CircTMEM87A promotes the tumorigenesis of gastric cancer by regulating the miR-1276/SLC7A11 axis.

BACKGROUND: Gastric cancer (GC) is a common malignancy with high incidence and mortality, and its pathogenesis involves the regulation of circular RNAs (circRNAs). However, the molecular mechanism of circTMEM87A in GC malignant progression is uncertain. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expressions of circTMEM87A, miR-1276, and solute carrier family 7 membrane 11 (SLC7A11). Western blot was applied to detect protein expression levels of EMT-related proteins (vimentin and E-cadherin) and SLC7A11. Cell counting kit-8 assay (CCK8) and thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) were performed to assess cell proliferation. Apoptosis was investigated using flow cytometry. Transwell assay and wound healing assay were carried out to examine the migration of MKN-7 and AGS cells. The Cellular ROS Assay Kit, Iron Assay Kit, and GSH/GSSG Ratio Detection Assay Kit were utilized to monitor lipid ROS level, iron level, and GSH/GSSG ratio, respectively. The interaction between miR-1276 and circTMEM87A or SLC7A11 was investigated using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. A xenograft mouse model was constructed to explore the function of circTMEM87A in tumor formation in vivo. RESULTS: CircTMEM87A and SLC7A11 were upregulated, while miR-1276 was downregulated in GC tissues and cells. Knockdown of circTMEM87A suppressed the proliferation and migration and promoted apoptosis and ferroptosis of GC cells. CircTMEM87A served as a sponge for miR-1276, and miR-1276 inhibitor relieved the circTMEM87A knockdown-induced effects on GC cell phenotypes. Similarly, SLC7A11, a downstream gene of miR-1276, rescued miR-1276 overexpression-induced effects on GC cell function. Furthermore, circTMEM87A knockdown inhibited GC cell tumor phenotypes in vivo. CONCLUSION: CircTMEM87A promoted the proliferation and migration and inhibited apoptosis and ferroptosis of GC cells by increasing SLC7A11 expression through binding to miR-1276.

circMSH3 is a potential biomarker for the diagnosis of colorectal cancer and affects the distant metastasis of colorectal cancer.

Objectives: To identify the most significantly differentially expressed circular RNAs (circRNAs) in colorectal cancer (CRC) tissues in terms of their expression levels and circularity, and to analyze the relationship between their expression levels and the clinical characteristics of patients. Methods: circRNA RNA-seq technology was used to screen differentially expressed circRNAs in CRC. Sanger sequencing was used to identify circRNA back-splice junction sites. The relative expression levels of hsa_circ_0003761 (circMSH3) in CRC tissues and cell lines were detected by quantitative real-time fluorescence PCR technology. An RNA-protein pull-down assay was used to detect protein binding to circRNAs. Dual-luciferase reporter gene vectors were constructed to verify that circRNAs bind to microRNAs. Results: Four hundred twenty circRNAs were found to be upregulated, and 616 circRNAs were downregulated. circMSH3 was derived from the MutS homolog 3 (MSH3) gene and was formed by a loop of exons 9, 10, 11, and 12. In 110 pairs of CRC and adjacent tissues, circMSH3 expression was 4.487-fold higher in CRC tissues. circMSH3 was also highly expressed in the HT-29 and LOVO CRC cell lines. The expression level of circMSH3 was associated with distant metastasis in CRC patients (P = 0.043); the area under the curve (AUC) of circMSH3 for CRC diagnosis was 0.75, with a sensitivity and specificity of 70.9% and 66.4%, respectively. circMSH3 could bind to a variety of proteins, mainly those involved in RNA transcription, splicing, cell cycle, and cell junctions. Furthermore, circMSH3 could bind to miR-1276, miR-942-5p, and miR-409-3p. Conclusion: circMSH3 is a potential biomarker for the diagnosis of CRC and affects the distant metastasis of CRC. Multiple RNA-binding protein binds to circMSH3, and circMSH3 binds to miR-1276, miR-942-5p, and miR-409-3p, thereby affecting the expression of circMSH3.

Hsa_circ_0063804 enhances ovarian cancer cells proliferation and resistance to cisplatin by targeting miR-1276/CLU axis.

PURPOSE: This article researched circ_0063804 effects on ovarian cancer (OC) development and resistance to cisplatin, aiming to provide a new target for OC therapy. METHODS: A total of 108 OC patients participated in this study. The circle structure of circ_0063804 was investigated using RNase R. Circ_0063804 expression in OC cells were up-regulated or down-regulated by transfection. Cell proliferation was assessed by cell counting kit-8 assay and colony formation assay. Flow cytometry was used to detect apoptosis. OC cells resistance to cisplatin was explored through MTT assay. Luciferase reporter assay was performed. qRT-PCR and Western blot was applied to research genes expression. Xenograft tumor experiment was conducted using nude mice. Ki67 expression in xenograft tumor was detected by immunohistochemistry. RESULTS: Circ_0063804 expression was up-regulated in OC patients and indicated poor prognosis (P < 0.05). Circ_0063804 had a stable circle structure. Circ_0063804 enhanced proliferation, resistance to cisplatin and reduced apoptosis of OC cells (P < 0.01). miR-1276 was down-regulated in OC patients and sponged by circ_0063804. CLU was directly inhibited by miR-1276 and up-regulated in OC patients. Circ_0063804 exacerbated malignant phenotype and resistance to cisplatin of OC cells in vitro by enhancing CLU expression via sponging miR-1276 (P < 0.01). Circ_0063804 silencing inhibited OC cells growth, resistance to cisplatin and Ki67 expression in vivo (P < 0.01). CONCLUSION: Circ_0063804 promoted OC cells proliferation and resistance to cisplatin by enhancing CLU expression via sponging miR-1276.

Circ_0002623 promotes bladder cancer progression by regulating the miR-1276/SMAD2 axis.

Circular RNAs (circRNAs) are key regulatory factors in the development of multiple cancers. This study is targeted at exploring the effect of circ_0002623 on bladder cancer (BCa) progression and its mechanism. Circ_0002623 was screened out by analyzing the expression profile of circRNAs in BCa tissues. Circ_0002623, miR-1276, and SMAD2 mRNA expression levels in clinical sample tissues and cell lines were detected through quantitative real-time polymerase chain reaction (qRT-PCR). After circ_0002623 had been overexpressed or silenced in BCa cells, the cell proliferation, migration, and cell cycle were evaluated by CCK-8, BrdU, Transwell assay, and flow cytometry. Tumor xenograft model was used to validate the biological function of circ_0002623 in vivo. Bioinformatics analysis and dual-luciferase reporter gene assay were conducted for analyzing and confirming, respectively, the targeted relationship between circ_0002623 and miR-1276, as well as between miR-1276 and SMAD2. The regulatory effects of circ_0002623 and miR-1276 on the expression levels of TGF-ß, WNT1, and SMAD2 in BCa cells were detected by Western blot. We reported that, in BCa tissues and cell lines, circ_0002623 was upregulated, whereas miR-1276 was downregulated. Circ_0002623 positively regulated BCa cell proliferation, migration, and cell cycle progression. Additionally, circ_0002623 could competitively bind with miR-1276 to increase the expression of SMAD2, the target gene of miR-1276. Furthermore, circ_0002623 could regulate the expression of TGF-ß and WNT1 via modulating miR-1276 and SMAD2. This study helps to better understand the molecular mechanism underlying BCa progression.

Circ_SMAD4 promotes gastric carcinogenesis by activating wnt/ß-catenin pathway.

OBJECTIVES: Circular RNAs (circRNAs) are essential participants in tumour progression. This study focused on investigating the mechanism of a novel functional circRNA in gastric cancer (GC). METHODS: Gene expression was detected by qRT-PCR or Western blot. Survival curves were generated via Kaplan-Meier method. In vitro and in vivo assays were used to investigate the impact of circ_SMAD4 on GC cell growth and tumorigenesis. Agarose gel electrophoresis assay, RNase R treatment and Sanger sequencing were utilized for confirming the circular structure of circ_SMAD4. Relationship between molecules was monitored by a series of mechanical experiments, as needed. RESULTS: Circ_SMAD4 expression was potentiated in GC. Circ_SMAD4 depletion impeded GC cell growth in vitro and restrained tumorigenesis in vivo. Mechanically, nuclear circ_SMAD4 recruited TCF4 to facilitate CTNNB1 transcription, while cytoplasmic circ_SMAD4 sequestered miR-1276 to prevent the silence of CTNNB1 mRNA, leading to activation of Wnt/ß-catenin pathway. Rescue experiments validated that circ_SMAD4 depended on miR-1276/TCF4-regulated CTNNB1 to elicit accelerating effects on GC cell growth. CONCLUSION: Circ_SMAD4 facilitated GC tumorigenesis by activating CTNNB1-dependent Wnt/ß-catenin pathway. Hopefully, the findings could provide new clues for improving GC prognosis and treatment.

MicroRNA-1276 Promotes Colon Cancer Cell Proliferation by Negatively Regulating LACTB.

PURPOSE: LACTB, regulated by a variety of microRNAs (miRNAs), is proven to be a tumor suppressor. However, there are few reports that LACTB in colon cancer cells is regulated by miRNA. Therefore, the aim of this study was to explore the miRNAs that regulate LACTB in colon cancer. PATIENTS AND METHODS: Data from TCGA were analyzed in starBase and GEPIA2, and Western blot and quantitative PCR (qPCR) were used to detect the expression of LACTB in colon cancer cell lines. MiRNAs targeting LACTB were predicted by MicroT-CDS, starBase, miRDB, mirDIP, and DIANA. The relationship between LACTB and miRNA was explored by dual-luciferase assay. MTT, propidium iodide (PI), Western blot, Annexin V-FITC/PI Kit, qPCR and transwell assay were used to detect the changes in cell proliferation, cell cycle, autophagy, apoptosis, epithelial-to-mesenchymal transition (EMT), cell migration, and invasiveness in colon cancer cells that overexpressed miR-1276 and/or LACTB. RESULTS: The results showed that the LACTB mRNA level was lower and the miR-1276 level was higher in colon cancer than in normal tissue. MiR-1276 inhibited the expression of LACTB. Furthermore, overexpression of miR-1276 in colon cancer cells increased proliferation, migration, invasiveness and EMT, and decreased autophagy and apoptosis. Supplementing LACTB suppressed these effects of miR-1276. CONCLUSION: In conclusion, miR-1276, which may be a potential therapy for colon cancer, inhibits cell growth and promotes apoptosis by targeting LACTB in colon cancer cells.

FOXC1-mediated LINC00301 facilitates tumor progression and triggers an immune-suppressing microenvironment in non-small cell lung cancer by regulating the HIF1α pathway.

BACKGROUND: Long non-coding RNAs (lncRNAs) are extensively intricate in the tumorigenesis and metastasis of various cancer types. Nevertheless, the detailed molecular mechanisms of lncRNA in non-small cell lung cancer (NSCLC) still remain mainly undetermined. METHODS: qPCR was performed to verify LINC00301 expression in NSCLC clinical specimens or cell lines. Fluorescence in situ hybridization (FISH) was conducted to identify the localization of LINC00301 in NSCLC cells. Chromatin immunoprecipitation (ChIP) was subjected to validate the binding activity between FOXC1 and LINC00301 promoters. RNA immunoprecipitation (RIP) was performed to explore the binding activity between LINC00301 and EZH2. RNA pull-down followed by dot-blot, protein domain mapping, and RNA electrophoresis mobility shift assay (EMSA) were conducted to identify the detailed binding regions between LINC00301 and EZH2. Alpha assay was conducted to quantitatively assess the interaction between LINC00301 and EZH2. RESULTS: LINC00301 is highly expressed in NSCLC and closely corelated to its prognosis by analyzing the relationship between differentially expressed lncRNAs and prognosis in NSCLC samples. in vitro and in vivo experiments revealed that LINC00301 facilitates cell proliferation, releases NSCLC cell cycle arrest, promotes cell migration and invasion, and suppresses cell apoptosis in NSCLC. In addition, LINC00301 increases regulatory T cell (Treg) while decreases CD8+ T cell population in LA-4/SLN-205-derived tumors through targeting TGF-ß. The transcription factor FOXC1 mediates LINC00301 expression in NSCLC. Bioinformatics prediction and in vitro experiments indicated that LINC00301 (83-123 nucleotide [nt]) can directly bind to the enhancer of zeste homolog 2 (EZH2) (612-727 amino acid [aa]) to promote H3K27me3 at the ELL protein-associated factor 2 (EAF2) promoter. EAF2 directly binds and stabilizes von Hippel-Lindau protein (pVHL), so downregulated EAF2 augments hypoxia-inducible factor 1 α (HIF1α) expression by regulating pVHL in NSCLC cells. Moreover, we also found that LINC00301 could function as a competing endogenous RNA (ceRNA) against miR-1276 to expedite HIF1α expression in the cytoplasm of NSCLC cells. CONCLUSIONS: In summary, our present research revealed the oncogenic roles of LINC00301 in clinical specimens as well as cellular and animal experiments, illustrating the potential roles and mechanisms of the FOXC1/LINC00301/EZH2/EAF2/pVHL/HIF1α and FOXC1/LINC00301/miR-1276/HIF1α pathways, which provides novel insights and potential theraputic targets to NSCLC.

Upregulation of CASP9 through NF-κB and Its Target MiR-1276 Contributed to TNFα-promoted Apoptosis of Cancer Cells Induced by Doxorubicin.

：Under some conditions, nuclear factor-κB (NF-κB) has a pro-apoptotic role, but the mechanisms underlying this function remain unclear. This study demonstrated that NF-κB directly binds to CASP9 and miR1276 in tumor necrosis factor α (TNFα)-treated HeLa and HepG2 cells. NF-κB upregulated CASP9 expression, whereas downregulated miR1276 expression in the TNFα-treated cells. The miR1276 repressed CASP9 expression in both cells. As a result, a typical NF-κB-mediated coherent feed-forward loop was formed in the TNFα-treated cells. It was proposed that the NF-κB-mediated loop may contribute to cell apoptosis under certain conditions. This opinion was supported by the following evidence: TNFα promoted the apoptosis of HeLa and HepG2 cells induced by doxorubicin (DOX). CASP9 was significantly upregulated and activated by TNFα in the DOX-induced cells. Moreover, a known inhibitor of CASP9 activation significantly repressed the TNFα promotion of apoptosis induced by DOX. These findings indicate that CASP9 is a new mediator of the NF-κB pro-apoptotic pathway, at least in such conditions. This study therefore provides new insights into the pro-apoptotic role of NF-κB. The results also shed new light on the molecular mechanism underlying TNFα-promotion of cancer cells apoptosis induced by some anticancer drugs such as DOX.

LncRNA HCG11 promotes proliferation and migration in gastric cancer via targeting miR-1276/CTNNB1 and activating Wnt signaling pathway.

BACKGROUND: Gastric cancer (GC) is one common cancer which occurs in the stomach leading to high mortality around the world. Long non-coding RNAs (lncRNAs) were found overexpressed or silenced in the occurrence and progression of multiple cancers including GC. METHOD: The gene expression level in GC tissues and cells were analyzed by RT-qPCR. CCK-8, colony formation, flow cytometry and transwell assays were performed for the function analysis of HLA complex group 11 (HCG11). The mechanism study for HCG11 was conducted using RIP, RNA pull down and luciferase reporter assays. RESULTS: HCG11 was discovered highly expressed in GC tissues and cells. Depletion experiments were used to evaluate HCG11 silence on cell proliferation, migration and apoptosis. Moreover, Wnt signaling pathway was found as a tumor promoter in GC. RIP assay, RNA pull down assay and luciferase reporter assay were performed to illustrate the relationship of HCG11, miR-1276 and CTNNB1. Rescue assays revealed that HCG11/miR-1276/CTNNB1 axis regulated the incidence and development of GC. Tumor formation in mice proved that HCG11 was negatively correlated with miR-1276 and had positively correlation with CTNNB1. CONCLUSION: Overall, HCG11 accelerated proliferation and migration in GC through miR-1276/CTNNB1 and Wnt signaling pathway, revealing that HCG11 could be a brand new target for GC.