scholarly journals SOX2 knockdown slows cholangiocarcinoma progression through inhibition of transcriptional activation of lncRNA PVT1

2020 ◽  
Vol 477 (18) ◽  
pp. 3527-3540
Author(s):  
Aijun Yu ◽  
Luwen Zhao ◽  
Qingmin Kang ◽  
Jian Li ◽  
Kai Chen ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
High Rate ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Loss Of Function ◽  
Luciferase Reporter Gene ◽  
Nude Mouse Model ◽  
Mortality And Morbidity ◽  
Gene Assay

Cholangiocarcinoma (CCA) has accounted for a high rate of mortality and morbidity in the recent years. Long non-coding RNAs (lncRNAs) play an important role in different cellular environments, including cancer. As such, they have been used as potential targets during CCA therapy. The objective of this study was to investigate the effects of lncRNA PVT1 on CCA and its mechanisms behind lncRNA PVT1 regulation. The interactions among SOX2, lncRNA PVT1, miR-186 and SEMA4D were verified by chromatin immunoprecipitation, RNA immunoprecipitation and dual luciferase reporter gene assay. Gain- and loss-of-function experiments were conducted to explore the modulatory effects of SOX2, lncRNA PVT1, miR-186 and SEMA4D on cell viability, migration and invasion of CCA by CCK-8 and Transwell assays. In vivo effects of lncRNA PVT1 or SEMA4D were studied in a nude mouse model. MiR-186 was poorly expressed while SOX2, lncRNA PVT1 and SEMA4D were highly expressed in CCA cells. SOX2 induced the transcriptional activation of lncRNA PVT1 expression to promote proliferation, migration and invasion of CCA cells. LncRNA PVT1 bound to miR-186 and miR-186 was found to target SEMA4D. The overexpression of lncRNA PVT1 and SEMA4D, as well as the inhibition of miR-186 led to elevated CCA cell proliferation, migration and invasion. In vivo experiments confirmed the inhibitory role of lncRNA PVT1 knockdown or SEMA4D knockdown in CCA. All in all, SOX2 down-regulated miR-186 through the transcriptional activation of lncRNA PVT1, whereas elevating SEMA4D expression, thus promoting the progression of CCA.

scholarly journals Identification of the ASPM-miR-26b-5p network associated with the aggressive traits of HCC cells

2020 ◽  
Author(s):  
Hong-Guang Li ◽  
Heng-Jun Gao ◽  
Fang-Feng Liu ◽  
Jun Liu
Keyword(s):  
Tumor Model ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Loss Of Function ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay ◽  
Hcc Cells

Abstract Background: Even though earlier reports have revealed that abnormal spindle-like microcephaly associated (ASPM) exert essential roles in diverse malignancies, its relationship between specific microRNAs (miRNAs) in regulation of hepatocellular carcinoma (HCC) progression has never been elaborated. Methods: Bioinformatics analysis detected differentially expressed genes in HCC and normal. qRT-PCR was performed to detect expression of miR-26b-5p in HCC tissues and cells. HCC cells were transfected with plasmids and their proliferative ability and colony formation were detected with loss-of-function assay. The invasion of HCC cells was determined using Transwell assay. The expression of ASPM was detected by western blotting. Luciferase reporter gene assay was performed to detect the interaction between miR-26b-5p and ASPM. ASMP silencing cells were injected into mice to establish xenograft tumor model.Results: Herein, we proved that ASPM was upregulated in HCC and higher level of ASPM was significantly associated with worse survival in HCC patients. ASPM silencing restrained HCC cell proliferation, migration and invasion capacities in vitro. In vivo, downregulation of ASPM also suppressed HCC cells growth. Mechanistic analyses illustrated that ASPM was a directly target of miR-26b-5p. The expression of ASPM was negatively modulated by miR-26b-5p. Rescues assays displayed that miR-26b-5p inhibited HCC cells growth and invasion via modulating the expression of ASPM. Conclusions: Our work validated that miR-26b-5p restrained the aggressiveness of HCC cells through targeting ASPM.

scholarly journals Hsa_circ_0013401 Accelerates the Growth, Metastasis and Prevents Apoptosis Autophagy of Neuroblastoma Cells by Sponging miR-195 to Release PAK2 

2021 ◽  
Author(s):  
Shibo Zhu ◽  
Xiangliang Tang ◽  
Xiaofeng Gao ◽  
Jingqi Zhang ◽  
Yanhong Cui ◽  
...  
Keyword(s):  
Neuroblastoma Cells ◽  
Circular Rna ◽  
Biological Properties ◽  
Tumor Formation ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Loss Of Function ◽  
Luciferase Reporter Gene ◽  
Gene Assay

Abstract Background: Circular RNA (circRNA) is a newly discovered non-coding RNA with a covalently closed loop structure. Recently, the increased circRNAs have been identified in a variety of cancers. While, the specific functions and mechanisms of some circRNAs in neuroblastoma (NB) are still largely unexplored.Materials and Methods: The levels of hsa_circ_0045997, hsa_circ_0080307, hsa_circ_0013401, hsa_circ_0077578 and microRNA-195 (miR-195) were confirmed by RT-qPCR assay in NB. Functionally, gain- and loss-of-function assays and the rescued experiment were conducted to determine the influences of hsa_circ_0013401, miR-195 and P21-activated kinase 2 (PAK2) on the proliferation, apoptosis, autophagy, migration and invasion of NB cells. Luciferase reporter gene assay was also applied to examine the relationships between hsa_circ_0013401 and miR-195, miR-195 and PAK2. Hsa_circ_0013401/miR-195/PAK2 axis was also identified by in vivo experiment study.Results: Hsa_circ_0013401 was highly expressed, miR-195 was lowly expressed, and there was a negative correlation between hsa_circ_0013401 and miR-195 in NB. Hsa_circ_0013401 significantly suppressed the proliferation, migration and invasion, and induced apoptosis and autophagy of NB. In mechanism, miR-195 was a direct target of hsa_circ_0013401, and PAK2 was the downstream target gene of miR-195. And the inhibitory effects of hsa_circ_0013401 knockdown on the malignant biological properties of NB can be achieved by targeting miR-195 to upregulated PAK2. Besides, in vivo evidences also revealed that hsa_circ_0013401 promotes tumor formation, and regulated miR-195 and PAK2. Conclusions: Hsa_circ_0013401 induced NB progression through miR-195 to enhance PAK2. Therefore, we might highlight a novel regulatory axis (hsa_circ_0013401/miR-195/PAK2) in NB.

scholarly journals Tumor-Suppressive Role of microRNA-202-3p in Hepatocellular Carcinoma Through the KDM3A/HOXA1/MEIS3 Pathway

2021 ◽  
Vol 8 ◽  
Author(s):  
Yijie Zhang ◽  
Qi Pan ◽  
Zigong Shao
Keyword(s):  
Hepatocellular Carcinoma ◽  
Normal Liver ◽  
Global Scale ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Patient Prognosis ◽  
Hcc Cells

Hepatocellular carcinoma (HCC) represents a malignant tumor predominantly arising in the setting of cirrhosis and is the third most common cause of cancer-associated death on a global scale. The heterogeneous nature of HCC and limited well-recognized biomarkers may contribute to poor patient prognosis and treatment failure. In this study, we identified expression pattern of microRNA-202-3p (miR-202-3p) in HCC and characterized its functional role as well as related mechanisms. First, we collected 50 HCC tissues and 38 normal liver tissues, and after bioinformatics prediction, the expression of miR-202-3p and KDM3A was determined in the tissues. We found lowly expressed miR-202-3p and overexpressed KDM3A in HCC tissues. Then, dual-luciferase reporter gene assay was employed to test the presence of miR-202-3p binding sites in the 3’UTR of KDM3A and chromatin immunoprecipitation (ChIP) assay to homeobox A1 (HOXA1) interaction with KDM3A and MEIS3. It has been confirmed that miR-202-3p negatively regulated KDM3A responsible for increasing the expression of HOXA1 by eliminating the histone H3 lysine 9 (H3K9)me2 in HCC cells. HOXA1 could evidently increase H3K4me1 and H3K27ac enrichment in the MEIS3 enhancer region and enhance the expression of MEIS3. Functional assays were also performed with the results showing that upregulated miR-202-3p or downregulated KDM3A retarded HCC cell viability, migration, and invasion. In addition, HepG2 cells were xenografted into nude mice, and we demonstrated that upregulated miR-202-3p reduced the growth of human HCC cells in vivo. Taken together, the present study elicits a novel miR-202-3p/KDM3A/HOXA1/MEIS3 pathway in HCC, potentiating an exquisite therapeutic target for HCC.

scholarly journals MicroRNA-137-mediated inhibition of lysine-specific demethylase-1 prevents against rheumatoid arthritis in an association with the REST/mTOR axis

2021 ◽  
Vol 17 ◽  
pp. 174480692110418
Author(s):  
Wei Sun ◽  
Yijun Zhang ◽  
Guanghui Wang
Keyword(s):  
Rheumatoid Arthritis ◽  
Mtor Pathway ◽  
Cell Counting ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Lysine Specific Demethylase ◽  
Gene Assay

Background It has been increasingly reported that microRNAs (miRNAs) are related to rheumatoid arthritis (RA) pathogenesis. This present research was conducted to analyze the functions of miR-137 and the underlying molecular mechanism in RA progression. Methods Differentially expressed miRNAs in RA patients were analyzed using microarray-based analyses. Next, experiments involving miR-137 overexpression were performed to analyze the role of miR-137 in human fibroblast-like synoviocytes-RA (HFLS-RA) using cell counting kit-8 (CCK-8) assay, EdU staining, Transwell assay and flow cytometry, respectively. The function of miR-137 in inflammation was determined using ELISA. The binding relationship between miR-137 and LSD1 was confirmed by dual-luciferase reporter gene assay and ChIP test. Besides, a rat model with RA was established for in vivo experiments. Results miR-137 was downregulated in RA tissues and cells, which was negatively correlated with inflammatory factors. Upregulated miR-137 suppressed growth, migration and invasion of HFLS-RA, but promoted apoptosis. Lysine-specific demethylase-1 (LSD1) was a target of miR-137 and could be negatively regulated by miR-137. Moreover, LSD1 could activate REST through demethylation, while the REST/mTOR pathway induced levels of pro-inflammatory factors in RA. We observed the similar results in our in vivo study. Conclusion This study suggested that miR-137 reduced LSD1 expression to inhibit the activation of REST/mTOR pathway, thus preventing against inflammation and ameliorating RA development. Our research may offer new insights into treatment of RA.

scholarly journals Contribution of microRNA-30d to the prevention of the thyroid cancer progression: mechanism and implications

2020 ◽  
Author(s):  
Yuan Shao ◽  
Shaoqiang Zhang ◽  
Xiaoxia Wang ◽  
Xin Sun ◽  
Jie Wu ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Cancer Progression ◽  
Endocrine Tumor ◽  
Luciferase Reporter ◽  
Tunel Staining ◽  
Loss Of Function ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Low Expression

Abstract Background Thyroid cancer is a major endocrine tumor and represents an emerging health problem worldwide. MicroRNAs (miRNAs) have been addressed to be associated with the pathogenesis and progression of thyroid cancer. However, it remains largely unknown what functions miR-30d may exert on thyroid cancer. This study herein aimed to identify the functional significance and mechanism of miR-30d in the progression of thyroid cancer. Methods The expression of miR-30d and ubiquitin-specific protease 22 (USP22) in cancerous tissues of patients with thyroid cancer was measured using RT-qPCR and Western blot analysis. In response to the gain- or loss-of-function of miR-30d and USP22, cell apoptosis was evaluated by flow cytometry and TUNEL staining in combination with the measurement of apoptosis-related proteins. The interactions among miR-30d, USP22, SIRT1, FOXO3a and PUMA were explored using a series of assays, including dual-luciferase reporter gene assay, Co-IP and ChIP assay. The effects of miR-30d and USP22 on thyroid tumorigenesis were finally validated in vivo. Results MiR-30b presented aberrant low expression in thyroid cancer tissues and this low expression correlated with poor prognosis of thyroid cancer patients. miR-30d promoted apoptosis of thyroid cancer cells through targeting USP22, an up-regulated gene in thyroid cancer. USP22 could enhance the stability of SIRT1 by inducing deubiquitination which consequently contributed to FOXO3a deacetylation-induced PUMA repression. It was verified that this regulatory mechanism was responsible for the pro-apoptotic effect of miR-30d by the in vivo tumorigenicity assay. Conclusion To conclude, the progression of thyroid cancer can be suppressed by miR-30d-mediated inhibition of USP22, provides a promising therapeutic target for thyroid cancer treatment.

scholarly journals miR-122 Inhibits Hepatocarcinoma Cell Progression by Targeting LMNB2

2020 ◽  
Vol 28 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Xiao-Na Li ◽  
Hong Yang ◽  
Tao Yang
Keyword(s):  
Cell Proliferation ◽  
Scratch Test ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Promote Cell Proliferation ◽  
Hepatocarcinoma Cell ◽  
Gene Assay ◽  
Cell Progression

In the present study, we investigated the role of miR-122 in hepatocarcinoma progression and explored the mechanism. In hepatocarcinoma tissues and cells, we used qRT-PCR to validate the miR-122 expression level. Next, we used colony formation by crystal violet staining assay to compare cell proliferation ability, and we used scratch test or Transwell assay to compare cell migration or invasion ability. We then conducted bioinformatics or luciferase reporter gene assay to prove the regulation effect of miR-122 on lamin B2 (LMNB2), and the biological function of LMNB2 was analyzed. We used nude mouse tumorigenicity assay to test the inhibition effect of miR-122 ASO therapy against hepatocarcinoma. miR-122 was reduced in hepatocarcinoma tissues compared to the paracarcinoma tissues, which was relatively low or high in hepatocarcinoma cell line SMMC7721 or Hep3B, and overexpressed miR-122 inhibited proliferation, migration, and invasion in hepatocarcinoma cells. Additionally, some reports showed that LMNB2 was regulated by miR-122, which inhibited the expression of LMNB2. Moreover, LMNB2 functioned to promote cell proliferation, migration, and invasion. We could achieve the inhibition of hepatocarcinoma using miR-122 therapy through decreasing LMNB2 expression in vivo. Our data indicated that miR-122 could inhibit hepatocellular carcinoma cell progression by targeting LMNB2 and as a therapeutic target for hepatocarcinoma treatment.

scholarly journals Inhibition of Microrna-766-5p Attenuates the Development of Cervical Cancer Through Regulating SCAI

2020 ◽  
Vol 19 ◽  
pp. 153303382098008
Author(s):  
Yongqin Cai ◽  
Kai Zhang ◽  
Liya Cao ◽  
Hong Sun ◽  
Honggang Wang
Keyword(s):  
Cervical Cancer ◽  
Roc Curve ◽  
Tumor Xenograft ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Growth Of Tumor

Purpose: MicroRNAs (miRNAs) are considered to play anti-tumor roles in cancers. This study is designed to illustrate the role and potential mechanism of miR-766-5p in cervical cancer (CC) progression. Methods: MiR-766-5p expression in tissues and serum of CC patients was detected by quantitative reverse-transcription PCR (qRT-PCR). Receiver operating characteristic (ROC) curve was performed to evaluate the diagnostic value of serum miR-766-5p in CC. The 5-ethynyl-2′-deoxyuridine (EdU) assay, flow cytometry, wound healing as well as transwell assay were utilized to detect the proliferation, apoptosis, migration and invasion of CC cells, respectively. The interaction between miR-766-5p and SCAI was confirmed by dual-luciferase reporter gene assay. Xenografted tumor model was established to measure the growth of tumor xenograft in vivo. Results: MiR-766-5p was significantly increased in tissues and serum of CC patients. ROC curve suggested that serum miR-766-5p could serve as a biomarker for the diagnosis of CC. Inhibition of miR-766-5p suppressed the proliferation, migration and invasion, and promoted the apoptosis of CC cells. SCAI was proved to be a target of miR-766-5p. Silencing of SCAI eliminated the inhibiting effects of miR-766-5p inhibitor on the proliferation, migration and invasion of CC cells in vitro. Additionally, down-regulation of SCAI also reversed the inhibitory effect of miR-766-5p inhibitor on the growth of tumor xenograft in vivo. Conclusions: Inhibition of miR-766-5p restrains the cell proliferation, migration and invasion, and promotes the apoptosis in CC through negatively regulating SCAI.

scholarly journals LncRNA PVT1 promotes cervical cancer progression by sponging miR-503 to upregulate ARL2 expression

2021 ◽  
Vol 16 (1) ◽  
pp. 1-13
Author(s):  
Weiwei Liu ◽  
Dongmei Yao ◽  
Bo Huang
Keyword(s):  
Cervical Cancer ◽  
Cancer Progression ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Nude Mouse Model ◽  
Western Blot Assay ◽  
Non Coding Rna ◽  
Long Non Coding Rna

Abstract Cervical cancer (CC) is a huge threat to the health of women worldwide. Long non-coding RNA plasmacytoma variant translocation 1 gene (PVT1) was proved to be associated with the development of diverse human cancers, including CC. Nevertheless, the exact mechanism of PVT1 in CC progression remains unclear. Levels of PVT1, microRNA-503 (miR-503), and ADP ribosylation factor-like protein 2 (ARL2) were measured by quantitative reverse transcription-polymerase chain reaction or western blot assay. 3-(4,5)-Dimethylthiazole-2-y1)-2,5-biphenyl tetrazolium bromide (MTT) and flow cytometry were used to examine cell viability and apoptosis, respectively. For migration and invasion detection, transwell assay was performed. The interaction between miR-503 and PVT1 or ARL2 was shown by dual luciferase reporter assay. A nude mouse model was constructed to clarify the role of PVT1 in vivo. PVT1 and ARL2 expressions were increased, whereas miR-503 expression was decreased in CC tissues and cells. PVT1 was a sponge of miR-503, and miR-503 targeted ARL2. PVT1 knockdown suppressed proliferation, migration, and invasion of CC cells, which could be largely reverted by miR-503 inhibitor. In addition, upregulated ARL2 could attenuate si-PVT1-mediated anti-proliferation and anti-metastasis effects on CC cells. Silenced PVT1 also inhibited CC tumor growth in vivo. PVT1 knockdown exerted tumor suppressor role in CC progression via the miR-503/ARL2 axis, at least in part.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

scholarly journals Blockade of LINC01605-enriched exosome generation in M2 macrophages impairs M2 macrophage-induced proliferation, migration, and invasion of human dermal fibroblasts

2021 ◽  
Vol 35 ◽  
pp. 205873842110167
Author(s):  
Zhensen Zhu ◽  
Bo Chen ◽  
Liang Peng ◽  
Songying Gao ◽  
Jingdong Guo ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
M2 Macrophage ◽  
M2 Macrophages ◽  
Luciferase Reporter Gene ◽  
Gene Assay

Activated M2 macrophages are involved in hypertrophic scar (HS) formation via manipulating the differentiation of fibroblasts to myofibroblasts having the proliferative capacity and biological function. However, the function of exosomes derived from M2 macrophages in HS formation is unclear. Thus, this study aims to investigate the role of exosomes derived by M2 in the formation of HS. To understand the effect of exosomes derived from M2 macrophages on formation of HS, M2 macrophages were co-cultured with human dermal fibroblast (HDF) cells. Cell Counting Kit-8 assay was performed to evaluate HDF proliferation. To evaluate the migration and invasion of HDFs, wound-healing and transwell invasion assays were performed, respectively. To investigate the interaction between LINC01605 and miR-493-3p, a dual-luciferase reporter gene assay was adopted; consequently, an interaction between miR-493-3p and AKT1 was detected. Our results demonstrated that exosomes derived from M2 macrophages promoted the proliferation, migration, and invasion of HDFs. Additionally, we found that long noncoding RNA LINC01605, enriched in exosomes derived from M2 macrophages, promoted fibrosis of HDFs and that GW4869, an inhibitor of exosomes, could revert this effect. Mechanistically, LINC01605 promoted fibrosis of HDFs by directly inhibiting the secretion of miR-493-3p, and miR-493-3p down-regulated the expression of AKT1. Exosomes derived from M2 macrophages promote the proliferation and migration of HDFs by transmitting LINC01605, which may activate the AKT signaling pathway by sponging miR-493-3p. Our results provide a novel approach and basis for further investigation of the function of M2 macrophages in HS formation.

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