scholarly journals miR-27a-3p attenuates induce autophagy-related cell death by suppressing Inhibitor of growth family 5 in cholangiocarcinoma

2020 ◽  
Author(s):  
Ming WAN ◽  
Fu-min Zhang ◽  
Peng-cheng Kang ◽  
Xing-ming Jiang ◽  
yunfu cui
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Immunofluorescence Analysis ◽  
Ki 67 ◽  
Western Blot Assay ◽  
Qrt Pcr ◽  
Related Cell ◽  
The Relationship

Abstract Background MicroRNAs (miRNAs) are abnormally expressed in human tumors, including cholangiocarcinoma (CCA). miR-27a-3p was observed up-regulated in CCA, but its functions in CCA are largely unknown.Methods CCK8 assay, Colony formation assays and Ki-67 staining was employed to detect the cell growth. The autophagy and proliferation relative-protein analyzed by western blot. The immunofluorescence staining was applied to analyze the expression level of LC3 I/II. Tumor xenografts was used to test the role of miR-27a-3p. Luciferase reporter assay, western bolt and qRT-PCR showed the relationship between miR-27a-3p and ING5.Results miR-27a-3p expression was increased in human CCA tissues. Inhibition of miR-27a-3p suppressed the proliferative capacity of CCA cells, silencing of miR-27a-3p dramatically induced cell death and suppressed tumor growth in vivo. The proteins, such as Beclin-1, p62, p21, p-p53, CDK4 and CDK6, were decreased upon miR-27a-3p inhibitor transfection. Western blot assay and immunofluorescence analysis were showed the induced-autophagy after transfecting with miR-27a-3p or inhibitor of growth family 5 (ING5) in RBE. ING5 as a direct miR-27a-3p target in CCA. Co-transfect of miR-27a-3p and ING5 can reverse CCA cell death which induced by miR-27a-3p inhibitor alone.Conclusions miR-27a-3p promotes oncogenesis of CCA by triggering autophagy-related cell death by interacting with ING5 directly.

scholarly journals Knockdown of lncRNA X inactive specific transcript (XIST) radiosensitizes non-small cell lung cancer (NSCLC) cells through regulation of miR-16-5p/WEE1 G2 checkpoint kinase (WEE1) axis

2021 ◽  
Vol 35 ◽  
pp. 205873842096608
Author(s):  
Ran Du ◽  
Feng Jiang ◽  
Yanhua Yin ◽  
Jinfen Xu ◽  
Xia Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Western Blot ◽  
Small Cell ◽  
Luciferase Reporter ◽  
Small Cell Lung ◽  
Luciferase Reporter Gene ◽  
G2 Checkpoint ◽  
Qrt Pcr ◽  
Specific Transcript

Long non-coding RNA (lncRNA) X inactive specific transcript (XIST) is reported to play an oncogenic role in non-small cell lung cancer (NSCLC). However, the role of XIST in regulating the radiosensitivity of NSCLC cells remains unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expressions of XIST and miR-16-5p in NSCLC in tissues and cells, and Western blot was used to assess the expression of WEE1 G2 checkpoint kinase (WEE1). Cell counting kit-8 (CCK-8), colony formation and flow cytometry assays were used to determine cell viability and apoptosis after NSCLC cells were exposed to different doses of X-rays. The interaction between XIST and miR-16-5p was confirmed by StarBase database, qRT-PCR and dual-luciferase reporter gene assays. TargetScan database was used to predict WEE1 as a target of miR-16-5p, and their targeting relationship was further validated by Western blot, qRT-PCR and dual-luciferase reporter gene assays. XIST was highly expressed in both NSCLC tissue and cell lines, and knockdown of XIST repressed NSCLC cell viability and cell survival, and facilitated apoptosis under the irradiation. MiR-16-5p was a target of XIST, and rescue experiments demonstrated that miR-16-5p inhibitors could reverse the role of XIST knockdown on radiosensitivity in NSCLC cells. WEE1 was validated as a target gene of miR-16-5p, and WEE1 could be negatively regulated by XIST. XIST promotes the radioresistance of NSCLC cells by regulating the expressions of miR-16-5p and WEE1, which can be a novel target for NSCLC therapy.

scholarly journals CircRNA_100290 Promotes GC Cell Proliferation And Invasion Via miR-29b-3p/ITGA11 Axis And Is Regulated By EIF4A3

2021 ◽  
Author(s):  
Gang Wang ◽  
Dan Sun ◽  
Wenhui Li ◽  
Yan Xin
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Qrt Pcr ◽  
Node Metastasis ◽  
Dual Luciferase Reporter Assay

Abstract Background: Circular RNA (circRNA) has been reported as an important regulator in the development and progression of various carcinomas. However, the role of circRNA_100290 in gastric cancer (GC) is still unclear. This study aimed to investigate the role of circRNA_100290 in GC invasion and metastasis and its possible mechanism.Methods: The expression of circRNA_100290 in GC cells and tissues were examined using quantitative real-time polymerase chain reaction (qRT-PCR). The role of circRNA_100290 in cell proliferation, migration, and invasion was evaluated on AGS and HGC-27 cell lines in vitro. Bioinformatics tools, dual-luciferase reporter assay, Western blot assay and qRT-PCR were used to explore the downstream pathways of circRNA_100290. The mechanism underlying the regulation of the expression of circRNA_100290 was explored using RNA immunoprecipitation, qRT-PCR, and Western blot assays.Results: The expression of circRNA_100290 was found significantly upregulated in GC cells and 102 GC tissues, high expression of circRNA_100290 in GC was closely related to Borrmann’s types, lymph node metastasis and tumor-node-metastasis staging. In vitro, knockdown of circRNA_100290 in AGS and HGC-27 cells significantly inhibited cell proliferation, migration, and invasion. Mechanistically, dual-luciferase reporter assay confirmed a direct binding between circRNA_100290 and miR-29b-3p, which targets ITGA11, an oncogene which is closely related to epithelial–mesenchymal transition (EMT). In addition, EIF4A3, one of RNA binding proteins (RBPs), could inhibit the formation of circRNA_100290 via enriching flanking sites of circRNA_100290. Low expression of EIF4A3 in GC was related to a worse prognosis.Conclusions: Elevated circRNA_100290 in GC promotes cell proliferation, invasion and EMT via miR-29b-3p/ITGA11 axi and might be regulated by EIF4A3. CircRNA_100290 might be a promising biomarker and target for GC therapy.

scholarly journals MicroRNA-486 promotes a more catabolic phenotype in chondrocyte-like cells by targeting SIRT6

2021 ◽  
Vol 10 (7) ◽  
pp. 459-466
Author(s):  
Jie Yang ◽  
Yunping Zhou ◽  
Xiaojun Liang ◽  
Bingfei Jing ◽  
Zandong Zhao
Keyword(s):  
Western Blot ◽  
Messenger Rna ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Luciferase Reporter Gene ◽  
Western Blot Assay ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Gene Assay ◽  
A Disintegrin And Metalloproteinase

Aims Osteoarthritis (OA) is characterized by persistent destruction of articular cartilage. It has been found that microRNAs (miRNAs) are closely related to the occurrence and development of OA. The purpose of the present study was to investigate the mechanism of miR-486 in the development and progression of OA. Methods The expression levels of miR-486 in cartilage were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN), matrix metalloproteinase (MMP)-13, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) in SW1353 cells at both messenger RNA (mRNA) and protein levels was determined by qRT-PCR, western blot, and enzyme-linked immunosorbent assay (ELISA). Double luciferase reporter gene assay, qRT-PCR, and western blot assay were used to determine whether silencing information regulator 6 (SIRT6) was involved in miR-486 induction of chondrocyte-like cells to a more catabolic phenotype. Results Compared with osteonecrosis, the expression of miR-486 was significantly upregulated in cartilage from subjects with severe OA. In addition, overexpressed miR-486 promoted a catabolic phenotype in SW1353 cells by upregulating the expressions of ADAMTS4 and MMP-13 and down-regulating the expressions of COL2A1 and ACAN. Conversely, inhibition of miR-486 had the opposite effect. Furthermore, overexpression of miR-486 significantly inhibited the expression of SIRT6, confirming that SIRT6 is a direct target of miR-486. Moreover, SW1353 cells were transfected with small interfering RNA (si)-SIRT6 and it was found that SIRT6 was involved in and inhibited miR-486-induced changes to SW1353 gene expression. Conclusion Our results indicate that miR-486 promotes a catabolic phenotype in SW1353 cells in OA by targeting SIRT6. Our findings might provide a potential therapeutic target and theoretical basis for OA. Cite this article: Bone Joint Res 2021;10(7):459–466.

scholarly journals CircRNA_100290 promotes GC cell proliferation and invasion via the miR-29b-3p/ITGA11 axis and is regulated by EIF4A3

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Gang Wang ◽  
Dan Sun ◽  
Wenhui Li ◽  
Yan Xin
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Rna Binding ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Qrt Pcr ◽  
Node Metastasis

Abstract Background Circular RNAs (circRNAs) have been reported to be important regulators of the development and progression of various carcinomas. However, the role of circRNA_100290 in gastric cancer (GC) is still unclear. This study aimed to investigate the role of circRNA_100290 in GC invasion and metastasis and the possible underlying mechanism. Methods The expression of circRNA_100290 in GC cells and tissues was examined using quantitative real-time polymerase chain reaction (qRT-PCR). The role of circRNA_100290 in cell proliferation, migration, and invasion was evaluated in the AGS and HGC-27 cell lines in vitro. Bioinformatics tools, dual-luciferase reporter assays, Western blot assays and qRT-PCR were used to explore the pathways downstream of circRNA_100290. The mechanism underlying the regulation of circRNA_100290 expression was explored using RNA immunoprecipitation, qRT-PCR, and Western blot assays. Results The expression of circRNA_100290 was significantly upregulated in GC cells and 102 GC tissues, and high circRNA_100290 expression in GC was closely related to Borrmann’s type, lymph node metastasis and tumour-node-metastasis stage. In vitro, knockdown of circRNA_100290 in AGS and HGC-27 cells significantly inhibited cell proliferation, migration, and invasion. Mechanistically, a dual-luciferase reporter assay confirmed the direct interaction between circRNA_100290 and miR-29b-3p, which targets ITGA11, an oncogene that is closely related to epithelial–mesenchymal transition (EMT). In addition, EIF4A3, an RNA-binding protein (RBP), could inhibit the formation of circRNA_100290 by binding to the flanking sites of circRNA_100290. Low EIF4A3 expression in GC was related to a poor prognosis. Conclusions Elevated circRNA_100290 expression in GC promotes cell proliferation, invasion and EMT via the miR-29b-3p/ITGA11 axis and might be regulated by EIF4A3. CircRNA_100290 might be a promising biomarker and target for GC therapy. Graphical abstract

scholarly journals LncRNA HOXA-AS2 promotes glioblastoma carcinogenesis by targeting miR-885-5p/RBBP4 axis

2020 ◽  
Author(s):  
Jixin Shou ◽  
Haidong Gao ◽  
Sen Cheng ◽  
Bingbing Wang ◽  
Haibo Guan
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Regulatory Mechanism ◽  
Luciferase Assay ◽  
Pcr Analysis ◽  
Western Blot Assay ◽  
Qrt Pcr ◽  
Brdu Assay ◽  
Rip Assay

Abstract Background: LncRNA HOXA-AS2 has been found in the literature to deteriorate glioblastoma. However, its regulatory mechanism is yet to be fully investigated. Our study focused chiefly on the interaction and role of the HOXA-AS2/miR-885-5p/RBBP4 axis in the development of glioblastoma. Methods: qRT-PCR analysis was performed to detect the expression of lncRNA, miRNA and mRNA in glioblastoma tissues and cells. Dual-luciferase assay, RIP assay and RNA pull-down assay were later carried out to reveal the interactions among HOXA-AS2, miR-885-5p and RBBP4. After that, CCK-8 assay, BrdU assay, nude mice xenografting assay, western blot assay, and flow cytometry were carried out to analyze the effect of the HOXA-AS2/miR-885-5p/RBBP4 axis on glioblastoma samples. Results: HOXA-AS2 and RBBP4 were found to be overexpressed in glioblastoma. Experimental results showed that HOXA-AS2 and RBBP4 contributed to the tumorigenesis of glioblastoma cells. However, miR-885-5p was observed to be downregulated in glioblastoma. Findings also indicated that HOXA-AS2 could negatively regulate miR-885-5p, thereby enhancing RBBP4 expression. Conclusion: Overall, HOXA-AS2 promoted the tumorigenesis of glioblastoma by targeting and regulating miR-885-5p to induce the expression of RBBP4.

scholarly journals EIF4A3-Mediated CircRNA_100290 Promotes GC Cell Proliferation, Invasion and EMT via miR-29b-3p/ITGA11 Axis

2020 ◽  
Author(s):  
Gang Wang ◽  
Dan Sun ◽  
Wenhui Li ◽  
Yan Xin
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Qrt Pcr ◽  
Node Metastasis ◽  
Dual Luciferase Reporter Assay

Abstract Background Circular RNA (circRNA) has been reported as an important regulator in the development and progression of various carcinomas. However, the role of circRNA_100290 in gastric cancer (GC) is still unclear. This study aimed to investigate the role of circRNA_100290 in GC invasion and metastasis and its possible mechanism.Methods The expression of circRNA_100290 in GC cells and tissues were examined using quantitative real-time polymerase chain reaction (qRT-PCR). The role of circRNA_100290 in cell proliferation, migration, and invasion was evaluated on AGS and HGC-27 cell lines in vitro. Bioinformatics tools, dual-luciferase reporter assay, Western blot assay and qRT-PCR were used to explore the downstream pathways of circRNA_100290. The mechanism underlying the regulation of the expression of circRNA_100290 was explored using RNA immunoprecipitation, qRT-PCR, and Western blot assays.Results The expression of circRNA_100290 was found significantly upregulated in GC cells and 102 GC tissues, high expression of circRNA_100290 in GC was closely related to Borrmann’s types, lymph node metastasis and tumor-node-metastasis staging. In vitro, knockdown of circRNA_100290 in AGS and HGC-27 cells significantly inhibited cell proliferation, migration, and invasion. Mechanistically, dual-luciferase reporter assay confirmed a direct binding between circRNA_100290 and miR-29b-3p, which targets ITGA11, an oncogene which is closely related to epithelial–mesenchymal transition (EMT). In addition, EIF4A3, one of RNA binding proteins (RBPs), could inhibit the formation of circRNA_100290 via enriching flanking sites of circRNA_100290. Low expression of EIF4A3 in GC was related to a worse prognosis.Conclusions Elevated circRNA_100290 in GC promotes cell proliferation, invasion and EMT via miR-29b-3p/ITGA11 axi and might be regulated by EIF4A3. CircRNA_100290 might be a promising biomarker and target for GC therapy.

scholarly journals miR-103 promotes the progression of non-Hodgkins lymphoma by inhibiting OTUD7B expression

2020 ◽  
Author(s):  
Chong Wang ◽  
Yating Wu ◽  
Mengya Li ◽  
Shujuan Wang ◽  
Yanfang Liu
Keyword(s):  
Western Blot ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Normal Tissues ◽  
Qrt Pcr ◽  
Non Hodgkin Lymphoma ◽  
Reporter Assays ◽  
Non Hodgkins Lymphoma ◽  
Polymerase Chain

Abstract Background MicroRNAs (miRNAs) are vital for regulating the malignant phenotypes of tumor cells. The purpose of this work is to investigate the function and downstream mechanism of miR-103 in the progression of non-Hodgkin lymphoma (NHL). Methods and Materials Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect miR-103 and OTU deubiquitinase 7B (OTUD7B) mRNA expressions in NHL tissues and cells. Immunohistochemistry and Western blot were used to detect the expression of OTUD7B in NHL tissues and cells. CCK-8 experiment, flow cytometry analysis, and Transwell experiment were used to detect the role of NHL cell proliferation, apoptosis, migration and invasion. Bioinformatics, qRT-PCR, Western blot and dual-luciferase reporter assays were used to validate the targeting relationship between miR-103 and OTUD7B. NF-κB p65 luciferase reporter assay and Western blot were applied to determine NF-κB activity and the expression of NF-κB targeted genes. Results Compared to normal tissues and cells, miR-103 expression levels were remarkably up-regulated in NHL tissues and cell lines. The up-regulation of miR-103 dramatically promoted the proliferation, migration and invasion of NHL cells and inhibited apoptosis. Conversely, down-regulating miR-103 significantly inhibited malignant phenotypes of the NHL cells. Additionally, OTUD7B was identified as a target gene of miR-103, and miR-103 increased NF-κB activity indirectly via repressing OTUD7B. Conclusion The miR-103/OTUD7B/NF-κB axis is involved in NHL progression.

scholarly journals MiR-30e-5p inhibits the migration and invasion of nasopharyngeal carcinoma via regulating the expression of MTA1

2020 ◽  
Vol 40 (5) ◽  
Author(s):  
Weiqun Hu ◽  
Wenfeng Yao ◽  
Haolin Li ◽  
Li Chen
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Western Blot ◽  
Cell Lines ◽  
Metastatic Cancer ◽  
Scratch Test ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Qrt Pcr ◽  
Related Proteins ◽  
The Relationship

Abstract The study explored the effect of miR-30e-5p on nasopharyngeal carcinoma (NPC). MiR-30e-5p levels in NPC cancer and adjacent normal samples, in metastatic and non-metastatic cancer samples of NPC, and in NP69 cell and five NPC cell lines were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between miR-30e-5p and MTA1 was confirmed by dual-luciferase reporter assay, Western blot and qRT-PCR. The viability, migration and invasion of 5-8F and 6-10B cells were determined by CCK-8, scratch test and transwell assays, respectively. The levels of migration-related proteins (vimentin and Snail) and invasion-related proteins (MMP2 and MMP3) in NPC cells were detected by Western blot. The results showed that low expression of miR-30e-5p was associated with HNSC cancer, NPC, metastasis of NPC and NPC cell lines. Overexpressed miR-30e-5p in HNSC cancer and NPC was predictive of a better prognosis of patients. In addition, the viability, migration and invasion were reduced by up-regulating miR-30e-5p in 5-8F cells, but promoted by down-regulated miR-30e-5p in 6-10B cells. MiR-30e-5p reversed the migration and invasion of NPC cells regulated by MTA1, and inhibited migration and invasion of NPC cells via regulating MTA1 expression.

scholarly journals HIF-1α and HIF-2α Mediated PARVB Expression Promotes Tumor Growth and Metastasis in Malignantmelanoma

2020 ◽  
Author(s):  
Ting Wang ◽  
Zhiqiang Wu ◽  
Yifeng Bi ◽  
Haitao Sun ◽  
Zhipeng Wu ◽  
...  
Keyword(s):  
Malignant Melanoma ◽  
Tumor Growth ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Human Malignant Melanoma ◽  
Qrt Pcr ◽  
Tumor Growth And Metastasis

Abstract BackgroundMalignant melanoma is the leading cause of skin cancer-related death. The role of PARVB in malignant melanoma remains unclear. Hypoxia is a hallmark of solid tumors including melanoma. But the regulation role of hypoxia in PARVB expression has not been reported.MethodsHuman malignant melanoma tissues, cell lines and their controls were collected. IHC staining, qRT-PCR and Western blot were performed to reveal the differential PARVB expression. The role of PARVB in tumor growth and metastasis of malignant melanoma was evaluated in vitro and in vivo. The regulation role and mechanism of hypoxia and HIFs in PARVB expression was validated by qRT-PCR, Western blot, ChIP-PCR and Luciferase reporter assays.ResultsPARVB was upregulated in malignant melanoma and correlated with patient survival. OverexpressionofPARVB promoted tumor growth and metastasis of malignant melanoma. Furthermore, hypoxia induced HIF-1α and HIF-2α expression activated PARVB transcription and expression through binding to the specific hypoxia-responsive element (HRE) in the promoter region of PARVB.ConclusionsIn malignant melanoma, Hypoxia induced HIF-1α and HIF-2α expression could directly activate PARVB expression, which further promoted tumor growth and metastasis, inducing poor prognosis. These results indicated that PARVB might be a potential therapeutic target for malignant melanoma.

scholarly journals Circ_001209 aggravates diabetic retinal vascular dysfunction through regulating miR-15b-5p/COL12A1

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Fang Wang ◽  
Meixia Zhang
Keyword(s):  
Diabetic Retinopathy ◽  
Western Blot ◽  
Vascular Endothelial Cells ◽  
Vascular Dysfunction ◽  
Tube Formation ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Tunel Staining ◽  
Western Blot Assay ◽  
Qrt Pcr

Abstract Objective Diabetic retinopathy, a common complication of diabetes mellitus and a major cause of blindness. circRNAs spongs target miRNA and thus influencing mRNA expression in DR. We investigated the mechanism of circ_001209 in regulating diabetic retinal vascular dysfunction. Methods QRT-PCR analysis was performed to detect the expression of miR-15b-5p, COL12A1 and circ_001209 in human retinal vascular endothelial cells (HRVECs) under high glucose conditions. Western blot assay, wound healing assay, transwell assay and tube formation were used to explore the roles of circ_001209/miR-15b-5p/COL12A1 in retinal vascular dysfunction. Bioinformatics analysis and luciferase reporter, RNA-FISH, and overexpression assays were performed to reveal the mechanisms of the circ_001209/miR-15b-5p/COL12A1 interaction. TUNEL staining and H&E staining were used to evaluate the pathological changes in streptozotocin (STZ)-induced DR in rats. Results Downregulation of miR-15b-5p under HG conditions promoted proliferation, migration, and tube formation of HRVECs. QRT-PCR and western blot results revealed that miR-15b-5p affected the HRVECs function through targeting COL12A1. Under HG conditions, circ_001209, which acts as a sponge of miR-15b-5p, is upregulated. Besides, overexpression of circ_001209 can affect HRVEC function and aggravate retinal injury in diabetic rats. Conclusion Upregulation of circ_001209 contributes to vascular dysfunction in diabetic retinas through regulating miR-15b-5p and COL12A1, providing a potential treatment strategy for diabetic retinopathy.

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