EREG silencing inhibits tumorigenesis via inactivating ERK/p38 MAPK pathway in pancreatic ductal adenocarcinoma.

Epiregulin (EREG) is a member of the epidermal growth factor (EGF) family. An increasing body of evidence has demonstrated the pivotal role of EREG in the pathogenesis and progression of various malignancies. However, the clinical significance and biological role of EREG in pancreatic ductal adenocarcinoma (PDAC) have yet to be fully elucidated. We found that EREG is highly expressed in PDAC tissues compared with paracancerous tissues through public databases and clinical samples. High EREG expression predicted worse overall survival (OS) and recurrence-free survival (RFS) in patients with PDAC. Multivariate analysis revealed that EREG can serve as an independent prognostic indicator. In addition, EREG silencing inhibited PDAC cell proliferation, migration, progression, altered cell cycle, facilitated apoptosis in vitro and suppressed tumor growth in vivo. Conversely, EREG overexpression facilitated the proliferation, migration, and invasion in PaTu-8988 t cell. Through transcriptome sequencing and experimental verification, we found EREG mediates PDAC tumorigenesis through ERK/p38 MAPK signaling pathway. Moreover, we found EREG expression is closely related to PD-L1 expression in PDAC tissues and cells. Therefore, EREG is expected to be a prospective prognostic and therapeutic marker for PDAC.

GARNL3 identified as a crucial target for overcoming temozolomide resistance in EGFRvIII-positive glioblastoma.

OBJECT: Amplification of the epidermal growth factor receptor (EGFR) and its active mutant type III (EGFRvIII), frequently occurr in glioblastoma (GBM), contributing to chemotherapy and radiation resistance in GBM. Elucidating the underlying molecular mechanism of temozolomide (TMZ) resistance in EGFRvIII GBM could offer valuable insights for cancer treatment. METHODS: To elucidate the molecular mechanisms underlying EGFRvIII-mediated resistance to TMZ in GBM, we conducted a comprehensive analysis using Gene Expression Omnibus and The cancer genome atlas (TCGA) databases. Initially, we identified common significantly differentially expressed genes (DEGs) and prioritized those correlating significantly with patient prognosis as potential downstream targets of EGFRvIII and candidates for drug resistance. Additionally, we analyzed transcription factor expression changes and their correlation with candidate genes to elucidate transcriptional regulatory mechanisms. Using estimate method and databases such as Tumor IMmune Estimation Resource (TIMER) and CellMarker, we assessed immune cell infiltration in TMZ-resistant GBM and its relationship with candidate gene expression. In this study, we examined the expression differences of candidate genes in GBM cell lines following EGFRvIII intervention and in TMZ-resistant GBM cell lines. This preliminary investigation aimed to verify the regulatory impact of EGFRvIII on candidate targets and its potential involvement in TMZ resistance in GBM. RESULTS: Notably, GTPase Activating Rap/RanGAP Domain Like 3 (GARNL3) emerged as a key DEG associated with TMZ resistance and poor prognosis, with reduced expression correlating with altered immune cell profiles. Transcription factor analysis suggested Epiregulin (EREG) as a putative upstream regulator of GARNL3, linking it to EGFRvIII-mediated TMZ resistance. In vitro experiments confirmed EGFRvIII-mediated downregulation of GARNL3 and decreased TMZ sensitivity in GBM cell lines, further supported by reduced GARNL3 levels in TMZ-resistant GBM cells. CONCLUSION: GARNL3 downregulation in EGFRvIII-positive and TMZ-resistant GBM implicates its role in TMZ resistance, suggesting modulation of EREG/GARNL3 signaling as a potential therapeutic strategy.

Prognostic significance of EGFR, AREG and EREG amplification and gene expression in muscle invasive bladder cancer.

Introduction: Muscle invasive bladder cancer (MIBC) remains a prevalent cancer with limited therapeutic options, obviating the need for innovative therapies. The epidermal growth factor receptor (EGFR) is a linchpin in tumor progression and presents a potential therapeutic target in MIBC. Additionally, the EGFR ligands AREG and EREG have shown associations with response to anti-EGFR therapy and improved progression-free survival in colorectal carcinoma. Materials and methods: We investigated the prognostic significance of EGFR, AREG, and EREG in MIBC. Gene expression and copy number analyses were performed via qRT-PCR on tissue samples from 100 patients with MIBC who underwent radical cystectomy at the University Hospital Mannheim (MA; median age 72, interquartile range [IQR] 64-78 years, 25% female). Results were validated in 361 patients from the 2017 TCGA MIBC cohort (median age 69, IQR 60-77 years, 27% female), in the Chungbuk and MDACC cohort. Gene expressions were correlated with clinicopathologic parameters using the Mann-Whitney test, Kruskal-Wallis- test and Spearman correlation. For overall survival (OS), cancer-specific survival (CSS) and disease-free survival (DFS) gene expression was analyzed with Kaplan-Meier and Cox-proportional hazard models. Results: Significant gene expression differences in EGFR, AREG, and EREG could be detected in all cohorts. In the TCGA cohort, EGFR expression was significantly elevated in patients with EGFR amplification and KRAS wildtype. High AREG expression independently predicted longer OS (HR = 0.35, CI 0.19 - 0.63, p = 0.0004) and CSS (HR = 0.42, CI 0.18 - 0.95, p = 0.0378) in the MA cohort. In the TCGA cohort, high EGFR, AREG, and EREG expression correlated with shorter OS (AREG: HR = 1.57, CI 1.12 - 2.20, p = 0.0090) and DFS (EGFR: HR = 1.91, CI 1.31 - 2.8, p = 0.0008). EGFR amplification was also associated with reduced DFS. Discussion: High EGFR and EREG indicate worse survival in patients with MIBC. The prognostic role of AREG should further be investigated in large, prospective series. Divergent survival outcomes between the four cohorts should be interpreted cautiously, considering differences in analysis methods and demographics. Further in vitro investigations are necessary to elucidate the functional mechanisms underlying the associations observed in this study.

Exosomal SOX21-AS1 Regulates EREG by Sponging miR-451a and Promotes the Malignancy of Pancreatic Ductal Adenocarcinoma.

The incidence and mortality of pancreatic ductal adenocarcinoma (PDAC) have increased. Exosomes, as a regulatory mode of intercellular communication, contain lncRNAs. SOX21-AS1 has been studied in other cancers, and its expression is elevated in PDAC, but its role in PDAC remains unclear. First, we analyzed the expression of lncRNAs in PDAC tissues and nontumor tissues through the TCGA database. Next, the results of the RT-qPCR experiment confirmed the prediction that the expression of SOX21-AS1 was elevated in PDAC tissues. In vivo and in vitro cell function assays confirmed that the degree of malignancy of PDAC was proportional to the expression of SOX21-AS1. In addition, through exosome isolation and uptake experiments, we first found that PDAC could secrete exosomal SOX21-AS1 and play an angiogenic role in HUVECs. Subsequently, the relationship between SOX21-AS1, miR-451a and epiregulin (EREG) was verified through database prediction and analysis and RIP assays. Finally, functional recovery assays in vivo and in vitro verified that SOX21-AS1 regulates the expression of EREG through combination with miR-451a and thus promotes the malignancy of PDAC. SOX21-AS1 was upregulated in PDAC. The upregulation of SOX21-AS1 can stimulate the proliferation, migration, invasion, stemness and epithelial-mesenchymal transition (EMT) progression of PDAC cells. Furthermore, PDAC cells secrete exosomal SOX21-AS1, which is absorbed by HUVECs and promotes angiogenesis. Our study first identified that SOX21-AS1 promotes the malignancy of PDAC through the SOX21-AS1/miR-451a/EREG axis, and also that exosomal SOX21-AS1 promotes angiogenesis in PDAC.

Development of a novel lncRNA-derived immune gene score using machine learning-based ensembles for predicting the survival of HCC.

BACKGROUND: Long noncoding RNAs (lncRNAs) are implicated in the tumor immunology of hepatocellular carcinoma (HCC). METHODS: HCC mRNA and lncRNA expression profiles were used to extract immune-related genes with the ImmPort database, and immune-related lncRNAs with the ImmLnc algorithm. The MOVICS package was used to cluster immune-related mRNA, immune-related lncRNA, gene mutation and methylation data on HCC from the TCGA. GEO and ICGC datasets were used to validate the model. Data from single-cell sequencing was used to determine the expression of genes from the model in various immune cell types. RESULTS: With this model, the area under the curve (AUC) for 1-, 3- and 5-year survival of HCC patients was 0.862, 0.869 and 0.912, respectively. Single-cell sequencing showed EREG was significantly expressed in a variety of immune cell types. Knockdown of the EREG target gene resulted in significant anti-apoptosis, pro-proliferation and pro-migration effects in HepG2 and HUH7 cells. Moreover, serum and liver tissue EREG levels in HCC patients were significantly higher than those of healthy control patients. CONCLUSION: We built a prognostic model with good accuracy for predicting HCC patient survival. EREG is a potential immunotherapeutic target and a promising prognostic biomarker.

A novel selective ERK1/2 inhibitor, Laxiflorin B, targets EGFR mutation subtypes in non-small-cell lung cancer.

Extracellular regulated protein kinases 1/2 (ERK1/2) are key members of multiple signaling pathways, including the ErbB axis. Ectopic ERK1/2 activation contributes to various types of cancer, especially drug resistance to inhibitors of RTK, RAF and MEK, and specific ERK1/2 inhibitors are scarce. In this study, we identified a potential novel covalent ERK inhibitor, Laxiflorin B, which is a herbal compound with anticancer activity. However, Laxiflorin B is present at low levels in herbs; therefore, we adopted a semi-synthetic process for the efficient production of Laxiflorin B to improve the yield. Laxiflorin B induced mitochondria-mediated apoptosis via BAD activation in non-small-cell lung cancer (NSCLC) cells, especially in EGFR mutant subtypes. Transcriptomic analysis suggested that Laxiflorin B inhibits amphiregulin (AREG) and epiregulin (EREG) expression through ERK inhibition, and suppressed the activation of their receptors, ErbBs, via a positive feedback loop. Moreover, mass spectrometry analysis combined with computer simulation revealed that Laxiflorin B binds covalently to Cys-183 in the ATP-binding pocket of ERK1 via the D-ring, and Cys-178 of ERK1 through non-inhibitory binding of the A-ring. In a NSCLC tumor xenograft model in nude mice, Laxiflorin B also exhibited strong tumor suppressive effects with low toxicity and AREG and EREG were identified as biomarkers of Laxiflorin B efficacy. Finally, Laxiflorin B-4, a C-6 analog of Laxiflorin B, exhibited higher binding affinity for ERK1/2 and stronger tumor suppression. These findings provide a new approach to tumor inhibition using natural anticancer compounds.

Integrated analysis of EREG expression, a gene associated with cervical cancer prognosis.

Cervical cancer (CC) is the fourth most gynecological malignancy in the world. The identification of predictive markers can provide a basis for personalized treatment and prognostic evaluation. Our aim was to identify a new predictive marker of epiregulin (EREG) gene and explore its functional characteristics of CC and other cancer types. Differentially highly expressed genes were obtained from Gene Expression Omnibus (GEO) databases. Key genes can be verified by the Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) data, and the functions of these genes were investigated through gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Survival analysis were performed to determine the key genes (EREG) related to the prognosis of CC. Then, the expression difference of EREG between tumor and normal tissue was evaluated by real-time polymerase chain reaction (PCR), western blotting, and immunohistochemistry. The relationship between EREG and prognosis of patients, immune microenvironment, immune checkpoint, immune therapy and angiogenesis was discussed in 33 tumor types. Finally, the regulatory mechanism of EREG on human umbilical vein endothelial cells (HUVECs) was also explored. The differential analysis results from multiple databases showed that EREG was significantly highly expressed in CC, which was further verified in Hela and Siha cell lines. Then, Survival analysis revealed that EREG was associated with the prognosis of CC and other tumor types, and high EREG expression was significantly associated with poor prognosis. In addition, in almost all tumor types, the expression of EREG was related to immune cells infiltration, immune checkpoint genes expression and immunotherapy. Further analysis exhibited that high EREG expression can promote the high expression of angiogenesis related genes. The experimental data demonstrated that EREG could promote the proliferative, migration, invasive and tube formation of HUVECs by interacting with receptors, such as epidermal growth factor receptor (EGFR and ERBB4). EREG may be an independent prognostic marker for predicting tumor prognosis and immunotherapy response of various cancers, and may be a potential target of tumor anti-angiogenic therapy in CC.

LncRNA CYP4A22-AS1 promotes the progression of lung adenocarcinoma through the miR-205-5p/EREG and miR-34c-5p/BCL-2 axes.

OBJECTIVES: Lung adenocarcinoma (LUAD) exhibits a higher fatality rate among all cancer types worldwide, yet the precise mechanisms underlying its initiation and progression remain unknown. Mounting evidence suggests that long non-coding RNAs (lncRNAs) exert significant regulatory roles in cancer development and progression. Nevertheless, the precise involvement of lncRNA CYP4A22-AS1 in LUAD remains incompletely comprehended. METHODS: Bioinformatics analyses evaluated the expression level of CYP4A22-AS1 in lung adenocarcinoma and paracancer. The LUAD cell line with a high expression of CYP4A22-AS1 was constructed to evaluate the role of CYP4A22-AS1 in the proliferation and metastasis of LUAD by CCK8, scratch healing, transwell assays, and animal experiments. We applied transcriptome and microRNA sequencing to examine the mechanism of CYP4A22-AS1 enhancing the proliferation and metastasis of LUAD. Luciferase reporter gene analyses, west-blotting, and qRT-PCR were carried out to reveal the interaction between CYP4A22-AS1, miR-205-5p/EREG, and miR-34c-5p/BCL-2 axes. RESULTS: CYP4A22-AS1 expression was significantly higher in LUAD tissues than in the adjacent tissues. Furthermore, we constructed a LUAD cell line with a high expression of CYP4A22-AS1 and noted that the high expression of CYP4A22-AS1 significantly enhanced the proliferation and metastasis of LUAD. We applied transcriptome and microRNA sequencing to examine the mechanism of CYP4A22-AS1 enhancing the proliferation and metastasis of LUAD. CYP4A22-AS1 increased the expression of EREG and BCL-2 by reducing the expression of miR-205-5p and miR-34-5p and activating the downstream signaling pathway of EGFR and the anti-apoptotic signaling pathway of BCL-2, thereby triggering the proliferation and metastasis of LUAD. The transfection of miR-205-5p and miR-34-5p mimics inhibited the role of CYP4A22-AS1 in enhancing tumor progression. CONCLUSION: This study elucidates the molecular mechanism whereby CYP4A22-AS1 overexpression promotes LUAD progression through the miR-205-5p/EREG and miR-34c-5p/BCL-2 axes.

ELF3 suppresses gallbladder cancer development through downregulation of the EREG/EGFR/mTOR complex 1 signalling pathway.

The prognosis of gallbladder cancer (GBC) remains poor, and a better understanding of GBC molecular mechanisms is important. Genome sequencing of human GBC has demonstrated that loss-of-function mutations of E74-like ETS transcription factor 3 (ELF3) are frequently observed, with ELF3 considered to be a tumour suppressor in GBC. To clarify the underlying molecular mechanisms by which ELF3 suppresses GBC development, we performed in vivo analysis using a combination of autochthonous and allograft mouse models. We first evaluated the clinical significance of ELF3 expression in human GBC tissues and found that low ELF3 expression was associated with advanced clinical stage and deep tumour invasion. For in vivo analysis, we generated Pdx1-Cre; KrasG12D ; Trp53R172H ; Elf3f/f (KPCE) mice and Pdx1-Cre; KrasG12D ; Trp53R172H ; Elf3wt/wt (KPC) mice as a control and analysed their gallbladders histologically. KPCE mice developed larger papillary lesions in the gallbladder than those developed by KPC mice. Organoids established from the gallbladders of KPCE and KPC mice were analysed in vitro. RNA sequencing showed upregulated expression of epiregulin (Ereg) in KPCE organoids, and western blotting revealed that EGFR/mechanical targets of rapamycin complex 1 (mTORC1) were upregulated in KPCE organoids. In addition, ChIP assays on Elf3-overexpressing KPCE organoids showed that ELF3 directly regulated Ereg. Ereg deletion in KPCE organoids (using CRISPR/Cas9) induced EGFR/mTORC1 downregulation, indicating that ELF3 controlled EGFR/mTORC1 activity through regulation of Ereg expression. We also generated allograft mouse models using KPCE and KPC organoids and found that KPCE organoid allograft tumours exhibited poorly differentiated structures with mTORC1 upregulation and mesenchymal phenotype, which were suppressed by Ereg deletion. Furthermore, EGFR/mTORC1 inhibition suppressed cell proliferation and epithelial-mesenchymal transition in KPCE organoids. Our results suggest that ELF3 suppresses GBC development via downregulation of EREG/EGFR/mTORC1 signalling. EGFR/mTORC1 inhibition is a potential therapeutic option for GBC with ELF3 mutation. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

NONHSAT021545/miR-330-3p/EREG: A Cooperative Axis in Breast Cancer Prognosis and Treatment.

Lymphatic metastasis is the most common form in breast cancer (BC) progression. Previously, we observed that lnc045874, a most conservative homology of Homo Sapiens NONHSAT021545 (lnc021545), miR-330-3p, and EREG may have some effects in mouse hepatocarcinoma cell lines with different lymphatic metastasis potentials. Through data from TCGA and GEO database analysis, we speculated that miR-330-3p might be a tumor promoter, while EREG could be a tumor suppressor in BC. MiR-330-3p was upregulated, while lnc021545 and EREG were downregulated in 50 BC tissues. MiR-330-3p advanced the metastatic behaviors of BC cells, whereas lnc021545 and EREG resulted in the opposite effects. The three molecules' expressions were correlated respectively and showed that miR-330-3p targeted lnc021545 and EREG to affect their expressions. Lnc021545/miR-330-3p axis affected BC metastasis by regulating EREG in epithelial-to-mesenchymal transition. In 50 BC patients, these three molecules and their cooperation are associated with aggressive tumor phenotypes, patient outcomes, and trastuzumab therapy. We finally discovered that lnc021545, miR-330-3p, and EREG formed a multi-gene co-regulation system that affected the metastasis of BC and the cooperation reflects the synergistic effects of the three molecules, recommending that their cooperation may provide a more accurate index for anti-metastasis therapeutic and prognostic evaluation of BC.

EREG is a risk factor for the prognosis of patients with cervical cancer.

Background: Cervical cancer continues to threaten women's health worldwide. Identifying critical oncogenic molecules is important to drug development and prognosis prediction for patients with cervical cancer. Recent studies have demonstrated that epiregulin (EREG) is upregulated in various cancer types, which contributes to cancer progression by triggering the EGFR signaling pathway. However, the role of EREG is still unclear. Methods: In this study, we first conducted a comprehensive biological analysis to investigate the expression of EREG in cervical cancer. Then, we investigated the correlations between EREG expression level and clinicopathological features. In addition, we validated the effects of EREG expression on the proliferation and apoptosis of cervical cancer cells. Results: Based on the public database, we found that the expression of EREG was higher in advanced cervical cancer samples. Survival analysis showed that EREG was a risk factor for the prognosis of cervical cancer. In vitro experiments demonstrated that EREG knockdown undermined proliferation and promoted apoptosis in cancer cells. Conclusion: EREG plays a vital role in the progression of cervical cancer, which contributes to hyperactive cell proliferation and decreased cell apoptosis. It might be a valuable target for prognosis prediction and drug development for cervical cancer in the future.

Fibroblast-derived EGF ligand neuregulin 1 induces fetal-like reprogramming of the intestinal epithelium without supporting tumorigenic growth.

Growth factors secreted by stromal fibroblasts regulate the intestinal epithelium. Stroma-derived epidermal growth factor (EGF) family ligands are implicated in epithelial regeneration and tumorigenesis, but their specific contributions and associated mechanisms remain unclear. Here, we use primary intestinal organoids modeling homeostatic, injured and tumorigenic epithelia to assess how the fibroblast-derived EGF family ligands neuregulin 1 (NRG1) and epiregulin (EREG) regulate the intestinal epithelium. NRG1 was expressed exclusively in the stroma, robustly increased crypt budding and protected intestinal epithelial organoids from radiation-induced damage. NRG1 also induced regenerative features in the epithelium, including a fetal-like transcriptome, suppression of the Lgr5+ stem cell pool and remodeling of the epithelial actin cytoskeleton. Intriguingly, unlike EGF and EREG, NRG1 failed to support the growth of pre-tumorigenic intestinal organoids lacking the tumor suppressor Apc, commonly mutated in human colorectal cancer (CRC). Interestingly, high expression of stromal NRG1 was associated with improved survival in CRC cohorts, suggesting a tumor-suppressive function. Our results highlight the power of stromal NRG1 in transcriptional reprogramming and protection of the intestinal epithelium from radiation injury without promoting tumorigenesis.

Epiregulin expression and secretion is increased in castration-resistant prostate cancer.

Introduction: In prostate cancer, long-term treatment directed against androgens often leads to the development of metastatic castration-resistant prostate cancer, which is more aggressive and not curatively treatable. Androgen deprivation results in elevated epiregulin expression in LNCaP cells which is a ligand of EGFR. This study aims to reveal the expression and regulation of epiregulin in different prostate cancer stages enabling a more specific molecular characterization of different prostate carcinoma types. Methods: Five different prostate carcinoma cell lines were used to characterize the epiregulin expression on the RNA and protein levels. Epiregulin expression and its correlation with different patient conditions were further analyzed using clinical prostate cancer tissue samples. Additionally, the regulation of epiregulin biosynthesis was examined at transcriptional, post-transcriptional and release level. Results: An increased epiregulin secretion is detected in castration-resistant prostate cancer cell lines and prostate cancer tissue samples indicating a correlation of epiregulin expression with tumor recurrence, metastasis and increased grading. Analysis regarding the activity of different transcription factors suggests the involvement of SMAD2/3 in the regulation of epiregulin expression. In addition, miR-19a, -19b, and -20b are involved in post-transcriptional epiregulin regulation. The release of mature epiregulin occurs via proteolytic cleavage by ADAM17, MMP2, and MMP9 which are increased in castration-resistant prostate cancer cells. Discussion: The results demonstrate epiregulin regulation by different mechanism and suggest a potential role as a diagnostic tool to detect molecular alterations in prostate cancer progression. Additionally, although EGFR inhibitors false in prostate cancer, epiregulin could be a therapeutic target for patients with castration-resistant prostate cancer.

Blocking EREG/GPX4 Sensitizes Head and Neck Cancer to Cetuximab through Ferroptosis Induction.

(1) Background: Epiregulin (EREG) is a ligand of EGFR and ErB4 involved in the development and the progression of various cancers including head and neck squamous cell carcinoma (HNSCC). Its overexpression in HNSCC is correlated with short overall survival and progression-free survival but predictive of tumors responding to anti-EGFR therapies. Besides tumor cells, macrophages and cancer-associated fibroblasts shed EREG in the tumor microenvironment to support tumor progression and to promote therapy resistance. Although EREG seems to be an interesting therapeutic target, no study has been conducted so far on the consequences of EREG invalidation regarding the behavior and response of HNSCC to anti-EGFR therapies and, more specifically, to cetuximab (CTX); (2) Methods: EREG was silenced in various HNSCC cell lines. The resulting phenotype (growth, clonogenic survival, apoptosis, metabolism, ferroptosis) was assessed in the absence or presence of CTX. The data were confirmed in patient-derived tumoroids; (3) Results: Here, we show that EREG invalidation sensitizes cells to CTX. This is illustrated by the reduction in cell survival, the alteration of cell metabolism associated with mitochondrial dysfunction and the initiation of ferroptosis characterized by lipid peroxidation, iron accumulation and the loss of GPX4. Combining ferroptosis inducers (RSL3 and metformin) with CTX drastically reduces the survival of HNSCC cells but also HNSCC patient-derived tumoroids; (4) Conclusions: The loss of EREG might be considered in clinical settings as a predictive biomarker for patients that might undergo ferroptosis in response to CTX and that might benefit the most from the combination of ferroptosis inducers and CTX.

Regulation of human oocyte maturation in vivo during the final maturation of follicles.

STUDY QUESTION: Which substances and signal transduction pathways are potentially active downstream to the effect of FSH and LH in the regulation of human oocyte maturation in vivo? SUMMARY ANSWER: The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. WHAT IS KNOWN ALREADY: Many studies in animal species have provided insight into the mechanisms that govern the final maturation of oocytes. Currently, these studies have identified several different mechanisms downstream to the effects of FSH and LH. Some of the identified mechanisms include the regulation of cAMP/cGMP levels in oocytes involving C-type natriuretic peptide (CNP), effects of epidermal growth factor (EGF)-related peptides such as amphiregulin (AREG) and/or epiregulin (EREG), effect of TGF-ß family members including growth differentiation factor 9 (GDF9) and morphogenetic protein 15 (BMP15), activins/inhibins, follicular fluid meiosis activating sterol (FF-MAS), the growth factor midkine (MDK), and several others. However, to what extent these pathways and mechanisms are active in humans in vivo is unknown. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the substances and signalling pathways potentially affecting human oocyte maturation in follicular fluid (FF) and granulosa cells (GCs) collected at five time points during the final maturation of follicles. Using ELISA measurement and proteomic profiling of FF and whole genome gene expression in GC, the following substances and their signal transduction pathways were collectively evaluated: CNP, the EGF family, inhibin-A, inhibin-B, activins, FF-MAS, MDK, GDF9, and BMP15. MAIN RESULTS AND THE ROLE OF CHANCE: All the evaluated substances and signal transduction pathways are potentially active in the regulation of human oocyte maturation in vivo except for GDF9/BMP15 signalling. In particular, AREG, inhibins, and MDK were significantly upregulated during the first 12-17 h after initiating the final maturation of follicles and were measured at significantly higher concentrations than previously reported. Additionally, the genes regulating FF-MAS synthesis and metabolism were significantly controlled in favour of accumulation during the first 12-17 h. In contrast, concentrations of CNP were low and did not change during the process of final maturation of follicles, and concentrations of GDF9 and BMP15 were much lower than reported in small antral follicles, suggesting a less pronounced influence from these substances. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Although GC and cumulus cells have many similar features, it is a limitation of the current study that information for the corresponding cumulus cells is not available. However, we seldom recovered a cumulus-oocyte complex during the follicle aspiration from 0 to 32 h. WIDER IMPLICATIONS OF THE FINDINGS: Delineating the mechanisms governing the regulation of human oocyte maturation in vivo advances the possibility of developing a platform for IVM that, as for most other mammalian species, results in healthy offspring with good efficacy. Mimicking the intrafollicular conditions during oocyte maturation in vivo in small culture droplets during IVM may enhance oocyte nuclear and cytoplasmic maturation. The primary outlook for such a method is, in the context of fertility preservation, to augment the chances of achieving biological children after a cancer treatment by subjecting oocytes from small antral follicles to IVM. Provided that aspiration of oocytes from small antral follicles in vivo can be developed with good efficacy, IVM may be applied to infertile patients on a larger scale and can provide a cheap alternative to conventional IVF treatment with ovarian stimulation. Successful IVM has the potential to change current established techniques for infertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, the Independent Research Fund Denmark (grant number 0134-00448), and the Interregional EU-sponsored ReproUnion network. There are no conflicts of interest to be declared.

Hsa_circ_0000078 Regulates miR-205-5p/EREG Pathway to Inhibit Cervical Cancer Progression.

It is well established that circular RNAs (circRNAs) play a role in tumor initiation and tumorigenesis. The goal of this study was to reveal the detailed functions and regulatory mechanisms of circ_0000078 in cervical cancer (CC). Circ_0000078, miR-205-5p, and epiregulin (EREG) mRNA expression levels were examined using RT-qPCR. Western blotting was performed to quantify EREG protein. Cell proliferation, apoptosis, migration, and invasion were examined by performing CCK-8, caspase 3 activity, wound healing, and transwell assays, respectively. The effect of circ_0000078 on tumor growth in vivo was confirmed in a xenograft model. The putative relationship between miR-205-5p and circ_0000078 or EREG, as predicted by bioinformatics analysis, was evaluated by dual-luciferase and RNA immunoprecipitation assays. Aberrant downregulation of circ_0000078 and EREG as well as upregulation of miR-205-5p were observed in cervical tumor samples and cancer cells. Ectopic expression of circ _0000078 not only restrained cancer cell growth, survival, migration, and invasiveness, but also decelerated tumor formation and development in a mouse model. miR-205-5p, acts as a target of circ_0000078 and directly binds to EREG to repress its expression. Overexpression of miR-205-5p reversed the inhibitory effects of circ_0000078 upregulation on cancer cell behavior and also partially abolished the anti-cancer effects of EREG upregulation in vitro. Circ_0000078 inhibits the growth of cancer by interfering with the miR-205-5p/EREG network, acting as a tumor suppressor in CC. These results provide a better understanding of the pathogenesis of this disease.

Micropeptide MIAC inhibits the tumor progression by interacting with AQP2 and inhibiting EREG/EGFR signaling in renal cell carcinoma.

BACKGROUND: Although, micropeptides encoded by non-coding RNA have been shown to have an important role in a variety of tumors processes, there have been no reports on micropeptide in renal cell carcinoma (RCC). Based on the micropeptide MIAC (micropeptide inhibiting actin cytoskeleton) discovered and named in the previous work, this study screened its tumor spectrum, and explored its mechanism of action and potential diagnosis and treatment value in the occurrence and development of renal carcinoma. METHODS: The clinical significance of MIAC in RCC was explored by bioinformatics analysis through high-throughput RNA-seq data from 530 patients with kidney renal clear cell carcinoma (KIRC) in the TCGA database, and the detection of clinical samples of 70 cases of kidney cancer. In vitro and in vivo experiments to determine the role of MIAC in renal carcinoma cell growth and metastasis; High-throughput transcriptomics, western blotting, immunoprecipitation, molecular docking, affinity experiments, and Streptavidin pulldown experiments identify MIAC direct binding protein and key regulatory pathways. RESULTS: The analysis of 600 renal carcinoma samples from different sources revealed that the expression level of MIAC is significantly decreased, and corelated with the prognosis and clinical stage of tumors in patients with renal carcinoma. Overexpression of MIAC in renal carcinoma cells can significantly inhibit the proliferation and migration ability, promote apoptosis of renal carcinoma cells, and affect the distribution of cells at various stages. After knocking down MIAC, the trend is reversed. In vivo experiments have found that MIAC overexpression inhibit the growth and metastasis of RCC, while the synthetized MIAC peptides can significantly inhibit the occurrence and development of RCC in vitro and in vivo. Further mechanistic studies have demonstrated that MIAC directly bind to AQP2 protein, inhibit EREG/EGFR expression and activate downstream pathways PI3K/AKT and MAPK to achieve anti-tumor effects. CONCLUSIONS: This study revealed for the first time the tumor suppressor potential of the lncRNA-encoded micropeptide MIAC in RCC, which inhibits the activation of the EREG/EGFR signaling pathway by direct binding to AQP2 protein, thereby inhibiting renal carcinoma progression and metastasis. This result emphasizes that the micropeptide MIAC can provide a new strategy for the diagnosis and treatment of RCC.

The homeostatic malfunction of a novel feedback pathway formed by lncRNA021545, miR-330-3p and epiregulin contributes in hepatocarcinoma progression via mediating epithelial-mesenchymal transition.

A better understanding of tumor metastasis is urgently required for the treatment and prognosis of hepatocarcinoma patients. Current work contributes a novel ceRNA feedback regulation pathway composed of epiregulin (EREG), microRNA-330-3p (miR-330-3p) and long non-coding RNA 021545 (lncRNA021545) in regulating hepatocarcinoma malignancy via epithelial-mesenchymal transition (EMT) process. Closely correlated, the deficiencies of EREG and lncRNA021545 and the overexpression of miR-330-3p were involved in the clinical progression of hepatocarcinoma. In vitro results showed that 1) lncRNA021545 downregulation promoted, 2) miR-330-3p dysexpression positively correlated, and 3) EREG dysexpression reversely correlated with the migratory and invasive properties of hepatocarcinoma HCCLM3 and Huh7 cell lines. By directly binding to EREG and lncRNA021545, miR-330-3p expression change reversely correlated with their expressions in HCCLM3 and Huh7 cells, which was also confirmed in primary tumors from HCCLM3-xenograft mice in responding to miR-330-3p change. LncRNA021545 and EREG positively regulated each other, and lncRNA021545 negatively regulated miR-330-3p, while, EREG dysregulation unchanged miR-330-3p expression in hepatocarcinoma cells. Furthermore, systemic in vitro cellular characterizations showed that the malfunctions of the three molecules mediated the invasiveness of hepatocarcinoma cells via EMT process through affecting the expressions of E-cadherin, N-cadherin, vimentin, snail and slug, which was further confirmed by in vivo miR-330-3p promotion on the tumorigenicity and metastasis of HCCLM3 bearing nude mice and by in vitro miR-330-3p promotion on the migration and invasion of hepatocarcinoma cells to be antagonized by EREG overexpression through acting on EMT process. Our work indicates, that by forming a circuit signaling feedback pathway, the homeostatic expressions of lncRNA021545, miR-330-3p and EREG are important in liver health. Its collapse resulted from the downregulations of lncRNA021545 and EREG together with miR-330-3p overexpression promote hepatocarcinoma progression by enhancing the invasiveness of tumor cells through EMT activation. These discoveries suggest that miR-330-3p/lncRNA021545/EREG axis plays a critical role in hepatocarcinoma progression and as a candidate for its treatment.

Clinical Significance of EREG Gene Expression in Gastric Cancer Tissue After Curative Surgery.

BACKGROUND/AIM: Epiregulin (EREG) is a ligand of the epidermal growth factor receptor (EGFR) and promotes tumour progression mainly by stimulating the EGF pathway. We investigated the clinical significance of EREG mRNA expression in cancer tissues from patients with gastric cancer (GC) in pathological (p) Stage II/III who have undergone curative surgery. PATIENTS AND METHODS: Expression of EREG mRNA was measured in cancer tissues obtained from 253 patients with pStage II/III GC who underwent curative surgery. Patients were divided into groups based on high or low expression of EREG mRNA. We examined the relationship between EREG mRNA expression levels and clinicopathological features and survival. RESULTS: Clinicopathological features did not vary between the high and low EREG mRNA expression groups. Overall survival was significantly lower in the high-expression group compared to that in the low-expression group (5-year survival probability: 55.0% vs. 73.0%; p=0.005). Multivariate analysis showed EREG mRNA expression to be an independent predictor of poor survival (hazard ratio=1.794; 95% confidence interval=1.186-2.712; p=0.006). CONCLUSION: Expression of EREG mRNA in cancer tissue from patients with pStage II/III GC may be a useful prognostic marker after curative surgery.

Single-Cell Transcriptomics Uncover Key Regulators of Skin Regeneration in Human Long-Term Mechanical Stretch-Mediated Expansion Therapy.

Tissue expansion is a commonly performed therapy to grow extra skin in vivo for reconstruction. While mechanical stretch-induced epidermal changes have been extensively studied in rodents and cell culture, little is known about the mechanobiology of the human epidermis in vivo. Here, we employed single-cell RNA sequencing to interrogate the changes in the human epidermis during long-term tissue expansion therapy in clinical settings. We also verified the main findings at the protein level by immunofluorescence analysis of independent clinical samples. Our data show that the expanding human skin epidermis maintained a cellular composition and lineage trajectory that are similar to its non-expanding neighbor, suggesting the cellular heterogeneity of long-term expanded samples differs from the early response to the expansion. Also, a decrease in proliferative cells due to the decayed regenerative competency was detected. On the other hand, profound transcriptional changes are detected for epidermal stem cells in the expanding skin versus their non-expanding peers. These include significantly enriched signatures of C-FOS, EMT, and mTOR pathways and upregulation of AREG and SERPINB2 genes. CellChat associated ligand-receptor pairs and signaling pathways were revealed. Together, our data present a single-cell atlas of human epidermal changes in long-term tissue expansion therapy, suggesting that transcriptional change in epidermal stem cells is the major mechanism underlying long-term human skin expansion therapy. We also identified novel therapeutic targets to promote human skin expansion efficiency in the future.