NACC-1 regulates hepatocellular carcinoma cell malignancy and is targeted by miR-760

Abstract Hepatocellular carcinoma (HCC) is the most prominent form of presentation in liver cancer. It is also the fourth most common cause of cancer-associated deaths globally. The role of nucleus accumbens associated protein-1 (NACC-1) has been evaluated in several cancers. This protein is a transcriptional regulator that regulates a number of significant cellular processes. In the current study, we aimed to understand the role of NACC-1 in HCC. Primarily, we measured the expression of NACC-1 using quantitative real time polymerase chain reaction and western blot analysis. We knocked down the expression of NACC-1 in HCC cell lines Huh7 and HepG2 by transferring a commercially synthesized small interfering RNA and explored the impact of NACC-1 knockdown on cellular growth, migration, invasion, and chemoresistance to doxorubicin. Through bioinformatic analysis, we identified NACC-1 as a potential target of miR-760. Using a dual reporter luciferase assay, we confirmed the predicted target and assessed miR-760-mediated regulation of NACC-1 and rescue of tumorigenic phenotypes. We observed increased expression of NACC-1 in HCC. Furthermore, knockdown of NACC-1 resulted in reduced cell proliferation and invasion and increased susceptibility to doxorubicin-mediated chemosensitivity. Overexpression of miR-760 in HCC cell lines rescued NACC-1-mediated migration and invasion. We revealed that miR-760 regulated NACC-1 expression in HCC. Our data indicated that both miR-760 and NACC-1 could be used as prognostic markers, and miR-760 may have therapeutic benefits for HCC and other cancers.

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Downregulation of CRABP2 Inhibit the Tumorigenesis of Hepatocellular Carcinoma In Vivo and In Vitro

BioMed Research International ◽

10.1155/2020/3098327 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Qingmin Chen ◽

Ludong Tan ◽

Zhe Jin ◽

Yahui Liu ◽

Ze Zhang

Keyword(s):

Hepatocellular Carcinoma ◽

Retinoic Acid ◽

Cell Lines ◽

Molecular Mechanisms ◽

Tumor Stage ◽

Migration And Invasion ◽

Hcc Cells

Cellular retinoic acid-binding protein 2 (CRABP2) binds retinoic acid (RA) in the cytoplasm and transports it into the nucleus, allowing for the regulation of specific downstream signal pathway. Abnormal expression of CRABP2 has been detected in the development of several tumors. However, the role of CRABP2 in hepatocellular carcinoma (HCC) has never been revealed. The current study aimed to investigate the role of CRABP2 in HCC and illuminate the potential molecular mechanisms. The expression of CRABP2 in HCC tissues and cell lines was detected by western blotting and immunohistochemistry assays. Our results demonstrated that the expression levels of CRABP2 in HCC tissues were elevated with the tumor stage development, and it was also elevated in HCC cell lines. To evaluate the function of CRABP2, shRNA-knockdown strategy was used in HCC cells. Cell proliferation, metastasis, and apoptosis were analyzed by CCK-8, EdU staining, transwell, and flow cytometry assays, respectively. Based on our results, knockdown of CRABP2 by shRNA resulted in the inhibition of tumor proliferation, migration, and invasion in vitro, followed by increased tumor apoptosis-related protein expression and decreased ERK/VEGF pathway-related proteins expression. CRABP2 silencing in HCC cells also resulted in the failure to develop tumors in vivo. These results provide important insights into the role of CRABP2 in the development and development of HCC. Based on our findings, CRABP2 may be used as a novel diagnostic biomarker, and regulation of CRABP2 in HCC may provide a potential molecular target for the therapy of HCC.

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Circular RNA hsa_circ_0001829 promotes gastric cancer progression through miR-155-5p/SMAD2 axis

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01790-w ◽

2020 ◽

Vol 39 (1) ◽

Author(s):

Qiuling Niu ◽

Zhijie Dong ◽

Min Liang ◽

Yuanwei Luo ◽

Hai Lin ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Lines ◽

Cancer Progression ◽

Cell Cycle Progression ◽

Luciferase Assay ◽

Migration And Invasion ◽

Circular Rnas ◽

Gain Of Function

Abstract Background Accumulating evidences have shown that circular RNAs (circRNAs) play important roles in regulating the pathogenesis of cancer. However, the role of circRNAs in gastric cancer (GC) remains largely unclear. Methods In this study, we identified a novel upregulated circRNA, hsa_circ_0001829, in chemically induced malignant transformed human gastric epithelial cells using RNA-seq. Subsequent qRT-PCR and ISH assays were performed to detect the expression level of hsa_circ_0001829 in GC cell lines and tissues. Functional roles of hsa_circ_0001829 in GC were then explored by loss- and gain-of- function assays. Bioinformatic prediction and luciferase assay were used to investigate potential mechanisms of hsa_circ_0001829. Finally, the mice xenograft and metastasis models were constructed to assess the function of hsa_circ_0001829 in vivo. Results We found that hsa_circ_0001829 was significantly upregulated in GC tissues and cell lines. Loss- and gain-of- function assays showed that hsa_circ_0001829 promotes GC cells proliferation, migration and invasion, and the affected cell cycle progression and apoptosis rates may account for the effect of hsa_circ_0001829 on GC proliferation. In addition, bioinformatic prediction and luciferase assay showed that hsa_circ_0001829 acts as a molecular sponge for miR-155-5p and that SMAD2 was a target gene of miR-155-5p; moreover, hsa_circ_0001829 sponges miR-155-5p to regulate SMAD2 expression and hsa_circ_0001829 promotes GC progression through the miR-155-5p–SMAD2 pathway. Finally, suppression of hsa_circ_0001829 expression inhibited tumor growth and aggressiveness in vivo. Conclusion Taken together, our findings firstly demonstrated a novel oncogenic role of hsa_circ_0001829 in GC progression through miR-155-5p–SMAD2 axis, and our study may offer novel biomarkers and therapeutic targets for GC.

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MiR-18a-5p promotes proliferation, migration and invasion of hepatocellular carcinoma by targeting CPEB3

10.21203/rs.3.rs-39546/v1 ◽

2020 ◽

Author(s):

Jianxing Zheng ◽

Dongyang Wu ◽

Libing Wang ◽

Fengzhi Qu ◽

Daming Cheng ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Wound Healing ◽

Expression Profiles ◽

Bioinformatic Analysis ◽

Luciferase Assay ◽

Migration And Invasion ◽

Promoting Effect ◽

Migration Ability ◽

Qrt Pcr ◽

Hcc Cells

Abstract Objective The study aims to explore the mechanism of miR-18a-5p targeting CPEB3 gene in regulating the occurrence and development of hepatocellular carcinoma (HCC). Methods Differential and survival analyses were conducted on HCC expression profiles from TCGA database to screen out target miRNAs on which targeted prediction was conducted. qRT-PCR was used to detect the expressions of miR-18a-5p and CPEB3. MTT assay examined the proliferation activity, wound healing assay analyzed the migration ability and Transwell assay detected the invasion ability of HCC cells after overexpressing miR-18a-5p.Dual luciferase assay verified the targeting relationship between miR-18a-5p and CPEB3. Meanwhile, MTT, wound healing and Transwellassays determined whether the overexpression of CPEB3 reversed the promoting effect of miR-18a-5p on HCC cells. Results Bioinformatic analysis showed that miR-18a-5p was significantly highly expressed in HCC tissues and its target binding site was found in CPEB3 genewith low expression.The qRT-PCR found that high miR-18a-5p expression was observed in HCC cells, and the expression of CPEB3 was significantly low. Overexpression of miR-18a-5p promoted proliferation, migration and invasion of HCC cells. Dual luciferase assay observed that miR-18a-5p inhibited the expression of CPEB3 while overexpression of CPEB3 reversed the promoting effect of miR-18a-5p on the growth of HCCcells. Conclusion miR-18a-5p promoted the proliferation and migration of HCC cells by inhibiting the expression of CPEB3. The role of miR-18a-5p /CPEB3 in HCCfound in this study provided a new potential target for the prognostic treatment of HCC patients.

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SPOCD1 regulated by miR-133a-3p promotes hepatocellular carcinoma invasion and metastasis

Journal of International Medical Research ◽

10.1177/03000605211053717 ◽

2022 ◽

Vol 50 (1) ◽

pp. 030006052110537

Author(s):

Tianying Zheng ◽

Xin Zhang ◽

Yonggang Wang ◽

Aijun Wang

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Cell Lines ◽

Colony Formation ◽

Quantitative Polymerase Chain Reaction ◽

The Cancer Genome Atlas ◽

Luciferase Assay ◽

Luciferase Reporter ◽

Downstream Target

Objective To investigate the tumorigenic role of spen paralogue and orthologue C-terminal domain-containing 1 (SPOCD1) in hepatocellular carcinoma (HCC) and identify the upstream regulatory mechanism. Methods We analyzed SPOCD1 and miR-133-3p expression in normal and HCC tissues from the Cancer Genome Atlas and UALCAN databases, and in normal hepatocytes and HCC cell lines by real-time quantitative polymerase chain reaction and western blot. We identified the miR-133a-3p-binding site on the SPOCD1 3ʹ-untranslated region using TargetScan. Hierarchical regulation was confirmed by luciferase assay and miR-133a-3p overexpression/silencing. Cell proliferation, migration, invasion, and colony formation were assessed by MTT, scratch, transwell, and clonogenic assays, respectively. Results SPOCD1 was highly expressed in HCC tissues and cell lines, while miR-133a-3p expression was significantly downregulated. Kaplan–Meier analysis indicated that high SPOCD1 expression was significantly associated with poor survival. TargetScan and luciferase reporter assay revealed that SPOCD1 was the downstream target of miR-133a-3p. Overexpression of miR-133a-3p significantly inhibited the expression of SPOCD1, while miR-133a-3p knockdown significantly increased SPOCD1 expression. Conclusion SPOCD1, regulated by miR-133a-3p, promotes HCC cell proliferation, migration, invasion, and colony formation. This study provides the first evidence for the role of the miR-133a-3p/SPOCD1 axis in HCC tumorigenesis.

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Silence of long non-coding RNA KCNQ1OT1 inhibits the proliferation, migration and invasion of hepatocellular carcinoma cells through PI3K/AKT signaling pathway

10.21203/rs.3.rs-21344/v1 ◽

2020 ◽

Author(s):

Hongliang Mei ◽

Zhiguo Yu ◽

Guanqi Zhang ◽

Zhiyuan Huang ◽

Hanjun Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Signaling Pathway ◽

Cell Lines ◽

Negative Impact ◽

Akt Signaling ◽

Cell Counting ◽

Migration And Invasion ◽

Akt Signaling Pathway ◽

Potential Biomarker

Abstract Background: KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) has been reported to be associated with hepatocellular carcinoma (HCC), which is considered as one of the most common cancers worldwide. However, the mechanism of action of KCNQ1OT1 in human HCC has not been fully explained. In this study, we aimed to explore the functional role and the potential mechanism of KCNQ1OT1 in human HCC.Methods: First, we analyzed the expression levels of KCNQ1OT1 in HCC tissues in starBase database and detected the expression of KCNQ1OT1 in HCC cell lines by quantitative real-time polymerase chain reaction assays. Next, we analyzed the role of KCNQ1OT1 in migration, invasion and proliferation of HCC by scratch wound healing, transwell and cell counting kit-8 assays. Finally, we analyzed the potential interrelationship between KCNQ1OT1 and PI3K/AKT signaling pathway through western blot assays.Results: Based on bioinformatics analyses, we found that KCNQ1OT1 was highly expressed in HCC tissues and its high expression was associated with a poor prognosis in HCC patients. We also confirmed an abnormal increase in the expression of KCNQ1OT1 in HCC cell lines. KCNQ1OT1 knockdown was found to have a negative impact on proliferation, migration and invasion of HCC cells. In addition, interference with the expression of KCNQ1OT1 reduced the phosphorylation level of AKT and the protein level of PI3K, indicating the association of KCNQ1OT1 with the PI3K/AKT signaling pathway.Conclusions: Collectively, this study confirmed the important role of KCNQ1OT1 in promoting HCC growth and revealed the inhibitory effect of KCNQ1OT1 on the PI3K/AKT signaling pathway. This work may contribute to a better understanding of HCC progression and provide a potential biomarker for HCC.

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Overexpressed Tumor Suppressor Exosomal miR-15a-5p in Cancer Cells Inhibits PD1 Expression in CD8+T Cells and Suppresses the Hepatocellular Carcinoma Progression

Frontiers in Oncology ◽

10.3389/fonc.2021.622263 ◽

2021 ◽

Vol 11 ◽

Author(s):

Hong-Yu Zhang ◽

Hong-Xia Liang ◽

Shu-Huan Wu ◽

He-Qing Jiang ◽

Qin Wang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

Cancer Cells ◽

Cd8 T Cells ◽

Hepg2 Cells ◽

Luciferase Assay ◽

Migration And Invasion ◽

Primary Liver Tumor ◽

High Fatality Rate

BackgroundHepatocellular carcinoma (HCC) is the most common primary liver tumor, and the main reason is the unclear pathogenesis of HCC, which leads to a high fatality rate of HCC. Therefore, it is of great clinical significance to explore the molecular mechanism of HCC and find a targeted therapeutic approach from the molecular level.Materials and MethodsMicroRNA-15a-5p (miR-15a-5p) expression level was measured by bioinformatics and qRT-PCR. Luciferase assay and RIP assays were used to verify the relationship between programmed cell death protein 1 (PD1) PD 1 with miR-15a-5p. Exosomes were identified using TEM, Zetasizer Nano ZS, and western blot. Edu, Transwell, and scratch assay were performed to explore the role of miR-15a-5p or exo-miR-15a-5p on HepG2 cells progression.ResultsMicroRNA-15a-5p (miR-15a-5p) was decreased in HCC tissues and cell lines, which indicated a poor prognosis. Overexpression of miR-15a-5p inhibited viability, proliferation, migration and invasion of HepG2 cells. Then, we isolated exosomes from cancer cells, and found that miR-15a-5p was packaged into exosomes from cancer cells. Furthermore, exo-miR-15a-5p was secreted into CD8+ T cells, then directly inhibited PD1 expression via targeted binding. Then, we co-cultured CD8+ T cells transfected with PD1 with HepG2 transfected with miR-15a-5p, PD1 remitted the inhibitory role of miR-15a-5p on HCC progression.ConclusionTogether, present study revealed exo-miR-15a-5p from cancer cells inhibited PD1 expression in CD8+ T cells, which suppressed the development of HCC.

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CircRNA hsa_circ_0110102 Function as Anti-Oncogenic Gene in Hepatocellular Carcinoma Through Modulating miR-580-5p/CCL2 Pathway

10.21203/rs.3.rs-62221/v1 ◽

2020 ◽

Author(s):

Xinxing Wang ◽

Wei Sheng ◽

Tao Xu ◽

Jiawen Xu ◽

Zhenhai Zhang

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Lines ◽

Molecular Mechanisms ◽

Luciferase Assay ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Dependent Manner ◽

Expression Levels ◽

Cox 2 ◽

Hcc Cells

Abstract Background: Circular RNAs (circRNAs) have been shown to have critical regulatory roles in tumor biology, whereas their contributions in hepatocellular carcinoma (HCC) still remains enigmatic. The purpose of this study was to investigate the molecular mechanisms involved in hsa_circ_0110102 in the occurrence and development of HCC. Methods: The expression levels of hsa_circ_0110102 in HCC cell lines and tissues were estimated by RT-qPCR assay. The proliferation, migration, and invasion of HCC cells were determined by CCK-8 and transwell assay. The western blot and ELISA were employed to examine the related-protein and cytokine expression. The association between miR-580-5p and hsa_circ_0110102 or CCL2 was predicted and affirmed by dual-luciferase reporter assay and RNA pull-down.Results: hsa_circ_0110102 was significantly down-regulated in HCC cell lines and tissues, low hsa_circ_0110102 expression levels were associated with poor prognosis. Knockdown hsa_circ_0110102 significantly inhibited cell proliferation, migration and invasion. In addition, luciferase assay and RNA pull-down assay indicating that hsa_circ_0110102 function as sponge for miR-580-5p. Moreover, miR-580-5p which could directly bind to the 3’-UTR of CCL2 and induce its expression, then active the COX-2/PGE2 pathway in macrophage via FoxO1 in p38 MAPK dependent manner. Furthermore, the Δ256 mutant of FoxO1 showed no activation effect. Conclusion: hsa_circ_0110102 act as a sponge for miR-580-5p and decreased CCL2 secretion in HCC cells, then inhibits pro-inflammatory cytokine release from activated macrophage by regulating the COX-2/PGE2 pathway. These results indicating that hsa_circ_0110102 serves as a potential prognostic predictor or therapeutic target for HCC.

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MicroRNA-20a Suppresses Tumor Proliferation and Metastasis in Hepatocellular Carcinoma by Directly Targeting EZH1

Frontiers in Oncology ◽

10.3389/fonc.2021.737986 ◽

2021 ◽

Vol 11 ◽

Author(s):

Qianqian Zhang ◽

Xiaohong Deng ◽

Xiuxin Tang ◽

Ying You ◽

Meihua Mei ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Lines ◽

Inhibitory Effect ◽

Luciferase Assay ◽

Migration And Invasion ◽

Tumor Proliferation ◽

Huh7 Cells ◽

Proliferation And Metastasis ◽

Tumor Proliferation And Metastasis

PurposeHepatocellular carcinoma (HCC), a worldwide leading cause of morbidity and mortality, is the most frequent primary liver tumor. Most HCC patients are diagnosed with advanced liver cancer, resulting in a very low 5-year survival rate. Thus, there is an urgent need for the development of targeted therapies. In this study, we aimed to investigate the effect and mechanism of the miR-20a/EZH1 axis on the proliferation and metastasis of HCC and the inhibitory effect of the EZH1/EZH2 inhibitor UNC1999 on HCC.Materials and MethodsThe expression of miR-20a in human HCC tissues and cell lines was detected using quantitative real-time PCR (qRT-PCR). The expressions of proteins were analyzed with immunohistochemistry and Western blotting. Luciferase assay was used to verify whether miR-20a targets EZH1 or EZH2. The effect of miR-20a on HCC progression was studied in vivo and in vitro. The tumor inhibitory effect of UNC1999 was confirmed in vivo. CCK8 assay, wound healing assay, cell migration and invasion assay were used to evaluate the synergistic effect of UNC1999 with sorafenib. RNA sequencing (RNA-seq) was performed to screen the differentially expressed genes in the Huh7 and SMMC7721 cell lines after UNC1999, sorafenib, and combination treatments.ResultsIn this study, miR-20a showed a lower expression in both HCC tissues and cell lines. MiR-20a inhibited the proliferation and migration of SMMC7721 and Huh7 cells. The results of the luciferase assay and Western blot analysis revealed that miR-20a directly targeted EZH1, a histone methyltransferase. We demonstrated that miR-20a negatively regulated the expression of EZH1 and inhibited the proliferation and metastasis of HCC by reducing H3K27 methylation. We found UNC1999 inhibited tumor cells proliferation and enhanced the inhibitory effect of sorafenib.ConclusionWe demonstrated that miR-20a suppresses the tumor proliferation and metastasis in HCC by directly targeting EZH1. UNC1999 can inhibit tumor proliferation in vivo and increase the sensitivity of hepatoma cell lines to sorafenib.

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Inhibiting roles of FOXA2 in hepatocellular carcinoma cell migration and invasion by transcriptionally suppressing microRNA-103a-3p and activating the GREM2/LATS2 axis

10.21203/rs.3.rs-26285/v1 ◽

2020 ◽

Author(s):

Guangzhen Ma ◽

Jirong Chen ◽

Tiantian Wei ◽

Jia Wang ◽

Wenshan Chen

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Migration ◽

Luciferase Assay ◽

Migration And Invasion ◽

Altered Expression ◽

Cell Migration And Invasion ◽

Invasion And Migration ◽

Forkhead Box ◽

And Migration

Abstract Background Forkhead box A2 (FOXA2) is a transcriptional activator for liver-specific genes. Hepatocellular carcinoma (HCC) is a prevalent fetal malignancy across the globe. This work focused on the role of FOXA2 in HCC cell migration and invasion and the involving molecules. Methods FOXA2 expression in HCC tissues and cells was determined using RT-qPCR. Altered expression of FOXA2 was introduced to identify its role in HCC cell migration and invasion using Transwell assays. The potential target microRNA (miRNA) of FOXA2 was predicted via online prediction and validated through a ChIP assay, and the mRNA target of miRNA-103a-3p was predicted and confirmed through a luciferase assay. The roles of miR-103a-3p and GREM2 in HCC cell invasion and migration were determined, and the downstream molecules mediated by GREM2 were analyzed. Results FOXA2 and GREM2 were poorly expressed while miR-103a-3p was abundant in HCC tissues and cells. Overexpression of FOXA2 or GREM2 suppressed migration and invasion of HepG2 and SK-HEP-1 cells, while up-regulation of miR-103a-3p led to reverse trends. FOXA2 transcriptionally suppressed miR-103a-3p to increase GREM2 expression, and silencing of GREM2 partially blocked the inhibitory effects of FOXA2 on cell migration and invasion. GREM2 increased LATS2 activity and YAP phosphorylation and degradation. Conclusion This study evidenced that FOXA2 inhibits migration and invasion potentials of HCC cell lines through suppressing miR-103a-3p transcription. The following upregulation of GREM2 plays key roles in migration inhibition by promoting LATS2 activity and YAP phosphorylation. This study may offer new insights into HCC treatment.

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Circular RNA hsa_circ_0001829 Promotes Gastric Cancer Progression through miR-155-5p/SMAD2 Axis

10.21203/rs.3.rs-57293/v1 ◽

2020 ◽

Author(s):

Qiuling Niu ◽

Zhijie Dong ◽

Min Liang ◽

Yuanwei Luo ◽

Hai Lin ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Lines ◽

Cancer Progression ◽

Cell Cycle Progression ◽

Luciferase Assay ◽

Migration And Invasion ◽

Circular Rnas ◽

Gain Of Function

Abstract Background: Accumulating evidences have shown that circular RNAs (circRNAs) play important roles in regulating the pathogenesis of cancer. However, the role of circRNAs in gastric cancer (GC) remains largely unclear. Methods: In this study, we identified a novel upregulated circRNA, hsa_circ_0001829, in chemically induced malignant transformed human gastric epithelial cells using RNA-seq. Subsequent qRT-PCR was performed to detect the expression level of hsa_circ_0001829 in GC cell lines and tissues. Functional roles of hsa_circ_0001829 in GC were then explored by loss- and gain-of- function assays. Bioinformatic prediction and luciferase assay were used to investigate potential mechanisms of hsa_circ_0001829. Finally, the mice xenograft models were constructed to assess the function of hsa_circ_0001829 in vivo.Results: We found that hsa_circ_0001829 was significantly upregulated in GC tissues and cell lines. Loss- and gain-of- function assays showed that hsa_circ_0001829 promotes GC cells proliferation, migration and invasion, and the affected cell cycle progression and apoptosis rates may account for the effect of hsa_circ_0001829 on GC proliferation. In addition, bioinformatic prediction and luciferase assay showed that hsa_circ_0001829 acts as a molecular sponge for miR-155-5p and that SMAD2 was a target gene of miR-155-5p; moreover, hsa_circ_0001829 sponges miR-155-5p to regulate SMAD2 expression and hsa_circ_0001829 promotes GC progression through the miR-155-5p–SMAD2 pathway. Finally, suppression of hsa_circ_0001829 expression inhibited subcutaneous tumor growth in vivo. Conclusion: Taken together, our findings firstly demonstrated a novel oncogenic role of hsa_circ_0001829 in GC progression, which is through miR-155-5p–SMAD2 axis, and our study may offer novel biomarkers and therapeutic targets for GC.

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