Decreased PTEN Expression Research Articles

PTEN downregulates WD repeat‑containing protein66 in salivary adenoid cystic carcinoma.

Salivary adenoid cystic carcinoma (SACC) is one of the most common types of salivary gland cancer that causes substantial morbidity and mortality. Despite the substantial health burden of SACC, the molecular mechanisms underlying its development and progression remain poorly understood. We previously reported the loss of phosphatase and tensin homolog (PTEN) expression to be common among SACC tumors, and the PTEN deficiency to be correlated with enrichment of epithelial-mesenchymal transition (EMT) genes based on expression array analysis. The aim of the present study was to investigate further the functional function of WD repeat-containing protein 66 (WDR66), one of the enriched EMT genes, in the context of PTEN deficiency and SACC pathogenesis. WDR66 was identified to be required to maintain the EMT phenotype and the expression of cancer stem cell genes in the context of PTEN deficiency. Furthermore, knockdown of WDR66 decreased cellular proliferation, migration and invasion. Finally, WDR66 expression was identified to be inversely associated with PTEN expression and negatively correlated with the overall survival of patients with SACC. Collectively, the results of the present study revealed a novel function of WDR66 in mediating the progression of PTEN-deficient SACCs, thereby suggesting WDR66 inhibition to be a potential therapeutic approach towards successful management of SACC disease progression, particularly against tumors with decreased PTEN expression levels.

CagA increases DNA methylation and decreases PTEN expression in human gastric cancer.

Gastric cancer is one of the leading causes of cancer-associated mortality worldwide. Cytotoxin-associated gene A (CagA) has been reported to be associated with gastric diseases. Phosphatase and tensin homolog (PTEN) and tet methylcytosine dioxygenase 1 (Tet1) are important tumor-suppressor genes. The present study aimed to investigate the underlying functions of CagA in human gastric cancer, and to explore the associations between CagA, PTEN and Tet1 in gastric cancer. For that purpose, CagA overexpression and Tet1 interference recombinant lentiviral plasmids were constructed. Quantitative polymerase chain reaction (qPCR) was utilized to screen gene expression in HGC-27 human gastric cancer cells overexpressing CagA. qPCR and western blotting were used to detect gene and protein expression, respectively. In addition, the methylation status of PTEN was detected by methylation-specific PCR. The expression levels of PTEN, Tet1, apolipoprotein B mRNA editing enzyme catalytic subunit (APOBEC)3A, APOBEC3C and APOBEC3F were significantly decreased in the CagA overexpression group compared with in the negative control group in HGC-27 cells. Compared with in the negative control group, the mRNA and protein expression levels of PTEN were markedly decreased in cells with Tet1 interference. The decreased expression of PTEN was associated with increased methylation levels in the cells. In addition, the protein expression levels of PTEN were significantly decreased in HGC-27 cells when CagA was overexpressed. The expression levels of PTEN and Tet1 were also markedly decreased in CagA+ gastric cancer tissues compared with in non-cancerous tissues. The decreased expression of PTEN in CagA+ gastric cancer tissues was associated with increased methylation levels. In conclusion, overexpression of CagA significantly decreased the expression of PTEN, Tet1, APOBEC3A, APOBEC3C and APOBEC3F in human gastric cancer. In addition, CagA increased DNA methylation and decreased PTEN expression, which was reversed by Tet1 overexpression. The present study may facilitate future therapeutic approaches targeting human gastric cancer.

Endothelial colony-forming cell-derived exosomes restore blood-brain barrier continuity in mice subjected to traumatic brain injury

Traumatic brain injury (TBI) tends to cause disruption of the blood-brain barrier (BBB). Previous studies have shown that intravenously or intracerebroventricularly infused human umbilical cord blood-derived endothelial colony-forming cells (ECFCs) can home to injury sites and improve outcomes in mice subjected to experimental TBI. Several reports have demonstrated that these cells did not incorporate directly into newly formed vasculature but instead stimulated the proliferation and migration of tissue-resident endothelial cells (ECs) via paracrine mechanisms. In the present study, exosomes, which range from 30 to 150 nm in diameter, were isolated from ECFC-conditioned medium. The exosomes were labeled with PKH67 ex vivo, and we observed that they were taken up by ECs with high efficiency after 12 h of incubation. Pretreatment with ECFC-derived exosomes promoted the migration of ECs subjected to scratch injury, and incubating ECs exposed to hypoxia with ECFC-derived exosomes decreased PTEN expression, stimulated AKT phosphorylation and increased tight junction (TJ) protein expression in the cells. Furthermore, in vivo delivery of ECFC-derived exosomes into TBI mice also inhibited PTEN expression and increased AKT expression, changes accompanied by reductions in Evans blue (EB) dye extravasation, brain edema and TJ degradation. These data demonstrated that ECFC-derived exosomes have beneficial effects on BBB integrity in mice with TBI.

Endothelial replicative senescence delayed by the inhibition of MTORC1 signaling involves MicroRNA-107.

Accumulation of senescent endothelial cells can contribute to endothelium dysfunction. Suppression of MTOR signaling has been shown to delay senescence but the mechanism that underpins this effect, particularly one that involves miRNAs, remains to be further defined. This study sought to identify miRNAs involved in MTORC1-mediated inhibition of replicative senescence in endothelial cells. Pre-senescent HUVECs were prolonged treated with low dose rapamycin (1 nM), an MTOR inhibitor. Rapamycin treatment down-regulated the phosphorylated MTOR, RPS6 and 4EBP1 expressions, which confirmed MTORC1 suppression. Prolonged low dose rapamycin treatment has significantly reduced the percentage of senescence-associated beta galactosidase (SA-β gal) positively stained senescent cells and P16INK4A expression in these cells. On the contrary, the percentage of BrdU-labelled proliferating cells has significantly increased. RPTOR, a positive regulator of MTORC1 was knockdown using RPTOR siRNA to inhibit MTORC1 activation. RPTOR knockdown was evidenced by significant suppressions of RPTOR mRNA and protein expression levels. In these cells, the expression of miR-107 was down-regulated whereas miR-145-5p and miR-217 were up-regulated. Target gene prediction revealed PTEN as the target of miR-107 and this was confirmed by biotin pull-down assay. Over-expression of miR-107 has decreased PTEN expression, increased MTORC1 activity, induced cell cycle arrest at G0/G1 phase and up-regulated P16INK4A expression but mitigated tube formation. Collectively, our findings revealed that delayed endothelial replicative senescence caused by the inhibition of MTORC1 activation could be modulated by miR-107 via its influence on PTEN.

Retracted: MicroRNA-92a promotes tumor growth and suppresses immune function through activation of MAPK/ERK signaling pathway by inhibiting PTEN in mice bearing U14 cervical cancer.

Cervical cancer is known as the possible outcome of genital infection, while the molecular mechanisms of initiation, development, and metastasis of cervical cancer have not yet been fully elucidated. Our study aims to investigate the effects of microRNA‐92a (miR‐92a) on tumor growth and immune function by targeting PTEN via the MAPK/ERK signaling pathway in tumor‐bearing mice. C57BL/6 female mice were used for tumor‐bearing mouse models and their tumor and adjacent normal tissues were collected, and normal cervical tissues were obtained from normal mice. Serum levels of tumor necrosis factor‐α (TNF‐α) and soluble interleukin‐2 receptor (sIL‐2R) were detected by ELISA. The cells were divided into the normal, blank, negative control (NC), miR‐92a mimic, miR‐92a inhibitor, siRNA‐PTEN, and miR‐92a inhibitor + siRNA‐PTEN groups. Dual‐luciferase reporter assay was adopted to determine the relationship between PTEN and miR‐92a. Expressions of miR‐92a, PTEN, TNF‐α, sIL‐2R, ERK1, and ERK2 were tested by RT‐qPCR and Western blotting. Cell proliferation was detected by cell count kit‐8 ( CCK‐8); cell cycle and apoptosis were detected by flow cytometry. Compared with the normal cervical tissues and adjacent normal tissues, the cervical cancer tissues exhibited increased expressions of miR‐92a, p‐ERK1/2, and serum levels of TNF‐α and sIL‐2R while decreased PTEN expression. PTEN was confirmed to be the target gene of miR‐92a. As compared with the blank and NC groups, expressions of miR‐92a, ERK1 and ERK2 increased, and expressions of PTEN decreased in the miR‐92a mimic group. The miR‐92a mimic group exhibited increased expression levels of TNF‐α and sIL‐2R, cell proliferation, and cell number in S phase but decreased cell apoptosis, and cell number in G0/G1 phase, while the miR‐92a inhibitor group followed opposite trends. miR‐92a promotes tumor growth and suppresses immune function by inhibiting PTEN via activation of the MAPK/ERK signaling pathway in mice bearing U14 cervical cancer.

Effects of microRNA-20a on the proliferation, migration and apoptosis of multiple myeloma via the PTEN/PI3K/AKT signaling pathway.

Multiple myeloma (MM) is a heterogeneous disease with a poor prognosis. Circulating microRNAs (miRNAs) have shown potential as non-invasive prognostic biomarkers for heterogeneous diseases. miR-20a has been shown involved in various human cancers, and the phosphatase and tensin homolog/phosphoinositide 3-kinase/protein kinase B (PTEN/P13K/Akt) signaling pathway plays a key role in cell proliferation, migration and apoptosis. Here, we investigated the effect of miR-20a on the PTEN/PI3K/Akt signaling pathway during MM cell proliferation, migration and apoptosis. Reverse transcription quantitative polymerase chain reaction was applied to detect miR-20a expression in plasma from 30 MM patients and MM cell lines. CCK-8 assays, Transwell assays, Annexin V/PI double-staining and western blotting were performed to examine the protein expressions of PTEN, PI3K and Akt during cellullar proliferation, migration, cycling, and apoptosis. Significant upregulation of miR-20a and deregulation of PTEN were observed in MM cells. We also identified PTEN as a downstream target gene of miR-20a, which bound to the 3'-untranslated region of PTEN. Overexpression of miR-20a was associated with decreased PTEN expression, and treatment with miR-20a inhibitors decreased cell proliferation, migration and clonogenicity and reduced the protein expressions of PI3K and p-Akt but increased PTEN protein expression compared with blank and negative control groups. Taken together, these results showed that inhibition of miR-20a suppresses MM progression by modulating the PTEN/PI3K/Akt signaling pathway. These findings suggest that miR-20a may be a novel molecular therapeutic target for the treatment of MM.

Reduced immunohistochemical PTEN staining is associated with higher progression rate and recurrence episodes in non-invasive low-grade papillary urothelial carcinoma of the bladder.

Non-invasive low-grade papillary urothelial carcinoma (NILGPUC) of the bladder is regarded as a relatively indolent disease. However, its propensity for frequent recurrences constitutes a major clinical problem. Additionally, there is a progression risk of 10-15% to either a higher grade and/or a higher stage disease in these tumors. The molecular factors that will predict recurrence and progression in low-grade pTa bladder carcinoma have not yet been elucidated. Herein, we investigated the association of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) alterations with recurrence and progression in NILGPUC using immunohistochemistry. Eighty-one cases of bladder cancer initially diagnosed as NILGPUC in a single institution with follow-up were encountered after searching medical records. Tissue microarrays (TMA) that contained both tumor and non-neoplastic mucosa from each case were constructed using paraffin blocks of transurethral resections. Sections from TMA blocks were stained immunohistochemically for PTEN protein and were evaluable in 76 cases. Any absence of staining was recorded and correlated with clinical findings. Ten patients (13.2%) showed progression and 41 (53.9%) showed recurrence. Reduced PTEN expression was observed in 29 cases (38.1%). Cases with reduced PTEN had higher progression rate compared to cases with intact PTEN (p = 0.026). Tumor relapse was more frequent in cases with reduced PTEN (65.5 vs 46.8%), but this difference was not statistically significant (p = 0.112). On the other hand, decreased PTEN expression was associated with higher number of recurrence episodes (p = 0.002). PTEN seems to have a link with the disease course in NILGPUC of the bladder.

Homocysteine-induced proliferation of vascular smooth muscle cells occurs via PTEN hypermethylation and is mitigated by Resveratrol

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in the development of atherosclerosis (AS), and the presence of homocysteine (Hcy) acts as an independent risk factor for AS. However, the underlying mechanisms remain to be elucidated. Phosphatase and tensin homologue on chromosome 10 (PTEN), is endogenously expressed in VSMCs and induces multiple signaling networks involved in cell proliferation, survival and inflammation, however, the specific role of PTEN is still unknown. The present study detected the proliferation ratio of VSMCs following treatment with Hcy and Resveratrol (RSV). In the 100µM Hcy group, the proliferation ratio increased, and treatment with RSV decreased the proliferation ratio induced by Hcy. Reverse transcription‑quantitative polymerase chain reaction and western blotting were used to analyze PTEN expression, RSV treatment was associated with decreased PTEN expression levels in VSMCs. PTEN levels were decreased in Hcy treated cells, and the proliferation ratio of VSMCs were increased following treated with Hcy. To study the mechanism of regulation of PTEN by Hcy, the present study detected PTEN methylation levels in VSMCs, and PTEN DNA methylation levels were demonstrated to be increased in the 100µM Hcy group, whereas treatment with RSV decreased the methylation status. DNA methyltransferase 1 is important role in the regulation of PTEN methylation. Overall, Hcy impacts the methylation status of PTEN, which is involved in cell proliferation, and induces the proliferation of VSMCs. This effect is alleviated by treatment with RSV, which exhibits an antagonistic mechanism against Hcy.

The prognostic value and potential drug target of phosphatase and tensin homolog in breast cancer patients: A meta-analysis.

Background:The prognostic significance of phosphatase and tensin homolog (PTEN) in patients with breast cancer (BC) remains controversial. The aims of our meta-analysis are to evaluate its association with clinicopathological characteristics and prognostic value in patients with breast cancer.Methods:PubMed, EMBASE, Web of Science, and China National Knowledge Infrastructure (CNKI) were systematically searched up to December 2016. The meta-analysis was performed using hazard ratio (HR), odds ratio (OR), and 95% confidence intervals (CI) as effect measures. A fixed or random effect model was used depending on the heterogeneity analysis. Statistical analysis was performed using Review manager software version 5.3.Results:Seventeen studies including 4343 patients with breast cancer were analyzed. The meta-analysis indicated that breast cancers with PTEN loss were significantly associated with the tumor size ≥2 cm group (ORFEM = 1.68, 95%CIFEM [1.34, 2.10]), negative expression of estrogen receptor (ORREM = 1.95, 95%CIREM [1.09, 3.49]), negative expression of progesterone receptor (ORFEM = 1.72, 95%CIFEM [1.43, 2.08]), the advanced stage (ORREM = 1.94, 95%CIREM [1.35, 2.80]), positive axillary lymph node metastasis (ORREM = 1.80, 95%CIREM [1.30, 2.50]), and the local recurrence (ORFEM = 1.70, 95%CIFEM [1.26, 2.28]). None of other clinicopathological parameters such as the HER2 status and distant metastasis were associated with PTEN loss. The decreased PTEN expression was significantly correlated with the overall survival (OS) of patients (HRREM = 1.83, 95%CIREM [1.32, 2.53]) and the disease-free survival (DFS) of patients (HRREM = 2.43, 95%CIREM [1.31, 4.53]).Conclusion:Our meta-analysis demonstrates that PTEN loss is of particular importance for predicting breast cancer aggressiveness and poor prognosis. PTEN is a potential drug target for the development of individualized treatment in BC patients.

P53 Maintains Baseline Expression of Multiple Tumor Suppressor Genes.

TP53 is the most commonly mutated tumor suppressor gene and its mutation drives tumorigenesis. Using ChIP-seq for p53 in the absence of acute cell stress, we found that wild-type but not mutant p53 binds and activates numerous tumor suppressor genes, including PTEN, STK11(LKB1), miR-34a, KDM6A(UTX), FOXO1, PHLDA3, and TNFRSF10B through consensus binding sites in enhancers and promoters. Depletion of p53 reduced expression of these target genes, and analysis across 18 tumor types showed that mutation of TP53 associated with reduced expression of many of these genes. Regarding PTEN, p53 activated expression of a luciferase reporter gene containing the p53-consensus site in the PTEN enhancer, and homozygous deletion of this region in cells decreased PTEN expression and increased growth and transformation. These findings show that p53 maintains expression of a team of tumor suppressor genes that may together with the stress-induced targets mediate the ability of p53 to suppress cancer development. p53 mutations selected during tumor initiation and progression, thus, inactivate multiple tumor suppressor genes in parallel, which could account for the high frequency of p53 mutations in cancer.Implications: In this study, we investigate the activities of p53 under normal low-stress conditions and discover that p53 is capable of maintaining the expression of a group of important tumor suppressor genes at baseline, many of which are haploinsufficient, which could contribute to p53-mediated tumor suppression. Mol Cancer Res; 15(8); 1051-62. ©2017 AACR.

Investigating the role of the IGF axis as a predictor of biochemical recurrence in prostate cancer patients post-surgery.

Between 20% and 35% of prostate cancer (PCa) patients who undergo treatment with curative intent (ie, surgery or radiation therapy) for localized disease will experience biochemical recurrence (BCR). Alterations in the insulin-like growth factor (IGF) axis and PTEN expression have been implicated in the development and progression of several human tumors including PCa. We examined the expression of the insulin receptor (INSR), IGF-1 receptor (IGF-1R), PTEN, and AKT in radical prostatectomy tissue of patients who developed BCR post-surgery. Tissue microarrays (TMA) of 130 patients post-radical prostatectomy (65 = BCR, 65 = non-BCR) were stained by immunohistochemistry for INSR, IGF-1R, PTEN, and AKT using optimized antibody protocols. INSR, IGF1-R, PTEN, and AKT expression between benign and cancerous tissue, and different Gleason grades was assessed. Kaplan-Meier survival curves were used to examine the relationship between proteins expression and BCR. INSR (P < 0.001), IGF-1R (P < 0.001), and AKT (P < 0.05) expression was significantly increased and PTEN (P < 0.001) was significantly decreased in cancerous versus benign tissue. There was no significant difference in INSR, IGF-1R, or AKT expression in the cancerous tissue of non-BCR versus BCR patients (P = 0.149, P = 0.990, P = 0.399, respectively). There was a significant decrease in PTEN expression in the malignant tissue of BCR versus non-BCR patients (P = 0.011). Combinational analysis of the tissue proteins identified a combination of decreased PTEN and increased AKT or increased INSR was associated with worst outcome. We found that in each case, our hypothesized worst group was most likely to experience BCR and this was significant for combinations of PTEN+INSR and PTEN+AKT but not PTEN+IGF-1R (P = 0.023, P = 0.028, P = 0.078, respectively). Low PTEN is associated with BCR and this association is strongly modified by high INSR and high AKT expression. Measurement of these proteins could help inform appropriate patient selection for postoperative adjuvant therapy and prevent BCR.

Altered PTEN expression as a diagnostic marker for the earliest endometrial precancerous changes

Introduction: Study was planned to investigate PTEN gene expression in endometrial hyperplasia and carcinoma as analyzed by immunohistochemistry. Material and Method: This study was conducted on 80 endometrial samples in the Department of Pathology, Gandhi Medical College, Bhopal (January 2012 to August 2016) Results: 5 out of 5 cases of proliferative endometrium, 3 out of 5 cases of secretory endometrium, 37 [84%] out of 44 cases of simple and complex endometrial hyperplasia without atypia, 8 out of 15 cases of simple and complex endometrial hyperplasia with atypia and 3 out of 11 cases of endometrial carcinoma showed positive PTEN expression. Conclusion: PTEN is a major gene involved in the pathogenesis of endometrial carcinoma. Our data suggest that decreased PTEN expression was a marker of earliest endometrial pre-cancer lesion and we propose that use of PTEN immunostaining in a clinical setting may be informative in identifying premalignant lesions that are likely to progress to carcinoma.

Effects of MicroRNA-19b on the Proliferation, Apoptosis, and Migration of Wilms' Tumor Cells Via the PTEN/PI3K/AKT Signaling Pathway.

Wilms' tumor (WT) is a most common renal cancer that occurs among children, and microRNA-19b (miR-19b) usually participates in various human cancers. Importantly, the PTEN/PI3K/Akt signaling pathway plays a key role in cell apoptosis, growth and proliferation. Thus, our present study aims to investigate the effect of miR-19b on the PTEN/PI3K/Akt signaling pathway during WT cell proliferation, migration, and apoptosis. WT tissues and adjacent normal tissues from WT patients were collected. qRT-PCR was applied to detect miR-19b expression in both the WT tissues and the adjacent normal tissues, immunohistochemistry was applied to detect the protein expressions of PTEN, P13K, and p-Akt, SK-NEP-1 cells were divided into the blank, negative control (NC), miR-19b mimics and miR-19b inhibitors groups. MTT assay, propidium iodide (PI) staining, Annexin-V/PI double-staining, Transwell assay and Western blotting were performed to examine cell proliferation, cycle, apoptosis, migration, and invasion, and the protein expressions of PTEN, P13K, Akt, and p-Akt. Increased miR-19b expression, positive expression rates of P13K and Akt, decreased PTEN expression rate, a negative correlation between PTEN expression and tumor lymph node metastasis, and a positive correlation between the expression of P13K and Akt and the clinical stages were observed in the WT tissues. The miR-19b inhibitors group exhibited decreased cell proliferation, cell cycle progression, migration and invasion, and protein expressions of PI3K and p-Akt but increased PTEN protein expression compared with the blank and NC groups. Thus, inhibition of miR-19b suppresses the progression of WT by modulating the PTEN/PI3K/AKT signaling pathway. J. Cell. Biochem. 118: 3424-3434, 2017. © 2017 Wiley Periodicals, Inc.

MicroRNA-181 inhibits proliferation and promotes apoptosis of chondrocytes in osteoarthritis by targeting PTEN.

To investigate the effects of microRNA-181 (miR-181) on the proliferation and apoptosis of chondrocytes in osteoarthritis (OA) by targeting PTEN. The chondrocytes in logarithmic growth phase were selected and divided into 6 test groups: the normal, blank, negative control, miR-181 mimic, miR-181 inhibitor, and miR-181 inhibitor + PTEN-siRNA groups. Reverse transcription qPCR was used to detect the expressions of miR-181 and PTEN mRNA. MTT assay and flow cytometry were performed to detect cell proliferation and apoptosis. The protein expressions of PARP and caspase-3 and the activity of MMP-2 and MMP-9 were detected by Western blotting and gelatin zymography assay. The miR-181 mimic group showed increased miR-181 expression and decreased PTEN expression compared with the other 5 groups. Also, by comparison with the other 5 groups, the cell proliferation rate declined and the rate of cell apoptosis was elevated in the miR-181 mimic group. The MiR-181 mimic group showed remarkably increased protein expression of caspase-3 and PARP compared with the other 5 groups. The activity of MMP-2 and MMP-9 was higher in the miR-181 mimic group than the other 5 groups. MiR-181 could up-regulate the expressions of caspase-3, PARP, MMP-2, and MMP-9, and thereby inhibit cell proliferation and promote apoptosis of chondrocytes in OA by targeting PTEN.

Sp1-mediated transcriptional activation of miR-205 promotes radioresistance in esophageal squamous cell carcinoma.

Radiotherapy for esophageal squamous cell carcinoma (ESCC) patients is limited by resistance to ionizing radiation (IR). However, the roles and mechanisms of microRNAs in radioresistance are obscure. Here, we investigated that microRNA-205 (miR-205) was upregulated in radioresistant (RR) ESCC cells compared with the parental cells. Overexpression of miR-205 promoted colony survival post-IR, whereas depletion of miR-205 sensitized ESCC cells to IR in vitro and in vivo. Further, we demonstrated that miR-205 promoted radioresistance by enhancing DNA repair, inhibiting apoptosis and activating epithelial-mesenchymal transition (EMT). Mechanistically, miR-205, upregulated post-IR, was demonstrated to be activated by Sp1 in parallel with its host gene, miR-205HG, both of which showed a perfect correlation. We also identified and validated phosphatase and tensin homolog (PTEN), as a target of miR-205 that promoted radioresistance via PI3K/AKT pathway. Lastly, increased miR-205 expression was closely associated with decreased PTEN expression in ESCC tissues and miR-205 expression predicted poor prognosis in patients with ESCC. Taken together, these findings identify miR-205 as a critical determinant of radioresistance and a biomarker of prognosis. The Sp1-mediated transcriptional activation of miR-205 promotes radioresistance through PTEN via PI3K/AKT pathway in ESCC. Inhibition of miR-205 expression may be a new strategy for radiotherapy in ESCC.

Analysis of genes involved in the PI3K/Akt pathway in radiation- and MNU-induced rat mammary carcinomas.

The PI3K/AKT pathway is one of the most important signaling networks in human breast cancer, and since it was potentially implicated in our preliminary investigations of radiation-induced rat mammary carcinomas, our aim here was to verify its role. We included mammary carcinomas induced by the chemical carcinogen 1-methyl-1-nitrosourea to determine whether any changes were radiation-specific. Most carcinomas from both groups showed activation of the PI3K/AKT pathway, but phosphorylation of AKT1 was often heterogeneous and only present in a minority of carcinoma cells. The negative pathway regulator Inpp4b was significantly downregulated in both groups, compared with in normal mammary tissue, and radiation-induced carcinomas also showed a significant decrease in Pten expression, while the chemically induced carcinomas showed a decrease in Pik3r1 and Pdk1. Significant upregulation of the positive regulators Erbb2 and Pik3ca was observed only in chemically induced carcinomas. However, no genes showed clear correlations with AKT phosphorylation levels, except in individual carcinomas. Only rare carcinomas showed mutations in PI3K/AKT pathway genes, yet these carcinomas did not exhibit stronger AKT phosphorylation. Thus, while AKT phosphorylation is a common feature of rat mammary carcinomas induced by radiation or a canonical chemical carcinogen, the mutation of key genes in the pathways or permanent changes to gene expression of particular signaling proteins do not explain the pathway activation in the advanced cancers. Although AKT signaling likely facilitates cancer development and growth in rat mammary carcinomas, it is unlikely that permanent disruption of the PI3K/AKT pathway genes is a major causal event in radiation carcinogenesis.

MiR-222 promotes drug-resistance of breast cancer cells to adriamycin via modulation of PTEN/Akt/FOXO1 pathway

Background and purposeAcquisition of resistance to adriamycin (ADR) is one of the most important clinical obstacles in the treatment of breast cancer, but the molecular mechanisms underlying sensitivity to ADR remain elusive. In our previous study, through miRNA microarray and experiments, we have emphasized that miR-222 could promote the ADR-resistance in breast cancer cells. The aim of this study was to explore the possible mechanism by which miR-222 affects sensitivity to ADR. MethodsThrough pathway enrichment analyses for miR-222, we found that PTEN/Akt/FOXO1 signaling pathway may be of importance. RT-qPCR analyses and western blot assays confirmed the relationship between miR-222 expression and target genes. Immunofluorescence further visually displayed the location of FOXO1. When blocking PTEN/Akt/FOXO1 signaling pathway, we demonstrated the effects of miR-222-mediated ADR resistance by MTT and apoptosis assays. ResultsRT-qPCR and Western blot results showed that miR-222 expression was negatively correlated with FOXO1 expression. In addition, the subcellular translocation of FOXO1 due to the altered expression of miR-222 was observed from immunofluorescence. Moreover, upregulation of miR-222 expression in MCF-7/S cells is associated with decreased PTEN expression levels and increased phospho-Akt (p-Akt) expression. Conversely in MCF-7/ADR cells, inhibition of miR-222 resulted in increased PTEN expression and decreased p-Akt expression. For further validation, results of the present study also demonstrated that PTEN/Akt/FOXO1 signaling was responsible for the ADR-resistance of breast cancer cells since LY294002, an inhibitor of Akt signaling, partially increased the sensitivity of MCF-7/S cells to ADR. More importantly, we postulated that high expression of miR-222 is closely related to poor overall survival by TCGA database validation. ConclusionsTaken together, these data elucidated that miR-222 mediated ADR-resistance of breast cancer cells partly through regulation of PTEN/Akt/FOXO1 signaling pathway and inhibition of miR-222 may improve the prognosis of breast cancer patients.

Effects of extracellular signal-regulated kinase/protein kinase B signal pathway in cholangiocarcinoma cells invasion and migration promoted by microRNA-21

Objective To observe the effects of extracellular signal-regulated kinase (ERK)1/2 and protein kinase B (Akt) signal pathway in cholangiocarcinoma cells invasion and migration promoted by microRNA-21(miR-21). Methods The experimental study was adopeted. QBC939 cholangiocarcinoma cells were cultured in vitro, through constructing and synthesizing unrelated sequence, miR-21 mimics and miR-21 inhibitor which were transfected into cells, and these cells were allocated into 4 groups, including growing naturally cells in the cell group, cells transfected by unrelated sequence in the 21-NC group, cells transfected by miR-21 mimics in the 21-M group and cells transfected by miR-21 inhibitor in the 21-I group. Besides, cells in the 21-M group were allocated again into the 2 groups, 20 μmol/L LY294002 and 10μmol/L U0126 were respectively added in order to dispose 48 hours for follow-up experiments. Indicatiors of the test: (1) real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-21 in each group of cholangiocarcinoma cells. (2) Werstern blot was performed to detect the relative expressions of PTEN, ERK and Akt proteins in each group of cholangiocarcinoma cells. (3) Scarification assay was executed to test the migration of each group of cholangiocarcinoma cells. Transwell experiment was conducted to examine the migration and invasion of each group of cholangiocarcinoma cells. The measurement data with normal distribution were presented by ±s. The means of the 2 groups were compared by the t test. The means among groups were compared by the ANOVA, and pairwise comparison was analyzed by the Bonferroni test.The repeated measurement data were analyzed by the repeated measures ANOVA. Results (1) The relative expression of miR-21 in the cell group, 21-NC group, 21-M group and 21-I group were 1.010±0.010, 0.980±0.050, 4.900±0.350 and 0.260±0.010, respectively, with a statistically significant difference among the 4 groups (F=78.23, P 0.05). There was increased expression between the 21-M group and cell group, decreased expression between the 21-I group and cell group and significant difference between 21-M group or 21-I group and cell group (P 0.05). Compared with cell group, there was decreased PTEN expression and increased p-ERK and p-Akt expressions in the 21-M group, showing statistically significant differences (P<0.05). Compared with cell group, there was increased PTEN expression and decreased p-ERK and p-Akt expressions in the 21-I group, showing statistically significant differences (P<0.05). (3) The change of migration rate of cells from 6 hours to 48 hours were from 12.0%±3.0% to 23.0%±5.0% in the cell group, from 21.0%±4.0% to 43.0%±7.0% in the 21-M group, from 6.0%±1.0% to 18.0%±4.0% in the miR-21+ LY294002 group and from 9.0%±2.0% to 26.0%±6.0% in the miR-21+ U0126 group, respectively. The migration rate of cells in the 21-M group at each time point was higher than that in the cell group (F=16.23, P<0.05). The migration rate of cells in the miR-21+ LY294002 group and miR-21+ U0126 group were lower than that in the 21-M group (F=25.21, P<0.05), and there was the interaction effects between the change of migration rate of cells of the 3 groups and time, with a statistically significant difference (F=35.31, P<0.05). (4) The numbers of migration cells in the cell group, 21-M group, miR-21+ LY294002 group and miR-21+ U0126 group were 198±32, 248±39, 187±23 and 174±28, respectively, with a statistically significant difference among the 4 groups (F=8.48, P<0.05) and between the 21-M group and cell group (t=4.13, P<0.05). Compared with the 21-M group, the numbers of migration cells in the miR-21+ LY294002 group and miR-21+ U0126 group were decreased (F=21.98, P<0.05). The numbers of invasion cells in the cell group, 21-M group, miR-21+ LY294002 group and miR-21+ U0126 group were 102±22, 211±36, 55±9 and 67±13, respectively, showing a statistically significant difference among the 4 groups (F=11.32, P<0.05) and between the 21-M group and cell group (t=6.67, P<0.05). Compared with the 21-M group, the numbers of invasion cells in the miR-21+ LY294002 group and miR-21+ U0126 group were decreased (F=36.23, P<0.05). Conclusion ERK and Akt signal pathway participate in the cholangio-carcinoma cells invasion and migration promoted by miR-21, PTEN could mediate the process of promoting cholangiocarcinoma cells invasion and migration through ERK and Akt signal pathway promoted by miR-21. Key words: Bile duct neoplasms; MicroRNA-21; Invasion; Migration; Extracellular signal-regulated kinase; Protein kinase B

PTEN expression is a prognostic marker for patients with non-small cell lung cancer: a systematic review and meta-analysis of the literature

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a known tumor suppressor in non-small cell lung cancer (NSCLC). By performing a systematic review and meta-analysis of the literature, we determined the prognostic value of decreased PTEN expression in patients with NSCLC. We comprehensively and systematically searched through multiple online databases up to May 22, 2016 for NSCLC studies reporting on PTEN expression and patient survival outcome. Several criteria, including the Newcastle-Ottawa Quality Assessment Scale (NOS), were used to discriminate between studies. In total, 23 eligible studies with a total of 2,505 NSCLC patients were included in our meta-analysis. Our results demonstrated that decreased expression of PTEN correlated with poor overall survival in NSCLC patients and was indicative of a poor prognosis for disease-free survival and progression-free survival in patients with NSCLC.

LncRNA FER1L4 suppresses cancer cell proliferation and cycle by regulating PTEN expression in endometrial carcinoma

BackgroundDysregulated long non-coding RNAs (lncRNAs) might exert key roles in pathways associated with endometrial carcinoma (EC) development. This study aims to investigate the new role of lncRNA FER1L4 in EC pathogenesis due to its correlation with phosphatase and tensin homolog (PTEN), one important indicator of EC progression. MethodsReal time PCR was performed to detect the expression of FER1L4 in thirty paired EC samples and two EC cell lines. Plasmid containing FER1L4 was transfected into HEC-50 cells with a relative lower level of FER1L4 expression, followed which PTEN expression and Akt phosphorylation were measured by western blotting. Cell proliferation was analyzed through MTT and colony-formation assays, while cell cycle and apoptosis were determined by flow cytometry. ResultsFER1L4 showed significantly downregulation in EC tissues compared to control, which was positively correlated with decreased PTEN expression. Moreover, FER1L4 could promote PTEN expression and inhibit Akt phosphorylation. Additionally, a significant decrease of cell proliferation was observed in FER1L4 overexpressing cells, along with cell cycle arrest at G0/G1 phase and increased proportion of apoptotic cells. ConclusionFER1L4 not only showed downregulation in EC tissues and cells, but also regulated PTEN expression and Akt signaling, which might contribute to its inhibition on cell proliferation. This study might provide a new potential therapeutic target for EC treatment.