Conventional tumor markers for non-invasive diagnosis of gastric cancer (GC) exhibit insufficient sensitivity and specificity to facilitate detection of early gastric cancer (EGC). We aimed to identify EGC-specific exosomal lncRNA biomarkers that are highly sensitive and stable for the non-invasive diagnosis of EGC. Hence, in the present study, exosomes from the plasma of five healthy individuals and ten stage I GC patients and from culture media of four human primary stomach epithelial cells and four gastric cancer cells (GCCs) were isolated. Exosomal RNA profiling was performed using RNA sequencing to identify EGC-specific exosomal lncRNAs. A total of 79 and 285 exosomal RNAs were expressed at significantly higher levels in stage I GC patients and GCCs, respectively, than that in normal controls. Through combinational analysis of the RNA sequencing results, we found two EGC-specific exosomal lncRNAs, lncUEGC1 and lncUEGC2, which were further confirmed to be remarkably up-regulated in exosomes derived from EGC patients and GCCs. Furthermore, stability testing demonstrates that almost all the plasma lncUEGC1 was encapsulated within exosomes and thus protected from RNase degradation. The diagnostic accuracy of exosomal lncUEGC1 was evaluated, and lncUEGC1 exhibited AUC values of 0.8760 and 0.8406 in discriminating EGC patients from healthy individuals and those with premalignant chronic atrophic gastritis, respectively, which was higher than the diagnostic accuracy of carcinoembryonic antigen. Consequently, exosomal lncUEGC1 may be promising in the development of highly sensitive, stable, and non-invasive biomarkers for EGC diagnosis.

Summary Wnt signalling through β-catenin and the lymphoid-enhancing factor 1/T-cell factor (LEF1/TCF) family of transcription factors maintains stem cell properties in both normal and malignant tissues; however, the underlying molecular pathway involved in this process has not been completely defined. Using a microRNA microarray screening assay, we identified let-7 miRNAs as downstream targets of the Wnt–β-catenin pathway. Expression studies indicated that the Wnt–β-catenin pathway suppresses mature let-7 miRNAs but not the primary transcripts, which suggests a post-transcriptional regulation of repression. Furthermore, we identified Lin28, a negative let-7 biogenesis regulator, as a novel direct downstream target of the Wnt–β-catenin pathway. Loss of function of Lin28 impairs Wnt–β-catenin-pathway-mediated let-7 inhibition and breast cancer stem cell expansion; enforced expression of let-7 blocks the Wnt–β-catenin pathway-stimulated breast cancer stem cell phenotype. Finally, we demonstrated that the Wnt–β-catenin pathway induces Lin28 upregulation and let-7 downregulation in both cancer samples and mouse tumour models. Moreover, the delivery of a modified lin28 siRNA or a let-7a agomir into the premalignant mammary tissues of MMTV-wnt-1 mice resulted in a complete rescue of the stem cell phenotype driven by the Wnt–β-catenin pathway. These findings highlight a pivotal role for Lin28/let-7 in Wnt–β-catenin-pathway-mediated cellular phenotypes. Thus, the Wnt–β-catenin pathway, Lin28 and let-7 miRNAs, three of the most crucial stem cell regulators, connect in one signal cascade.

Recent studies have unequivocally identified multipotent stem/progenitor cells in mammary glands, offering a tractable model system to unravel genetic and epigenetic regulation of epithelial stem/progenitor cell development and homeostasis. In this study, we show that Pygo2, a member of an evolutionarily conserved family of plant homeo domain–containing proteins, is expressed in embryonic and postnatal mammary progenitor cells. Pygo2 deficiency, which is achieved by complete or epithelia-specific gene ablation in mice, results in defective mammary morphogenesis and regeneration accompanied by severely compromised expansive self-renewal of epithelial progenitor cells. Pygo2 converges with Wnt/β-catenin signaling on progenitor cell regulation and cell cycle gene expression, and loss of epithelial Pygo2 completely rescues β-catenin–induced mammary outgrowth. We further describe a novel molecular function of Pygo2 that is required for mammary progenitor cell expansion, which is to facilitate K4 trimethylation of histone H3, both globally and at Wnt/β-catenin target loci, via direct binding to K4-methyl histone H3 and recruiting histone H3 K4 methyltransferase complexes.

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that play important roles in multiple biological processes. MiR-20b has been reported to participate in breast cancer tumorigenic progression, however, the functional roles are still unclear and under debating. The aim of this study is to explicit the molecular mechanism of miR-20b underlying breast cancer tumorigenesis. In the present study, we showed that miR-20b was overexpressed in human breast cancer tissues and cell lines compared with paired adjacent normal tissues and normal cell lines, respectively. We identified PTEN, a well-known tumor suppressor, as the functional downstream target of miR-20b. Luciferase assays confirmed that miR-20b could directly bind to the 3′ untranslated region(UTR) of PTEN and suppress translation. Alteration of miR-20b expression changed PTEN protein level but not mRNA expression in ZR-75-30 and MCF-7 breast cancer cells, suggesting miR-20b regulates PTEN gene expression at the posttranscriptional level. Furthermore, upregulation of miR-20b significantly promoted the proliferation, colony formation and DNA synthesis of ZR-75-30 and MCF-7 breast cancer cells. Conversely, knockdown of miR-20b expression inhibited the growth of breast cancer cells in vitro and in vivo. Dysregulation of miR-20b plays critical roles in the breast cancer tumorigenesis, at least in part via targeting the tumor suppressor PTEN. This microRNA may serve as a potential diagnostic marker and therapeutic target for breast cancer.

Summary Wnt–β-catenin signaling participates in the epithelial–mesenchymal transition (EMT) in a variety of cancers; however, its involvement in hepatocellular carcinoma (HCC) and downstream molecular events is largely undefined. HNF4α is the most prominent and specific factor maintaining the differentiation of hepatic lineage cells and a potential EMT regulator in HCC cells. However, the molecular mechanisms by which HNF4α maintains the differentiated liver epithelium and inhibits EMT have not been completely defined. In this study, we systematically explored the relationship between Wnt–β-catenin signaling and HNF4α in the EMT process of HCC cells. Our results indicated that HNF4α expression was negatively regulated during Wnt–β-catenin signaling-induced EMT through Snail and Slug in HCC cells. In contrast, HNF4α was found to directly associate with TCF4 to compete with β-catenin but facilitate transcription co-repressor activities, thus inhibiting expression of EMT-related Wnt–β-catenin targets. Moreover, HNF4α may control the switch between the transcriptional and adhesion functions of β-catenin. Overexpression of HNF4α was found to completely compromise the Wnt–β-catenin-signaling-induced EMT phenotype. Finally, we determined the regulation pattern between Wnt–β-catenin signaling and HNF4α in rat tumor models. Our studies have identified a double-negative feedback mechanism controlling Wnt–β-catenin signaling and HNF4α expression in vitro and in vivo, which sheds new light on the regulation of EMT in HCC. The modulation of these molecular processes may be a method of inhibiting HCC invasion by blocking Wnt–β-catenin signaling or restoring HNF4α expression to prevent EMT.

Great progress has been achieved in the study of Hippo signaling in regulating tumorigenesis; however, the downstream molecular events that mediate this process have not been completely defined. Moreover, regulation of Hippo signaling during tumorigenesis in hepatocellular carcinoma (HCC) remains largely unknown. In the present study, we systematically investigated the relationship between Yes‐associated protein/TEA domain family member (YAP‐TEAD) and hepatocyte nuclear factor 4‐alpha (HNF4α) in the hepatocarcinogenesis of HCC cells. Our results indicated that HNF4α expression was negatively regulated by YAP1 in HCC cells by a ubiquitin proteasome pathway. By contrast, HNF4α was found to directly associate with TEAD4 to compete with YAP1 for binding to TEAD4, thus inhibiting the transcriptional activity of YAP‐TEAD and expression of their target genes. Moreover, overexpression of HNF4α was found to significantly compromise YAP‐TEAD‐induced HCC cell proliferation and stem cell expansion. Finally, we documented the regulatory mechanism between YAP‐TEAD and HNF4α in rat and mouse tumor models, which confirmed our in vitro results. Conclusion: There is a double‐negative feedback mechanism that controls TEAD‐YAP and HNF4α expression in vitro and in vivo, thereby regulating cellular proliferation and differentiation. Given that YAP acts as a dominant oncogene in HCC and plays a crucial role in stem cell homeostasis and tissue regeneration, manipulating the interaction between YAP, TEADs, and HNF4α may provide a new approach for HCC treatment and regenerative medicine. (Hepatology 2017;65:1206‐1221).