High expression of Chemokine receptor 4 (CXCR4) is important in tumor invasion, metastasis, drug-resistance and maintenance of stemness in non-small cell lung cancer (NSCLC). We therefore studied the molecular characteristics of drug-resistant CXCR4-positive cells on epithelial-mesenchymal transition (EMT) for the future identification of the tumor cells with the properties of both EMT and stemness. EMT RT2 Profier PCR Array was performed to determine the expression levels of mRNA genes in A549 with TGF-β1 induced EMT (A549/TGF-β1) and gefitinib-resistant CXCR4-positive cells (A549/GR). TCGA database on the cBio Cancer Genomics Portal website and Gene Network Central (GNC) Pro Tutorial were used to analyze their clinical relevance and pathway interactions. CXCR4 was up-regulated both in TGF-β induced EMT cells and in gefitinib-resistant cells. In 84 mRNA genes related to EMT, 17 mRNA genes were up-regulated in CXCR4-positive population of A549/GR when compared to those in CXCR4 negative fraction, while 66 mRNA genes were up-regulated during TGF-β induced EMT. ITGA5, BMP7, MMP3, VIM, RGS2, ZEB2, TCF3, SNAI2, VCAN, PLEK2, WNT5A, COL3A1, SPARC and FOXC2 were doubly up-regulated during the two biological processes. Kaplan-Meier analysis indicated that the doubly up-regulated ITGA5, RGS2, SNAI2 and PLEK2 mRNA genes were related to poor overall survival in lung adenocarcinoma patients (P=9.291e-6, 0.0090, 3.81e-7 and 0.0013, respectively). In GNC analysis, SNAI2 mRNA gene but not ITGA5, RGS2 and PLEK2 was dependent on the signaling pathway of CXCR4. The molecular characteristics of drug-resistant CXCR4-positive cells have a crosstalk with EMT, which has the potential to find the marker with prognostic value on multiple signaling pathways in NSCLC.

miR-18a is a member of primary transcript called miR-17-92a (C13orf25 or MIR17HG) which also contains five other miRNAs: miR-17, miR-19a, miR-20a, miR-19b and miR-92a. This cluster as a whole shows specific characteristics, where miR-18a seems to be unique. In contrast to the other members, the expression of miR-18a is additionally controlled and probably functions as its own internal controller of the cluster. miR-18a regulates many genes involved in proliferation, cell cycle, apoptosis, response to different kinds of stress, autophagy and differentiation. The disturbances of miR-18a expression are observed in cancer as well as in different diseases or pathological states. The miR-17-92a cluster is commonly described as oncogenic and it is known as 'oncomiR-1', but this statement is a simplification because miR-18a can act both as an oncogene and a suppressor. In this review we summarize the current knowledge about miR-18a focusing on its regulation, role in cancer biology and utility as a potential biomarker.

Keywords: 5-FU, 5-fluorouracyl; ACVR2A, activin A receptor type 2A; AKT, AKT serine/threonine kinase; AR, androgen receptor; ATG7, autophagy related 7; ATM, ATM serine/threonine kinase; BAX, BCL2 associated Xapoptosis regulator; BCL2, BCL2 apoptosis regulator; BCL2L10, BCL2 like 10; BDNF, brain derived neurotrophic factor; BLCA, bladder urothelial carcinoma; BRCA, breast cancer; Biomarker; Bp, base pair; C-myc (MYCBP), MYC binding protein; CASC2, cancer susceptibility 2; CD133 (PROM1), prominin 1; CDC42, cell division cycle 42; CDKN1, Bcyclin dependent kinase inhibitor 1B; COAD, colon adenocarcinoma; Cancer; Circulating miRNA; DDR, DNA damage repair; E2F family (E2F1, E2F2, E2F3), E2F transcription factors; EBV, Epstein-Barr virus; EMT, epithelial-to-mesenchymal transition; ER, estrogen receptor; ERBB (EGFR), epidermal growth factor receptor; ESCA, esophageal carcinoma; FENDRR, FOXF1 adjacent non-coding developmental regulatory RNA; FER1L4, fer-1 like family member 4 (pseudogene); GAS5, growth arrest–specific 5; HIF-1α (HIF1A), hypoxia inducible factor 1 subunit alpha; HNRNPA1, heterogeneous nuclear ribonucleoprotein A1; HNSC, head and neck squamous cell carcinoma; HRR, homologous recombination-based DNA repair; IFN-γ (IFNG), interferon gamma; IGF1, insulin like growth factor 1; IL6, interleukin 6; IPMK, inositol phosphate multikinase; KIRC, clear cell kidney carcinoma; KIRP, kidney renal papillary cell carcinoma; KRAS, KRAS proto-oncogene, GTPase; LIHC, liver hepatocellular carcinoma; LMP1, latent membrane protein 1; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; Liquid biopsy; MAPK, mitogen-activated protein kinase; MCM7, minichromosome maintenance complex component 7; MET, mesenchymal-to-epithelial transition; MTOR, mechanistic target of rapamycin kinase; N-myc (MYCN), MYCN proto-oncogene, bHLH transcription factor; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NOTCH2, notch receptor 2; Oncogene; PAAD, pancreatic adenocarcinoma; PERK (EIF2AK3), eukaryotic translation initiation factor 2 alpha kinase 3; PI3K (PIK3CA), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PIAS3, protein inhibitor of activated STAT 3; PRAD, prostate adenocarcinoma; RISC, RNA-induced silencing complex; SMAD2, SMAD family member 2; SMG1, SMG1 nonsense mediated mRNA decay associated PI3K related kinase; SNHG1, small nucleolar RNA host gene 1; SOCS5, suppressor of cytokine signaling 5; STAD, stomach adenocarcinoma; STAT3, signal transducer and activator of transcription 3; STK4, serine/threonine kinase 4; Suppressor; TCGA; TCGA, The Cancer Genome Atlas; TGF-β (TGFB1), transforming growth factor beta 1; TGFBR2, transforming growth factor beta receptor 2; THCA, papillary thyroid carcinoma; TNM, Classification of Malignant Tumors: T - tumor / N - lymph nodes / M – metastasis; TP53, tumor protein p53; TP53TG1, TP53 target 1; TRIAP1, p53-regulating inhibitor of apoptosis gene; TSC1, TSC complex subunit 1; UCA1, urothelial cancer associated 1; UCEC, uterine corpus endometrial carcinoma; UTR, untranslated region; WDFY3-AS2, WDFY3 antisense RNA 2; WEE1, WEE1 G2 checkpoint kinase; WNT family, Wingless-type MMTV integration site family/Wnt family ligands; ZEB1/ZEB2, zinc finger E-box binding homeobox 1 and 2; ceRNA, competitive endogenous RNA; cncRNA, protein coding and non-coding RNA; lncRNA, long-non coding RNA; miR-17-92a; miR-18a; miRNA.

Objective: MicroRNAs play vital roles in the development of endometriosis. It is reported that miR-200b-3p is downregulated in endometriosis, although its mechanisms in this disease remain still unclear. Therefore, the purpose of this study was to explore the function and potential regulatory network of miR-200b-3p in endometriosis through database analysis.

Methods: The endometriosis gene expression profiles were downloaded from the GEO database to screen differentially expressed genes (DEGs). The predicted and validated target genes of miR-200b-3p were obtained from miRWalk and miRTarBase database. Then, a comparison was performed between miR-200b-3p target genes and DEGs. GO enrichment and KEGG pathway analysis of the target genes was performed using clusterProfiler package. STRING was used to predict the protein-protein interaction among the proteins encoded by the target genes. Then, TransmiR, LncBase, StarBase, PROMO, and AnimalTFDB were employed to identify interactive transcription factors and lncRNAs of miR-200b-3p.

Results: miR-200b-3p was associated with the transcription factors DNMT1, EZH2, HNF1B, JUN, MYB, ZEB1, and ZEB2 during the pathogenesis of endometriosis. The downstream 110 target genes were involved in the biological processes of positive regulation of MAPK cascade, muscle cell proliferation, organ growth, vasculogenesis, and axon development. KEGG analysis revealed that the main pathways related to miR-200b-3p were microRNAs in cancer, PI3K-Akt signaling pathway, colorectal cancer, and tight junction. In addition, four lncRNAs such as MALAT1, NEAT1, SNHG22, and XIST interacted with miR-200b-3p and were associated with transcription factors FOXP3 and YY1.

Conclusion: The predicted target genes and molecular regulatory network of miR-200b-3p in endometriosis not only revealed its biological function but also provided a valuable guideline for further research.

Large and giant congenital melanocytic nevi (CMN) are rare melanocytic lesions mostly caused by post-zygotic NRAS alteration. Molecular characterization is usually focused on NRAS and BRAF genes in a unique biopsy of the CMN. However, large/giant CMN may exhibit phenotypic differences among distinct areas, and patients differ in features such as presence of multiple CMN or Spilus-like lesions. Herein, we have characterized a series of 21 large/giant CMN including Spilus-type nevus patients (9/21 cases, 42.8%). Overall, 53 fresh-frozen biopsies corresponding to 40 phenotypically characterized areas of large/giant CMNs and 13 satellite lesions were analyzed by a multigene panel and RNA sequencing (RNA-seq). Mutational screening showed mutations in 76.2% (16/21) of large/giant CMN. A NRAS mutation was found in 57.1% (12/21) of cases, and mutations in other genes such as BRAF, KRAS, APC and MET were detected in 14.3% (3/21) of patients. RNA-seq revealed the fusion transcript ZEB2-ALK and SOX5-RAF1 in large/giant CMN from two patients without missense mutations. Both alterations were not detected in unaffected skin and were detected in different areas of affected skin. These findings suggest that large/giant CMN may result from distinct molecular events in addition to NRAS mutations including point mutations and fusion transcripts.

This study aims to find the difference of genomewide DNA methylation in Schwann cells (SCs) before and after peripheral nerve system (PNS) injury by Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq) and seek meaningful differentially methylated genes related to repairment of injured PNS. SCs harvested from sciatic nerve were named as activated Schwann cells (ASCs), and the ones harvested from brachial plexus were named as normal Schwann cells (NSCs). Genomic DNA of ASCs and NSCs were isolated and MeDIP-Seq was conducted. Differentially methylated genes and regions were discovered and analyzed by bioinformatic methods. MeDIP-Seq analysis showed methylation differences were identified between ASCs and NSCs. The distribution of differentially methylated regions (DMRs) peaks in different components of genome was mainly located in distal intergenic regions. GO and KEGG analysis of these methylated genes were also conducted. The expression patterns of hypermethylated genes (Dgcr8, Zeb2, Dixdc1, Sox2, and Shh) and hypomethylated genes (Gpr126, Birc2) detected by qRT-PCR were opposite to the MeDIP analysis data with significance (p < 0.05), which proved MeDIP analysis data were real and believable. Our data serve as a basis for understanding the injury-induced epigenetic changes in SCs and the foundation for further studies on repair of PNS injury.

Metabolic reprogramming is a molecular hallmark of cancer. Recently, we have reported the overexpression of glyoxalase 1 (encoded by GLO1), a glutathione-dependent enzyme involved in detoxification of the reactive glycolytic byproduct methylglyoxal, in human malignant melanoma cell culture models and clinical samples. However, the specific role of GLO1 in melanomagenesis remains largely unexplored. Here, using genetic target modulation, we report the identification of GLO1 as a novel molecular determinant of invasion and metastasis in malignant melanoma. First, A375 human malignant melanoma cells with GLO1 deletion (A375-GLO1_KO) were engineered using CRISPR/Cas9, and genetic rescue clones were generated by stable transfection of KO clones employing a CMV-driven GLO1 construct (A375-GLO1_R). After confirming GLO1 target modulation at the mRNA and protein levels (RT-qPCR, immunodetection, enzymatic activity), phenotypic characterization indicated that deletion of GLO1 does not impact proliferative capacity while causing significant sensitization to methylglyoxal-, chemotherapy-, and starvation-induced cytotoxic stress. Employing differential gene expression array analysis (A375-GLO1_KO versus A375-GLO1_WT), pronounced modulation of epithelial--mesenchymal transition (EMT)-related genes [upregulated: CDH1, OCLN, IL1RN, PDGFRB, SNAI3; (downregulated): BMP1, CDH2, CTNNB1, FN1, FTH1, FZD7, MELTF, MMP2, MMP9, MYC, PTGS2, SNAI2, TFRC, TWIST1, VIM, WNT5A, ZEB1, and ZEB2 (up to tenfold; p < 0.05)] was observed-all of which are consistent with EMT suppression as a result of GLO1 deletion. Importantly, these expression changes were largely reversed upon genetic rescue employing A375-GLO1_R cells. Differential expression of MMP9 as a function of GLO1 status was further substantiated by enzymatic activity and ELISA analysis; phenotypic assessment revealed the pronounced attenuation of morphological potential, transwell migration, and matrigel 3D-invasion capacity displayed by A375-GLO1_KO cells, reversed again in genetic rescue clones. Strikingly, in a SCID mouse metastasis model, lung tumor burden imposed by A375-GLO1_KO cells was strongly attenuated as compared to A375-GLO1_WT cells. Taken together, these prototype data provide evidence in support of a novel function of GLO1 in melanoma cell invasiveness and metastasis, and ongoing investigations explore the function and therapeutic potential of GLO1 as a novel melanoma target.

Keywords: CMV-driven re-expression; CRISPR/Cas9; SCID mouse metastasis model; epithelial–mesenchymal transition; glyoxalase 1; malignant melanoma; matrix metalloproteinase 9; transwell invasion.

Background: Esophageal squamous cell carcinoma (ESCC), one of the leading causes of cancer mortality worldwide, is a heterogeneous cancer with diverse clinical manifestations. However, little is known about the epigenetic heterogeneity and its clinical relevance for this prevalent cancer. Methods: We generated 7.56 Tb single-base resolution whole-genome bisulfite sequencing data for 84 ESCC and paired paraneoplastic tissues. The analysis identified inter- and intratumor DNA methylation (DNAm) heterogeneity, epigenome-wide DNAm alterations together with the functional regulators involved in the hyper- or hypomethylated regions, and their association with clinical features. We then validated the correlation between the methylation level of specific regions and clinical outcomes of 96 ESCC patients in an independent cohort. Results: ESCC manifested substantial inter- and intratumor DNAm heterogeneity. The high intratumor DNAm heterogeneity was associated with lymph node metastasis and worse overall survival. Interestingly, hypermethylated regions in ESCC were enriched in promoters of numerous transcription factors, and demethylated noncoding regions related to RXR transcription factor binding appeared to contribute to the development of ESCC. Furthermore, we identified numerous DNAm alterations associated with carcinogenesis and lymph node metastasis of ESCC. We also validated three novel prognostic markers for ESCC, including one each in the promoter of CLK1, the 3' untranslated region of ZEB2, and the intergenic locus surrounded by several lncRNAs. Conclusions: This study presents the first population-level resource for dissecting base-resolution DNAm variation in ESCC and provides novel insights into the ESCC pathogenesis and progression, which might facilitate diagnosis and prognosis for this prevalent malignancy.

Gastric cancer (GC) is the most common epithelial malignancy worldwide. Basic transcription factor 3 (BTF3) plays a crucial role in the regulation of various biological processes. We designed experiments to investigate the molecular mechanism underlying the role of BTF3 in GC cell proliferation and metastasis. We confirmed that BTF3 expression was decreased in GC tissues and several GC cell lines. Lentivirus-mediated downregulation of BTF3 reduced cell proliferation, induced S and G2/M cell cycle arrest, and increased apoptosis. Knockdown of BTF3 significantly reduced the expression of Forkhead box M1 (FOXM1). Upregulation of FOXM1 significantly inhibited the decrease in cell proliferation due to BTF3 silencing, S and G2/M cell cycle arrest, and increase in apoptosis. Knockdown of BTF3 decreased Ki-67 and PCNA expression, whereas it increased p27 expression, which was inhibited by upregulation of FOXM1. Knockdown of BTF3 significantly decreased the ability to invade and migrate. Moreover, knockdown of BTF3 increased E-cadherin expression, whereas it decreased N-cadherin and ZEB2 expression, indicating a decrease in epithelial-mesenchymal transition (EMT). Phosphorylation of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) was significantly inhibited by knockdown of BTF3. IL-6-stimulated phosphorylation of STAT3 and JAK2 markedly suppressed inhibition of EMT due to BTF3 silencing. Silencing of BTF3 decreased tumor volume and weight and reduced peritoneal nodules in implanted tumors. Our findings provide a novel understanding of the mechanism of GC and highlight the important role of BTF3/FOXM1 in tumor growth and BTF3/JAK2/STAT3 in EMT and metastasis.

Mowat-Wilson syndrome (MWS) is a recently delineated mental retardation; a multiple congenital anomaly syndrome characterised by a typical facial gestalt, Hirschsprung disease or severe constipation, genitourinary anomaly, congenital heart defects, agenesis of corpus callosum and eye defects. Some cases also present with epilepsy, growth retardation with microcephaly and speech impairment. MWS was first described in 1998 by Mowat et al, and approximately 180 cases have been reported as of August 2008. The syndrome occurs as a result of heterozygous mutations or deletions in the zinc finger E-box-binding homeobox 2 gene, ZEB2, previously called ZFHX1B (SIP1). Most cases reported so far were sporadic occurrences; however, rare cases of sibling recurrence have been cited. The facial phenotype is particularly important for the initial clinical diagnosis and provides the hallmark, warranting ZEB2 mutational analysis even in the absence of Hirschsprung disease. We present the first two molecularly confirmed Malaysian MWS patients, one of whom has a novel mutation.

Rubinstein-Taybi syndrome (RSTS) is a rare autosomal dominant disorder characterised by facial dysmorphisms, growth and psychomotor development delay, and skeletal defects. The known genetic causes are point mutations or deletions of the CREBBP (50-60%) and EP300 (5%) genes. To detect chromosomal rearrangements indicating novel positional candidate RSTS genes, we used a-CGH to study 26 patients fulfilling the diagnostic criteria for RSTS who were negative at fluorescence in situ hybridisation analyses of the CREBBP and EP300 regions, and direct sequencing analyses of the CREBBP gene. We found seven imbalances (27%): four de novo and three inherited rearrangements not reported among the copy number variants. A de novo 7p21.1 deletion of 500 kb included the TWIST1 gene, a suggested candidate for RSTS that is responsible for the Saethre-Chotzen syndrome, an entity that enters in differential diagnosis with RSTS. A similar issue of differential diagnosis was raised by a large 4.3 Mb 2q22.3q23.1 deletion encompassing ZEB2, the gene responsible for the Mowat-Wilson syndrome, whose signs may overlap with RSTS. Positional candidate genes could not be sought in the remaining pathogenetic imbalances, because of the size of the involved region (a 9 Mb 2q24.3q31.1 deletion) and/or the relative paucity of suitable genes (a 5 Mb 3p13p12.3 duplication). One of the inherited rearrangements, the 17q11.2 379Kb duplication, represents the reciprocal event of the deletion underlying an overgrowth syndrome, both being mediated by the NF1-REP-P1 and REP-P2 sub-duplicons. The contribution of this and the other detected CNVs to the clinical RSTS phenotype is difficult to assess.

Repetitive stimulation by persistent pathogens such as human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV) induces the differentiation of natural killer (NK) cells. This maturation pathway is characterized by the acquisition of phenotypic markers, CD2, CD57, and NKG2C, and effector functions-a process regulated by Tim-3 and orchestrated by a complex network of transcriptional factors, involving T-bet, Eomes, Zeb2, promyelocytic leukemia zinc finger protein, and Foxo3. Here, we show that persistent immune activation during chronic viral co-infections (HCMV, hepatitis C virus, and HIV) interferes with the functional phenotype of NK cells by modulating the Tim-3 pathway; a decrease in Tim-3 expression combined with the acquisition of inhibitory receptors skewed NK cells toward an exhausted and cytotoxic phenotype in an inflammatory environment during chronic HIV infection. A better understanding of the mechanisms underlying NK cell differentiation could aid the identification of new immunological targets for checkpoint blockade therapies in a manner that is relevant to chronic infection and cancer.

OBJECTIVE

To present a seven-cases serie of Mowat-Wilson syndrome (MWS).

METHODS

All patients with positive mutation for the ZEB2 were evaluated by a geneticist and a neurologist, with clinical and laboratorial characterization.

RESULTS

A peculiar facies and mental retardation were present in all patients. The Denver II scale showed intense delay in all aspects, especially fine motor and adaptive. Acquired microcephaly was observed in five patients. Only one patient did not present epilepsy. Epilepsy was focal and predominating in sleep, with status epilepticus in three patients. The initial seizure was associated with fever in most patients (4/6). The EEG showed epileptic focal activity (5/7). The imaging studies revealed total agenesis (4/7) and partial agenesis of the corpus callosum (1/7).

CONCLUSIONS

Physicians who care for patients with mental retardation and epilepsy should be aware of SMW.

BACKGROUND

Hirschsprung's disease (HSCR) is cited as a classical component in the constellation of features found in children with Mowat-Wilson syndrome (MWS), which is caused by a mutation of the ZEB2 gene. The prevalence and phenotype of HSCR in those with MWS has yet to be determined. Similarly, it is not known if children with MWS who undergo a curative pull-through operation experience similar functional outcomes. We aimed to delineate the clinical features of those with MWS and HSCR and to determine if these patients experience unfavourable outcomes following pull-through surgery.

METHODS

A systematic review of the literature using the key search term "Mowat Wilson" was performed using three online databases. Clinical data were collected on all patients with a diagnosis of MWS confirmed by ZEB2 analysis. Data regarding bowel function in children with biopsy-proven HSCR were recorded where available. Statistical analysis was performed using SPSS (v. 20.0).

RESULTS

Fifty-two articles were reviewed in the final analysis, incorporating data on 256 patients with a diagnosis of MWS. HSCR was diagnosed in 111 patients (43.4%). Males with HSCR had a slightly increased risk of genital tract anomalies (e.g. hypospadias) compared to those without HSCR (RR 1.79, p = 0.05). Data pertaining to disease phenotype and functional outcome were only available on 42 and 13 patients, respectively. Rectosigmoid aganglionosis was the most common sub-type of HSCR, being described 26 patients (66.7%), albeit accounting for a lower proportion than would normally be expected in an HSCR population. Only two patients (15.4%) were described as having normal bowel function at follow-up with the remainder having terminal stomas, or experiencing troublesome persistent bowel symptoms and recurrent enterocolitis.

CONCLUSIONS

Hirschsprung's disease is present in approximately 45% of patients diagnosed with MWS. Although there is a relative lack of data available on the clinical phenotype of HSCR in this group and their functional outcome following pull-through operation, our data suggest an increased prevalence of long-segment aganglionosis and an increased risk of clinically significant persistent bowel symptoms following pull-through surgery, in many cases necessitating terminal stoma formation.

Patients with mediastinal lymph node metastasis (N2) in squamous cell carcinoma (SqCC) of the lung have poor prognosis after surgical resection of the primary tumor. The aim of this study was to clarify predictive factors of the recurrence of pathological lung SqCC with N2 focusing on the biological characteristics of both cancer cells and cancer-associated fibroblasts (CAFs) in primary and metastatic lymph node tumors. We selected 64 patients with pathological primary lung N2 SqCC who underwent surgical complete resection and investigated the expressions of four epithelial-mesenchymal transition-related markers (caveolin, clusterin, E-cadherin, ZEB2), three cancer stem cell-related markers (ALDH-1, CD44 variant6, podoplanin) of cancer cells, and four markers of CAFs (caveolin, CD90, clusterin, podoplanin) in both primary and matched metastatic lymph node tumors in the N2 area. In the primary tumors, the expressions of all the examined molecules were not related to recurrence. However, in the metastatic lymph node tumors, high clusterin and ZEB2 expressions in the cancer cells and high podoplanin expression in the CAFs were significantly correlated with recurrence (P = 0.03, 0.04, and 0.007, respectively). In a multivariate analysis, only podoplanin expression in the CAFs in metastatic lymph node tumors was identified as a significantly independent predictive factor of recurrence (P = 0.03). Our study indicated that the immunophenotypes of both cancer cells and CAFs in metastatic lymph node tumors, but not primary tumors, provide useful information for predicting the recurrence of pathological N2 lung SqCC.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies around the world. The poor prognosis and high recurrence rate of HCC are largely the result of the high frequencies of intrahepatic and extrahepatic metastases. However, the treatment of metastasis is very limited. Ultrasound-targeted microbubble destruction (UTMD) technology has been recognized as a promising technology for drug and gene delivery in vivo and in vitro. Long noncoding RNA activated by transforming growth factor-β (TGF-β; lncRNA-ATB) was recently identified, which was upregulated in HCC metastases and associated with poor prognosis of HCC patients. In this study, we used microbubbles for UTMD-mediated siRNA transfection to silence lncRNA-ATB expression. We found that UTMD-mediated siRNA transfection significantly inhibited lncRNA-ATB expression and ZEB1 and ZEB2 expression and suppressed cell migration and invasion. We also demonstrated that transfecting siRNA against lncRNA-ATB by using UTMD was more efficient than that by using lipidosome. UTMD-mediated siRNA transfection against lncRNA-ATB may be a promising therapy for HCC metastasis.

Previous studies have demonstrated that microRNA (miR)-205-5p expression is significantly increased in non‑small cell lung cancer tissues and is associated with tumor differentiation grade. The aim of the present study was to explore the effects of miR‑205‑5p on viability, apoptosis and invasion of lung cancer A549 cells. The hsa‑miR‑205‑5p small interfering RNA (siRNA) inhibitor was transfected into A549 cells and expression of miR‑205‑5p was detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Cell viability, apoptosis and invasion were assayed by Cell Counting kit‑8, Annexin V/propidium iodide double staining and Transwell assay, respectively. Target genes of miR‑205‑5p were predicted using bioinformatics analysis. Expression of mRNA and protein levels of candidate target genes following miR‑205‑5p inhibition were detected using RT‑qPCR and western blot analysis respectively. The results demonstrated that relative survival rates of A549 cells were significantly inhibited in miR‑205‑5p siRNA‑transfected cells at 24 and 48 h compared with control cells. Apoptosis was markedly increased in the miR‑205‑5p siRNA cells compared with control cells. The number of invaded cells following miR‑205‑5p siRNA silencing was significantly decreased compared with control cells. Bioinformatics analysis revealed that erb‑B2 receptor kinase 3 (erbB3), zinc finger E‑box binding homeobox 2 (ZEB2), clathrin heavy chain (CLTC) and mediator complex subunit 1 (MED1) may be potential target genes of miR‑205‑5p. Reduced expression of miR‑205‑5p significantly increased the expression of ZEB2 mRNA and protein, inhibited the expression of erbB3 protein, but had no significant effect on the expression levels of CLTC and MED1. In summary, reduced expression of miR‑205‑5p promoted apoptosis and inhibited proliferation and invasion in lung cancer A549 cells through upregulation of ZEB2 and downregulation of erbB3. The present results suggested that the increased miR‑205‑5p expression observed in non‑small cell lung cancer tissues may contribute to increased proliferation and invasion of lung cancer cells and thus to cancer progression.

Ovarian cancers present a mild clinical course when diagnosed early but an aggressive pathway when diagnosed in the peri- and postmenopausal periods. However, the predictability of tumor progression is stochastic and is difficult to predict. In the present study, we hypothesized to examine the key pathways that are dysregulated to promote epithelial-mesenchymal transition in serous ovarian carcinoma. Examination of these steps would help to identify ascitic fluid with cells poised for metastasis or otherwise. We focused on examining the Akt2 expression, mainly because of its report as being overamplified in the aggressive variants of ovarian cancer, as well as TGFbeta-sensitivity of Akt2 that forms the key basis for metastasis initiation of most kinds of carcinoma. We obtained primary ovarian carcinoma samples as well as ascitic fluid and distantly metastatic ovarian carcinoma to examine the expression of Akt2. The results of the study demonstrated that in malignant exfoliated ovarian cancer cells, Smad4 expression was tremendously increased in the nuclei, suggesting nuclear translocation of Smad, which thereafter may have activated ZEB2, and thereafter genomically affected the expression of E-cadherin, myosin II, and vimentin, key components for initiating and sustaining metastasis. All of these may have been stimulated by increased cellular expression of Akt2 in metastatic variants of the serous ovarian carcinoma. The reliance on Akt2 and TGF beta signaling may also potentiate the case for Akt inhibitors or small molecule inhibitors of TGFbeta signaling like doxycycline as adjunct chemotherapy in serous ovarian carcinoma, especially the metastatic variants.

Although the incidence and mortality of gastric cancer (GC) are slowly decreasing, the overall prognosis of GC patients with distal metastasis remains dismal. Long non-coding RNA PVT1 has been verified to function as a tumor promoter in several types of cancer. However, the role of PVT1 in GC metastasis remains obscure. Herein, we found that PVT1 was highly expressed in GC tissues and high PVT1 level was associated with tumor stage, lymph node metastasis, and poor prognosis. Overexpression of PVT1 significantly elevated epithelial-to-mesenchymal transition (EMT) marker (N-cadherin, ZEB1, and ZEB2) levels and promoted GC cell EMT process and tumor metastasis in vitro and in vivo. Mechanistically, Snail was identified as a direct target of miR-30a. PVT1 could bind with miR-30a and increase the expression of Snail by acting as a competing endogenous RNA, whereas re-expression of miR-30a in GC cells rescued the EMT markers, decreased Snail level, and inhibited GC cell migration. Taken together, these findings provide a new light on PVT1 in the pathogenesis and development of GC and an important implication for future therapy of the GC.

Keywords: Snail; gastric cancer; long non-coding RNA PVT1; metastasis; miR-30a.

The transition between epithelial and mesenchymal phenotypes (EMT) occurs in a variety of contexts. It is critical for mammalian development and it is also involved in tumor initiation and progression. Master transcription factor (TF) regulators of this process are conserved between mouse and human.

From a computational analysis of a variety of high-throughput sequencing data sets we initially inferred that TFAP2A is connected to the core EMT network in both species. We then analysed publicly available human breast cancer data for TFAP2A expression and also studied the expression (by mRNA sequencing), activity (by monitoring the expression of its predicted targets), and binding (by electrophoretic mobility shift assay and chromatin immunoprecipitation) of this factor in a mouse mammary gland EMT model system (NMuMG) cell line.

We found that upon induction of EMT, the activity of TFAP2A, reflected in the expression level of its predicted targets, is up-regulated in a variety of systems, both murine and human, while TFAP2A's expression is increased in more "stem-like" cancers. We provide strong evidence for the direct interaction between the TFAP2A TF and the ZEB2 promoter and we demonstrate that this interaction affects ZEB2 expression. Overexpression of TFAP2A from an exogenous construct perturbs EMT, however, in a manner similar to the downregulation of endogenous TFAP2A that takes place during EMT.

Our study reveals that TFAP2A is a conserved component of the core network that regulates EMT, acting as a repressor of many genes, including ZEB2.

This article has been reviewed by Dr. Martijn Huynen and Dr. Nicola Aceto.

BACKGROUND Hyperglycemia has been confirmed to damage endothelial function of vascular and microvascular. The regulation of zinc finger E-box binding protein 2 (ZEB2) on vascular endothelial cells (VECs) is reported rarely. Our study investigates the role of ZEB2 on the apoptosis of VECs induced by high glucose through MAPK pathway. MATERIAL AND METHODS Downregulated and upregulated expression of ZEB2 in human umbilical vein endothelial cells (HUVECs) were performed by plasmids transfection. HUVECs are respectively treated with different concentrations of glucose (5.5 mM, 33 mM). The expression of mRNA and protein were detected by real-time quantified PCR and western blotting. Apoptotic cells were measured by flow cytometry. Proliferation and migration of HUVECs were detected by MTT assay and wound healing assay. RESULTS The apoptosis of HUVECs detected by flow cytometry and western blot revealed that ZEB2 overexpression distinctly suppressed the apoptosis of HUVECs induced by high glucose. ZEB2 overexpression promoted the proliferative and migration activity of HUVECs. Besides, ZEB2 overexpression specifically accelerated the phosphorylation level of JNK, and suppressed the apoptosis and promoted the proliferative of VECs via JNK pathway. CONCLUSIONS ZEB2 suppress apoptosis of VECs induced by high glucose through MAPK pathway activation, which provides a novel insight and therapeutic target for endothelial injury.

OBJECTIVE

To establish a miRNA signature for metastasis in an animal model of esophageal adenocarcinoma (EAC).

BACKGROUND

The incidence of esophageal adenocarcinoma (EAC) has dramatically increased and esophageal cancer is now the sixth leading cause of cancer deaths worldwide. Mortality rates remain high among patients with advanced stage disease and esophagectomy is associated with high complication rates. Hence, early identification of potentially metastatic disease would better guide treatment strategies.

METHODS

The modified Levrat's surgery was performed to induce EAC in Sprague-Dawley rats. Primary EAC and distant metastatic sites were confirmed via histology and immunofluorescence. miRNA profiling was performed on primary tumors with or without metastasis. A unique subset of miRNAs expressed in primary tumors and metastases was identified with Ingenuity Pathway Analysis (IPA) along with upstream and downstream targets. miRNA-linked gene expression analysis was performed on a secondary cohort of metastasis positive (n=5) and metastasis negative (n=28) primary tumors.

RESULTS

The epithelial origin of distant metastasis was established by IF using villin (VIL1) and mucin 5AC (MUC5AC) antibodies. miRNome analysis identified four down-regulated miRNAs in metastasis positive primary tumors compared to metastasis negative tumors: miR-92a-3p (p=0.0001), miR-141-3p (p=0.0022), miR-451-1a (p=0.0181) and miR133a-3p (p=0.0304). Six target genes identified in the top scoring networks by IPA were validated as significantly, differentially expressed in metastasis positive primary tumors: Ago2, Akt1, Kras, Bcl2L11, CDKN1B and Zeb2.

CONCLUSIONS

In vivo metastasis was confirmed in the modified Levrat's model. Analysis of the primary tumor identified a distinctive miRNA signature for primary tumors that metastasized.

Regulation of metastasis continues to remain enigmatic despite our improved understanding of cancer. Identification of microRNAs associated with metastasis in the recent past has provided a new hope. Here, we show how microRNA-101 (miR-101) regulates two independent processes of cellular metastasis by targeting pro-metastatic upstream regulatory transcription factors, ZEB1 and ZEB2, and downstream effector-actin modulators, RHOA and RAC1, providing a single target for therapeutic intervention. Further, we depict how down-regulation of miR-101 by extracellular signal-regulated kinase-2 (ERK2) is vital for MAP kinase pathway induced cellular migration and mesenchymal transition. Importantly, EKR2 induced expression of ZEB1 seems essential for down-regulation of miR-101-1 and induction of EMT. Given the role of EMT in metastasis, we also observe a significant correlation between miR-101 expression and lymph node metastasis; and identify the ERK2-ZEB1-miR-101-1 pathway active in breast cancer tissues, with an apparent clinicopathological implication.