luciferase reporter assay
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2022 ◽  
Vol 12 (4) ◽  
pp. 747-755
Shengyong Liu ◽  
Xiangcheng Li

Background: Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide with a poor prognosis. Amounting studies revealed that long non-coding RNAs (lncRNAs) show important roles in various biological processes. The purpose of this study was to explore the biological function and potential molecular mechanism of CASC7 in HCC. Methods: CASC7 expression in HCC cell lines was detected by qRT-PCR. The expressions of CASC7 and miR-340-5p were changed by transfection of miR-340-5p mimic, the CASC7 overexpression and knockdown plasmids. The interaction between CASC7 and miR-340-5p was assessed by a Dual-Luciferase reporter assay. The biological functions of CASC7 were evaluated by CCK-8, colony formation assay, ROS assay kit, immunofluorescence and flow cytometry (FCM). Results: CASC7 was upregulated in HCC cell lines. CASC7 overexpression significantly promoted cell proliferation, as well as inhibited apoptosis and oxidative stress. In contrast, CASC7 knockdown could reverse these above changes. The result of the Dual-luciferase reporter assay revealed that CASC7 directly targeted miR-340-5p and negatively regulated its expression. In addition, CASC7 promoted proliferation and inhibited apoptosis of HCC cells through activating Nrf2 pathway by downregulating miR-340-5p. Conclusions: In summary, CASC7 promotes HCC tumorigenesis and progression through the Nrf2 pathway by targeting miR-340-5p, which may provide a new target for therapy of HCC.

2022 ◽  
Vol 12 (3) ◽  
pp. 461-470
Gang Quan ◽  
Bo Ren ◽  
Jian Xu ◽  
Jie Zhou ◽  
Guo Wu ◽  

<sec> <title>Objective:</title> This study was designed to probe the influence and mechanism of lncRNA HOTAIR on migration, apoptosis and proliferation of hepatocellular carcinoma (HCC) cells. </sec> <sec> <title>Methods:</title> We evaluated LncRNA HOTAIR expression in HCC tissues and adjacent tissues, and serum of HCC patients and healthy controls. Later, we knocked down lncRNA HOTAIR, and utilized CCK-8 to determine Hep3B cell proliferation, flow cytometry for prospecting Hep3B cell apoptosis, and cell scratch assay for observing Hep3B cell migration.We anticipated the direct target of lncRNA HOTAIR, and adopted luciferase reporter assay to verify. Moreover, we inhibitedmiR-126-5p expression, and rescue experiment for evaluating the influence of si-HOTAIR+miR-126-5p inhibitors on Hep3B cell migration, apoptosis as well as proliferation. </sec> <sec> <title>Results:</title> Our results showed that lncRNA HOTAIR expression in tumor tissues and serum was significantly increased. Moreover, lncRNA HOTAIR inhibition significantly decreased the Hep3B cell proliferation rate, elevated Hep3B cell apoptosis rate, and inhibited Hep3B cell migration. Luciferase reporter assay suggested that miR-126-5p was the direct target of lncRNA HOTAIR. Furthermore, co-transfection of si-HOTAIR+miR-126-5p inhibitor could diminishthe effects of HOTAIR silencing on apoptosis, proliferation and migration. </sec> <sec> <title>Conclusion:</title> Silencing of lncRNA-HOTAIR can inhibit the HCC cell migration and proliferation, and increase the apoptosis by up-regulating miR-126-5p expression. </sec>

2022 ◽  
Vol 8 (1) ◽  
Dianbo Long ◽  
Yiyang Xu ◽  
Guping Mao ◽  
Ruobing Xin ◽  
Zengfa Deng ◽  

AbstracttRNA-derived fragments (tRFs) are new noncoding RNAs, and recent studies have shown that tRNAs and tRFs have important functions in cell metabolism via posttranscriptional regulation of gene expression. However, whether tRFs regulate cellular metabolism of the anterior cruciate ligament (ACL) remains elusive. The aim of this study was to investigate the role and action mechanism of tRFs in ACL cell metabolism. A tRF array was used to determine tRF expression profiles in different human ACL cells, and quantitative real-time polymerase chain reaction and fluorescence in situ hybridisation were used to determine TRF365 expression. ACL cells were transfected with a TRF365 mimic or a TRF365 inhibitor to determine whether TRF365 regulates IKBKB expression. A rescue experiment and dual-luciferase reporter assay were conducted to determine whether the 3′-untranslated region (UTR) of IKBKB has a TRF365-binding site. TRF365 was weakly expressed in osteoarthritis (OA) ACL and interleukin-1β-treated ACL cells. IKBKB was highly expressed in OA ACL and interleukin-1β-treated ACL cells; transfection with the TRF365 mimic suppressed IKBKB expression, whereas transfection with the TRF365 inhibitor had the opposite effect. A dual-luciferase reporter assay showed that TRF365 silenced the expression of IKBKB by binding to its 3′-UTR. Thus, TRF365 regulates the metabolism of ACL cells by targeting IKBKB. In summary, TRF365 may provide a new direction for the study of ACL degeneration and on the pathophysiological process of OA.

Xiaolei Gao ◽  
Xuan Zheng ◽  
Yixin Zhang ◽  
Liying Dong ◽  
Liangjie Sun ◽  

Background: p53 mutations are highly frequent in various human cancers and are reported to contribute to tumor malignance and chemoresistance. In this study, we explored the mechanism by which mutant p53 promotes carcinogenesis and chemoresistance and provided novel insights into cancer therapy.Materials and methods: A total of 409 patients with colorectal carcinoma from TCGA database were subdivided into two groups according to the p53 status, namely, mutant p53 and wild-type p53, following with GSEA analysis. The differences of the clinicopathologic index were also analyzed. Two HCT116 cell lines containing hot spots at codons R273H and R248W of p53 were constructed based on HCT116 with knockout p53, respectively. Cell viability, mobility, clonogenesis, and stemness were detected by CCK8, transwell migration and invasion, colonogenic, and sphere formation assays. Resistance to 5-FU was examined by live-dead staining and flow cytometry. qPCR, Western blot, and luciferase reporter assay were performed to identify that deficient or mutant p53 promoted chemoresistance of the colorectal carcinoma cell line HCT116 through the TCF21/CD44 signaling pathway, with the following rescue assays by overexpression of TCF21 and knockdown of CD44.Results: Patients with recurrence harbor a higher frequency of mutant p53 than those without recurrence (p &lt; 0.05). The mutant p53 group developed a larger tumor than the wild-type one. GSEA analysis showed that oncogenic signatures were enriched in the mutant p53 group. Extracellular assays showed that cancer cells with deficient or mutant p53 (R273H and R248W, respectively) promoted colon cancer cell growth, migration, invasion, and stemness. The mutant cancer cells were also observed to be significantly resistant to 5-FU. Xenografts also confirmed that HCT116 cells harboring deficient or mutant p53 promoted cancer growth and 5-FU tolerance. Luciferase reporter assay showed that deficient or mutant p53 R237H and R248W endowed cancer cells with chemoresistance by activating CD44 via repressing the nuclear transcription factor TCF21 expression. Overexpression of TCF21 or knockdown of CD44 could rescue the sensitivity to 5-FU in deficient and mutant p53 HCT116 cell lines.Conclusion: Our results, for the first time, reveal a novel deficient or mutant p53/TCF21/CD44 signaling pathway which promotes chemoresistance in colorectal carcinoma. The axis could be an effective therapeutic strategy against deficient- or mutant p53-driven chemoresistance.

2022 ◽  
Vol 24 (1) ◽  
Lingjiao Meng ◽  
Sheng Chang ◽  
Yang Sang ◽  
Pingan Ding ◽  
Liuxin Wang ◽  

Abstract Background A growing body of evidence indicates that abnormal expression of circular RNAs (circRNAs) plays a crucial role by acting as molecular sponges of microRNAs (miRNAs) in various diseases, including cancer. In this study, we explored whether circCCDC85A could function as a miR-550a-5p sponge and influence breast cancer progression. Methods We detected the expression of circCCDC85A in breast cancer tissues and cells using fluorescence in situ hybridization (FISH) and quantitative reverse transcription polymerase chain reaction (qRT-PCR). CCK-8 and colony formation assay were used to detect the proliferative ability of breast cancer cells. Wound healing assay and transwell migration and invasion assays were used to detect the migrative and invasive abilities of breast cancer cells. We also examined the interactions between circCCDC85A and miR-550a-5p using FISH, RNA-binding protein immunoprecipitation (RIP), and luciferase reporter assay. Moreover, we performed luciferase reporter assay, qRT-PCR, and Western blot to confirm the direct targeting of miR-550a-5p to MOB1A. Results The expression of circCCDC85A in breast cancer tissues was obviously lower than that in normal breast tissues. Over-expression of circCCDC85A substantially inhibited the proliferative, migrative, and invasive ability of breast cancer cells, while knocking down of circCCDC85A enhanced the aforementioned properties of breast cancer cells. Moreover, enforced expression of circCCDC85A inhibits the oncogenic activity of miR-550a-5p and increases the expression of MOB1A targeted by miR-550a-5p. Further molecular mechanism research showed that circCCDC85A may act as a molecular sponge for miR-550a-5p, thus restoring miR-550a-5p-mediated targeting repression of tumor suppressor MOB1A in breast cancer cells. Conclusion Our findings provide novel evidence that circCCDC85A inhibits the progression of breast cancer by functioning as a molecular sponge of miR-550a-5p to enhance MOB1A expression.

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Xu Niu ◽  
Haitao Sun ◽  
Feng Qiu ◽  
Jing Liu ◽  
Tianchi Yang ◽  

Objective. To analyze the function of miR-10b-5p in suppressing the invasion and proliferation of primary hepatic carcinoma cells by downregulating erythropoietin-producing hepatocellular receptor A2 (EphA2). Material and Methods. Eighty-six hepatic carcinoma (HCC) tissue specimens and 86 corresponding adjacent tissue specimens were collected, and the mRNA expression of miR-10b-5p and Ephrin type-A receptor 2 (EphA2) in the specimens was determined using a reverse transcription-polymerase chain reaction (RT-PCR) assay. Western blot was employed to quantify EphA2, B-cell chronic lymphocytic leukemia/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-3 in the cells, and CCK8, Transwell assay, and flow cytometry were applied to evaluate the proliferation, invasion, and apoptosis of cells, respectively. Moreover, the dual luciferase reporter assay was utilized for correlation analysis between miR-10b-5p and EphA2. Results. miR-10b-5p was lowly expressed in HCC, while EphA2 was highly expressed. Cell experiments revealed that miR-10b-5p overexpression or EphA2 knockdown could reduce cell proliferation, accelerate apoptosis, strongly upregulate Bax and Caspase-3, and downregulate Bcl-2. In contrast, miR-10b-5p knockdown or EphA2 overexpression gave rise to reverse biological phenotypes. Furthermore, dual luciferase reporter assay verified that miR-10b-5p was a target of EphA2, and the rescue experiment implied that transfection of pCMV-EphA2 or Si-EphA2 could reverse EphA2 expression and cell biological functions caused by miR-10b-5p overexpression or knockdown. Conclusions. miR-10b-5p reduced HCC cell proliferation but accelerate apoptosis by regulating EphA2, suggesting it has the potential to be a clinical target for HCC.

2021 ◽  
Vol 13 (1) ◽  
Zhiliang Chen ◽  
Zaosong Zheng ◽  
Yingwei Xie ◽  
Qiyu Zhong ◽  
Wentai Shangguan ◽  

AbstractCircular RNAs (circRNAs) are a type of covalently closed circular-formed RNAs and play crucial roles in the oncogenesis and progression of various human cancers. Here we identified a novel circRNA, circPPP6R3, to be highly expressed both in clear cell renal cell carcinoma (ccRCC) tissues and cell lines based on analyzing high-throughput sequencing data and qRT-PCR analysis. Highly expressed circPPP6R3 was positively correlated with higher histological grade, T stage, and M stage as well as advanced clinical stage of ccRCC patients. Functionally, knockdown of circPPP6R3 attenuated the proliferation, migration, and invasion of ccRCC cells whereas overexpression had the reverse effects. Mechanistically, the biotin-labeled pull-down assay and dual-luciferase reporter assay revealed that circPPP6R3 directly interacted with miR-1238-3p. miR-1238-3p inhibitors had a rescue effect on the proliferative and metastatic capacities by knockdown of circPPP6R3. Moreover, RNA-sequencing analysis and dual-luciferase reporter assay indicated that circPPP6R3 upregulated CD44, a cell-surface glycoprotein contributed to the cell adhesion and metastasis, via sponging to miR-1238-3p. Further investigation revealed that MMP9 and Vimentin were regulated by CD44 in ccRCC. Our study thus provided evidence that the regulatory network involving circPPP6R3/miR-1238-3p/CD44 axis might provide promising biomarkers as well as a therapeutic approach for ccRCC.

2021 ◽  
Vol 20 (11) ◽  
pp. 2279-2285
Shenglin Wu ◽  
Shan Nie ◽  
Jian Wang

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.

2021 ◽  
Vol 22 (1) ◽  
Pan Jiang ◽  
Fan Li ◽  
Zilong Liu ◽  
Shengyu Hao ◽  
Jian Gao ◽  

Abstract Background Growing evidence suggests that cancer stem cells (CSCs) are responsible for cancer initiation in tumors. Bach1 has been identified to contribute to several tumor progression, including lung cancer. The role of Bach1 in CSCs remains poorly known. Therefore, the function of Bach1 on lung CSCs was focused currently. Methods The expression of Bach1, CD133, CD44, Sox2, Nanog and Oct4 mRNA was assessed using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). Protein expression of Bach1, CD133, CD44, Sox2, Nanog, Oct4, p53, BCL2, BAX, p-p38, p-AKT1, c-Fos and c-Jun protein was analyzed by western blotting. 5-ethynyl-29-deoxyuridine (EdU), colony formation, Flow cytometry analysis and transwell invasion assay were carried out to analyze lung cancer cell proliferation, apoptosis and invasion respectively. Tumor sphere formation assay was utilized to evaluate spheroid capacity. Flow cytometry analysis was carried out to isolate CD133 or CD44 positive lung cancer cells. The relationship between Bach1 and CD44 was verified using ChIP-qPCR and dual-luciferase reporter assay. Xenograft tumor tissues were collected for hematoxylin and eosin (HE) staining and IHC analysis to evaluate histology and Ki-67. Results The ratio of CD44 + CSCs from A549 and SPC-A1 cells were significantly enriched. Tumor growth of CD44 + CSCs was obviously suppressed in vivo compared to CD44− CSCs. Bach1 expression was obviously increased in CD44 + CSCs. Then, via using the in vitro experiment, it was observed that CSCs proliferation and invasion were greatly reduced by the down-regulation of Bach1 while cell apoptosis was triggered by knockdown of Bach1. Loss of Bach1 was able to repress tumor-sphere formation and tumor-initiating CSC markers. A repression of CSCs growth and metastasis of shRNA-Bach1 was confirmed using xenograft models and caudal vein injection. The direct interaction between Bach1 and CD44 was confirmed by ChIP-qPCR and dual-luciferase reporter assay. Furthermore, mitogen-activated protein kinases (MAPK) signaling pathway was selected and we proved the effects of Bach1 on lung CSCs were associated with the activation of the MAPK pathway. As manifested, loss of Bach1 was able to repress p-p38, p-AKT1, c-Fos, c-Jun protein levels in lung CSCs. Inhibition of MAPK signaling remarkably restrained lung CSCs growth and CSCs properties induced by Bach1 overexpression. Conclusion In summary, we imply that Bach1 demonstrates great potential for the treatment of lung cancer metastasis and recurrence via activating CD44 and MPAK signaling.

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