LncRNA SNHG15 contributes to doxorubicin resistance of osteosarcoma cells through targeting the miR-381-3p/GFRA1 axis

AbstractBackgroundOsteosarcoma is a common primary malignant bone cancer. Long noncoding RNA small nucleolar RNA host gene 15 (SNHG15) has been reported to play an oncogenic role in many cancers. Nevertheless, the role of SNHG15 in the doxorubicin (DXR) resistance of osteosarcoma cells has not been fully addressed.MethodsCell Counting Kit-8 assay was conducted to measure the half-maximal inhibitory concentration value of DXR in osteosarcoma cells. Western blotting was carried out to examine the levels of autophagy-related proteins and GDNF family receptor alpha-1 (GFRA1). Quantitative reverse transcription-polymerase chain reaction was performed to determine the levels of SNHG15, miR-381-3p, and GFRA1. The proliferation of osteosarcoma cells was measured by MTT assay. The binding sites between miR-381-3p and SNHG15 or GFRA1 were predicted by Starbase bioinformatics software, and the interaction was confirmed by dual-luciferase reporter assay. Murine xenograft model was established to validate the function of SNHG15 in vivo.ResultsAutophagy inhibitor 3-methyladenine sensitized DXR-resistant osteosarcoma cell lines to DXR. SNHG15 was upregulated in DXR-resistant osteosarcoma tissues and cell lines. SNHG15 knockdown inhibited the proliferation, DXR resistance, and autophagy of osteosarcoma cells. MiR-381-3p was a direct target of SNHG15, and GFRA1 bound to miR-381-3p in osteosarcoma cells. SNHG15 contributed to DXR resistance through the miR-381-3p/GFRA1 axis in vitro. SNHG15 depletion contributed to the inhibitory effect of DXR on osteosarcoma tumor growth through the miR-381-3p/GFRA1 axis in vivo.ConclusionsSNHG15 enhanced the DXR resistance of osteosarcoma cells through elevating the autophagy via targeting the miR-381-3p/GFRA1 axis. Restoration of miR-381-3p expression might be an underlying therapeutic strategy to overcome the DXR resistance of osteosarcoma.

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Silencing of Long Noncoding RNA LINC01132 Alleviates the Oncogenicity of Epithelial Ovarian Cancer by Regulating the microRNA-431-5p/SOX9 Axis

10.21203/rs.3.rs-219226/v1 ◽

2021 ◽

Author(s):

Wei Zhu ◽

Xiangming Xiao ◽

Jinqin Chen

Keyword(s):

Ovarian Cancer ◽

Epithelial Ovarian Cancer ◽

Noncoding Rna ◽

Xenograft Model ◽

Cell Counting ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Molecular Events

Abstract Background: To date, long intergenic nonprotein coding RNA 1132 (LINC01132) expression in epithelial ovarian cancer (EOC) and the underlying mechanisms have not been explored. In this study, we measured LINC01132 expression in EOC and assessed the effects of LINC01132 on the malignant behaviours of EOC cells in vitro and in vivo. Additionally, mechanistic studies were performed to elucidate the molecular events that occurred downstream of LINC01132 in EOC cells. Methods: Reverse-transcription quantitative PCR (RT-qPCR) was utilized to verify LINC01132 expression in EOC. The effects of LINC01132 on the malignant behaviours of EOC cells were determined using a Cell Counting Kit-8 assay, flow cytometry analysis, cell migration and invasion assays and a tumour xenograft model. The targeting interaction among LINC01132, microRNA-431-5p (miR-431-5p) and SRY-Box 9 (SOX9) was verified by RNA immunoprecipitation and luciferase reporter assays. Results: LINC01132 was overexpressed in EOC and was obviously associated with poor patient prognosis. Functionally, cell experiments revealed that LINC01132 depletion could inhibit EOC cell proliferation, migration and invasion and promote cell apoptosis in vitro. Additionally, loss of LINC01132 attenuated tumour growth in vivo. Mechanistically, LINC01132 acted as a competing endogenous RNA by sequestering miR-431-5p and thereby increasing SOX9 expression in EOC cells, forming a LINC01132/miR-431-5p/SOX9 axis. In rescue experiments, miR-431-5p inhibition or SOX9 re-expression eliminated the inhibitory effects of LINC01132 silencing on the pathological behaviours of EOC cells. Conclusions: Generally, LINC01132 exhibited oncogenic activities in EOC cells in vitro and in vivo by regulating the outcome of the miR-431-5p/SOX9 axis, providing an effective target for EOC diagnosis, therapy and prognosis evaluation.

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LINC00202 promotes retinoblastoma progression by regulating cell proliferation, apoptosis, and aerobic glycolysis through miR-204-5p/HMGCR axis

Open Life Sciences ◽

10.1515/biol-2020-0047 ◽

2020 ◽

Vol 15 (1) ◽

pp. 437-448

Author(s):

Aimin Wu ◽

Xuewei Zhou ◽

Linglong Mi ◽

Jiang Shen

Keyword(s):

Cell Proliferation ◽

Noncoding Rna ◽

Caspase 3 ◽

Aerobic Glycolysis ◽

Xenograft Model ◽

Cell Counting ◽

Luciferase Reporter ◽

Reaction Cell

AbstractLINC00202 is a newly identified long noncoding RNA (lncRNA) and has been demonstrated to involve in the progression of retinoblastoma (RB). Here, we further explored the role and the underlying molecular mechanism of LINC00202 on RB malignant properties and glycolysis. LINC00202, microRNA (miR)-204-5p, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) mRNA were detected by a quantitative real-time polymerase chain reaction. Cell proliferation and apoptosis were analyzed using cell counting kit-8 assay and colony formation assay and flow cytometry, respectively. Glucose metabolism was calculated by measuring the extracellular acidification rate (ECRA). Western blot was used to detect the levels of HMGCR, ki67, pro-caspase-3, cleaved-caspase-3, and lactate dehydrogenase A chain (LDHA). The interaction between miR-204-5p and LINC00202 or HMGCR was analyzed by the dual-luciferase reporter assay. Murine xenograft model was established to conduct in vivo experiments. LINC00202 expression was upregulated in RB tumor tissues and LINC00202 knockdown inhibited RB cell proliferation, glycolysis, and stimulated apoptosis in vitro as well as impeded tumor growth in vivo. MiR-204-5p directly bound to LINC00202 and HMGCR in RB cells, and LINC00202 functioned as a competing endogenous RNA in regulating HMGCR through competitively binding to miR-204-5p. More importantly, the regulation of malignant properties and glycolysis of RB cells mediated by LINC00202 could be reversed by abnormal miR-204-5p or HMGCR expression in RB cells. In all, LINC00202 promoted RB cell proliferation, glycolysis, and suppressed apoptosis by regulating the miR-204-5p/HMGCR axis, suggesting a novel therapeutic target for patients with RB.

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LINC00958 promotes the proliferation of TSCC via miR-211-5p/CENPK axis and activating the JAK/STAT3 signaling pathway

Cancer Cell International ◽

10.1186/s12935-021-01808-z ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Bo Jia ◽

Junfeng Dao ◽

Jiusong Han ◽

Zhijie Huang ◽

Xiang Sun ◽

...

Keyword(s):

Noncoding Rna ◽

Xenograft Model ◽

Tumor Xenograft ◽

Luciferase Reporter ◽

Cell Cycle Regulators ◽

Normal Tissues ◽

Stat3 Signaling ◽

Long Intergenic Noncoding Rna

Abstract Background Tongue squamous cell carcinoma (TSCC) is one of the most common oral tumors. Recently, long intergenic noncoding RNA 00958 (LINC00958) has been identified as an oncogene in human cancers. Nevertheless, the role of LINC00958 and its downstream mechanisms in TSCC is still unknown. Methods The effect of LINC00958 on TSCC cells proliferation and growth were assessed by CCK-8, colony formation, 5-Ethynyl-2′-deoxyuridline (EdU) assay and flow cytometry assays in vitro and tumor xenograft model in vivo. Bioinformatics analysis was used to predict the target of LINC00958 in TSCC, which was verified by RNA immunoprecipitation and luciferase reporter assays. Results LINC00958 was increased in TSCC tissues, and patients with high LINC00958 expression had a shorter overall survival. LINC00958 knockdown significantly decreased the growth rate of TSCC cells both in vitro and in vivo. In mechanism, LINC00958 acted as a ceRNA by competitively sponging miR-211-5p. In addition, we identified CENPK as a direct target gene of miR-211-5p, which was higher in TSCC tissues than that in adjacent normal tissues. Up-regulated miR-211-5p or down-regulated CENPK could abolish LINC00958-induced proliferation promotion in TSCC cells. Furthermore, The overexpression of CENPK promoted the expression of oncogenic cell cycle regulators and activated the JAK/STAT3 signaling. Conclusions Our findings suggested that LINC00958 is a potential prognostic biomarker in TSCC.

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LINC00958 promotes the proliferation of TSCC via miR-211-5p/CENPK axis and activating the JAK/STAT3 signaling pathway

10.21203/rs.3.rs-78207/v2 ◽

2021 ◽

Author(s):

Bo Jia ◽

Junfeng Dao ◽

Jiusong Han ◽

Zhijie Huang ◽

Xiang Sun ◽

...

Keyword(s):

Noncoding Rna ◽

Xenograft Model ◽

Tumor Xenograft ◽

Luciferase Reporter ◽

Cell Cycle Regulators ◽

Normal Tissues ◽

Stat3 Signaling ◽

Long Intergenic Noncoding Rna

Abstract Background: Tongue squamous cell carcinoma (TSCC) is one of the most common oral tumors. Recently, long intergenic noncoding RNA 00958 (LINC00958) has been identified as an oncogene in human cancers. Nevertheless, the role of LINC00958 and its downstream mechanisms in TSCC is still unknown. Methods: The expression levels of LINC00958 in human TSCC tissues and adjacent normal tissues were detected. The effect of LINC00958 on TSCC cells proliferation and growth were assessed by CCK-8, colony formation, 5-Ethynyl-2’-deoxyuridline (EdU) assay, and flow cytometry assays in vitro and tumor xenograft model in vivo. Bioinformatics analysis was used to predict the target of LINC00958 in TSCC, which was verified by RNA immunoprecipitation and luciferase reporter assays. Results: We found LINC00958 was increased in TSCC tissues, and patients with high LINC00958 expression had a shorter overall survival. LINC00958 knockdown significantly decreased the growth rate of TSCC cells both in vitro and in vivo . In mechanism, LINC00958 acted as a competing endogenous RNA (ceRNA) by competitively sponging miR-211-5p. In addition, we identified centromere protein K (CENPK) as a direct target gene of miR-211-5p, which was higher in TSCC tissues than that in adjacent normal tissues. Up-regulated miR-211-5p or down-regulated CENPK could abolish LINC00958-induced proliferation promotion in TSCC cells. Conclusion: Furthermore, CENPK promoted the expression of oncogenic cell cycle regulators and activated the JAK/STAT3 signaling. Our findings suggest that LINC00958 is a potential prognostic biomarker in TSCC.

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Mutant NPM1-regulated lncRNA HOTAIRM1 promotes leukemia cell autophagy and proliferation by targeting EGR1 and ULK3

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02122-2 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Yipei Jing ◽

Xueke Jiang ◽

Li Lei ◽

Meixi Peng ◽

Jun Ren ◽

...

Keyword(s):

Noncoding Rna ◽

Animal Studies ◽

Leukemia Cell ◽

Cell Counting ◽

Luciferase Reporter ◽

Promising Therapeutic Target ◽

Rna Immunoprecipitation ◽

Incorporation Assay

Abstract Background Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1), which displays a distinct long noncoding RNA (lncRNA) expression profile, has been defined as a unique subgroup in the new classification of myeloid neoplasms. However, the biological roles of key lncRNAs in the development of NPM1-mutated AML are currently unclear. Here, we aimed to investigate the functional and mechanistic roles of the lncRNA HOTAIRM1 in NPM1-mutated AML. Methods The expression of HOTAIRM1 was analyzed with a public database and further determined by qRT-PCR in NPM1-mutated AML samples and cell lines. The cause of upregulated HOTAIRM1 expression was investigated by luciferase reporter, chromatin immunoprecipitation and ubiquitination assays. The functional role of HOTAIRM1 in autophagy and proliferation was evaluated using western blot analysis, immunofluorescence staining, a Cell Counting Kit-8 (CCK-8) assay, a 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, flow cytometric analyses and animal studies. The action mechanism of HOTAIRM1 was explored through RNA fluorescence in situ hybridization, RNA pulldown and RNA immunoprecipitation assays. Results HOTAIRM1 was highly expressed in NPM1-mutated AML. High HOTAIRM1 expression was induced in part by mutant NPM1 via KLF5-dependent transcriptional regulation. Importantly, HOTAIRM1 promoted autophagy and proliferation both in vitro and in vivo. Mechanistic investigations demonstrated that nuclear HOTAIRM1 promoted EGR1 degradation by serving as a scaffold to facilitate MDM2-EGR1 complex formation, while cytoplasmic HOTAIRM1 acted as a sponge for miR-152-3p to increase ULK3 expression. Conclusions Taken together, our findings identify two oncogenic regulatory axes in NPM1-mutated AML centered on HOTAIRM1: one involving EGR1 and MDM2 in the nucleus and the other involving the miR-152-3p/ULK3 axis in the cytoplasm. Our study indicates that HOTAIRM1 may be a promising therapeutic target for this distinct leukemia subtype.

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Preclinical Evaluation of the Pan-FGFR Inhibitor LY2874455 in FRS2-Amplified Liposarcoma

Cells ◽

10.3390/cells8020189 ◽

2019 ◽

Vol 8 (2) ◽

pp. 189 ◽

Cited By ~ 3

Author(s):

Robert Hanes ◽

Else Munthe ◽

Iwona Grad ◽

Jianhua Han ◽

Ida Karlsen ◽

...

Keyword(s):

Cell Lines ◽

Xenograft Model ◽

Inhibitory Effect ◽

Growth Inhibitory Effect ◽

Growth Inhibitory ◽

Signaling Components ◽

Fgfr Inhibition ◽

Model Expression

Background: FGFR inhibition has been proposed as treatment for dedifferentiated liposarcoma (DDLPS) with amplified FRS2, but we previously only demonstrated transient cytostatic effects when treating FRS2-amplified DDLPS cells with NVP-BGJ398. Methods: Effects of the more potent FGFR inhibitor LY2874455 were investigated in three DDLPS cell lines by measuring effects on cell growth and apoptosis in vitro and also testing efficacy in vivo. Genome, transcriptome and protein analyses were performed to characterize the signaling components in the FGFR pathway. Results: LY2874455 induced a stronger, longer-lasting growth inhibitory effect and moderate level of apoptosis for two cell lines. The third cell line, did not respond to FGFR inhibition, suggesting that FRS2 amplification alone is not sufficient to predict response. Importantly, efficacy of LY2874455 was confirmed in vivo, using an independent FRS2-amplified DDLPS xenograft model. Expression of FRS2 was similar in the responding and non-responding cell lines and we could not find any major difference in downstream FGFR signaling. The only FGF expressed by unstimulated non-responding cells was the intracellular ligand FGF11, whereas the responding cell lines expressed extracellular ligand FGF2. Conclusion: Our study supports LY2874455 as a better therapy than NVP-BGJ398 for FRS2-amplified liposarcoma, and a clinical trial is warranted.

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Long Noncoding RNA HOXA-AS2 Functions as an Oncogene by Binding to EZH2 and Suppressing LATS2 in AML

10.21203/rs.3.rs-42525/v1 ◽

2020 ◽

Author(s):

Yubin Feng ◽

shuang Hu ◽

lanlan Li ◽

xiaoqing Peng ◽

Feihu Chen

Keyword(s):

Noncoding Rna ◽

Cell Counting ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Large Tumor ◽

Hoxa Cluster ◽

The Public

Abstract BackgroundLong noncoding RNAs (lncRNAs) plays an important role in the development of physiology and pathology. Many reports have shown that LncRNA HOXA cluster antisense RNA 2 (HOXA-AS2) is a carcinogen and plays an important role in many tumors, but there are few reports on its role in Acute myeloid leukemia (AML). MethodsThe expression of HOXA-AS2 in AML cell line was detected by qRT-PCR. AML cases from the public database (GEPIA) were also included in this study. Cell counting kit-8 (CCK-8) assay, flow cytometry, immunofluorescence and Western blot were used to detect the role of HOXA-AS2 in AML cells. Luciferase reporter gene detection, RIP, RNA pull-down and RNA-ChIP detection were used to demonstrate the molecular biological mechanism of HOXA-AS2 in AML.ResultsOur results show that HOXA-AS2 was upregulated in AML cell lines and tissues, and the overexpression of HOXA-AS2 is negatively correlated with the survival of patients. Silencing HOXA-AS2 can inhibit the proliferation and induce differentiation of AML cells in vitro and in vivo. After overexpressing HOXA-AS2, it will show the opposite result. Moreover, more in-depth mechanism studies show that HOXA-AS2 exerts its carcinogenicity mainly by binding with the epigenetic inhibitor Enhancer of zeste homolog 2 (EZH2) and then inhibiting the expression of Large Tumor Suppressor 2 (LATS2). ConclusionsTaken together, our results highlight the important role of HOXA-AS2 in AML, suggesting that HOXA-AS2 may be an effective therapeutic target for patients with AML.

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Inhibitory Effects of Targeted Regulatory LKB1 Gene on the Proliferation and Invasion of Osteosarcoma Cells

International Journal of Human Genetics ◽

10.31901/245666330.2021/21.01.770 ◽

2021 ◽

Vol 21 (01) ◽

Author(s):

Jie Liu

Keyword(s):

Transcription Factor ◽

Xenograft Model ◽

Mtor Inhibitors ◽

Regulatory Genes ◽

Osteosarcoma Cell ◽

Osteosarcoma Cells ◽

Inhibitory Effects ◽

Proliferation And Invasion

In this study, Immunodeficiency Nude Mouse Osteosarcoma Xenograft Model was subjected to the drug intervention to explore the effect of bexarotene on the proliferation and invasion of osteosarcoma cell lines in vitro. The inhibitory effects of targeted regulatory genes on the proliferation and invasion of osteosarcoma cells were studied through various in vitro experiments include bioinformatics combined with tissue microarray research, transcription factor prediction combined with co-expression analysis to predict the transcription factor of targeted regulatory genes in osteosarcoma. The RXR protein family, LKB1, AMPK pathway, and mTOR are closely related to the body’s immune regulation. The oral administration of Bexarotene could inhibit the proliferation and able to up-regulate the expression of LKB1 gene in living osteosarcoma tissue. The xenograft model of immunodeficiency nude mice used in this study was reason for reduced the potential immunoregulatory effect of drug targeted LKB1 therapy to a certain extent. However, overexpression of LKB1 in vivo, and combined immunotherapy may become an important immunotherapy approach for osteosarcoma. LKB1 targeted therapy can potentially be used as one of the alternative treatments for mTOR inhibitors.

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LncRNA HOXD-AS1 functions as a ceRNA to promote hepatocellular carcinoma metastasis in zebrafish xenograft models

10.21203/rs.3.rs-232919/v1 ◽

2021 ◽

Author(s):

Ying Zhang ◽

Xiaolu Wang ◽

Feng Qin ◽

Shaochang Jia

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Lines ◽

Noncoding Rna ◽

Xenograft Model ◽

Cerna Network ◽

Xenograft Models ◽

Proliferation And Invasion ◽

Hcc Cells

Abstract Background: A few studies have shown that long noncoding RNA (lncRNA) HOXD cluster antisense RNA 1 (HOXD-AS1) plays an important role in hepatocellular carcinoma (HCC) metastasis as a competing endogenous RNA (ceRNA), but there is little in vivo evidence. This study aims to explore the zebrafish HCC xenograft as an in vivo metastasis model to verify the ceRNA network of HOXD-AS1. Methods: The quantitative reverse transcription PCR (qRT-PCR) assay was used to assess the expression level of HOXD-AS1 in HCC cell lines. Knockdown of HOXD-AS1 or miR-130a-3p was performed by transfecting small interfering RNA (siRNA) or microRNA (miRNA) inhibitor, respectively. The proliferation and invasion of HCC cells in vitro were analyzed by CCK-8 and transwell assays. The growth and metastasis of HCC cells in vivo were assessed by zebrafish xenograft models.Results: We verified that HOXD-AS1 was overexpressed in all tested HCC cell lines than the normal hepatic cells. Silence of HOXD-AS1 suppressed cell proliferation and invasion in Hep3B and Huh7 HCC cell lines in vitro. In zebrafish xenograft models, knockdown of HOXD-AS1 also reduced the growth and metastasis of the two HCC cells. Moreover, downregulation of miR-130a-3p not only increased the HCC metastasis, but also rescued the metastasis which inhibited by silence of HOXD-AS1 in vitro and in vivo.Conclusions: Our study demonstrates the metastasis role of the HOXD-AS1/miR-130a-3p ceRNA network in HCC cells in vitro and in vivo, and these findings suggest that zebrafish xenograft model could be used for ceRNA mechanism verification in tumor metastasis.

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Long noncoding RNA AFAP1-AS1 promotes tumor progression and invasion by regulating the miR-2110/Sp1 axis in triple-negative breast cancer

Cell Death and Disease ◽

10.1038/s41419-021-03917-z ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Xiaohui Zhang ◽

Fangyuan Li ◽

Yidong Zhou ◽

Feng Mao ◽

Yan Lin ◽

...

Keyword(s):

Breast Cancer ◽

Triple Negative Breast Cancer ◽

Noncoding Rna ◽

Triple Negative ◽

Quantitative Polymerase Chain Reaction ◽

Cell Counting ◽

Luciferase Reporter ◽

Migration And Invasion

AbstractLong noncoding ribonucleic acids (LncRNAs) have been found to be involved in the proliferation, apoptosis, invasion, migration, and other pathological processes of triple-negative breast cancer (TNBC). Expression of the lncRNA actin filament-associated protein 1 antisense RNA1 (AFAP1-AS1) has been found to be significantly higher in TNBC than in other subtypes or in normal tissue samples, but the specific mechanism by which AFAP1-AS1 affects the occurrence and development of TNBC is yet to be revealed. In this study, we used Cell Counting Kit-8 (CCK-8), colony formation, wound healing migration, Transwell invasion, and nude mouse xenograft assays to confirm the role of AFAP1-AS1 in the proliferation, migration of TNBC cells in vitro and in vivo. In addition, we performed bioinformatics analyses, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), western blot (WB), and dual-luciferase reporter assays (dual-LRA) to confirm interaction among AFAP1-AS1, micro-RNA 2110 (miR-2110), and Sp1 transcription factor (Sp1). We found that silencing AFAP1-AS1 and Sp1 or upregulating miR-2110 suppressed the proliferation, migration, and invasion of MDA–MB-231 and MDA–MB-468 cells in vitro as well as tumor growth in vivo. Mechanistically, the dual-LRA highlighted that miR-2110 was an inhibitory target of AFAP1-AS1, and that AFAP1-AS1 functioned as a miR-2110 sponge to increase Sp1 expression. AFAP1-AS1 silencing led to a reduction in Sp1 mRNA and protein levels, which could be reversed by joint transfection with miR-2110 inhibitor. Our findings demonstrated that AFAP1-AS1 could modulate the progression of breast cancer cells and affect tumorigenesis in mice by acting as a miR-2110 sponge, resulting in regulation of Sp1 expression. Therefore, AFAP1-AS1 could play a pivotal role in the treatment of TNBC.

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