MiR 206 inhibits reorganization of the cytoskeleton in melanoma cells by targeting DDX5

Purpose: To investigate the role and mechanism of microRNA-206 (miR-206) in cytoskeleton reorganization in melanoma cells. Methods: MiR-206 and RNA helicase p68 (DDX5) expression levels were measured in A375, A875, and HEM-M cells by quantitative real time polymerase chain reaction (qRT-PCR). A DDX5 overexpression cell line was constructed, and DDX5 overexpression, A375, and A875 cells were transfected with miR-206 mimic or DDX5 small interfering RNA (siRNA). Transwell assay was used to assess cell migration and invasion of A375 and A875 cells, while Luciferase reporter assay was used to determine the putative target of miR-206. DDX5, miR-206, vinculin, coronin3, and ezrin expression levels were evaluated by qRT-PCR. Protein expressions of DDX5, vinculin, coronin3, and ezrin were evaluated by western blot analysis. Results: DDX5 expression was higher and miR-206 expression lower in A375 and A875 cells when compared to HEM-M cells (p < 0.05). Knockdown of DDX5 and overexpression of miR-206 repressed invasion and migration, and inhibited expression of vinculin, coronin3, and ezrin in A375 and A875 cells (p < 0.05). However, overexpression of DDX5 reversed the effect of miR-206 on cytoskeletal protein expression. Luciferase reporter assay data confirmed that DDX5 is a direct target of miR-206 (p < 0.05). Conclusion: MiR-206 suppresses reorganization of the cytoskeleton in melanoma cells by targeting DDX5, and is thus, a promising target for the treatment of melanoma.

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MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

Technology in Cancer Research & Treatment ◽

10.1177/1533033820985868 ◽

2021 ◽

Vol 20 ◽

pp. 153303382098586

Author(s):

Xuhui Wu ◽

Gongzhi Wu ◽

Huaizhong Zhang ◽

Xuyang Peng ◽

Bin Huang ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Cell Invasion ◽

Target Gene ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Reporter Assay ◽

Invasion And Migration ◽

And Migration ◽

Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

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MiR-18a-5p Promotes NPC Cell Proliferation, Invasion, Migration, and EMT by Targeting SMAD2

10.21203/rs.3.rs-36707/v1 ◽

2020 ◽

Author(s):

Pengcheng Li ◽

Junhui Xing ◽

Jianwu Jiang ◽

Xinyu Tian ◽

Xuemeng Liu ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Invasion And Migration ◽

Metastasis Model ◽

And Migration ◽

Dual Luciferase Reporter Assay

Abstract Background: Nasopharyngeal carcinoma (NPC) is the most common malignant tumor in the head and neck that is characterized by high local malignant invasion and distant metastasis. miR-18a-5p reportedly plays an important role in tumorigenesis and development. However, little is known about the mechanism underlying miR-18a-5p’s role in NPC.Methods：Quantitative real-time PCR was used to detect the expression of miR-18a-5p in NPC tissues and cell lines. MTT assay and plate clone formation assay were used to detect the effect of miR-18a-5p on NPC cell proliferation. Woundhealing assays and Transwell assays were used to detect the effect of miR-18a-5p on NPC cell invasion and migration. The expressions of epithelialmesenchymal transition (EMT)-related proteins N-cadherin, Vimentin, and E-cadherin were detected by Westernblot. Bioinformatics and dual-luciferase reporter assay were used to detect the targeting interaction between miR-18a-5p and SMAD2. Xenotransplantation and metastasis model were used to detect the effect of miR-18a-5p on NPC growth and metastasis in vivo.Results:miR-18a-5p was highly expressed in NPC tissues and cell lines. Overexpression of miR-18a-5p promotedNPC cell proliferation, invasion, migration, and EMT process, whereas inhibition of miR-18a-5p expression led to the oppositeresults. Results of dual-luciferase reporter assay showed that SMAD2 was the target gene of miR-18a-5p, and SMAD2 could reverse the effect of miR-18a-5p on NPC cell line. Xenotransplantation and metastasis model experiments in nude mice showed that miR-18a-5p promotesNPC growth and metastasis in vivo.Conclusions:Targeting SMAD2 downregulated miR-18a-5p expression, thereby promoting NPC cell proliferation, invasion, migration, and EMT.

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Circular RNA hsa_circ_0004872 inhibits gastric cancer progression via the miR-224/Smad4/ADAR1 successive regulatory circuit

Molecular Cancer ◽

10.1186/s12943-020-01268-5 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Cunying Ma ◽

Xiaoying Wang ◽

Fenghua Yang ◽

Yichen Zang ◽

Jiansong Liu ◽

...

Keyword(s):

Gastric Cancer ◽

Nude Mice ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Invasion And Migration ◽

Potential Biomarker ◽

And Migration

Abstract Background Emerging evidence has shown that circular RNAs (circRNAs) play a crucial regulatory role in the occurrence and development of cancer. Exploring the roles and mechanisms of circRNAs in tumorigenesis and progression may help to identify new diagnostic markers and therapeutic targets. In the present study, we investigated the role and regulatory mechanism of hsa_circ_0004872 in gastric cancer (GC). Methods qRT-PCR was used to determine the expression of hsa_circ_0004872 in GC tissues and cells. EdU, CCK-8, transwell and scratch wound healing assays were used to assess the role of hsa_circ_0004872 in GC cell proliferation, invasion and migration, respectively. Subcutaneous and tail vein tumor injections in nude mice were used to assess the role of hsa_circ_0004872 in vivo. RIP assay, biotin-coupled probe pull-down assay, FISH and luciferase reporter assay were performed to confirm the relationship between hsa_circ_0004872 and the identified miRNA. ChIP assay, luciferase reporter assay and western blot were used to determine the direct binding of Smad4 to the promoter of the ADAR1 gene. Results In this study, we found that hsa_circ_0004872 was dramatically downregulated in GC tissues compared with adjacent noncancerous tissues. The expression level of hsa_circ_0004872 was associated with tumor size and local lymph node metastasis. Enforced expression of hsa_circ_0004872 inhibited the proliferation, invasion and migration of GC cells, whereas knockdown of hsa_circ_0004872 had the opposite effects. Nude mice experiments showed that ectopic expression of hsa_circ_0004872 dramatically inhibited tumor growth and metastasis in vivo. Moreover, we demonstrated that hsa_circ_0004872 acted as a “molecular sponge” for miR-224 to upregulate the expression of the miR-224 downstream targets p21 and Smad4. Importantly, we found that the RNA-editing enzyme ADAR1 inhibited hsa_circ_0004872 expression and further led to the upregulation of miR-224. Smad4, the downstream target of miR-224, could further affect hsa_circ_0004872 levels by directly binding to the promoter region of ADAR1 to inhibit ADAR1 expression. Conclusions Our findings showed that hsa_circ_0004872 acted as a tumor suppressor in GC by forming a negative regulatory loop consisting of hsa_circ_0004872/miR-224/Smad4/ADAR1. Thus, hsa_circ_0004872 may serve as a potential biomarker and therapeutic target for GC.

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MicroRNA-214-5p Inhibits the Invasion and Migration of Hepatocellular Carcinoma Cells by Targeting Wiskott-Aldrich Syndrome Like

Cellular Physiology and Biochemistry ◽

10.1159/000488734 ◽

2018 ◽

Vol 46 (2) ◽

pp. 757-764 ◽

Cited By ~ 12

Author(s):

Hongdan Li ◽

Haoqi Wang ◽

Zhen Ren

Keyword(s):

Hepatocellular Carcinoma ◽

Carcinoma Cells ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Hepatocellular Carcinoma Cells ◽

Invasion And Migration ◽

Wiskott Aldrich Syndrome ◽

Normal Tissues ◽

Qrt Pcr ◽

And Migration

Background/Aims: This study aims to explore the effects of microRNA-214-5p (miR-214-5p) on the invasion and migration of Hepatocellular Carcinoma cells (HCC). Methods: Hepatocellular Carcinoma tissues and adjacent normal tissues from 44 hepatocellular carcinoma patients were prepared for this study. The HepG2 and BEL-7402 cells were transfected with miR-214-5p mimic and inhibitor. qRT-PCR was performed to detect the expressions of miR-214-5p. Transwell assays were used to detect the invasion and migration assays in HepG2 and BEL-7402 cells. A dual-luciferase reporter assay was conducted to examine the effect of miR-214-5p on Wiskott-Aldrich Syndrome Like (WASL/ N-WASP). Western blot and qRT-PCR were used to measure the expressions of the E-cadherin, N-cadherin and Vimentin proteins. Transwell chamber assays were performed to detect cell invasion and migration. Results: Compared with normal tissues, HCC tissues demonstrated significantly lower expression of miR-214-5p. Overexpression of miR-214-5p significantly inhibited the migration and invasion of HCC cells and inhibition of miR-214-5p promoted the migration and invasion. Additionally, miR-214-5p suppressed the epithelial-mesenchymal transition (EMT). Further study showed WASL was a putative target gene of miR-214-5p. Up-regulating the expression of WASL could reverse the inhibition effect of miR-214-5p on invasion and migration. Conclusion: Our data suggested that miR-214-5p inhibited the invasion and migration of HepG2 and BEL-7402 by targeting WASL in Hepatocellular carcinoma.

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Long non-coding RNA HOTTIP promotes prostate cancer cells proliferation and migration by sponging miR-216a-5p

Bioscience Reports ◽

10.1042/bsr20180566 ◽

2018 ◽

Vol 38 (5) ◽

Cited By ~ 13

Author(s):

Bin Yang ◽

Ge Gao ◽

Zhixin Wang ◽

Daju Sun ◽

Xin Wei ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Proliferation ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Reporter Assay ◽

Online Programs ◽

Real Time Quantitative Pcr ◽

And Migration ◽

And Function

Long non-coding RNAs (lncRNAs) are a class of ncRNAs with >200 nts in length that regulate gene expression. The HOXA transcript at the distal tip (HOTTIP) lncRNA plays an important role in carcinogenesis, however, the underlying role of HOTTIP in prostate cancer (PCa) remains unknown. The aim of the present study was to evaluate the expression and function of HOTTIP in PCa. In the present study, we analyzed HOTTIP expression levels of 86 PCa patients in tumor and adjacent normal tissue by real-time quantitative PCR (qPCR). Knockdown or overexpression of HOTTIP was performed to explore its roles in cell proliferation, migration, invasion, and cell cycle. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOTTIP and miR-216a-5p in PCa cells. Our results found that HOTTIP was up-regulated in human primary PCa tissues with lymph node metastasis. Knockdown of HOTTIP inhibited PCa cell proliferation, migration, and invasion. Overexpression of HOTTIP promoted cell proliferation, migration, and invasion of PCa cells. Bioinformatics online programs predicted that HOTTIP sponge miR-216a-5p at 3′-UTR with complementary binding sites, which was validated using luciferase reporter assay. HOTTIP could negatively regulate the expression of miR-216a-5p in PCa cells. Above all, the knockdown of HOTTIP could represent a rational therapeutic strategy for PCa.

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MicroRNA-449b-5p suppresses cell proliferation, migration and invasion by targeting TPD52 in nasopharyngeal carcinoma

The Journal of Biochemistry ◽

10.1093/jb/mvz057 ◽

2019 ◽

Vol 166 (5) ◽

pp. 433-440 ◽

Cited By ~ 6

Author(s):

Wei Yin ◽

Lei Shi ◽

Yanjiao Mao

Keyword(s):

Cell Proliferation ◽

Nasopharyngeal Carcinoma ◽

Cell Lines ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Reporter Assay ◽

Western Blot Assay ◽

Qrt Pcr ◽

Dual Luciferase Reporter Assay

Abstract Nasopharyngeal carcinoma (NPC) is an important type of head and neck malignant cancer with geographical distribution. MicroRNA-449b-5p (miR-449b-5p) is related to the development of various cancers, while its function in NPC remains unknown. The present study aimed to investigate the role and target gene of miR-449b-5p in NPC. Expressions of miR-449b-5p in NPC cell lines and clinical tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was determined by MTT and colony formation assays. Migration and invasion abilities after different treatment were evaluated by wound healing and Transwell assays, respectively. Dual-luciferase reporter assay was performed to explore the relationship between miR-449b-5p and tumour protein D52 (TPD52). TPD52 expression was determined by qRT-PCR and western blot assay. miR-449b-5p was significantly downregulated in NPC cell lines and clinical tissues than the matched control. Overexpression of miR-449b-5p inhibited proliferation, migration and invasion of NPC cells. Dual-luciferase reporter assay indicated that miR-449b-5p directly targeted TPD52. Furthermore, shRNA-mediated downregulation of TPD52 rectified the promotion of cell migration and invasion by miR-449b-5p inhibition. In conclusion, the present study suggests that miR-449b-5p, as a novel tumour-suppressive miRNA against NPC, inhibits proliferation, migration and invasion of NPC cells via inhibiting TPD52 expression.

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Long non-coding RNA MCM3AP-AS1 promotes growth and migration through modulating FOXK1 by sponging miR-138-5p in pancreatic cancer

Molecular Medicine ◽

10.1186/s10020-019-0121-2 ◽

2019 ◽

Vol 25 (1) ◽

Cited By ~ 4

Author(s):

Ming Yang ◽

Shijuan Sun ◽

Yao Guo ◽

Junjie Qin ◽

Guangming Liu

Keyword(s):

Pancreatic Cancer ◽

Suppressive Effect ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Reporter Assay ◽

Non Coding Rna ◽

And Migration ◽

Long Non Coding Rna ◽

Dual Luciferase Reporter Assay

Abstract Background Pancreatic cancer (PC) is a type of malignant gastrointestinal tumor. Long non-coding RNA MCM3AP antisense RNA 1 (MCM3AP-AS1) has been reported to stimulate proliferation, migration and invasion in several types of tumors. However, the role of MCM3AP-AS1 in PC remains unclear. Methods MCM3AP-AS1, microRNA miR-138-5p (miR-138-5p) and FOXK1 levels were detected using quantitative real time PCR. Cell proliferation, migration and invasion were analyzed. Dual luciferase reporter assay was used to confirm the relationship between MCM3AP-AS1 and miR-138-5p, between miR-138-5p and FOXK1. Protein levels were identified using western blot analysis. Results MCM3AP-AS1 overexpression promoted proliferation, migration and invasion in PC cells. MCM3AP-AS1 silencing showed a suppressive effect on cell growth in PC cells. Moreover, MCM3AP-AS1 knockdown suppressed tumor growth in mice. Dual luciferase reporter assay demonstrated MCM3AP-AS1 could sponge microRNA-138-5p (miR-138-5p), and FOXK1 could bind with miR-138-5p. Positive correlation between MCM3AP-AS1 and FOXK1 was testified, as well as negative correlation between miR-138-5p and FOXK1. MCM3AP-AS1 promoted FOXK1 expression by targeting miR-138-5p, and MCM3AP-AS1 facilitated growth and invasion in PC cells by FOXK1. Conclusion MCM3AP-AS1 promoted growth and migration through modulating miR-138-5p/FOXK1 axis in PC, providing insights into MCM3AP-AS1/miR-138-5p/FOXK1 axis as novel candidates for PC therapy from bench to clinic.

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MicroRNA-450a/PPM1L Axis Contributes to The Malignant Phenotype of Glioma

10.21203/rs.3.rs-100631/v1 ◽

2020 ◽

Author(s):

Jian You ◽

Xiufen Chen ◽

Jie Zhou ◽

Lilei Peng ◽

Yang Ming ◽

...

Keyword(s):

Glioma Cells ◽

Tumor Formation ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Malignant Phenotype ◽

Invasion And Migration ◽

Direct Target ◽

And Migration

Abstract MicroRNAs play an important role in glioma, and the function of miR-450a in glioma is unknown. We aimed to clarify the role and underlying mechanism of miR-450a in glioma. We analyzed the miR-450a expression and prognosis data of glioma patients from TCGA and CGGA databases. CCK8, colony formation, nude tumor formation, transwell and wound healing assay investigated glioma cells transfected with miR-450a inhibitor growth, invasion and migration, respectively. Bioinformatics and luciferase reporter assay were applied to predict and verify the direct target gene of miR-450a. In this study, we found that miR-450a expression was significantly higher in glioma tissues and cells than the normal tissues and cells, and there is a positive correlation between miR-450a expression and histopathological grade of gliomas. In addition, the glioma patients with high miR-450a expression exhibited poorer prognosis. Downregulation of miR-450a remarkedly suppressed glioma cells growth in vitro and vivo, and inhibited glioma cells invasion and migration. Luciferase reporter assay verified that PPMIL is a direct target of miR-450a. PPM1L silencing partially rescued miR-450a knockdown-induced suppressive effects on glioma cells. Therefore, our data demonstrated that miR-450a/PPM1L axis could mediate the malignant phenotype of glioma, which provided a feasible target for glioma therapy.

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Silencing of miR-200a-3p Inhibits Proliferation, Invasion and Migration of Breast Cancer Cells by Targeting Ephrin-A5

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2690 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1227-1235

Author(s):

Yongmei Zhang ◽

Huayi Zhang ◽

Gang Guo

Keyword(s):

Breast Cancer ◽

Cell Proliferation ◽

Cell Lines ◽

The Cancer Genome Atlas ◽

Luciferase Reporter Assay ◽

Cell Counting ◽

Luciferase Reporter ◽

Reporter Assay ◽

Invasion And Migration ◽

And Migration

Increasing evidence suggests microRNAs (miRs/miRNAs) exert considerable functions in the pathogenesis of malignancies, including breast cancer (BC). The miR-200a-3p has previously been reported to promote tumorigenesis in different types of cancers. The present study aimed to investigate the potential role of and possible mechanisms of miR-200a-3p in BC. In this study miR-200a-3p and ephrin-A5 (EFNA5) expression in tissues of patients with BC was analyzed using The Cancer Genome Atlas (TCGA) database. And several BC cell lines were employed to determine the expression levels of miR-200a-3p and EFNA5. Then, miR-200a-3p expression was silenced by transfection with miR-200a-3p inhibitor. Cell proliferation was evaluated using a cell counting kit-8 kit and colony formation assay, whilst cell invasion and migration were detected using Transwell and wound healing assays, respectively. Then, the potential interaction between miR-200a-3p and EFNA5 was verified using luciferase reporter assay. Subsequently, rescue assays were conducted by co-transfection with miR-200a-3p inhibitor and short hairpin RNA (shRNA) targeted against EFNA5 (shRNA-EFNA5) to study the effects of TTN-AS1 and miR-211-5p on BC development. Results indicated that miR-200a-3p expression was significantly upregulated while EFNA5 was notably downregulated in BC tissues and cell lines. Cells transfected with miR-200a-3p inhibitor presented lower abilities of cell proliferation, invasion and migration. Moreover, the luciferase reporter assay confirmed that EFNA5 was a direct target of miR-200a-3p. And EFNA5 silencing reversed the inhibitory effects of miR-200a-3p inhibitor on proliferation, invasion and migration of BC cells. Taken together, these findings revealed that miR-200a-3p silencing inhibits proliferation, invasion and migration of BC cells by targeting EFNA5, which provides insights into the regulatory mechanism of BC and new strategies for developing therapeutic interventions for this disease.

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Up-regulation of miR-1825 inhibits the progression of glioblastoma by suppressing CDK14 though Wnt/β-catenin signaling pathway

10.21203/rs.3.rs-18711/v1 ◽

2020 ◽

Author(s):

Fengqin Lu ◽

Chunhong Li ◽

Yuping Sun ◽

Ting Jia ◽

Na Li ◽

...

Keyword(s):

Signaling Pathway ◽

Luciferase Reporter ◽

Reporter Assay ◽

Potential Mechanism ◽

Transwell Assay ◽

Invasion And Migration ◽

Qrt Pcr ◽

Kaplan Meier ◽

And Migration ◽

Tissue Specimens

Abstract Mounting evidences displayed that miRNAs modulated glioblastoma (GBM) development. Here, our goal was to investigate whether miR-1825 could regulate GBM development and explore its potential mechanism. Our findings displayed that miR-1825 was decreased in GBM tissue specimens by qRT-PCR and it confirmed as a prognostic marker of GBM by Kaplan-Meier survival analysis. Moreover, we also found that miR-1825 up-regulation suppressed GBM cell viability, tumor growth, invasion and migration by MTT assay, Xenograft assay and Transwell assay. Furthermore, CDK14 was first identified as the target of miR-1825 by Luciferase reporter assay and CDK14 acted as an oncogene in GBM development by Immunohistochemistry. In addition, Western blot analysis demonstrated that Wnt/β-catenin signaling pathway took part in GBM development modulating by miR-1825. In conclusion, miR-1825 up-regulation suppressed GBM progression by targeting CDK14 through Wnt/β-catenin pathway.

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