Long noncoding RNA ATB promotes the epithelial-mesenchymal transition by upregulating the miR-200c/Twist1 axe and predicts poor prognosis in breast cancer.

Recent studies indicate that the long noncoding RNA ATB (lncATB) can induce the epithelial-mesenchymal transition (EMT) in cancer cells, but the specific cellular targets of lncATB require further investigation. In the present study, the upregulation of lncATB in breast cancer cells was validated in a TGF-ß-induced EMT model. Gain- and loss-of-function studies demonstrated that lncATB enhanced cell migration, invasion and clonogenicity in vitro and in vivo. LncATB promoted the EMT by acting as a sponge for the miR-200 family and restoring Twist1 expression. Subsequently, the clinical significance of lncATB was investigated in a cohort of breast cancer patients (N = 131). Higher lncATB expression was correlated with increased nodal metastasis (P = 0.036) and advanced clinical stage (P = 0.011) as well as shorter disease-free survival (P = 0.043) and overall survival (P = 0.046). These findings define Twist1 as a major target of lncATB in the induction of the EMT and highlight lncATB as a biomarker in breast cancer patients.

Notch3 inhibits epithelial-mesenchymal transition in breast cancer via a novel mechanism, upregulation of GATA-3 expression.

Notch3 and GATA binding protein 3 (GATA-3) have been, individually, shown to maintain luminal phenotype and inhibit epithelial-mesenchymal transition (EMT) in breast cancers. In the present study, we report that Notch3 expression positively correlates with that of GATA-3, and both are associated with estrogen receptor-α (ERα) expression in breast cancer cells. We demonstrate in vitro and in vivo that Notch3 suppressed EMT and breast cancer metastasis by activating GATA-3 transcription. Furthermore, Notch3 knockdown downregulated GATA-3 and promoted EMT; while overexpression of Notch3 intracellular domain upregulated GATA-3 and inhibited EMT, leading to a suppression of metastasis in vivo. Moreover, inhibition or overexpression of GATA-3 partially reversed EMT or mesenchymal-epithelial transition induced by Notch3 alterations. In breast cancer patients, high GATA-3 expression is associated with higher Notch3 expression and lower lymph node metastasis, especially for hormone receptor (HR) positive cancers. Herein, we demonstrate a novel mechanism whereby Notch3 inhibit EMT by transcriptionally upregulating GATA-3 expression, at least in part, leading to the suppression of cancer metastasis in breast cancers. Our findings expand our current knowledge on Notch3 and GATA-3's roles in breast cancer metastasis.

Notch3 Maintains Luminal Phenotype and Suppresses Tumorigenesis and Metastasis of Breast Cancer via Trans-Activating Estrogen Receptor-α.

The luminal A phenotype is the most common breast cancer subtype and is characterized by estrogen receptor α expression (ERα). Identification of the key regulator that governs the luminal phenotype of breast cancer will clarify the pathogenic mechanism and provide novel therapeutic strategies for this subtype of cancer. ERα signaling pathway sustains the epithelial phenotype and inhibits the epithelial-mesenchymal transition (EMT) of breast cancer. In this study, we demonstrate that Notch3 positively associates with ERα in both breast cancer cell lines and human breast cancer tissues. We found that overexpression of Notch3 intra-cellular domain, a Notch3 active form (N3ICD), in ERα negative breast cancer cells re-activated ERα, while knock-down of Notch3 reduced ERα transcript and proteins, with alteration of down-stream genes, suggesting its ability to regulate ERα. Mechanistically, our results show that Notch3 specifically binds to the CSL binding element of the ERα promoter and activates ERα expression. Moreover, Notch3 suppressed EMT, while suppression of Notch3 promoted EMT in cellular assay. Overexpressing N3ICD in triple-negative breast cancer suppressed tumorigenesis and metastasis in vivo. Conversely, depletion of Notch3 in luminal breast cancer promoted metastasis in vivo. Furthermore, Notch3 transcripts were significantly associated with prolonged relapse-free survival in breast cancer, in particular in ERα positive breast cancer patients. Our observations demonstrate that Notch3 governs the luminal phenotype via trans-activating ERα expression in breast cancer. These findings delineate the role of a Notch3/ERα axis in maintaining the luminal phenotype and inhibiting tumorigenesis and metastasis in breast cancer, providing a novel strategy to re-sensitize ERα negative or low-expressing breast cancers to hormone therapy.

Analysis of Epithelial-Mesenchymal Transition Induced by Overexpression of Twist.

Breast cancer, the most common malignancy among women worldwide, is a heterogeneous disease, and it therefore has remarkably different biological characteristics and clinical behavior. Breast cancer has been divided into several different molecular subtypes based on the status of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 (HER2, also named as ErbB2) status. Her2 is a member of EGFR family of transmembrane tyrosine kinase-type receptors, and is involved in the activation of its downstream signaling cascades, which could promote cell proliferation, metastasis, and angiogenesis in tumors. In addition, Twist, a transcriptional factor has been shown to associate with ErbB2 signaling to increase the proliferation and the number of cells, and to induce epithelial-mesenchymal transition. Deregulated cell proliferation can result in hyperplasia and even malignancies. Actually, the proliferative or survival ability of cells can be measured by a variety of methods. Clonogenic assay and CCK8 assay can serve as useful tools to test whether the clonogenic survival ability of tumor cells can be enhanced or reduced upon stimulation of appropriate mitogenic signals or a given cancer therapy respectively. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. Moreover, migration and invasion assay, in some degree, represents the potential for EMT promotion. Here, we introduce colony formation assay; CCK8 proliferation assay; soft agar; and migration and invasion assay using overexpression of ErbB2 and EGFR receptors as an example.

MCAM/CD146 promotes tamoxifen resistance in breast cancer cells through induction of epithelial-mesenchymal transition, decreased ERα expression and AKT activation.

Tamoxifen resistance presents a prominent clinical challenge in endocrine therapy for hormone sensitive breast cancer. However, the underlying mechanisms that contribute to tamoxifen resistance are not fully understood. In this study, we established a tamoxifen resistant MCF-7 cell line (MCF-7-Tam-R) by continuously incubating MCF-7 cells with 4-OH-tamoxifen. We found that melanoma cell adhesion molecule (MCAM/CD146), a unique epithelial-to-mesenchymal transition (EMT) inducer, was significantly up-regulated at both mRNA and protein levels in MCF-7-Tam-R cells compared to parental MCF-7 cells. Mechanistic research demonstrated that MCAM promotes tamoxifen resistance by transcriptionally suppressing ERα expression and activating the AKT pathway, followed by induction of EMT. Elevated MCAM expression was inversely correlated with recurrence-free and distant metastasis-free survival in a cohort of 4142 patients with breast cancer derived from a public database, particularly in the subgroup only treated with tamoxifen. These results demonstrate a novel function of MCAM in conferring tamoxifen resistance in breast cancer. Targeting MCAM might be a promising therapeutic strategy to overcome tamoxifen resistance in breast cancer patients.

Notch3 overexpression causes arrest of cell cycle progression by inducing Cdh1 expression in human breast cancer cells.

Uncontrolled cell proliferation, genomic instability and cancer are closely related to the abnormal activation of the cell cycle. Therefore, blocking the cell cycle of cancer cells has become one of the key goals for treating malignancies. Unfortunately, the factors affecting cell cycle progression remain largely unknown. In this study, we have explored the effects of Notch3 on the cell cycle in breast cancer cell lines by 3 methods: overexpressing the intra-cellular domain of Notch3 (N3ICD), knocking-down Notch3 by RNA interference, and using X-ray radiation exposure. The results revealed that overexpression of Notch3 arrested the cell cycle at the G0/G1 phase, and inhibited the proliferation and colony-formation rate in the breast cancer cell line, MDA-MB-231. Furthermore, overexpressing N3ICD upregulated Cdh1 expression and resulted in p27(Kip) accumulation by accelerating Skp2 degradation. Conversely, silencing of Notch3 in the breast cancer cell line, MCF-7, caused a decrease in expression levels of Cdh1 and p27(Kip) at both the protein and mRNA levels, while the expression of Skp2 only increased at the protein level. Correspondingly, there was an increase in the percentage of cells in the G0/G1 phase and an elevated proliferative ability and colony-formation rate, which may be caused by alterations of the Cdh1/Skp2/p27 axis. These results were also supported by exposing MDA-MB-231 cells or MCF-7 treated with siN3 to X-irradiation at various doses. Overall, our data showed that overexpression of N3ICD upregulated the expression of Cdh1 and caused p27(Kip) accumulation by accelerating Skp2 degradation, which in turn led to cell cycle arrest at the G0/G1 phase, in the context of proliferating breast cancer cell lines. These findings help to illuminate the precision therapy targeted to cell cycle progression, required for cancer treatment.

Role of N-acyl homoserine lactone (AHL)-based quorum sensing (QS) in aerobic sludge granulation.

N-acyl homoserine lactone (AHL)-based quorum sensing (QS) has been recognized to play an important role in the formation of biofilm. However, aerobic granular sludge is considered as a special biofilm, and its biological implication and role of AHL-based QS still remain unclear. This study investigated the role of AHL-based QS in aerobic granulation. Results showed that AHLs were necessary to the typical aerobic granulation, and AHL-associated coordination of bacteria in sludge aggregation was sludge density dependent only when it reached a threshold of 1.010 g/mL; AHL-based QS was activated to regulate aerobic granulation. Furthermore, a quorum quenching method was firstly adopted to investigate the role of AHLs in aerobic granules. Results showed inhibition of AHL by acylase that reduced the AHL content in aerobic granules and further weakened its attachment potential, which proved that AHLs play an important role in the formation of aerobic granules. Additionally, the assay of quorum quenching not only proved that AHL-based QS could regulate EPS production but also provided additional evidence for the role of AHLs in aerobic granulation by regulating EPS content and its component proportion.

Alpha-crystallin promotes rat olfactory ensheathing cells survival and proliferation through regulation of PI3K/Akt/mTOR signaling pathways.

Transplantation of cultured olfactory ensheathing cells (OECs) into lesions can promote axonal regeneration. However, the acutely injured CNS environment affects the survival and proliferation of OECs which might impair its therapy effects. To investigate whether α-crystallin can promote the survival and proliferation of OECs, OECs were cultured with α-crystallin. The survival of OECs was assessed by counting the numbers of p75-labeled OECs. Cellular proliferative activity was estimated by flow cytometry and quantification of BrdU-labeled cells. Phosphorylated p85, Akt and mammalian target of rapamycin (mTOR) were detected when OECs were culture for 7 days. Our results showed that the numbers of p75-labeled or Brdu-labeled OECs in α-crystallin group were much more than that in control group. And α-crystallin increased the phosphorylation of both p85, Akt and mTOR. LY294002 abrogated the ability of α-crystallin to phosphorylate Akt and mTOR, and decreased the percentage of cells in S and G2/M stage which were treated with α-crystallin. These findings indicated that α-crystallin positively regulated the activation of PI3K/Akt/mTOR signaling pathway and promote the proliferation and survival of cultured OECs.

Transplanted olfactory ensheathing cells reduce retinal degeneration in Royal College of Surgeons rats.

PURPOSE OF THE STUDY: Retinitis pigmentosa (RP) is a group of genetic disorders and a slow loss of vision that is caused by a cascade of retinal degenerative events. We examined whether these retinal degenerative events were reduced after cultured mixtures of adult olfactory ensheathing cells (OECs) and olfactory nerve fibroblasts (ONFs) were transplanted into the subretinal space of 1-month-old RCS rat, a classic model of RP. MATERIALS AND METHODS: The changes in retinal photoreceptors and Müller cells of RCS rats after cell transplantation were observed by the expression of recoverin and glial fibrillary acidic protein (GFAP), counting peanut agglutinin (PNA)-positive cone outer segments and calculating the relative apoptotic area. The retinal function was also evaluated by Flash electroretinography (ERG). To further investigate the mechanisms, by which OECs/ONFs play important roles in the transplanted retinas, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF) secretion of the cultured cells were analyzed by ELISA. The ability of OECs/ONFs to ingest porcine retinal outer segments and the amount of phagocytosis were compared with retinal pigment epithelium (RPE) cells. RESULTS: Our research showed that the transplantation of OECs/ONFs mixtures restored recoverin expression, protected retinal outer segments, increased PNA-positive cone outer segments, reduced caspase-positive apoptotic figures, downregulated GFAP, and maintained the b-wave of the ERG. Cultured OECs/ONFs expressed and secreted NGF, BDNF, and bFGF which made contributions to assist survival of the photoreceptors. An in vitro phagocytosis assay showed that OECs, but not ONFs, phagocytosed porcine retinal outer segments, and the phagocytic ability of OECs was even superior to that of RPE cells. CONCLUSIONS: These findings demonstrate that transplantation of OECs/ONFs cleaned up the accumulated debris in subretinal space, and provided an intrinsic continuous supply of neurotrophic factors. It suggested that transplantation of OECs/ONFs might be a possible future route for protection of the retina and reducing retinal degeneration in RP.

[Inducement effect of recombinant human TNF-alpha on apoptosis of breast cancer cell line ZR75-1 and Its mechanism].

BACKGROUND & OBJECTIVE: Tumor necrosis factor alpha (TNF-alpha) is an important agent for tumor biotherapy because of its strong antitumor activity, but its clinical application is limited because of its severe fatal systemic toxicity. A new recombinant human (rh) mutein, mutant type (mt) 471rhTNF-alpha, was produced to decrease toxicity while keep antitumor activity of wild type (wt) TNF-alpha. This study was to compare the antitumor activities of mt-471rhTNF-alpha and wt-rhTNF-alpha, and analyze the mechanism of tumor cell apoptosis induced by mt-471rhTNF-alpha. METHODS: Breast cancer cell line ZR75-1 was treated with 100 microg/L of mt-471rhTNF-alpha or wt-rhTNF-alpha for 12 h. Cell apoptosis was analyzed with 2% agarose gel electrophoresis, and cell cycle distribution was analyzed by flow cytometry. The activation of nuclear factor-kappaB (NF-kappaB) p65 in ZR75-1 cells treated with 10 microg/L, 50 microg/L, and 100 microg/L of mt-471rhTNF-alpha or wt-rhTNF-alpha for 4 h were detected using Trans AMTM NF-kappaB p65 kit based on ELISA. RESULTS: ZR75-1 cells treated with mt-471rhTNF-alpha had typical ladders of DNA fragmentation, whereas ZR75-1 cells treated with wt-rhTNF-alpha only had weak ladders; the apoptosis rate of ZR75-1 cells was significantly higher in mt-471rhTNF-alpha group than in wt-rhTNF-alpha group [(44.6+/-3.6)% vs. (24.7+/-2.7)%, P<0.05]. The proportion of G1 phase cells was significantly higher in mt-471rhTNF-alpha group than in control group and wt-rhTNF-alpha group [(66.6+/-4.2)% vs. (34.8+/-3.1)% and (46.2+/-3.8)%, P<0.05]; the proportions of S phase and G2 phase cells were significantly lower in mt-471rhTNF-alpha group than in control group and wt-rhTNF-alpha group [(33.2+/-2.9)% vs. (50.1+/-3.8)% and (45.7+/-2.9)%, P<0.05; (0.2+/-0.0)% vs. (15.1+/-2.2)% and (8.1+/-3.1)%, P<0.05]. When treated with 50 microg/L rhTNF-alpha for 4 h, NF-kappaB activation was significantly higher in wt-rhTNF-alpha group than in mt-471rhTNF-alpha group (3.3+/-0.1 vs. 1.5+/-0.2, P<0.05 ). CONCLUSIONS: The antitumor ability of mt-471rhTNF-alpha is more stronger than that of wt-rhTNF-alpha. The explanation for the enhanced apoptosis-inducible ability of mt-471rhTNF-alpha could be that NF-kappaB activation is inhibited in tumor cells treated with mt-471rhTNF-alpha.

Construction of influenza virus siRNA expression vectors and their inhibitory effects on multiplication of influenza virus.

Three plasmid constructs were prepared that express small interfering RNAs (siRNAs) targeted to sequences encoding the ribonucleoprotein member, nucleoprotein (NP) and/or PA, of influenza virus genome. The antiviral properties of siRNAs against the H5N1 strain of influenza virus were studied by evaluating their capacity to silence expression of target genes as well as their effect on influenza virus-induced apoptosis in Madin-Darby canine kidney cells, chicken embryo fibroblast cells, and embryonated chicken eggs in a transient replication model. The results demonstrated that all three siRNAs expressing plasmids efficiently transcribed the short hairpin RNAs and inhibited expression of the NP or PA proteins measured by northern blot and western blot analyses, respectively, in the transfected cells. We also found that the integrated siRNA expression plasmid pEGFP/NP+PA, which we constructed for the first time to synchronously target NP and PA segments of the influenza virus genome, could more efficiently inhibit synthesis of influenza virus detected by cytopathogenic effects, hemagglutinin, and plaque-forming unit assays in the transfected cells. Furthermore, the integrated siRNA expression plasmid pEGFP/NP+PA could remarkably interrupt the cellular apoptotic course caused by influenza virus, which protected infected cells from apoptotic damage. In contrast, a control siRNA expression plasmid, pEGFP/HK, could neither inhibit the protein expression and production of influenza virus nor interrupt the cell apoptotic course mediated by influenza virus. These results demonstrate that RNA interference (RNAi) can be used to inhibit protein expression and replication of influenza virus and that RNAi treatment holds potential as a new approach to prevent avian influenza.

[Study on the preliminary purification and bioactivity of recombinant human TNF-alpha mutein 471].

AIM: To purify preliminary recombinant human TNF-alpha mutein 471 and detect its bioactivity on the basis of the TNF-alpha mutein 471 expressed in prokaryotic express system. METHODS: The expression of recombinant human TNF-alpha mutein 471 in engineering bacteria strains E.coil was induced under the condition of optimal fermentation and expression. After cultured E.coil cells were collected and broken by using an ultrasonic disintegrator, the TNF-alpha mutein 471 existed in the form of inclusion body was extracted and purified, and then the effects of denaturation and protein concentration on protein folding were examined. The bioactivities of wild type TNF-alpha and the TNF-alpha mutein 471 were detected by MTT colorimetry. RESULTS: The TNF-alpha mutein 471 was folded and polymerized successfully to from a trimer with bioactivity under the condition of proper denaturation and renaturation. The cytotoxic activity of the TNF-alpha mutein 471 to the L929 cells was 15 times as much as wild type TNF-alpha. CONCLUSION: The TNF-alpha mutein 471 expressed in prokaryotic expression system possesses significantly bioactivity after renaturation, which lays the foundation for further animal experiment and clinical experimental researches.

[Construction and expression of eukaryotic expression plasmid pcDNA3.1/hIL-18].

AIM: To construct an eukaryotic expression plasmid pcDNA3.1/hIL-18 and express it in mammalian cells. METHODS: cDNA encoding mature hIL-18 was cleavaged by enzyme digestion from mesomeric clone vector pGEM-T/hIL-18 and inserted into an eukaryotic expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pcDNA3.1/hIL-18. Then the constructed plasmid was transfected into COS-7 and Rlc310 cells by liposome-mediated gene transfer method. hIL-18 expressed in transfected COS-7 and Rlc310 cells was detected by immunohistochemical staining and level of hIL-18 mRNA in transfected Rlc310 cells was assayed by RT-PCR. RESULTS: A recombinant eukaryotic expression plasmid pcDNA3.1/hIL-18 was successfully constructed and expressed transiently in COS-7 cells and stably in Rlc310 cells. CONCLUSION: The construction and expression of pcDNA3.1/hIL-18 have been achieved successfully, which lays a foundation for further research on anti-tumor effect of IL-18.