PTEN Protein Expression Levels Research Articles

Overexpression of SP1 restores autophagy to alleviate acute renal injury induced by ischemia-reperfusion through the miR-205/PTEN/Akt pathway

BackgroundAcute kidney injury (AKI) is a major kidney disease with poor clinical outcome. SP1, a well-known transcription factor, plays a critical role in AKI and subsequent kidney repair through the regulation of various cell biologic processes. However, the underlying mechanism of SP1 in these pathological processes remain largely unknown.MethodsAn in vitro HK-2 cells with anoxia-reoxygenation injury model (In vitro simulated ischemic injury disease) and an in vivo rat renal ischemia-reperfusion injury model were used in this study. The expression levels of SP1, miR-205 and PTEN were detected by RT-qPCR, and the protein expression levels of SP1, p62, PTEN, AKT, p-AKT, LC3II, LC3I and Beclin-1 were assayed by western blot. Cell proliferation was assessed by MTT assay, and the cell apoptosis was detected by flow cytometry. The secretions of IL-6 and TNF-α were detected by ELISA. The targeted relationship between miR-205 and PTEN was confirmed by dual luciferase report assay. The expression and positioning of LC-3 were observed by immunofluorescence staining. TUNEL staining was used to detect cell apoptosis and immunohistochemical analysis was used to evaluate the expression of SP1 in renal tissue after ischemia-reperfusion injury in rats.ResultsThe expression of PTEN was upregulated while SP1 and miR-205 were downregulated in renal ischemia-reperfusion injury. Overexpression of SP1 protected renal tubule cell against injury induced by ischemia-reperfusion via miR-205/PTEN/Akt pathway mediated autophagy. Overexpression of SP1 attenuated renal ischemia-reperfusion injury in rats.ConclusionsSP1 overexpression restored autophagy to alleviate acute renal injury induced by ischemia-reperfusion through the miR-205/PTEN/Akt pathway.

Transferred by exosomes-derived MiR-19b-3p targets PTEN to regulate esophageal cancer cell apoptosis, migration and invasion.

The present study aims to investigate the relationship between miR-19b-3p and esophageal cancer (ESCA), and to detect the effects of miR-19b-3p transferred by exosomes on the phenotype of EC9706 cells. The expression of miR-19b-3p was detected by starBase analysis and real-time quantitative PCR (RT-qPCR). The target genes of miR-19b-3p were predicted by TargetScan and further verified by luciferase analysis. The mRNA and protein expression levels of PTEN and EMT-related genes were detected by RT-qPCR and Western blotting. The effects of miR-19b-3p transferred by exosomes and its target genes on the apoptosis, migration and invasion of EC9706 cells were studied by establishing a co-culture model of donor cells. The expression of miR-19b-3p in ESCA plasma, cells and exosomes was significantly up-regulated. miR-19b-3p transferred by exosomes could significantly reduce EC9706 cells apoptosis rate, promote cell migration and invasion, and could target the inhibition of PTEN expression. PTEN overexpression promoted apoptosis, inhibited cell migration and invasion, down-regulated the expression of MMP-2 and vimentin, and up-regulated E-cadherin expression; however, these effects could be partially reversed by miR-19b-3p. In summary, our results reveal that miR-19b-3p transferred by exosomes can target PTEN to regulate ESCA biological functions in the receptor EC9706 cells.

LINC00861 inhibits the progression of cervical cancer cells by functioning as a ceRNA for miR‑513b‑5p and regulating the PTEN/AKT/mTOR signaling pathway.

Long non-coding RNAs (lncRNAs) have been discovered to serve important roles in a variety of types of cancer, including cervical cancer. The low expression of lncRNA long intergenic non-protein coding RNA 861 (LINC00861) is related to poor prognosis in ovarian cancer. However, the effects and underlying mechanisms of LINC00861 in cervical cancer remain largely unknown. The present study aimed to examine the role of LINC00861 in the development and progression of ovarian cancer and its underlying mechanisms. The expression levels of LINC00861 and microRNA (miR)-513b-5p were analyzed using reverse transcription-quantitative PCR analysis. Cell proliferation, migration and invasion were measured by using Cell Counting Kit-8, colony formation, wound healing and Transwell assays, respectively. A luciferase assay was used to determine whether miR-513b-5p targeted LINC00861 and PTEN. The expression of protein was measured by using western blot assay. The results of the present study discovered that LINC00861 expression levels were significantly downregulated in cervical cancer tissues and CaSki and ME-180 cell lines. Downregulated LINC00861 expression levels were identified to be associated with an advanced-stage, lymph node metastasis and the poor survival of patients with cervical cancer. Gene Set Enrichment Analysis revealed that the PI3K/AKT/mTOR signaling pathway was significantly enriched in cervical tumors expressing low expression levels of LINC00861 compared with tumors expressing high levels of LINC00861. The overexpression of LINC00861 reduced cervical cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) processes, upregulated PTEN protein expression levels and downregulated phosphorylated (p)-AKT and p-mTOR protein expression levels. The regulatory relationship between LINC00861, microRNA (miR)-513b-5p and PTEN was validated using a dual luciferase reporter gene assay. PTEN expression levels were significantly downregulated in the miR-513b-5p mimic group and significantly upregulated in the miR-513b-5p inhibitor group compared with the mimic NC and inhibitor NC in both cell lines. Furthermore, LINC00861 was suggested to serve as a competing endogenous RNA by sponging miR-513b-5p and consequently upregulating the expression levels of PTEN in cervical cancer cells. The expression of PTEN, the phosphorylation of Akt and mTOR and and the EMT phenotype were rescued following co-transfection with LINC00861 and miR-513b-5p mimics. In conclusion, the findings of the present study indicated that the LINC00861/miR-513b-5p axis may inhibit the progression of cervical cancer cells through the PTEN/AKT/mTOR signaling pathway to suppress the EMT process.

Low PTEN expression and overexpression of phosphorylated AktSer473 and AktThr308 are associated with poor overall survival in upper tract urothelial carcinoma.

The PI3K/Akt signaling pathway serves an essential role in various cellular processes, including cell growth, survival, cell motility, angiogenesis and cell metabolism. Loss of PTEN expression and hyperactivation of Akt can result in tumorigenesis. Previous studies observed expression of the Akt protein and absence of the PTEN protein in bladder cancer and non-small cell lung carcinoma tissues. The aim of the present study was to evaluate the expression status and prognostic value of PTEN and the PI3K/Akt signaling pathway in Taiwanese patients with upper tract urothelial carcinoma (UTUC). Archival formalin-fixed, paraffin-embedded (FFPE) tissues from 65 UTUC cases were stained via immunohistochemistry for PTEN, phosphorylated (p)Akt serine (Ser)473 and pAkt threonine (Thr)308. The expression levels of each protein were significantly correlated with clinicopathological parameters. PTEN, pAktSer473 and pAktThr308 protein expression levels were higher in adjacent normal tissues compared with those in tumor tissues. Cytoplasmic PTEN protein expression levels were lower in high-stage tumors compared with those in low-stage tumors, and nuclear and cytoplasmic pAktThr308 protein expression levels were higher in high-grade tumors compared with those in low-grade tumors. Univariate analysis showed that high pathological tumor stage (pT2-4) [P=0.01; hazard ratio (HR)=3.40; 95% confidence interval (CI), 1.34–8.60], metastatic status (P=0.003; HR=3.55, 95% CI, 1.55–8.11), low cytoplasmic PTEN protein expression levels (P=0.016; HR=3.14; 95% CI, 1.24–7.95) and high cytoplasmic pAktSer473 protein expression levels (P=0.019, HR=2.71, 95% CI, 1.18–6.21) were predictive of poor overall survival. However, only metastatic status (P=0.031; HR=2.73; 95% CI, 1.10–6.78), low cytoplasmic PTEN protein expression levels (P=0.017; HR=3.29; 95% CI, 1.24–8.73) and high cytoplasmic pAktSer473 protein expression levels (P=0.027; HR=2.64; 95% CI, 1.12–6.23) remained significant in the multivariate analysis. Kaplan-Meier survival analysis showed that high T stage, metastasis, low expression levels of cytoplasmic PTEN protein and high expression levels of cytoplasmic pAktSer473 protein were significantly associated with poor survival (P=0.006, 0.001, 0.011 and 0.014, respectively). Co-expression of PTENlow/pAktSer473/high and pAktThr308/high phenotypes was associated with a less favorable overall survival (P=0.001). Overall, the present findings demonstrated that low expression levels of PTEN and high expression levels of pAktSer473 and pAktThr308 were predictors for poor overall survival in patients with UTUC.

Up-Regulation of PI3K/Akt Signaling Pathway in H929 Cell Line by Doxycycline Not Beneficial to Its Anti-Myeloma Effect

To investigate the effect and possible mechanism of up-regulation of p-Akt by doxycycline (DOX) on myeloma cell line H929. Multiple myeloma cell line H929 was treated with DOX at different concentrations for different times, and cell proliferation rate was measured by CCK-8 assay. The protein expression level of p-Akt, PTEN, p-PDK1, p-mTOR, p-GSK-3β, and p-BAD was analyzed by Western blot. The mRNA levels of mTOR, BCL-2, and NF-κB was analyzed by RT-PCR. PI3K inhibitor Wortmannin was used to antagonize the up-regulation of p-Akt, and the cell proliferation and p-Akt protein expression level were analyzed by CCK-8 assay and Western blot respectively. DOX could inhibit the proliferation of H929 cells and up-regulate the expression of p-Akt at the same time. The protein levels of both p-PDK1 and PTEN in H929 cells did not alter significantly during DOX treatment. The expressions of p-BAD and p-GSK-3β were up-regulated in H929 cells after treated with DOX, but the expression of p-mTOR was not altered. The mRNA levels of mTOR, BCL-2, and NF-κB in H929 were all down-regulated in H929 cells during DOX treatment. The effect up-regulating p-Akt level by DOX was suppressed when DOX combined with PI3K inhibitor Wortmannin and Wortmannin could enhance the inhibitory effect of DOX in H929 cells. DOX can activate PI3K/Akt signaling pathway in H929 cells, and antagonizing this effect of DOX may enhance its cytotoxicity to myeloma cells.

Effect of salvianolic acid B on high-glucose induced renal tubular epithelial-mesenchymal transition in rats and its mechanism

The aim of this paper was to observe the effect of salvianolic acid B(Sal B) on high-glucose induced renal tubular epithelial-mesenchymal transition(EMT) in rats, and to explore its possible mechanisms of prevention and treatment of diabetic nephropathy. The rat renal tubular epithelial NRK-52 E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+10 μmol·L~(-1)Sal B group(Sal B), the above 3 groups were set at 6, 12, 24 and 48 h for dynamic observation; high glucose+Sal B different concentration(1, 5, 10 μmol·L~(-1)) groups, high glucose+5.0 μmol·L~(-1) pioglitazone group, high glucose+10 μmol·L~(-1)Sal B+5 μmol·L~(-1)GW9662 group. The protein expression levels of PPARγ, PTEN, α-SMA, E-cadherin and PI3 K/Akt signaling molecules were determined by Western blot. The mRNA expression of PPARγ and PTEN were detected by Real-time PCR. The viabi-lity of NRK52 E cells was determined by MTT assay. The results showed that as compared with control group, the mRNA and protein expression levels of PPARγ and PTEN in high glucose group gradually reduced, the protein expression levels of α-SMA and p-Akt~((Thr308))gradually increased, and the protein expression of E-cadherin gradually reduced(P<0.05). As compared with high glucose group, when increases in Sal B doses, the mRNA and protein expression levels of PPARγ, PTEN in high glucose + different concentrations of Sal B groups gradually increased, the protein expression levels of α-SMA and p-Akt~((Thr308)) gradually reduced, and the protein expression of E-cadherin gradually increased(P<0.05), however, the effect of 1 μmol·L~(-1)concentration of Sal B on the expression of PPARγ mRNA and protein and PTEN mRNA was not significantly different. As compared with high glucose group, the mRNA and protein expression levels of PPARγ mRNA(except 6 h) and protein(except 6 h), PTEN mRNA(except 6 h) and protein(except 6, 12 h) kept increasing, the protein expression levels of α-SMA and p-Akt~((Thr308))(except 6 h) continued to reduce, the protein expression of E-cadherin kept increasing in high glucose+10 μmol·L~(-1) Sal B dynamic observation group(P<0.05). As compared with high glucose group, Sal B and the pioglitazone(PIO) can greatly enhance the expression of PPARγ, PTEN at mRNA and protein levels, enhance the expression of E-cadherin at protein levels, and reduce the expression of α-SMA, p-Akt~((Thr308))protein level(P<0.05), there was no significant difference between the two groups. However, the expression levels of PPARγ and PTEN mRNA and protein, E-cadherin, α-SMA and p-Akt(Thr308) protein in the Sal B+GW9662 control group were not statistically significant compared with the high glucose group. The effect of Sal B was blocked by the PPARγ antagonist GW9662. It can be concluded that Sal B can suppress the NRK52 E cells induced by high-glucose EMT. The mechanism may be related to the activation of PPARγ with Sal B, and the up-regulation of PTEN expression, and thereby inhibiting the fibrosis effect of PI3 K/Akt signaling pathway.

MTMR14 protects against cerebral stroke through suppressing PTEN-regulated autophagy

The phosphoinositide phosphatase, myotubularinrelated protein 14 (MTMR14), plays a critical role in the regulating autophagy. However, its functional contribution to neuronal autophagy is still unclear. In the present study, we attempted to explore the effects of MTMR14 on ischemic stroke progression, as well as the underlying molecular mechanisms. Oxygen-glucose deprivation/reoxygenation (OGDR)-induced primary cortical neurons and pheochromocytoma (PC12) cells, and middle cerebral artery occlusion (MCAO)-operated mice were used to establish cerebral ischemia/reperfusion (I/R) injury in vitro and in vivo, respectively. OGDR treatment markedly decreased the expression of MTMR14 expression from mRNA and protein levels in the cultured primary neurons and PC12 cells. Functional analysis showed that OGDR-reduced cell viability was further accelerated by MTMR14 knockdown. On the contrary, MTMR14 over-expression significantly rescued the cell survival in OGDR-exposed cells. Moreover, autophagic markers including LC3BII and Beclin 1 were highly up-regulated in OGDR-incubated neurons and PC12 cells, while being further exacerbated by MTMR14 deletion. However, promoting MTMR14 dramatically alleviated LC3BII and Beclin 1 expression levels stimulated by OGDR. Importantly, we found that MTMR14-regulated autophagy was through its interactions with phosphatase and tensin homolog (PTEN). MTMR14 negatively modulated PTEN protein expression levels in OGDR-exposed cells. In vivo, MCAO-operated mice exhibited significantly reduced expression of MTMR14 in the ischemic penumbra tissues. After MCAO operation, MTMR14 over-expression effectively reduced infarct volume and neurological deficits scores, along with decreased activation of LC3B in neurons. Consistently, MCAO-increased PTEN, LC3BII and Beclin 1 were repressed by MTMR14 in mice. An interaction between MTMR14 and PTEN in response to MCAO was confirmed in vivo. Together, these results indicated the neuroprotective effects of MTMR14 on modulating PTEN-dependent excessive autophagy during cerebral I/R injury. Thus, targeting MTMR14 may provide feasible therapy for ischemic stroke onset and progression.

Ginsenoside Rg3 inhibits the biological activity of SGC-7901.

AimTo explore the suppressive effects of ginsenoside Rg3 on the biological activities of gastric cancer and the mechanisms responsible therein, by conducting an in vitro study.Materials and MethodsSGC‐7901 gastric cancer cells were divided into NC, DMSO, Gin‐Low (10 mg/L), Gin‐Middle (20 mg/L), and Gin‐High (40 mg/L) groups. Using MTT, flow cytometry, transwell, and wound‐healing assays, the cell biological activities in the different groups were evaluated; the protein expression levels of PTEN, p‐PI3K, AKT, and P53 were measured by Western blot assay, and p‐PI3K nuclear volume was evaluated by immunofluorescence.ResultsThe SGC‐7901 cell proliferation rate was depressed significantly, and cell apoptosis increased significantly while cells were arrested in the G1 phase (p < .05) with ginsenoside Rg3 treatment in a dose‐dependent manner (p < .05). Meanwhile, the SGC‐7901 cell invasion number and wound‐healing rate of ginsenoside Rg3‐treated groups were significantly downregulated compared with those of the NC group, also in a dose‐dependent manner (p < .05). PTEN and P53 protein expression levels were significantly increased, and p‐PI3K and AKT protein expression levels were significantly depressed in ginsenoside Rg3‐treated groups in a dose‐dependent manner (p < .05).ConclusionGinsenoside Rg3 suppresses gastric cancer via regulation of the PTEN/p‐PI3K/AKT pathway.

Anti-Cancer Effects of miR-181/PTEN/PI3K/Akt Signaling Pathway Suppressed Cell Proliferation and Migration of Human Cervical Cancer

Cervical cancer ranks the second in the incidence of common women malignant tumors, which is the commonest malignant tumor in women's reproductive organs. In present research, we evaluated the possible antitumor effect of miR-181 on cervical cancer and explored the molecular mechanism of this phenomenon. Briefly, MTT, LDH activity kit and flow cytometry were used to detect cell growth, LDH activity and cell apoptosis rate of HeLa cells. The experiment correlation protein expression levels of PTEN, phosphatidylinositol-3-hydroxykinase (PI3K) and phosphorylation (p)-Akt (protein kinase B) were measured by Western blot analysis. In present research, this research observed that miR-181 weakened the cell proliferation activity of cervical cancer cells and induced their caspase3/9 to promotion of cell apoptosis. PTEN expression was induced at the genetic and protein levels, and the PI3K/Akt expressions were inhibited at the protein levels in cervical cancer cells. SiPTEN weakened the antitumor effect of miR-181 on cervical cancer by activation of PI3K/Akt signaling pathway.

LncRNA MEG3 inhibits proliferation and promotes apoptosis of synovial cells in rats with knee osteoarthritis by regulating PTEN.

To study the influences of long non-coding ribonucleic acid (lncRNA) maternally expressed gene 3 (MEG3) on the proliferation and apoptosis of synovial cells in rats with knee osteoarthritis by regulating phosphate and tension homology deleted on chromosome ten (PTEN). In this experiment, rat synovial cell (RSC)-364 cells were cultured in vitro. Then, they were treated with PBS or lncRNA MEG3 overexpression lentiviruses and divided into normal control (NC) group and lncRNA MGE3 overexpression group (LncRNA MEG3 group). The messenger RNA (mRNA) expression levels of lncRNA MEG3 and PTEN in rat synovial cells were measured via qRT-PCR in each group, and Western blotting (WB) was performed to determine the protein levels of PTEN, cyclin D1, P21, B-cell lymphoma 2 (Bcl-2) and tubulin in rat synovial cells in both groups. The proliferation of rat synovial cells was detected via MTT assay, and the apoptosis was evaluated using FITC/PI double staining and flow cytometer. Compared with NC group, LncRNA MEG3 group had notably overexpressed lncRNA MEG3 in RSC-364 cells (p<0.01), and an extremely substantially elevated mRNA level of PTEN (p<0.05). Besides, it was found through WB that the protein expression level of PTEN had a consistent trend with that of the mRNA level. The proliferation ability of cells was weakened (p<0.05), and the number of apoptotic cells was increased (p<0.05) in LncRNA MEG3 group compared with those in NC group. Finally, LncRNA MEG3 group had remarkably lower protein levels of cyclin D1 and Bcl-2, but a markedly higher protein level of P21 than NC group (p<0.05). LncRNA MEG3 can raise the level of PTEN to weaken the proliferation ability but elevate the apoptosis level of RSC-364 cells.

MicroRNA-23a knockdown attenuates angiotensin Ⅱ induced hypertrophy in rat H9c2 cells via activating PTEN and AMPK pathway

Objective: To investigate if microRNA (miR) -23a knockdown could attenuate angiotensin Ⅱ(AngⅡ) induced cardiac hypertrophy by activating phosphatase and tensin homolog deleted on chromosome ten(PTEN) and AMP-activated protein kinase(AMPK) pathway. Methods: Rat H9c2 cells were cultured in DMEM high glucose medium and put in 5% CO(2) incubator at 37 ℃(normal group). After 48 hours of culture, H9c2 cells were stimulated with 10 nmol/L AngⅡ to establish cell hypertrophy model (AngⅡgroup). The H9c2 cells were inoculated in a 6-well cell culture plate and cultured in an incubator at 37 ℃. When the confluence degree of cell growth was about 70%, the cells were transfected with different reagents, and 24 hours after transfection, 10 nmol/L AngⅡ was used to interfere with the cells. The H9c2 cells were divided into different groups according to the reagents, namely AngⅡ+anti-miR group(transfected with miR-23a inhibitor), Ang Ⅱ+NC group(transfected with miR-23a inhibitor negative control), Ang Ⅱ+anti-miR+si-PTEN group(cotransfected with miR-23a inhibitor and PTEN small interference RNA(siRNA)), and AngⅡ+anti-miR+si-NC group(cotransfected with miR-23a inhibitor and PTEN siRNA negative control). The surface area of single cell was measured by Image J software.The mRNA expression levels of α-actin 1 (ACTA1) and β-myosin heavy chain (β-MHC) and miR-23a were detected by quantitative real-time PCR(qRT-PCR). The expression levels of PTEN and AMPK signal pathway related proteins were detected by Western blot. In order to verify whether miR-23a targets PTEN gene, double luciferase reporter gene experiment was performed. The luciferase reporter gene vector recombinant plasmids of wild type pGL-WT-PTEN and mutant pGL-MUT-PTEN were constructed and prepared after normal sequencing. H9c2 cells was inoculated into 24-well cell culture plate and cultured overnight in 37 ℃ incubator. The cells were co-transfected with miR-23a mimic or miR-23a mimic negative control and wild type or mutant reporter gene recombinant plasmid. Forty-eight hours after transfection, firefly luciferase activity and sea kidney luciferase activity were measured, and the ratio of them was recorded as relative luciferase activity. Results: Compared with the normal group, the cell surface area, the mRNA expression levels of ACTA1, β-MHC and miR-23a were significantly higher, while the protein expression levels of PTEN and p-AMPK were significantly lower in the Ang Ⅱ group(all P<0.05). The results of double luciferase reporter gene assay showed that the relative luciferase activity of cells co-transfected with miR-23a mimic and wild-type reporter gene recombinant plasmid was lower than that of miR-23a mimic negative control (P<0.05), and PTEN served as the target gene of miR-23a. In AngⅡ+anti-miR group the mRNA expression levels of miR-23a, ACTA1 and β-MHC were lower, and the cell surface area was smaller, while the protein expression levels of PTEN and p-AMPK were higher than that in AngⅡ group and AngⅡ+NC group(all P<0.05). Compared with AngⅡ+anti-miR group, the cell surface area was bigger, the expression of ACTA1 and β-MHC mRNA was up-regulated, and the protein expression levels of PTEN and p-AMPK were down-regulated in Ang Ⅱ+anti-miR+si-PTEN group(all P<0.05). Conclusion: Inhibition of miR-23a can attenuate Ang Ⅱ-induced hypertrophy in H9c2 cells through targeting PTEN and activating AMPK signaling pathway.

Overexpression of PTEN regulated by miR-19b and miR-494 in the villous of recurrent spontaneous abortion patients

Recurrent spontaneous abortion (RSA) can be attributed to multiple factors, and abnormal invasion and apoptosis of trophoblast cells have attracted extensive attention. Our study aimed to investigate the expression of PTEN and miRNAs with potential regulatory relationships in the placental villi of RSA patients. Nineteen RSA patients and sixteen healthy women at reproductive age undergoing induced abortion (IA) were enrolled in the present study. The expression of PTEN and miRNAs were investigated using real-time polymerase chain reaction (PCR) and Western blotting, further verification between PTEN and potential miRNAs used cell culture and transfection, and luciferase activity assays were used to determine whether PTEN is directly regulated by potential miRNA. The results indicated that both PTEN mRNA and protein expression levels were upregulated in RSA patients, but a significant difference was only observed in protein expression level (p < 0.001). Through real-time PCR pre-scanning, the results of nine potential miRNAs revealed three significantly upregulated miRNAs (miR-494, miR-146a, and miR-21) and one significantly downregulated miRNA (miR-19b). The results of further verification regarding miR-19b and miR-494 suggested that upregulated miR-19b, cooperating with downregulated miRNA-494, could inhibit PTEN expression. In conclusion, the findings suggest that the overexpression of PTEN plays an important role in the pathogenesis of RSA, with miR-19b directly regulating PTEN and working with miR-494, all of which participating in abnormal effects of villous’ trophoblastic cell may be a critical event.

MicroRNA‑28 promotes the proliferation of non‑small‑cell lung cancer cells by targeting PTEN.

Non-small-cell lung cancer (NSCLC) is the fundamental form of lung cancer and the leading cause of cancer‑related mortality in humans. Numerous studies have identified a role for microRNAs (miRs) in cell proliferation, invasion and metastasis in numerous types of cancer, including lung cancer. In the present study, the functional roles and molecular mechanisms of miR‑28 in NSCLC tumorigenesis were investigated. Reverse transcription‑quantitative PCR (RT‑qPCR) was used to measure miR‑28 expression levels in NSCLC tumor tissues and cell lines. A dual‑luciferase assay was performed to observe the direct interaction between miR‑28 and PTEN in A549cells. Furthermore, the effect of miR‑28 on the mRNA and protein expression levels of PTEN was examined by RT‑qPCR and western blotting, respectively. A Cell Counting kit‑8 assay was performed to identify the relationship between the miR‑28/PTEN axis and tumor cell proliferation using cells infected with lentivirus (LV)‑anti‑miR‑28 or LV‑anti‑miR‑28+short hairpin RNA‑PTEN. miR‑28 expression was upregulated in NSCLC tumor tissues and cell lines compared with the control groups. PTEN was identified as the downstream gene of miR‑28 in NSCLC and was negatively regulated by miR‑28. In addition, miR‑28 knockdown suppressed the proliferation of A549 and H292cells. Cells infected with LV‑anti‑miR‑28+short hairpin RNA‑PTEN promoted tumor cell proliferation in A549 and H292cells compared with cells infected with LV‑anti‑miR‑28. Taken together, the present study suggested that miR‑28 might serve as the promoter in the development of NSCLC by targeting PTEN. Therefore, the miR‑28/PTEN axis may serve as a potential diagnostic and therapeutic target for NSCLC.

Study on the mechanism of inhibiting invasion of human laryngeal squamous cell carcinoma Hep-2 and TU212 cells after the downregulation of miRNA-106b

Objective To investigate the effect of miRNA-106b (miR-106b) on human laryngeal squamous cell carcinoma Hep-2 and TU212 cells and its mechanism. Methods Hep-2 and TU212 cells were divided into miR-106b inhibitory sequence transfected group (the experimental group), miR-106b competitive negative sequence transfected group (the negative control group) and non-intervention group (the blank group). The inhibitory effect of miR-106b inhibitory sequence on the expression of miR-106b was verified by using reverse transcription quantitative polymerase chain reaction (qRT-PCR). Whether phosphatase and tensin homolog (PTEN) was the target gene of miR-106b was analyzed by using bioinformatics and luciferase report vector. PTEN small interfering RNA (siRNA) was used to inhibit the expression of PTEN in Hep-2 and TU212 cells. Transwell method and Western blot were used to detect the change of invasion ability of Hep-2 and TU212 cells after miR-106b silencing or the PTEN intervening, and the expression change of PTEN, epithelial cadherin and vimentin. Results The relative expression levels of miR-106b in Hep-2 and TU212 cells in the experimental group were 0.110 ± 0.037 and 0.074 ± 0.009, respectively, which were lower than those in the negative control group (1.013±0.059 and 1.035±0.062, respectively; all P < 0.05). In Transwell experiments, the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group was less than that in the negative control group [(37.09±4.02) vs. (95.65±4.77), (29.16±2.49) vs. (103.19±6.08), all P < 0.05]. The bioinformatics analysis results showed that 3'-UTR region of PTEN mRNA was complementary to 3'-UTR region of miR-106b. Dual-luciferase reporter system analysis showed that the luciferase reporter activity of wild-type PTEN gene transfected with miR-106b was decreased to (22.84±2.68)%, and that of mutant PTEN gene transfected with miR-106b was almost unchanged [(92.08±3.44)%], and the difference was statistically significant (P < 0.001). The expression level of PTEN protein of Hep-2 and TU212 cells in the experimental group was higher than that in the negative control group. Transwell method showed that the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group with the inhibition of PTEN expression was more than that in the experimental group without the inhibition of PTEN expression [(65.08±3.57) vs. (26.72±2.58), (57.38±4.96) vs. (31.81±2.97), all P < 0.05]. Western blot showed that the expression level of epithelial-cadherin was up-regulated and vimentin was down-regulated of Hep-2 and TU212 cells in the experimental group with the inhibition of PTEN expression. Conclusions The human laryngeal squamous cell carcinoma Hep-2 and TU212 cell miR-106b can influence the downstream invasion-related protein of PTEN and change the cell invasion ability through the targeted regulation of PTEN expression. Key words: Laryngeal neoplasms; Carcinoma, squamous cell; Neoplasm invasiveness; RNA interference; miRNA-106b; Phosphatase and tensin homolog

Molecular Characteristics, Phylogeny and Expression Profile of the PTEN Gene in Goats

Phosphatase and the tensin homologue deleted on chromosome ten (PTEN) has pleiotropic effects on cell growth, organ development, glucose metabolism and insulin resistance in mammals. In the present study, we investigated the molecular characteristics, phylogeny and expression profile of the PTEN gene in different tissues of Jianzhou Daer goats. In this study, eight different tissues from E90, E135 and D90 female goats were collected to quantify the expression pattern of the PTEN gene using quantitative real-time PCR (qPCR), western blotting and FISH. In addition, the dynamic expression of PTEN was also determined during the differentiation of goat precursor adipose cells. A 1212-bp fragment (accession number MG923848), encoding a 403-amino acid protein with a putative molecular weight of 47.14kDa, was identified in Jianzhou Daer goats by reverse-transcription polymerase chain reaction (RT-PCR). The phylogenetic tree showed that caprine PTEN had a relatively close relationship with ovine PTEN and bovine PTEN. qPCR revealed that PTEN was highly expressed in the liver, lung and spleen, while the lowest expression levels were observed in muscle tissues (P < 0.05). Moreover, the expression of the PTEN gene showed a decreasing trend during the differentiation of goat precursor adipose cells. RNA in situ hybridization yielded a consistent result with the qPCR data. Indeed, low protein expression was found in psoas major muscle and longissimus dorsi muscle, as well as in kidney and liver. However, PTEN protein was expressed at the highest level in the brain. The expression levels of PTEN mRNA and protein were inconsistent with each other, possibly because of post-transcriptional regulation. The findings obtained in our study lay a foundation for further investigations examining the caprine PTEN gene in embryo and organ development.

MiR‑21 promotes osteoclastogenesis through activation of PI3K/Akt signaling by targeting Pten in RAW264.7 cells.

The present study aimed to investigate the effects of microRNA (miR)-21 on osteoclastogenesis and its underlying molecular mechanisms. The expression levels of tartrate-resistant acid phosphatase (TRAP) and miR-21 were detected during osteoclastogenesis in receptor activator of NF-κB ligand (RANKL)-induced RAW264.7 cells via reverse transcription-quantitative PCR. Bioinformatics and dual luciferase reporter assays were performed to analyze the association between miR-21 and Pten. RANKL-induced RAW264.7 cells were divided into the following groups: MiR-negative control (NC), miR-21 mimic, miR-21 inhibitor and miR-21 mimic + LY294002. The effects of miR-21 on osteoclastogenesis and bone resorption were then detected using TRAP staining and a bone resorption assay. Pten, phosphorylated-Akt and nuclear factor of activated T cell (NFATc1) expression levels were measured by western blotting to analyze the effects of miR-21 on the PI3K/Akt signaling pathway. The present study revealed that miR-21 was upregulated during osteoclastogenesis in RANKL-induced RAW264.7 cells. Furthermore, miR-21 negatively regulated Pten. Compared with the miR-negative control (NC) group, the number of osteoclasts and the percentage of bone resorption were increased in the miR-21 mimic group, whereas they were decreased in the miR-21 inhibitor group. The number of osteoclasts and the percentage of bone resorption in the miR-21 mimic + LY294002 group were lower than in the miR-21 mimic group. Compared with the miR-NC group, the protein expression levels of Pten were decreased, whereas p-Akt and NFATc1 were increased in the miR-21 mimic group. Conversely, Pten protein expression was increased, whereas p-Akt and NFATc1 were decreased in the miR-21 inhibitor group. In the miR-21 mimic + LY294002 group, Pten protein expression was higher, and p-Akt and NFATc1 were lower than in the miR-21 mimic group. In conclusion, miR-21 is upregulated during osteoclastogenesis, and may promote osteoclastogenesis and bone resorption through activating the PI3K/Akt signaling pathway via targeting Pten.

IRS1/PI3K/AKT pathway signal involved in the regulation of glycolipid metabolic abnormalities by Mulberry (Morus alba L.) leaf extracts in 3T3-L1 adipocytes

BackgroundMulberry (Morus alba L.) leaf tea benefits the control of diabetes in Asian nations. This study was aim to investigate if the flavonoids, which extracts from mulberry leaves, could regulate the metabolism of glycolipid, and to investigate if flavonoids could regulate IRS1/PI3K/AKT pathway signal to affect the expression of FAS and membrane transfer capacity GLUT4 in 3T3-L1 adipocytes.ResultsResults revealed that flavonoids decreased the levels of free fatty acid and increased the glucose consumption and the levels of adiponectin and leptin in a dose-dependent manner, and remarkably increased the protein expression levels of p-IRS1, p-PI3K, p-Akt, total GLUT4, and membrane GLUT4, and decreased the protein expression levels of PTEN and FAS in 3T3-L1 adipocytes IR model. On the other hand, wortmannin (2 nM), a selective and irreversible PI3K inhibitor, significantly decreased the glucose consumption and the adiponectin and leptin levels, and increased the free fatty acid level in flavonoids treated 3T3-L1 adipocytes IR model. Furthermore, wortmannin (2 nM) partly eliminated the activation of PI3K/AKT signaling, the suppression of FAS, and the up-regulated membrane transfer capacity of GLUT4 in flavonoids treated 3T3-L1 adipocytes IR model.ConclusionIn conclusion, our results illustrated that mulberry leaf extracts flavonoids alleviated the glycolipid metabolic abnormalities in 3T3-L1 adipocytes IR model, and the effect was associated with the activation of IRS1/PI3K/AKT pathway, the suppression of FAS, and the up-regulation of membrane transfer capacity of GLUT4.

Inhibition of PTEN Gene Expression by Small Interfering RNA on PI3K/Akt/FoxO3a Signaling Pathway in Human Nasopharyngeal Carcinoma.

The objective of this article is to study the effect of inhibiting phosphatase and tensin homolog deleted chromatosome 10 gene on phosphoinositide 3-kinase/protein kinase B (Akt)/Forkhead homeobox O3a signaling pathway in human nasopharyngeal carcinoma HK-1 cells. Nasopharyngeal carcinoma HK-1 cell lines were divided into PTEN gene interference group (siPTEN), nonspecific small interfering RNA group (siNC), empty vector group (Vector), and no transfection control group (Normal). The mRNA and protein expression levels of PTEN, PI3K, p-Akt, and FoxO3a were detected by real-time fluorescence quantitative polymerase chain reaction and Western blot. Immunofluorescence was used to detect the subcellular localization of PTEN, PI3K, p-Akt, and FoxO3a in HK-1 cells. The proliferation of HK-1 cells was detected by MTT assay, and the apoptosis of HK-1 cells was detected by flow cytometry. Compared with the siNC group, the expression levels of PTEN, FoxO3a messenger RNA, and protein in the siPTEN group were significantly decreased (P < .05), while the expression levels of PI3K, p-Akt messenger RNA, and protein were significantly increased (P < .05). The growth rate of HK-1 cells in the siPTEN group was significantly higher than the siNC group (P < .05), while the apoptosis rate was significantly lower than that of the siNC group (P < .05). Small interfering RNA can inhibit the expression of PTEN in HK-1 cells, and PTEN can participate in the development of NPC by affecting PI3K/Akt/FoxO3a signaling pathway.

Effects of miR-21 on proliferation and apoptosis of WT cells via PTEN/Akt pathway.

Micro ribonucleic acid (miR)-21 in the proliferation and apoptosis of Wilms' tumor (WT) cells was explored. SK-NEP-1 cells were transfected with miR-21 inhibitor to silence the expression of miR-21. Then, the effects of miR-21 silencing on the proliferation and apoptosis of WT SK-NEP-1 cells were detected through cell counting kit-8 (CCK-8), colony formation assay and flow cytometry. The targets of miR-21 were analyzed via TargetScan database. Fluorescence real-time quantitative polymerase chain reaction (RT-qPCR) assay and western blot analysis were conducted to detect the changes in messenger RNA (mRNA) and protein expression levels of gene of phosphate and tension homology deleted on chromosome ten (PTEN) after silencing miR-21. Whether miR-21 directly binds to PTEN was examined by activity detection via dual luciferase reporter gene assay. Western blotting was employed to detect the correlation of miR-21 with PTEN and protein kinase B (Akt). Compared with normal control (NC) group, miR-21 inhibitor group had significantly inhibited proliferation of SK-NEP-1 cells (P<0.05), notably reduced number of clones (P<0.05) and overtly raised proportion of apoptotic cells (P<0.05). The suppression of miR-21 expression upregulated the mRNA and protein expression levels of PTEN, and the results of activity detection via dual luciferase reporter gene assay indicated that miR-21 bound to PTEN 3′-untranslated region (UTR) to repress its expression (P<0.05). PTEN silencing increased phosphorylated Akt (p-Akt) level in SK-NEP-1 cells, but there was no changes in Akt protein level. After silencing both PTEN and miR-21, the decrease in p-Akt was reversed, thereby reversing the inhibitory effect of miR-21 on the proliferation of SK-NEP-1 cells (P<0.05). miR-21 affects the proliferation and apoptosis of WT SK-NEP-1 cells via the PTEN/Akt pathway.

Integrating serum exosomal microRNA and liver microRNA profiles disclose the function role of autophagy and mechanisms of Fructus Meliae Toosendan-induced hepatotoxicity in mice

Herb-induced liver injury (HILI) is a growing clinical and economic problem worldwide. However, the underlying mechanism of HILI remains largely unknown, which hinders the prevention and treatment of this disease. Recently evidence supports that microRNAs (miRNAs) in circulating exosomes and cells play an important role in the pathology of liver diseases. Thus, using Fructus Meliae Toosendan (FMT) as an example of hepatoxic herbal medicine, the aim of this study was to reveal the mechanisms of FMT-induced liver injury (FILI) through integrated analysis of serum exosomal miRNAs and liver miRNAs profiles on FMT ethyl acetate extract (FMT for short)-exposed mice. Two dosages of FMT (20 and 40 g/kg) were involved in this study, while only high-dose exposure induced obvious liver injury in mice. Pathway analysis of 209 differentially expressed miRNAs (DEMs) in serum exosomes between high-dose FMT and control groups exhibited that FILI might be regulated by apoptosis-related pathways, such as p53 signaling, PI3K/Akt signaling, and PTEN signaling. Integrated analysis of the mRNA targets of serum exosomal DEMs and liver DEMs of high-dose FMT group showed that autophagy was significantly enriched as one of the top canonical pathways in FILI. Hepatocyte apoptosis was then proved by TUNEL assay in the liver tissue of high-dose FMT-treated mice. Moreover, in vivo validation studies suggested that the protein expression levels of PTEN, p-AKT, p53, and BAX were indeed regulated in the mouse liver after high-dose FMT administration, indicating hepatocyte apoptosis may be mediated by these three pathways mentioned above. Intriguingly, PINK1/Parkin-mediated mitophagy was activated in high-dose FMT-treated mouse liver and the protective effect of autophagy in FILI was validated in vitro with an autophagic flux inhibitor. In addition, serum exosomal miR-222, the most downregulated miRNAs between low- and high-dose FMT treatments, might be an important event in the hepatocyte apoptosis by regulating PTEN and PPP2R2A. In conclusion, integrated analysis of microRNA profiles in mouse serum exosomes and liver cells provides insights into the hepatotoxicity mechanisms of FMT and discloses the protective role of autophagy in FILI, suggesting this method could contribute to deeply understand the mechanism of HILI and activation of autophagy may be a potentially therapeutic strategy for FILI even HILI.