Stabilization of UCA1 by N6-methyladenosine RNA methylation modification promotes colorectal cancer progression.

BACKGROUND: UCA1 is frequently upregulated in a variety of cancers, including CRC, and it can play an oncogenic role by various mechanisms. However, how UCA1 is regulated in cancer is largely unknown. In this study, we aimed to determine whether RNA methylation at N6-methyladenosine (m6A) can impact UCA1 expression in colorectal cancer (CRC). METHODS: qRT-PCR was performed to detect the level of UCA1 and IGF2BP2 in CRC samples. CRISPR/Cas9 was employed to knockout (KO) UCA1, METTL3 and WTAP in DLD-1 and HCT-116 cells, while rescue experiments were carried out to re-express METTL3 and WTAP in KO cells. Immunoprecipitation using m6A antibody was performed to determine the m6A modification of UCA1. In vivo pulldown assays using S1m tagging combined with site-direct mutagenesis was carried out to confirm the recognition of m6A-modified UCA1 by IGF2BP2. Cell viability was measured by MTT and colony formation assays. The expression of UCA1 and IGF2BP2 in TCGA CRC database was obtained from GEPIA ( http://gepia.cancer-pku.cn ). RESULTS: Our results revealed that IGF2BP2 serves as a reader for m6A modified UCA1 and that adenosine at 1038 of UCA1 is critical to the recognition by IGF2BP2. Importantly, we showed that m6A writers, METTL3 and WTAP positively regulate UCA1 expression. Mechanically, IGF2BP2 increases the stability of m6A-modified UCA1. Clinically, IGF2BP2 is upregulated in CRC tissues compared with normal tissues. CONCLUSION: These results suggest that m6A modification is an important factor contributing to upregulation of UCA1 in CRC tissues.

RETRACTED: lncRNA RMST Enhances DNMT3 Expression through Interaction with HuR

Large bodies of studies have shown that the CRISPR/Cas9-based library screening is a very powerful tool for the identification of gene functions. However, most of these studies have focused on protein-coding genes, and, furthermore, very few studies have used gene reporters for screening. In the present study, we generated DNA methyltransferase 3B (DNMT3B) reporter and screened a CRISPR/Cas9 synergistic activation mediator (SAM) library against a focused group of lncRNAs. With this screening approach, we identified Rhabdomyosarcoma 2-Associated Transcript (RMST) as a positive regulator for DNMT3B. This was confirmed by activation of the endogenous RMST by SAM or ectopic expression of RMST. Moreover, RMST knockout (KO) suppresses DNMT3, while rescue with RMST in the KO cells restores the DNMT3 level. Finally, RMST KO suppresses global DNA methylation, leading to the upregulation of methylation-regulated genes. Mechanistically, RMST promotes the interaction between the RNA-binding protein HuR and DNMT3B 3' UTR, increasing the DNMT3B stability. Together, these results not only provide the feasibility of a reporter system for CRISPR library screening but also demonstrate the previously uncharacterized factor RMST as an important player in the modulation of DNA methylation.

PRMT5 promotes epithelial-mesenchymal transition via EGFR-ß-catenin axis in pancreatic cancer cells.

Protein arginine methyltransferase 5 (PRMT5) has been implicated in the development and progression of human cancers. However, few studies reveal its role in epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. In this study, we find that PRMT5 is up-regulated in pancreatic cancer, and promotes proliferation, migration and invasion in pancreatic cancer cells, and promotes tumorigenesis. Silencing PRMT5 induces epithelial marker E-cadherin expression and down-regulates expression of mesenchymal markers including Vimentin, collagen I and ß-catenin in PaTu8988 and SW1990 cells, whereas ectopic PRMT5 re-expression partially reverses these changes, indicating that PRMT5 promotes EMT in pancreatic cancer. More importantly, we find that PRMT5 knockdown decreases the phosphorylation level of EGFR at Y1068 and Y1172 and its downstream p-AKT and p-GSK3ß, and then results in down-regulation of ß-catenin. Expectedly, ectopic PRMT5 re-expression also reverses the above changes. It is suggested that PRMT5 promotes EMT probably via EGFR/AKT/ß-catenin pathway. Taken together, our study demonstrates that PRMT5 plays oncogenic roles in the growth of pancreatic cancer cell and provides a potential candidate for pancreatic cancer treatment.

The oncogenic potentials and diagnostic significance of long non-coding RNA LINC00310 in breast cancer.

Recent studies have revealed that long non-coding RNAs (lncRNAs) are involved in different physiological processes and human diseases. However, to date, the function and overall clinical significance of the vast majority of lncRNAs in breast cancer remain largely unexplored. Here, we focused on LINC00310 by interrogating the breast invasive carcinoma data set of the Cancer Genome Atlas (TCGA). The results showed that LINC00310 was increased as breast cancer progressed, and the deregulation of LINC00310 was significantly associated with patients' survival. Experiments with knockout (KO) approach by CRISPR/Cas9 system and the subsequent rescue experiments revealed that LINC00310 promoted cell proliferation by regulating c-Myc expression in vitro. Nude mouse xenograft assay demonstrated that LINC00310 KO significantly suppressed tumour growth in vivo. Furthermore, we found that serum LINC00310 expression was significantly up-regulated in patients with breast cancer, and receiver operating characteristic (ROC) curve analysis indicated that LINC00310 had a powerful capability of distinguishing patients with breast cancer from healthy individuals (the area under curve 0.828). Taken together, these results provide a more intuitive approach to explore the clinical relevance and functional roles of lncRNAs. As a result, lncRNAs, such as LINC00310, may be used in clinical applications as circulating markers for breast cancer.

LncRNA UCA1 promotes migration and invasion in pancreatic cancer cells via the Hippo pathway.

Although overexpression of the long non-coding RNA (lncRNA) UCA1 has been implicated in several human cancers, its biological function in pancreatic cancer remains to be clarified. In this study, we reported that UCA1 expression was significantly increased in pancreatic cancer tissues and correlated with clinicopathological features, tumor stage, and poorer patient outcome. We further showed that UCA1 promoted cell migration and invasion of pancreatic cancer cells. Importantly, we found that UCA1 overexpression inhibited YAP phosphorylation, and increased YAP expression. Mechanistically, UCA1 interacted with MOB1, Lats1, and YAP, forming shielding composites. Moreover, we demonstrated that UCA1 increased YAP nuclear localization and stabilization, and improved TEAD luciferase activity. In turn, YAP promotes UCA1 expression. Collectively, the present study provides insights into the mechanistic regulation of UCA1 promoting pancreatic cancer progression through the Hippo signaling pathway. UCA1 may serve as a candidate biomarker for poor prognosis and a target for new pancreatic cancer therapies.

Linc-RoR promotes MAPK/ERK signaling and confers estrogen-independent growth of breast cancer.

BACKGROUND: The conversion from estrogen-dependent to estrogen-independent state of ER+ breast cancer cells is the key step to promote resistance to endocrine therapies. Although the crucial role of MAPK/ERK signaling pathway in estrogen-independent breast cancer cell growth is well established, the underlying mechanism is not fully understood. METHODS: In this study, we profiled lncRNA expression against a focused group of lncRNAs selected from lncRNA database. CRISPR/Cas9 was employed to knockout (KO) linc-RoR in MCF-7 cells, while rescue experiments were carried out to re-express linc-RoR in KO cells. Colony formation and MTT assays were used to examine the role of linc-RoR in estrogen-independent growth and tamoxifen resistance. Western blot and qRT-PCR were used to determine the change of protein and lncRNA levels, respectively. The expression of DUSP7 in clinical specimens was downloaded from Oncomine ( www.oncomine.org ) and the dataset from Kaplan-Meier Plotter ( http://kmplot.com ) was used to analyze the clinical outcomes in relation to DUSP7. RESULTS: We identified that linc-RoR functions as an onco-lncRNA to promote estrogen-independent growth of ER+ breast cancer. Under estrogen deprivation, linc-RoR causes the upregulation of phosphorylated MAPK/ERK pathway which in turn activates ER signaling. Knockout of linc-RoR abrogates estrogen deprivation-induced ERK activation as well as ER phosphorylation, whereas re-expression of linc-RoR restores all above phenotypes. Moreover, we show that the ERK-specific phosphatase Dual Specificity Phosphatase 7 (DUSP7), also known as MKP-X, is involved in linc-RoR KO-induced repression of MAPK/ERK signaling. Interestingly, linc-RoR KO increases the protein stability of DUSP7, resulting in repression of ERK phosphorylation. Clinical data analysis reveal that DUSP7 expression is lower in ER+ breast cancer samples than that in ER- breast cancer. Moreover, downregulation of DUSP7 expression is associated with poor patient survival. CONCLUSION: Taken together, these results suggest that linc-RoR promotes estrogen-independent growth and activation of MAPK/ERK pathway of breast cancer cells by regulating the ERK-specific phosphatase DUSP7. Thus, this study might help not only in establishing a role for linc-RoR in estrogen-independent and tamoxifen resistance of ER+ breast cancer, but also suggesting a link between linc-RoR and MAPK/ERK pathway.

Methyl-CpG-binding domain 3 inhibits epithelial-mesenchymal transition in pancreatic cancer cells via TGF-ß/Smad signalling.

BACKGROUND: Methyl-CpG-binding domain 3 (MBD3) is an aberrant expression in human malignancies. However, the role of MBD3 in pancreatic cancer progression remains to be clarified. In this study, we investigated the effects of MBD3 on the epithelial-mesenchymal transition (EMT), and the underlying mechanism in pancreatic cancer cells. METHODS: The abilities of migration and invasion were examined by transwell and BD Matrigel invasion assays. EMT and TGF-ß/Smad signalling were evaluated. RESULTS: First, we find that MBD3 expression is lower in pancreatic cancer tissues than that in non-tumour tissues, and patients with lower MBD3 levels survive significantly less than those with higher levels. Subsequently, we find that MBD3 knockdown promotes the abilities of migration and invasion, while MBD3 overexpression inhibits the above abilities. Also, MBD3 knockdown remarkably increases mesenchymal markers expression of Vimentin, α-SMA, Snail, N-cadherin, ß-catenin, and downregulates epithelial markers expression of E-cadherin. On the contrary, MBD3 overexpression results in the opposite effects. Further evidence reveals that MBD3 knockdown up-regulates expression of TGF-ß, and then activates p-Smad2 and p-Smad3, while MBD3 overexpression results in downregulation of TGF-ß, p-Smad2, and p-Smad3. CONCLUSIONS: MBD3 inhibits EMT in pancreatic cancer cells probably via TGF-ß/Smad signalling, and may be a new candidate target for diagnostics and prognosis of pancreatic cancer.

FoxM1-mediated RFC5 expression promotes temozolomide resistance.

Although methylguanine-DNA-methyltransferase (MGMT) plays an important role in resistance to temozolomide (TMZ) in glioma, 40% of gliomas with MGMT inactivation are still resistant to TMZ. The underlying mechanism is not clear. Here, we report that forkhead box M1 (FoxM1) transcriptionally activates the expression of DNA repair gene replication factor C5 (RFC5) to promote TMZ resistance in glioma cells independent of MGMT activation. We showed that RFC5 expression is positively correlated with FoxM1 expression in human glioma cells and FoxM1 is able to transcriptionally activate RFC expression by interaction with the RFC5 promoter. Furthermore, knockdown of FoxM1 or RFC5 partially re-sensitizes glioma cells to TMZ. Consistently, thiostrepton, a FoxM1 inhibitor, in combination with TMZ significantly inhibits proliferation and promotes apoptosis in glioma cells. Taken together, these findings suggest that the FoxM1-RFC5 axis may mediate TMZ resistance and thiostrepton may serve as a potential therapeutic agent against TMZ resistance in glioma cells.

Egr-1 promotes hypoxia-induced autophagy to enhance chemo-resistance of hepatocellular carcinoma cells.

Previous studies suggest that early growth response gene-1 (Egr-1) plays an important role in hypoxia-induced drug-resistance. However, the mechanism still remains to be clarified. Herein, we investigated the role of Egr-1 in hypoxia-induced autophagy and its resulted hypoxia-driven chemo-resistance in Hepatocellular Carcinoma (HCC) cells. Our data demonstrated that Egr-1 was overexpressed in HCC tissues and cells and conferred them drug resistance under hypoxia. Mechanistically, Egr-1 transcriptionally regulated hypoxia-induced autophagy by binding to LC3 promoter in HCC cells, which resulted in resistance of HCC cells to chemotherapeutic agents; while dominant negative Egr-1 could inhibit autophagy level, and thus enhanced the sensitivity of HCC cells to chemotherapeutic agents, indicating that hypoxia-induced Egr-1 expression enhanced drug resistance of HCC cells likely through autophagy. Accordingly, it is suggested that a mechanism of hypoxia/Egr-1/autophagy axis might be involved in drug resistance in HCC.

Scoparone Protects Against Pancreatic Fibrosis via TGF-ß/Smad Signaling in Rats.

BACKGROUND/AIMS: This study was to investigate the influence of scoparone on pancreatic fibrosis in vitro and in vivo. METHODS: Pancreatic stellate cells (PSCs) were isolated from pancreas tissue blocks, and cultured for 3-5 generations for the experiment. PSCs were treated with scoparone in different doses as experimental groups, salvianolic acid B as a positive control and PBS as a blank group. We measured the effects of scoparone on cellular proliferation, oxidative stress, epithelial-mesenchymal transition (EMT), and pancreatic fibrosis. Cellular oxidative stress was detected by using commercially available kits. The impact of scoparone on EMT and fibrosis was detected through immunofluorescence or western blotting. RESULTS: Compared with the control group, scoparone significantly inhibited stellate cell proliferation, and reduced MDA, the expression of mesenchymal makers, and increased the levels of SOD and the expression of E-cadherin (P < 0.05). Western blot analysis showed that scoparone downregulated the expression of TGF-ß and p-smad2/3, and upregultated the expression of smad7 (P < 0.05). CONCLUSION: Scoparone can reduce the levels of oxidative stress, repress pancreatic stellate cells activation, and alleviate fibrosis by regulating TGF-ß/Smad pathway.

Aging alters histone H3 lysine 4 methylation in mouse germinal vesicle stage oocytes.

Decreasing oocyte competence with maternal aging is a major factor in mammalian infertility. One of the factors contributing to this infertility is changes to chromatin modifications, such as histone acetylation in old MII stage oocytes. Recent studies indicate that changes in histone acetylation at MII arise at the germinal vesicle (GV) stage. We hypothesised that histone methylation could also change in old GV oocytes. To test this hypothesis, we examined mono-, di- and trimethylation of histone H3 lysine 4 (H3K4 me1, me2 and me3, respectively) in young and older oocytes from 6-8- and 42-44-week-old mice, respectively. We found that H3K4 me2 and me3 decreased in older compared with young GV oocytes (100% vs. 81% and 100% vs. 87%, respectively; P<0.05). H3K4 me2 later increased in older MII oocytes (21% vs. 56%; P<0.05). We also examined the expression of genes encoding the H3K4 demethylases lysine (K)-specific demethylase 1A (Kdm1a) and retinol binding protein 2 (Rbp2). Expression of Kdm1a increased at both the mRNA and protein levels in older GV oocytes, but decreased in older MII oocytes (P<0.05), and was negatively correlated with H3K4 me2 levels. Conversely, expression of Rbp2 mRNA and protein decreased in older GV oocytes (P<0.05), and this was not correlated with H3K4 me3 levels. Finally, we showed that inhibition of Kdm1a of older oocytes at the GV stage restored levels of H3K4 me2 at the MII stage to those seen in 'young' oocytes (41% vs. 38%; P>0.05). These results suggest that changes in expression of H3K4 me2 and Kdm1a in older GV oocytes may represent a molecular mechanism underlying human infertility caused by aging.

Autophagy contributes to ING4-induced glioma cell death.

Previous studies suggest that ING4, a novel member of ING (inhibitor of growth) family, can inhibit brain tumor growth. However, whether autophagy is involved in ING4-induced cell death still remains unknown. In this study, we found that in addition to apoptosis, autophagy also contributed to cell death induced by ING4. Autophagy levels were elevated following the exposure to Ad-ING4, including enhanced fluorescence intensity of monodansylcadervarine (MDC), a specific in vivo marker for autophagic vacuoles, and increased expression levels of the LC3-II and Beclin-1, wheras the autophagic levels were attenuated following the pretreatment of 3-MA, the inhibitor of autophagy, which significantly decreased the Ad-ING4-induced cell death compared with caspase inhibitor zVAD. Furthermore, ING4 also induced mitochondrial dysfunction, such as mitophagy, collapse of mitochondrial membrane potential and the intracellular ROS, which indicated that mitochondria might be associated with the process of autophagic cell death of glioma cells. Finally, the relationship among Bax, Bcl-2, Beclin-1 and caspase family proteins levels were analyzed in glioma cells U251MG and LN229 infected with Ad-ING4 or Ad-lacZ. It is suggested that both autophagy and apoptosis could contribute to ING4-induced glioma cell death, and mitochondria might play an important role in this process. Our findings reveal novel aspects of the autophagy in glioma cells that underlie the cytotoxic action of ING4, possibly providing new insights in the development of combinatorial therapies for gliomas.

Corrigendum to "N6-methyladenosine modified LINC00901 promotes pancreatic cancer progression through IGF2BP2/MYC axis" [Genes & Diseases 10 (2023) 554-567].

[This corrects the article DOI: 10.1016/j.gendis.2022.02.014.].

Perioperative complication incidence and risk factors for retroperitoneal neuroblastoma in children: analysis of 571 patients.

BACKGROUND: Surgery plays an important role in the treatment of neuroblastoma. Perioperative complications may impact the course of neuroblastoma treatment. To date, comprehensive analyses of complications and risk factors have been lacking. METHODS: Patients with retroperitoneal neuroblastoma undergoing tumor resection were retrospectively analyzed between 2014 and 2021. The data collected included clinical characteristics, operative details, operative complications and postoperative outcomes. Risk factors for perioperative complications of retroperitoneal neuroblastoma were analyzed. RESULTS: A total of 571 patients were enrolled in this study. Perioperative complications were observed in 255 (44.7%) patients. Lymphatic leakage (28.4%), diarrhea (13.5%), and injury (vascular, nerve and organ; 7.5%) were the most frequent complications. There were three operation-related deaths (0.53%): massive hemorrhage (n = 1), biliary tract perforation (n = 1) and intestinal necrosis (n = 1). The presence of image-defined risk factors (IDRFs) [odds ratio (OR) = 2.09, P < 0.01], high stage of the International Neuroblastoma Risk Group staging system (INRGSS) (OR = 0.454, P = 0.04), retroperitoneal lymph node metastasis (OR = 2.433, P = 0.026), superior mesenteric artery encasement (OR = 3.346, P = 0.003), and inferior mesenteric artery encasement (OR = 2.218, P = 0.019) were identified as independent risk factors for perioperative complications. CONCLUSIONS: Despite the high incidence of perioperative complications, the associated mortality rate was quite low. Perioperative complications of retroperitoneal neuroblastoma were associated with IDRFs, INRGSS, retroperitoneal lymph node metastasis and vascular encasement. Patients with high-risk factors should receive more serious attention during surgery but should not discourage the determination to pursue total resection of neuroblastoma. Video Abstract (MP4 94289 KB).

Effective and Efficient Porous CeO2 Adsorbent for Acid Orange 7 Adsorption.

A porous CeO2 was synthesized following the addition of guanidine carbonate to a Ce3+ aqueous solution, the subsequent addition of hydrogen peroxide and a final hydrothermal treatment. The optimal experimental parameters for the synthesis of porous CeO2, including the amounts of guanidine carbonate and hydrogen peroxide and the hydrothermal conditions, were determined by taking the adsorption efficiency of acid orange 7 (AO7) dye as the evaluation. A template-free hydrothermal strategy could avoid the use of soft or hard templates and the subsequent tedious procedures of eliminating templates, which aligned with the goals of energy conservation and emission reduction. Moreover, both the guanidine carbonate and hydrogen peroxide used in this work were accessible and eco-friendly raw materials. The porous CeO2 possessed rapid adsorption capacities for AO7 dye. When the initial concentration of AO7 was less than 130 mg/L, removal efficiencies greater than 90.0% were obtained, achieving a maximum value of 97.5% at [AO7] = 100 mg/L and [CeO2] = 2.0 g/L in the first 10 min of contact. Moreover, the adsorption-desorption equilibrium between the porous CeO2 adsorbent and the AO7 molecule was basically established within the first 30 min. The saturated adsorption amount of AO7 dye was 90.3 mg/g based on a Langmuir linear fitting of the experimental data. Moreover, the porous CeO2 could be recycled using a NaOH aqueous solution, and the adsorption efficiency of AO7 dye still remained above 92.5% after five cycles. This study provided an alternative porous adsorbent for the purification of dye wastewater, and a template-free hydrothermal strategy was developed to enable the design of CeO2-based catalysts or catalyst carriers.

Roles of lncRNAs in childhood cancer: Current landscape and future perspectives.

According to World Health Organization (WHO), cancer is the leading cause of death for children and adolescents. Leukemias, brain cancers, lymphomas and solid tumors, such as neuroblastoma, ostesarcoma and Wilms tumors are the most common types of childhood cancers. Approximately 400,000 children and adolescents between the ages of 0 and 19 are diagnosed with cancer each year worldwide. The cancer incidence rates have been rising for the past few decades. Generally, the prognosis of childhood cancers is favorable, but the survival rate for many unresectable or recurring cancers is substantially worse. Although random genetic mutations, persistent infections, and environmental factors may serve as contributing factors for many pediatric malignancies, the underlying mechanisms are yet unknown. Long non-coding RNAs (lncRNAs) are a group of transcripts with longer than 200 nucleotides that lack the coding capacity. However, increasing evidence indicates that lncRNAs play vital regulatory roles in cancer initiation and development in both adults and children. In particular, many lncRNAs are stable in cancer patients' body fluids such as blood and urine, suggesting that they could be used as novel biomarkers. In support of this notion, lncRNAs have been identified in liquid biopsy samples from pediatric cancer patients. In this review, we look at the regulatory functions and underlying processes of lncRNAs in the initiation and progression of children cancer and discuss the potential of lncRNAs as biomarkers for early detection. We hope that this article will help researchers explore lncRNA functions and clinical applications in pediatric cancers.