GATA-2 Regulates Dendritic Cell Differentiation

Abstract (Background) Dendritic cells (DCs) are critical regulators of the immune response, but their differentiation mechanism remains unclear. Heterozygous germline GATA-2 mutations in humans cause MonoMAC syndrome, characterized by monocytopenia and predisposition to myelodysplasia/acute myeloid leukemia. In this syndrome, DC count decreases profoundly, with an increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. In the present study, we analyzed the role of GATA-2 in DC differentiation and the underlying molecular mechanisms. (Method) Gata2 haploinsufficient mice (Gata2+/−: Tsai et al. Nature 1994) and tamoxifen-inducible Gata2-knockout mice (Gata2flox/flox/ER-Cre: Charles et al. Molecular Endocrinology 2006) were used. To generate conditional Gata2 knockouts in vivo, Gata2flox/flox/ER-Cre mice were intraperitoneally injected with 1-μg tamoxifen on days 1-3 and 8-10 and evaluated on days 20-22. Isolation of splenic DCs and bone marrow (BM) precursors, including LSK (Lin- Sca1+ Kit+ cell), CMP (common myeloid-restricted progenitor), GMP (granulocyte-macrophage progenitor), CLP (common lymphoid-restricted progenitor), and CDP (common dendritic cell precursor), were separated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For the in vitro analysis of Gata2-knockout, BM cells were cultured with CD45.1+ BM feeder cells from SJL mice (The Jackson Laboratory) with FLT3L (200 ng/mL) and 4-hydroxytamoxifen (Sigma). For transcription profiling, SurePrint G3 mouse GE microarray (Agilent) was used, and the data was subsequently analyzed with ImmGen database (http://www.immgen.org). Promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using CMP fraction and erythroid-myeloid-lymphoid (EML) hematopoietic precursor cell line (ATCC) with antibodies to GATA-2 (sc-9008, Santa Cruz Biotechnology). (Results) Quantitative RT-PCR analysis showed abundant Gata2 expression in LSK and CMP fractions, with detectable expression in GMP, CLP, and CDP fractions and in vitro differentiated DCs. Although the DC count did not change in Gata2 haploinsufficient mice, it significantly and profoundly decreased in Gata2 conditional knockout mice. To examine the role of GATA-2 during DC differentiation, we knocked out Gata2 during in vitro DC differentiation, starting from LSK, CMP, GMP, CLP, and CDP fractions obtained from Gata2flox/flox/ER-Cre mice. Gata2 knockout significantly decreased CD11c+ DC counts from LSK, CMP, and CDP fractions, while those from CLP and GMP were unaffected, implying the importance of GATA-2 during DC differentiation in the pathway from LSK to CDP via CMP, not via CLP nor GMP. To elucidate the underlying molecular mechanisms, we performed expression profiling with control and Gata2 -knockout DC progenitors from CMP of Gata2flox/flox/ER-Cre mice. Gata2 knockout caused >5-fold upregulation and downregulation of 67 and 63 genes, respectively. Although genes critical for the DC differentiation, e.g., Spi1, Ikzf1, and Gfi1, were not detected among the GATA-2-regulated gene ensemble, we found significant enrichment of myeloid-related and T lymphocyte-related genes among the downregulated and upregulated gene ensembles, respectively. We focused on Gata3 upregulation (7.33-fold) as a potential key mechanism contributing to Gata2 knockout-related impaired DC differentiation. Quantitative ChIP analysis with both CMP fraction and EML cell line demonstrated obvious GATA-2 chromatin occupancy at the consensus GATA-binding motif within Gata3+190 kb, which was conserved with human. Furthermore, addition of Gata3 +190 kb region to the Gata3 promoter (~0.5 kb) significantly decreased luciferase activity, which was significantly recovered by the deletion of GATA sequence within Gata3 +190 kb, in EML cells. (Conclusion) GATA-2 seems to play an important role for cell fate specification toward myeloid versus T lymphocytes, and thus contributing to the DC differentiation. Our data offer a better understanding of the pathophysiology of MonoMAC syndrome. Disclosures Fujiwara: Chugai Pharmaceuticals. Co., Ltd.: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Ishizawa:GSK: Research Funding; Takeda: Research Funding; Celgin: Speakers Bureau; Kyowa Kirin: Research Funding; Celgin: Research Funding; Janssen: Research Funding; Takeda: Speakers Bureau; Kyowa Kirin: Speakers Bureau; Pfizer: Speakers Bureau.

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Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line

International Journal of Molecular Sciences ◽

10.3390/ijms22031163 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1163

Author(s):

Gaia Palmini ◽

Cecilia Romagnoli ◽

Simone Donati ◽

Roberto Zonefrati ◽

Gianna Galli ◽

...

Keyword(s):

Cell Line ◽

Molecular Mechanisms ◽

Osteogenic Sarcoma ◽

Cancer Cell Line ◽

Microscopic Features ◽

In Vitro Establishment ◽

Profile Characteristic ◽

First Time

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

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MAZ-LINC00645-GP73 Axis Promotes Hepatocellular Carcinoma Proliferation and Metastasis

10.21203/rs.3.rs-65401/v1 ◽

2020 ◽

Author(s):

Yang Chen ◽

Huiyan Li ◽

Chunxun Liu ◽

Yongmei Han ◽

Yubao Zhang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Molecular Mechanisms ◽

Tumor Promoter ◽

Transcriptional Analysis ◽

Luciferase Reporter ◽

Chip Assay ◽

Bioinformatics Analyses

Abstract BACKGROUND: Long non-coding RNAs (lncRNA) have been shown to play important roles in the development and progression of hepatocellular carcinoma (HCC). In this report, we examined the role of lncRNA LINC00645 in HCC. MATERIAL AND METHODS: Based on public databases and integrating bioinformatics analyses, the over-expression of LINC00645 in HCC tissues was detected and further validated in a cohort of liver tissues. A series of in vitro and in vivo functional experiments were executed to investigate the role of LINC00645 in the carcinogenesis and development of HCC. Comprehensive transcriptional analysis, chromatin immunoprecipitation (ChIP) assay, dual-luciferase reporter assay and western blot etc. were performed to explore the molecular mechanisms underlying the functions of LINC00645. RESULTS: LINC00645 was significantly upregulated in HCC cell lines and HCC tissues, which was correlated with poor prognosis in HCC patients. LINC00645 knockdown remarkably suppressed tumor growth in vitro and in vivo. Mechanistically, LINC00645 could competitively bind with miR-141-3p to prevent the degradation of its target gene GP73, which acts as a tumor-promoter in HCC. Furthermore, the ChIP assay showed that the transcription factor MAZ could bind to the LINC00645 promoter and increase its transcription. CONCLUSIONS: Collectively, this study demonstrated that LINC00645 plays a critical regulatory role in hepatocellular carcinoma cells and LINC00645 may serve as a potential diagnostic biomarker and therapeutic target of HCC. Thus, targeting MAZ/LINC00645/miR-141-3p/GP73 signaling axis may prevent the progression of HCC.

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lncRNA SNHG15 Induced by SOX12 Promotes the Tumorigenic Properties and Chemoresistance in Cervical Cancer via the miR-4735-3p/HIF1a Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2022/8548461 ◽

2022 ◽

Vol 2022 ◽

pp. 1-15

Author(s):

Jiang Yang ◽

Mei Yang ◽

Huabing Lv ◽

Min Zhou ◽

Xiaogang Mao ◽

...

Keyword(s):

Cervical Cancer ◽

Molecular Mechanisms ◽

Therapeutic Strategies ◽

Luciferase Reporter ◽

Major Health ◽

Tumor Tissues ◽

High Prevalence ◽

Reporter Assays

Cervical cancer (CC) is one of the most common malignancies in females, with high prevalence and mortality globally. Despite advances in diagnosis and therapeutic strategies developed in recent years, CC is still a major health burden worldwide. The molecular mechanisms underlying the development of CC need to be understood. In this study, we aimed to demonstrate the role of lncRNA SNHG15 in CC progression. Using qRT-PCR, we determined that lncRNA SNHG15 is highly expressed in CC tumor tissues and cells. lncRNA SNHG15 knockdown also reduces the tumorigenic properties of CC in vitro, as determined using the MTT, EdU, flow cytometry, and transwell assays. Using bioinformatics analysis, RNA pull-down, ChIP, and luciferase reporter assays, we verified the molecular mechanisms of lncRNA SNHG15 in CC progression and found that lncRNA SNHG15 expression in CC cells is transcriptionally regulated by SOX12; moreover, lncRNA SNHG15 promotes CC progression via the miR-4735-3p/HIF1a axis. This study can provide a potential target for CC diagnosis or therapeutic strategies in the future.

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MiR-624-5p enhances cell resistance against cisplatin via PDGFRA/Stat3/PI3K axis in ovarian cancer

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v19i4.3 ◽

2020 ◽

Vol 19 (4) ◽

pp. 691-698

Author(s):

Lin I-Ju ◽

Tian YongJie

Keyword(s):

Ovarian Cancer ◽

Cell Viability ◽

Cell Line ◽

Cell Lines ◽

Clinical Stage ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay

Purpose: The purpose of this study was to evaluate the role of miR-624-5p in ovarian cancer.Methods: MiR-624-5p expression in ovarian cancer {OC) cell lines and normal cells (NCs) was evaluated and compared the differential miR-624-5p in OC A2780 cells and cisplatin-resistant OC cell line (A2780/DDP). CCK-8 was used to evaluate changes in cell viability of the A2780 and A2780/DDP cell lines as well as silenced miR-624-5p. Western Blot examined the Stat3 and phosphorylated Pi3k. The binding between PDGFRA and miR-624-5p was predicted on Targetscan and verified through Luciferase Reporter Assay. The role of PDGFRA in A2780/DDP by overexpressing PDGFRA was evaluated by RT-qPCR and CCK-8 assays. RT-qPCR assay also measured miR-624-5p expression responsive to different dosages of cisplatin and CCK8 examined viability levels correspondingly. In addition, the interplay of PDGFRA and miR-624-5p by combined downregulation of both miR-624-5pand PDGFRA were evaluated.Results: OC cells had higher miR-624-5p expression than NCs but lower compared to cisplatinresistant A2780/DDP cells. A2780/DDP cells had higher viability than OC cell line A2780. Stat3 and phosphorylated PI3K were activated in A2780/DDP cells. Silencing miR-624-5p led to lower viability inA2780/DDP cells. miR-624-5p expression dropped as the cisplatin concentration increased, resulting in decreasing viability respectively. Luciferase Reporter assay validated the binding of miR-624-5p and PDGFRA in A2780/DDP cells. Overexpressed PDGFRA induced lower cell viability in A2780/DDP cells. Downregulation of PDGFRA partially restored the lowered viability and inhibited Stat3 as well as phosphorylated Pi3k induced by miR-624-5p inhibitor.Conclusion: MiR-624-5p could add to the cellular resistance to cisplatin in OC in-vitro model, which indicated that it might help unveil the mystery of drug-resistance in clinical stage of ovarian cancer. Keywords: MiR-624-5p, resistance, cisplatin, PDGFRA/Stat3/PI3K, ovarian cancer

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Hsa_circ_0000839 Inhibits Papillary Thyroid Carcinoma Proliferation by Targeting CDC27

10.21203/rs.3.rs-638595/v1 ◽

2021 ◽

Author(s):

Jiahui Guo ◽

Tingting Liu ◽

Zhongyan Shan ◽

Weiping Teng

Keyword(s):

Papillary Thyroid Carcinoma ◽

Thyroid Carcinoma ◽

Molecular Mechanisms ◽

Circular Rna ◽

Luciferase Reporter ◽

Papillary Thyroid ◽

Potential Biomarker

Abstract Background: Circular RNA (circRNA) has been reported to play multiple roles in a variety of cancers. However, the role of circRNA in papillary thyroid carcinoma (PTC) remains mostly unknown. Methods: The expression, function and potential molecular mechanisms of hsa_circ_0000839 in PTC in vitro were evaluated by quantitative RT-PCR, western blot, flow cytometry, CCK8, Edu, RNA-sequencing, luciferase reporter, and RNA immunoprecipitation assay. The function of hsa_circ_0000839 in PTC in vivo was evaluated by xenograft tumors assay.Results: Hsa_circ_0000839 was significantly downregulated in PTC tissues and plasma from patients with PTC, and its downregulation was correlated with larger tumor size in patients with PTC. The role of hsa_circ_0000839 in the proliferation of PTC cell lines was evaluated in both vitro and in vivo. Mechanistically, hsa_circ_0000839 regulated the level of CDC27 via sponging miR-149-5p in PTC. Conclusions: Hsa_circ_0000839 might act as a tumor suppressor of PTC through the hsa_circ_0000839/miR-149-5p/CDC27 axis. Hsa_circ_0000839 could serve as a potential biomarker and therapeutic target for patients with PTC.

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The Krüppel-like factor 15-NFATc1 axis ameliorates podocyte injury: a novel rationale for using glucocorticoids in proteinuria diseases

Clinical Science ◽

10.1042/cs20200075 ◽

2020 ◽

Vol 134 (12) ◽

pp. 1305-1318

Author(s):

Caoshuai Dou ◽

Hong Zhang ◽

Guibao Ke ◽

Li Zhang ◽

Zhiwen Lian ◽

...

Keyword(s):

Critical Role ◽

Luciferase Reporter ◽

Protective Effects ◽

Podocyte Injury ◽

Direct Target ◽

In Vitro Treatment ◽

Chip Analysis ◽

Dual Luciferase Reporter Assay

Abstract Podocyte injury and loss contribute to proteinuria, glomerulosclerosis and eventually kidney failure. Recent studies have demonstrated that the loss of Kruppel-like factor 15 (KLF15) in podocytes increases the susceptibility to injury; however, the mechanism underlying the protective effects on podocyte injury remains incompletely understood. Herein, we showed that KLF15 ameliorates podocyte injury through suppressing NFAT signaling and the salutary effects of the synthetic glucocorticoid dexamethasone in podocyte were partially mediated by the KLF15–NFATc1 axis. We found that KLF15 was significantly reduced in glomerular cells of proteinuric patients and in ADR-, LPS- or HG-treated podocyets in vitro. Overexpression of KLF15 attenuated podocyte apoptosis induced by ADR, LPS or HG and resulted in decreased expression of pro-apoptotic Bax and increased expression of anti-apoptotic Bcl-2. Conversely, the flow cytometry analysis and TUNEl assay demonstrated that loss of KLF15 accelerated podocyte apoptosis and we further found that 11R-VIVIT, a specific NFAT inhibitor, and NFATc1–siRNA rescued KLF15-deficient induced podocyte apoptosis. Meanwhile, Western blot and RT-qPCR showed that the expression of NFATc1 was up-regulated in KLF15 silenced podocytes and reduced in KLF15 overexpressed podocytes. Mechanistically, ChIP analysis showed that KLF15 bound to the NFATc1 promoter region -1984 to -1861base pairs upstream of the transcription start site and the binding amount was decreased after treatment with LPS. The dual-luciferase reporter assay indicated that NFATc1 was a direct target of KLF15. In addition, we found that in vitro treatment with dexamethasone induced a decrease of NFATc1 expression in podocytes and was abrogated by knockdown of KLF15. Hence, our results identify the critical role of the KLF15–NFATc1 axis in podocyte injury and loss, which may be involved in mediating the salutary effects of dexamethasone in podocytes.

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Unleashing Cell-Intrinsic Inflammation As a Strategy to Kill AML Blasts

Blood ◽

10.1182/blood-2021-151511 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3305-3305

Author(s):

Jana M Ellegast ◽

Gabriela Alexe ◽

Amanda Hamze ◽

Shan Lin ◽

Maxim Pimkin ◽

...

Keyword(s):

Cell Death ◽

Inflammatory Response ◽

Cell Line ◽

Research Funding ◽

Gene Set Enrichment Analysis ◽

Luciferase Reporter ◽

Inflammatory Signaling ◽

Inflammatory Pathways

Abstract Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as cell-intrinsic, self-directed immunotherapy. To test the hypothesis, we analyzed three large, independent data collections from primary acute myeloid leukemia (AML) samples and identified an AML subgroup of approximately 40% of all samples enriched for immune and inflammatory pathways. Moreover, we observed a positive correlation between the enrichment of inflammatory response and a monocytic lineage signature in these primary AML samples. To discover genetic vulnerabilities in AML cells implicated in inflammatory pathways, we integrated data from the Cancer Dependency Map on 789 cancer cell lines with two independent genome-wide screens. We identified the immune modulator interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a selective dependency in AML. We validated AML cell dependency on IRF2BP2 with orthogonal genetic and degradation approaches in vitro and in vivo. Genetic perturbation and degradation of IRF2BP2 translated into a reduction of AML cell line viability and decreased colony formation capacity in vitro and impaired leukemia progression in vivo in AML cell line xenografts. Moreover, inducible knock-out of IRF2BP2 significantly attenuated disease burden in multiple patient-derived xenograft models of AML and prolonged survival. Mechanistically, IRF2BP2 repression induced cell death with hallmarks of apoptosis, evidenced by an increase in annexin V/PI-positive cells and induction of cleaved caspase 3 and 8. To decipher the role of IRF2BP2 in inflammatory signaling, we studied its localization on chromatin and its relationship to histone marks and RNA polymerase II binding in AML cells. We determined that IRF2BP2 binds at both enhancers and promotors. Global gene expression profiling, performed six hours after degradation of IRF2BP2, identified significantly more genes increased than decreased in expression. Most upregulated genes were bound by IRF2BP2 at baseline and gained H3K27ac at enhancers with IRF2BP2 degradation. All told, our data support a role for IRF2BP2 as a transcriptional repressor in AML. Gene set enrichment analysis of the genes directly regulated by IRF2BP2 identified immune response signatures as the top enriched gene sets, with genes regulated by NF-kB in response to TNFa being the most significantly enriched. We thus hypothesized that IRF2BP2 is a repressor of NF-kB mediated TNFa signaling that, when acutely perturbed, leads to leukemia cell death. Indeed, we observed an activation of NF-κB signaling in a luciferase reporter assay, an increase in nuclear NF-kB (RELA) protein levels, and a gain in NF-κB chromatin binding following degradation of IRF2BP2 in AML cells. Moreover, a mutant "super-repressor" allele of IkBa rescued the impaired cell growth upon IRF2BP2 perturbation in AML cells, supporting cell death associated with IRF2BP2 loss being mediated through activation of NF-kB signaling. In addition, we identified IL-1ß as an enhancer of the inflammatory response repressed by IRF2BP2. In summary, we have demonstrated that IRF2BP2 represses IL-1ß/TNFα signaling via NF-κB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival. Disclosures Dharia: Genentech: Current Employment. Benajiba: Gilead: Research Funding; Pfizer: Research Funding. Gray: Gatekeeper, Syros, Petra, C4, Allorion, Jengu, B2S, Inception and Soltego: Consultancy, Research Funding. Stegmaier: Auron Therapeutics, Kronos Bio, AstraZeneca, Novartis Institute of Biomedical Research: Consultancy, Research Funding.

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Loss of Gab1 adaptor protein in hepatocytes aggravates experimental liver fibrosis in mice

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00289.2014 ◽

2015 ◽

Vol 308 (7) ◽

pp. G613-G624 ◽

Cited By ~ 8

Author(s):

Takashi Kizu ◽

Yuichi Yoshida ◽

Kunimaro Furuta ◽

Satoshi Ogura ◽

Mayumi Egawa ◽

...

Keyword(s):

Liver Fibrosis ◽

Tyrosine Kinases ◽

Molecular Mechanisms ◽

Adaptor Protein ◽

Conditional Knockout ◽

Hepatocyte Proliferation ◽

Duct Ligation ◽

Liver Fibrogenesis

Grb2-associated binder 1 (Gab1) adaptor protein amplifies signals downstream of a broad range of growth factors/receptor tyrosine kinases. Although these signals are implicated in liver fibrogenesis, the role of Gab1 remains unclear. To elucidate the role of Gab1, liver fibrosis was examined in hepatocyte-specific Gab1-conditional knockout (Gab1CKO) mice upon bile duct ligation (BDL). Gab1CKO mice developed exacerbated liver fibrosis with activation of hepatic myofibroblasts after BDL compared with control mice. The antifibrotic role of hepatocyte Gab1 was further confirmed by another well-established mouse model of liver fibrosis using chronic injections of carbon tetrachloride. After BDL, Gab1CKO mice also displayed exacerbated liver injury, decreased hepatocyte proliferation, and enhanced liver inflammation. Furthermore, cDNA microarray analysis was used to investigate the potential molecular mechanisms of the Gab1-mediated signal in liver fibrosis, and the fibrosis-promoting factor chemokine (C-C motif) ligand 5 ( Ccl5) was identified as upregulated in the livers of Gab1CKO mice following BDL. Interestingly, in vitro studies using primary hepatocytes isolated from control and Gab1CKO mice revealed that the loss of Gab1 resulted in increased hepatocyte CCL5 synthesis upon lipopolysaccharide stimulation. Finally, pharmacological antagonism of CCL5 reduced BDL-induced liver fibrosis in Gab1CKO mice. In conclusion, our results demonstrate that hepatocyte Gab1 is required for liver fibrosis and that hepatocyte CCL5 could be an important contributor to this process. Thus, we present a novel antifibrotic function of hepatocyte Gab1 in liver fibrogenesis.

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Hsa_circ_0005273 facilitates breast cancer tumorigenesis by regulating YAP1-hippo signaling pathway

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01830-z ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Xuehui Wang ◽

Changle Ji ◽

Jiashu Hu ◽

Xiaochong Deng ◽

Wenfang Zheng ◽

...

Keyword(s):

Breast Cancer ◽

Signaling Pathway ◽

Cell Lines ◽

Luciferase Reporter ◽

Circular Rnas ◽

Hippo Signaling ◽

Hippo Signaling Pathway ◽

Potential Biomarker

Abstract Background Circular RNAs (circRNAs), a novel class of endogenous RNAs, have shown to participate in the development of breast cancer (BC). Hsa_circ_0005273 is a circRNA generated from several exons of PTK2. However, the potential functional role of hsa_circ_0005273 in BC remains largely unknown. Here we aim to evaluate the role of hsa_circ_0005273 in BC. Methods The expression level of hsa_circ_0005273 and miR-200a-3p were examined by RT-qPCR in BC tissues and cell lines. The effect of knocking down hsa_circ_0005273 in BC cell lines were evaluated by examinations of cell proliferation, migration and cell cycle. In addition, xenografts experiment in nude mice were performed to evaluate the effect of hsa_circ_0005273 in BC. RNA immunoprecipitation assay, RNA probe pull-down assay, luciferase reporter assay and fluorescence in situ hybridization were conducted to confirm the relationship between hsa_circ_0005273, miR-200a-3p and YAP1. Results Hsa_circ_0005273 is over-expressed in BC tissues and cell lines, whereas miR-200a-3p expression is repressed. Depletion of hsa_circ_0005273 inhibited the progression of BC cells in vitro and in vivo, while overexpression of hsa_circ_0005273 exhibited the opposite effect. Importantly, hsa_circ_0005273 upregulated YAP1 expression and inactivated Hippo pathway via sponging miR-200a-3p to promote BC progression. Conclusions Hsa_circ_0005273 regulates the miR-200a-3p/YAP1 axis and inactivates Hippo signaling pathway to promote BC progression, which may become a potential biomarker and therapeutic target.

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Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01882-1 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Zizhen Si ◽

Lei Yu ◽

Haoyu Jing ◽

Lun Wu ◽

Xidi Wang

Keyword(s):

Colorectal Cancer ◽

Rna Binding ◽

Xenograft Model ◽

Luciferase Reporter ◽

Cancer Proliferation ◽

Mouse Xenograft ◽

Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

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