-29a relative expression levels were significantly decreased in the 20 μmol·L−1 Al(mal)3 group (0.74±0.09) and the 40 μmol·L−1 Al(mal)3 group (0.62±0.12) (P < 0.05); the relative expression levels of PTEN mRNA were significantly increased in the 20 μmol·L−1 Al(mal)3 group (1.32±0.12) and the 40 μmol·L−1 Al(mal)3 group (1.48±0.11) (P < 0.05); the PTEN protein relative expression levels (1.29±0.12 and 1.82±0.10, respectively) were also significantly increased (P < 0.05). By overexpressing miR-29a in mouse primary hippocampal neurons, the spike frequency, burst frequency, and network burst frequency were significantly higher in the miR-29a group compared with the mNG group (The changes were 252.80%±62.03%, 171.65%±56.30%, and 197.75%±27.12%, respectively, P<0.05). The mNG+20 μmol·L−1 Al(mal)3 group showed a significant decrease in all indicators of neuronal network electrical activity (The changes were 123.28%±47.31%, 66.62%±31.53%, 70.60%±12.48%, and 52.86%±20.26%, respectively, P < 0.05). Compared with the mNG+20 μmol·L−1 Al(mal)3 group, the electrical activity indicators of neuronal network were significantly higher in the miR-29a+20 μmol·L−1 Al(mal)3 group (The changes were 161.41%±42.13%, 101.16%±30.63%, 127.02%±29.58%, and 109.73%±15.61%, respectively, P < 0.05). Compared with the mNG group (1.00), the neuronal PTEN mRNA relative expression (0.67±0.11) and the PTEN protein expression (0.75±0.08) were decreased in the miR-29a group (P < 0.05); the PTEN mRNA relative expression (1.32±0.12) and the PTEN protein relative expression (1.46±0.15) in the mNG+20 μmol·L−1 Al(mal)3 group were increased (P < 0.05). Compared with the mNG+20 μmol·L−1 Al(mal)3 group, the PTEN mRNA relative expression (0.93±0.06) and the PTEN protein relative expression (0.92±0.09) were decreased in the miR-29a+20 μmol·L−1 Al(mal)3 group (P < 0.05). Conclusion Aluminum significantly inhibits the electrical activity of hippocampal neuronal networks, and miRNA-29a may be involved in the aluminum-induced impairment of hippocampal neuronal network electrical activity by regulating PTEN expression.
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ObjectiveTo study the mechanism of Shenbai Jiedu prescription inhibiting the proliferation of HCT116 colorectal cancer （CRC） cells by regulating the phosphatase and tensin homolog deleted on chromosome ten （PTEN）/phosphatidylinositol 3-kinase （PI3K）/ protein kinase B （Akt） signaling pathway. MethodShenbai Jiedu prescription was extracted by water extraction and alcohol precipitation to prepare freeze-dried powder. HCT116 cells were cultured in vitro， and treated with different concentrations of Shenbai Jiedu prescription （2， 4， 8， 16 g·L-1）. The inhibitory effect of Shenbai Jiedu prescription on the proliferation of HCT116 cells was tested by methyl thiazolyl tetrazolium （MTT）. Real-time quantitative PCR was used to detect the mRNA expression levels of PTEN， PI3K， Akt， glycogen synthase kinase-3β （GSK-3β）， c-Myc， survivin and Cyclin D1. Western blot was employed to measure the protein expression levels of PTEN， phosphorylated PTEN （p-PTEN）， PI3K， Akt， phosphorylated Akt （p-Akt）， GSK-3β， phosphorylated GSK-3β （p-GSK-3β）， c-Myc， survivin and Cyclin D1， β-catenin nuclear import was explored by immunofluorescence assay. ResultCompared with the control group， Shenbai Jiedu prescription inhibited the proliferation of HCT116 cells in a dose-dependent manner （P<0.01）. Compared with the control group， the mRNA expression levels of PTEN and GSK-3β were up-regulated whereas those of PI3K， Akt， c-Myc， survivin and CyclinD1 were down-regulated after treatment with Shenbai Jiedu prescription （P<0.01）. The protein expression levels of PTEN， p-PTEN and GSK-3β were up-regulated whereas those of PI3K， Akt， p-Akt， GSK-3β， p-GSK-3β， c-Myc， survivin and CyclinD1 were down-regulated （P<0.05， P<0.01）. Immunofluorescence assay showed that Shenbai Jiedu prescription suppressed β-catenin nuclear import in HCT116 cells. ConclusionShenbai Jiedu prescription inhibited the proliferation of HCT116 cells via the mechanism of regulating the PTEN/PI3K/Akt signaling pathway.
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Objective To investigate the expression and significance of bone morphogenetic protein?2 (BMP?2) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in incipient,recurrent and malignant salivary gland pleomorphic adenoma??Methods A total of 93 cases of salivary gland pleomorphic adenoma in Kailuan general hospital from 2008 to 2017 were collected,including 54 in incipient group (group A,47 cases in benign group A1,7 cases in malignant group A2),39 cases in recurrent group (group B,26 cases in benign group B1,13 cases in malignant group B2)??The expression of BMP?2 and PTEN were detected by immunohistochemical detection and western blot,the correlation of BMP?2 and PTEN was analyzed by Spearman analysis??Results The immunohistochemical and western blot analysis both showed expression of BMP?2 in recurrent group was significantly higher than that in incipient cases((129??03 ±15??52) vs??(87??88±18??11),t＝－8??094,P＝0??000),and it was lower in malignant cases than that in benign cases(( 100??24 ± 25??07) vs ( 116??66 ± 26??19), t＝2??125, P＝0??040)??There was no significant difference in PTEN expression between incipient and recurrent groups (( 89??15 ± 13??92 ) vs??( 96??19 ±28??02),t＝1??055,P＝0??279),but lower PTEN expression was found in malignant cases than benign cases ((76??06±11??16) vs??(109??28±17??05),t＝7??543,P＝0??000)??BMP?2 was positively correlated with PTEN expression (r＝0??313,P＜0??05)??Conclusion BMP?2 is associated with the recurrence of salivary gland pleomorphic adenoma, and both BMP?2 and PTEN are associated with malignant in the salivary gland pleomorphic adenoma??
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Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important negative regulatory molecule in the development and progression of primary liver cancer and other cancers. TCM has unique advantages in the prevention and treatment of primary liver cancer. This article reviewed the research progress in PTEN as target spot in TCM for prevention and treatment of primary liver cancer in the past 10 years, and provided references for further research and clinical application.
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Objective To explore the expression and clinical significance of PTEN and CyclinD1 protein in patients with PTC,and to provide theoretic evidence on prognosis evaluation and clinical personalized treatment.Methods 76 patients diagnosed with PCT,treated in Quhua Hospital in Zhejiang Province,from Jan.2015 to Jan.2017,were enrolled.The expression of PTEN and CyclinD1 protein was detected by EnVision two-step immunohistochemical method,and their correlation with clinical pathologic characteristic of PTC was analyzed.Results The negative rate of PTEN of the patients enrolled was 73.68％(56/76),and compared with negative rate of tumor-adjacent tissue(19.74％,15/76),demonstrated statistic significance (x2=44.429,P＜0.05).The down regulation of PTEN expression was positively related with pathological stage,tumor lymph node metastasis and sex (P＜0.05).The positive rate of CyclinD1 was 76.32％(58/76),and compared with positive rate of tumoradjacent tissue(13.16％,10/76),demonstrated statistic significance(x2=61.311,P＜0.05).The up regulation of CyclinD1 protein was positively related with pathological stage and tumor lymph node metastasis (P＜0.05).Conclusions The down regulation of PTEN and up regulation of CyclinD1 are closely related with PTC pathological stage and tumor lymph node metastasis.Detection of PTEN and CyclinD1 would be benefit to early evaluation on prognosis of PTC patients.
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OBJECTIVE@#To study whether miR-200a and miR-200b target PTEN gene expression to regulate the endometrial cancer cell growth in vitro.@*METHODS@#Endometrial cancer cells ECC-1 were cultured and transfected with the miR-200a and miR-200b mimics and inhibitors as well as the negative control mimics and inhibitors, and then the cell proliferation activity as well as the expression of PTEN and downstream genes in cells was determined; after transfection of miR-200a and miR-200b mimics as well as PTEN-3'UTR luciferase report gene plasmids, the fluorescence activity of luciferase reporter gene was determined.@*RESULTS@#12 h, 24 h and 48 h after transfection, the cell proliferation activity of miR-200a mimics group and miR-200b mimics group were significantly higher than those of NC mimics group while the cell proliferation activity of miR-200a inhibitor group and miR-200b inhibitor group were significantly lower than those of NC inhibitor group; 48 h after transfection, PTEN expression in cells and PTEN-3'UTR luciferase reporter gene fluorescence activity of miR-200a mimics group and miR-200b mimics group were significantly lower than those of NC mimics group while p-PI3K and p-Akt expression were significantly higher than those of NC mimics group; PTEN expression in cells and PTEN-3'UTR luciferase reporter gene fluorescence activity of miR-200a inhibitor group and miR-200b inhibitor group were significantly higher than those of NC inhibitor group while p-PI3K and p-Akt expression were significantly lower than those of NC inhibitor group.@*CONCLUSION@#miR-200a and miR-200b can promote the endometrial cancer cell growth in vitro by targeted inhibition of PTEN gene expression.
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Objective@#To explore the effect and mechanism of over-expression of miR-17-92 gene cluster on the biological characteristics of prostate cancer cells.@*Methods@#DU145 cells were transfected with miR-17-92 gene expression plasmid and clones with stable ectopic miR-17-92 overexpression were established. The cell viabilities of DU145-17-92 and DU145-control cells were monitored by xCELLigence system. Cell proliferation and apoptosis were analyzed by Ki-67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Cell cycle was detected by flow cytometry. Expression levels of proteins involved in apoptosis and Akt pathway were determined by western blotting.@*Results@#xCELLigence RTCA array data showed that the growth rate of DU145-17-92 cells was significantly higher than that of DU145-control cells after 24 h of seeding (P<0.01). The Ki-67-positive rates of the DU145-control group at 24, 48 and 72 hours were (56.57±1.68)%, (85.48±0.26)% and (90.85±2.08)%, respectively. While the Ki-67 positive rates of the DU145-17-92 group at the desired time points were (73.64±0.68)%, (93.43±1.23)% and (97.36±0.86)%, respectively, with a statistically significant difference at 24 hours (P<0.01). The percentages of apoptotic cells of the DU145-control group at 24, 48 and 72 hours were (6.76±0.09)%, (14.51±0.86)% and (20.73±1.64)%, respectively, while the apoptotic percentages of the DU145-17-92 group were (1.86±0.15)%, (7.90±0.40)% and (4.92±0.48)%, respectively. The percentages of apoptotic cells of the DU145-control group at different time were significantly higher than those of DU145-17-92 group (P<0.01 for all). The result of western blotting showed that the protein expression levels of Bcl-2 interacting mediator of cell death (BIM) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in DU145-control cells were 0.83±0.00 and 0.91±0.00, respectively, significantly higher than 0.16±0.00 and 0.13±0.00 of DU145-17-92 cells (both P<0.01). Overexpression of miR17-92 induced the phosphorylation of protein kinase B (Akt) at Ser473 while no appreciable effect on the phosphorylation of Akt at Thr308. The phosphorylated level of extracellular regulated protein kinases (ERK) in DU145-control cells was 0.21±0.01, significantly lower than 0.72±0.01 of DU145-17-92 cells (P<0.01).@*Conclusions@#Overexpression of miR-17-92 gene plays a pivotal role in growth, proliferation, apoptosis and cell cycle of DU145 cells through down-regulation of apoptotic protein BIM and tumor suppressor PTEN and activation of Akt and ERK signaling pathway.
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Objective To study whether miR-200a and miR-200b target PTEN gene expression to regulate the endometrial cancer cell growth in vitro. Methods Endometrial cancer cells ECC-1 were cultured and transfected with the miR-200a and miR-200b mimics and inhibitors as well as the negative control mimics and inhibitors, and then the cell proliferation activity as well as the expression of PTEN and downstream genes in cells was determined; after transfection of miR-200a and miR-200b mimics as well as PTEN-3′UTR luciferase report gene plasmids, the fluorescence activity of luciferase reporter gene was determined. Results 12 h, 24 h and 48 h after transfection, the cell proliferation activity of miR-200a mimics group and miR-200b mimics group were significantly higher than those of NC mimics group while the cell proliferation activity of miR-200a inhibitor group and miR-200b inhibitor group were significantly lower than those of NC inhibitor group; 48 h after transfection, PTEN expression in cells and PTEN-3′UTR luciferase reporter gene fluorescence activity of miR-200a mimics group and miR-200b mimics group were significantly lower than those of NC mimics group while p-PI3K and p-Akt expression were significantly higher than those of NC mimics group; PTEN expression in cells and PTEN-3′UTR luciferase reporter gene fluorescence activity of miR-200a inhibitor group and miR-200b inhibitor group were significantly higher than those of NC inhibitor group while p-PI3K and p-Akt expression were significantly lower than those of NC inhibitor group. Conclusion miR-200a and miR-200b can promote the endometrial cancer cell growth in vitro by targeted inhibition of PTEN gene expression.
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AIM:To investigate the role of extracellular signal-regulated kinase 5(ERK5) in platelet aggrega-tion in vitro and arterial thrombosis in vivo. METHODS:The expression and phosphorylation levels of ERK5 in human platelet were detected by Western blot. The effects of ERK5 selective inhibitor XMD8-92 on platelet aggregation and dense granule secretion were detected by Chrono-Log aggregometer. The effect of ERK5 on in vivo thrombosis was analyzed using an FeCl3artery thrombosis model. The effects of XMD8-92 on protein kinase B (PKB/Akt) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) phosphorylation levels were determined by Western blot. RESULTS:ERK5 was stably expressed in human platelets and its phosphorylation level increased significantly after platelet activation (P<0.05). XMD8-92,a selective inhibitor of ERK5,inhibited platelet aggregation and dense granule secretion in response to several platelet stimulators (P<0.05). The results of Western blot showed that XMD8-92 inhibited Akt phosphorylation level by down-regulating PTEN Ser370 phosphorylation and enhancing PTEN activity. The pathway was further confirmed u-sing platelet specific PTEN deficiency mice. The first occlusion time was obviously extended in the mice intravenously given XMD8-92 in the FeCl3-induced carotid artery injury model. CONCLUSION:ERK5 plays a role in platelet activation and arterial thrombosis by influencing PTEN and Akt phosphorylation.
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Objective To analyze the expression of extracellular matrix metalloproteinase inducer (CD147)and phosphatase and tensin homolog deleted on chromosome ten(PTEN)in non small cell lung cancer (NSCLC) ,and to explore the correlations be‐tween expressions of CD147 and PTEN and those with clinicopathological factors .Methods The expressions of CD147 and PTEN proteins in tissues of 64 cases of patients with NSCLC and 10 cases of normal paracancerous tissues were determined by using im‐munohistochemical SP method .The correlations between expressions of CD147 and PTEN with clinicopathological factors were ana‐lysed ,as well .Results The expression of CD147 in NSCLC tissues(75 .00% )was significantly higher than that in paracancerous tissues(0 .00% ,P<0 .05) .The expression of CD147 was strongly associated with degrees of differentiation ,lymph node metastasis and TNM stage(P<0 .05) .The expression of PTEN in NSCLC tissues (32 .81% )was significantly lower than that in paracancerous tissues(80 .00% ,P<0 .05) .Expression of PTEN was strongly associated with TNM stage (P<0 .05) .Spearman correlation analy‐sis shown that CD147 expression was negatively correlated with PTEN expression (r= -0 .442 ,P<0 .05) .Conclusion The abnor‐mal expression of CD147 and PTEN might play an important role in the malignant progression of NSCLC .
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Objective To study the expressions of phosphatese and tensin homolog deletedin chromosom ten (PTEN),Fas/FasL system and matrix metalloproteinnases-2 (MMP-2) in human gastric cancer.Methods Seventy-five cases of gastric carcinoma were selected from paraffin wax embodied specimens with full clinicopathological data,and another 15 cases of normal gastric mucosa specimens were selected as the control group.SP immunohistochemistry was used to measure the expressions of PTEN,Fas/FasL and MMP-2 in them.The data was statistically analyzed by ?2 test and relative analysis.Results The expressions of PTEN,Fas/FasL and MMP-2 were correlated with the lymphatic metastasis,degree of infiltration,clinical TMN stage and pathological histological differentiated degree of gastric cancer (P
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0.05) .The expression of PTEN and p27 in gastric cancer correlated with each other(r=0.3043,P