Antagonizing adipose tissue-derived exosome miR-103-hepatocyte phosphatase and tensin homolog pathway alleviates autophagy in non-alcoholic steatohepatitis:A trans-cellular crosstalk

BACKGROUND Obesity plays a vital role in the occurrence and development of non-alcoholic steatohepatitis(NASH).However,the underlining mechanism is still unclear,where adipose tissue(AT)derived exosomes may actively participate.MicroRNAs(miRNAs)are commonly secreted from exosomes for cell communication.Though the regulation of miR-103 on insulin sensitivity has been reported,the specific role of AT-derived exosomes miR-103 in NASH is still vague and further investigation may provide novel therapeutic choices.AIM To determine the specific role of AT-derived exosomes miR-103 in developing NASH through various methods.METHODS The expression levels of miR-103 in the AT-derived exosomes and livers were detected and compared between NASH mice and control.The effect of miR-103 on NASH progression was also explored by antagonizing miR-103,including steatosis and inflammation degree changes.The interaction between miR-103 and the autophagy-related gene phosphatase and tensin homolog(PTEN)was confirmed by dual-luciferase reporter assay.The role of the interaction between miR-103 and PTEN on autophagy was verified in NASH-like cells.Finally,the effects of miR-103 from adipose-derived exosomes on NASH and autophagy were analyzed through animal experiments.RESULTS The expression of miR-103 was increased in NASH mice,compared to the control,and inhibition of miR-103 could alleviate NASH.The results of the dual-luciferase reporter assay showed miR-103 could interact with PTEN.MiR-103-anta decreased p-AMPKa,p-mammalian target of rapamycin(mTOR),and p62 but increased the protein levels of PTEN and LC3-II/I and the number of autophagosomes in NASH mice.Similar results were also observed in NASH-like cells,and further experiments showed PTEN silencing inhibited the effect of miR-103-anta.AT derivedexosome miR-103 aggravated NASH and increased the expressions of p-AMPKa,p-mTOR,and p62 but decreased the protein levels of PTEN and LC3-II/I and the number of autophagosomes in mice.CONCLUSION AT derived-exosome increased the levels of miR-103 in the liver,and miR-103 aggravated NASH.Mechanically,miR-103 could interact with PTEN and inhibit autophagy.

Phosphatase and tensin homology deleted in chromosome 10,hypoxia-inducible factor-1 alpha gene expression in colorectal adenoma and adenocarcinoma and their relation to vascular endothelial growth factor protein expression

To examine phosphatase and tensin homology deleted in chromosome 10 (PTEN),hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene expressions and their relation to vascular endothelial growth factor(VEGF) protein expression in the patients with human colorectal adenomas and adenocarcinomas.Methods The expression of PTEN,HIF-1 alpha gene was detected by using in situ hybridization,and the VEGF expression levels by immunohistochemistry in colorectal adenomas and primary colorectal adenocarcinoma.Results Strong expression of HIF-1 alpha was detectable in the majority of colorectal dadenocarcinoma,particularly surrounding areas of necrosis in adenocarcinoma.PTEN,HIF-1 alpha mRNA and VEGF protein were positive in 51.6%,67.7% and 59.7% respectively in 62 cases of adenocarcinomas,and 77.8%,44.4% and 33.3% respectively in 18 cases of adenomas.The positive rate of VEGF was higher in the patients with colorectal adenocarcinomas than that in those with adenomas,whereas that of PTEN mRNA was contrary.HIF-1 mRNA expression was correlated significantly with lymph node metastasis,liver metastasis,Duke’s stage and recurrence.During colorectal tumor progression,the expression of HIF-1 alpha mRNA was positively correlated with the VEGF protein expression (χ2= 4.751 ,P<0.05),but negatively with the PTEN mRNA expression(χ2=21.84,P<0.01).Conclusion The absence or low expression of PTEN and the increased levels of HIF-1α and VEGF may paly an important role in carcinogenesis and progression of colorectal carcinoma.These results suggest that VEGF upregulated by HIF-1 alpha gene may be involved in angiogenesis of colorectal adenocarcinoma.4 refs,1 tab.

Phosphatase and Tensin Homologue Genetic Polymorphisms and their Interactions with Viral Mutations on the Risk of Hepatocellular Carcinoma

Background： Chronic hepatitis B virus （HBV） infection is the major cause of hepatocellular carcinoma （HCC）. Some HBV mutants and dysregulation of phosphatase and tensin homolog （PTEN） may promote the development of HCC synergistically. We aimed to test the effects of PTEN genetic polymorphisms and their interactions with important HBV mutations on the development of HCC in HBV-infected subjects. Methods： Quantitative po[ymerase chain reaction was applied to genotype PTEN polymorphisms （rs1234220, rs2299939, rsl234213） in 1012 healthy controls, 302 natural clearance subjects, and 2011 chronic H BV-infected subjects including 1021 HCC patients. HBV mutations were determined by sequencing. The associations of PTEN polymorphisms and their interactions with HBV mutations with HCC risk were assessed using multivariate logistic regression analysis. Results： Rs1234220 C allele was significantly associated with HCC risk compared to healthy controls （adjusted odds ratio [AOR] = 1.35, 95% confidence interval [CI] = 1,07-1.69） and HCC-free HBV-infected subjects （AOR = 1.27, 95% CI = 1.01-1 .57）. rsl234220 C allele was significantly associated with increased frequencies of HCC-risk A 1652G, C 1673T, and C 1730G mutations in genotype B H BV-in fected subjects. Rs2299939 GT genotype was inversely associated with HCC risk in HBV-infected patients （AOR 0.75, 95% CI 0.62-0.92）. The interaction of rs2299939 variant genotypes （GT＋TT） with A3054T mutation significantly increased HCC risk （AOR = 2.41, 95% CI = 1.08-5.35）; whereas its interaction with C3116T mutation significantly reduced HCC risk （AOR = 0.34, 95% CI 0.18-0.66）. These significant effects were only evident in males alter stratification. Conclusions： PTEN polymorphisms and their interactions with HBV mutations may contribute to hepatocarcinogenesis in males. The host-virus interactions are important in identifying HBV-infected subjects who are more likely to develop HCC.

Nuclear miR-665 aggravates heart failure via suppressing phosphatase and tensin homolog transcription

Although numerous miRNAs have been discovered,their functions in the different subcellular organelles have remained obscure.In this study,we found that miR-665 was enriched in the nucleus of cardiomyocytes,and then investigated the underlying role of nuclear miR-665 in heart failure.RNA fluorescence in situ hybridization assays in human heart tissue sections and primary cardiomyocytes showed that miR-665 was localized in the nucleus of cardiomyocytes.Increased expression of nuclear miR-665 was observed not only in the cardiomyocytes isolated from the heart of mice treated in vivo by transverse aortic constriction(TAC),but also in phenylephrine(PE)-treated cultured cardiomyocytes in vitro.To further explore the role of miR-665 in heart failure,a type 9 recombinant adeno-associated virus(rAAV)system was employed to manipulate the expression of miR-665 in mice.Overexpression of miR-665 aggravated TAC-induced cardiac dysfunction,while down-expression of miR-665 showed opposite effects.Bioinformatic prediction and biological validation confirmed that the PTEN(phosphatase and tensin homolog)gene was one of the targets of miR-665 in the nucleus.Furthermore,restoring PTEN expression significantly eliminated the destructive effects of miR-665 over-expression in TAC-induced cardiac dysfunction.Our data showed that nuclear miR-665 aggravates heart failure via inhibiting PTEN expression,which provided a therapeutic approach for heart failure.

MiR-106b-5p Inhibits Tumor Necrosis Factor-α-induced Apoptosis by Targeting Phosphatase and Tensin Homolog Deleted on Chromosome 10 in Vascular Endothelial Cells

Background： Apoptosis of endothelial cells （ECs） plays a key role in the development of atherosclerosis and there are also evidence indicated that phosphatase and tensin homolog deleted on chromosome 10 （PTEN） is a viable target in therapeutic approaches to prevent vascular ECs apoptosis. Aberrant miR-106b-5p expression has been reported in the plasma of patients with unstable atherosclerotic plaques. However, the role and underlying mechanism of miR-106-5p in the genesis of atherosclerosis have not been addressed. In this study, we explored the anti-apoptotic role of miR-106-5p by regulating PTEN expression in vascular ECs. Methods： Real-time reverse transcription polymerase chain reaction （RT-PCR） was performed to detect the expression levels of miR-106b-5p in human atherosclerotic plaques and normal vascular tissues. Human umbilical vein endothelial cells （HUVEC） were transfected with miR-106b-5p mimic or negative control mimic, and apoptosis was induced by serum starvation and tumor necrosis factor-α （TN F-α） treat. Western blotting and real-time RT-PCR experiments were used to detect PTEN expression levels and TN F-α-induced apoptosis was evaluated by the activation of caspase-3 and cell DNA fragmentation levels in HUVEC. Results： The expression ofmiR-106b-5p was significantly downregulated in plaques than in normal vascular tissues. TNF-α significantly downregulated miR-106b-5p expression levels and upregulated activation of caspase-3 and cell DNA fragmentation levels in HUVEC. Overexpression ofmiR-106b-5p with miR-106b-5p mimic inhibited PTEN expression and TNF-α-induced apoptosis in HUVEC. Luciferase reporter assays confirmed that miR-106b-5p binds to PTEN mRNA 3＇ untranslated region site, Conclusion： MiR-106b-5p could inhibit the expression of PTEN in vascular ECs, which could block TNF-α-induced activation of caspase-3, thus prevent ECs apoptosis in atherosclerosis diseases.

PTEN in liver diseases and cancer

The phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/Akt axis is a key signal transduction node that regulates crucial cellular functions, including insulin and other growth factors signaling, lipid and glucose metabolism, as well as cell survival and apoptosis. In this pathway, PTEN acts as a phosphoinositide phosphatase, which terminates PI3Kpropagated signaling by dephosphorylating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. However, the role of PTEN does not appear to be restricted only to PI3K signaling antagonism, and new functions have been recently discovered for this protein. In addition to the well-established role of PTEN as a tumor suppressor, increasing evidence now suggests that a dysregulated PTEN expression and/or activity is also linked to the development of several hepatic pathologies. Dysregulated PTEN expression/activity is observed with obesity, insulin resistance, diabetes, hepatitis B virus/hepatitis C virus infections, and abusive alcohol consumption, whereas mutations/deletions have also been associated with the occurrence of hepatocellular carcinoma. Thus, it appears that alterations of PTEN expression and activity in hepatocytes are common and recurrent molecular events associated with liver disorders of various etiologies. These recent f indings suggest that PTEN might represent a potential common therapeutic target for a number of liver pathologies.

MiR-200a and miR-200b target PTEN to regulate the endometrial cancer cell growth in vitro

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 mi R-200 a 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-200 a 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-200 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-200 a and miR-200b can promote the endometrial cancer cell growth in vitro by targeted inhibition of PTEN gene expression.

Upregulated DJ-1 Promotes Renal Tubular EMT by Suppressing Cytoplasmic PTEN Expression and Akt Activation

Recently,phosphatase and tensin homolog deleted on chromosome 10（PTEN） is suggested as a new agent in the fighting against fibrogenesis.In tumor,DJ-1 is identified as a negative regulator of PTEN.But the expression of DJ-1 and the regulation of PTEN in fibrosis are unclear.Renal fibrosis was induced in 5/6 subtotal nephrectomy rat model.Human proximal tubular epithelial cells（HKC） were treated with transforming growth factor-beta 1（TGF-β1）,or transfected with DJ-1 or PTEN.Confocal microscope was used to investigate the localization of DJ-1 and PTEN.The selective phosphoinositide-3 kinase（PI3K） inhibitor,LY294002,was administered to inhibit PI3K pathway.The DJ-1 and PTEN expression,markers of epithelial-mesenchymal transition（EMT） and Akt phosphorylation were measured by RT-PCR,Western blotting or immunocytochemistry.In vitro,after HKC cells were stimulated with 10 ng/mL TGF-β1 for 72 h,the expression of DJ-1 was increased,and that of PTEN was decreased.In vivo,the same results were identified in 5/6-nephrectomized rats.In normal HKC cells,most of DJ-1 protein localized in cytoplasm,and little in nucleus.TGF-β1 upregulated DJ-1 expression in both cytoplasma and nuclei.In contrary,TGF-β1 emptied cytoplasmic PTEN protein into nucleus.Overexpression of DJ-1 decreased the expression of PTEN,promoted the activation of Akt and the expression of vimentin,and also led to the loss of cytoplasmic PTEN.Contrarily,overexpression of PTEN protected HKC cells from TGF-β1-induced EMT.In conclusion,DJ-1 is upregulated in renal fibrosis and DJ-1 mediates EMT by suppressing cytoplasmic PTEN expression and Akt activation.

PTEN and Ki67 expression is associated with clinicopathologic features of non-small cell lung cancer

Phosphatase and tensin homolog deleted on chromosome 10（PTEN） and the proliferating antigen Ki67 have been widely studied in several tumors.However,their role as indicator in non-small cell lung cancer（NSCLC）remains unknown.Here,we investigated the expression of PTEN and Ki67 in NSCLC tissues and paired normal lung tissues to identify whether these proteins are associated with lung cancer development and survival.Immunohistochemistry for PTEN and Ki67 was performed on 67 lung cancer tissues and 41 paired adjacent normal lung tissues to detect the expression of these two proteins.The expression of PTEN in NSCLC tissues（32.8%） was significantly lower than that in normal tissues（82.9%,P 〈 0.05）.In contrast,the expression of Ki67 in NSCLC tissues（76.1%） was significantly higher than that in normal tissues（27.3%,P 〈 0.05）.Expression of both PTEN and Ki67 were strongly associated with tumor histology,clinical stage,lymph node metastasis,differentiation and4-year postoperative survival rate（P 〈 0.05）.However,PTEN expression was negatively correlated with Ki67 expression（r =-0.279,P 〈 0.05）.In conclusion,low PTEN expression and Ki67 overexpression are associated with malignant invasion and lymph node metastasis of NSCLC.These proteins may serve as diagnostic and prognostic biomarkers of NSCLC.

Oscillation and bistable switching dynamical behavior of p53 regulated by PTEN upon DNA damage

The tumor suppressor p53 plays a key regulatory role in the response of cells to various stresses.It have experimentally shown that p53 can exhibit rich dynamic behaviors under DNA damage.In order to study the meticulous mechanism,we construct a coupling model including p53-murine double minute 2(Mdm2)negative feedback loop(NFL)and p53-phosphatase and tensin homolog(PTEN)-Mdm2 positive feedback loop(PFL).By making use of bifurcation analysis and Binomial r-leap algorithm,we confirm that PTEN is a essential condition for p53 oscillation or bistable switching dynamic behaviours.We investigate the p53 dynamics affected by PFL through studying the p53-dependent PTEN synthesis rate.The results suggest that PFL may enrich the dynamic behaviors of the p53 system.This work can promote the understanding of p53 dynamics mediated by PTEN and provide clues for cancer therapy.

Prognostic value of PTEN in de novo diagnosed metastatic prostate cancer

The purpose of our study is to investigate the prognostic value of phosphatase and tensin homolog on chromosome 10(PTEN)expression in patients with de novo metastatic castration naive prostate cancer(mCNPC).A total of 205 patients with mCNPC at Fudan University Shanghai Cancer Center(Shanghai,China)were retrospectively examined.Immunohistochemical staining of PTEN was performed on prostate biopsy samples of these patients.Associations among clinicopathological features,patient survival and PTEN protein expression were analyzed.PTEN loss occurred in 58 of 205(28.3%)patients.Loss of PTEN was significantly correlated with high metastatic volume(P=0.017).No association between PTEN expression and Gleason score was observed.Patients with PTEN loss had significantly shorter progression-free survival(PFS,P<0.001)and overall survival(OS,P<0.001)compared with patients with intact PTEN expression.Multivariate analysis showed that elevated alkaline phosphatase,high metastatic volume and PTEN loss were independent poor prognostic factors for PFS.The Eastern Cooperative Oncology Group performance status(ECOG PS)≥2 and PTEN loss were independent poor prognostic factors for OS.The adjusted hazard ratio of PTEN loss for PFS and OS was 1.67(95%confidence interval[CI]:1.14–2.43,P=0.008)and 1.95(95%CI:1.23–3.10,P=0.005),respectively.PTEN loss was also significantly associated with shorter PFS(P=0.025)and OS(P<0.001)in patients with low-volume metastatic disease.Our data showed that PTEN loss is an independent predictor for shorter PFS and OS in patients with de novo mCNPC.