Long Non-Coding RNA CASC7 Promotes Proliferation and Inhibits Apoptosis of Hepatocellular Carcinoma Cells via Downregulating miR-340-5p CASC7/miR-340-5p Axis in Hepatocellular Carcinoma

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.

2019 ◽  
Vol 25 (1) ◽  
Ming Yang ◽  
Shijuan Sun ◽  
Yao Guo ◽  
Junjie Qin ◽  
Guangming Liu

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.

2020 ◽  
Yifeng Cui ◽  
Han Lin ◽  
Yunzheng Zhao ◽  
Jiaming Liu ◽  
Chengpeng Zhang ◽  

Abstract Background: Hepatocellular carcinoma (HCC) is a common malignancy of the digestive system. A novel long non-coding RNA, ENSG00000236199 (lncRNA-199) , whose role in tumors has not been reported, especially in HCC. Methods: The expression of lncRNA-199 and miR-576-5p were detected by Real-time quantitative polymerase chain reaction (RT-qPCR), NRIP1 was measured by Western blotting. HCC cell proliferation and metastasis of HCC were examined using functional tests. Luciferase reporter assay was performed to confirm the relationship between lncRNA-199 and miR-576-5p, between miR-576-5p and NRIP1. Results: LncRNA-199 was frequently down-regulated in HCC and this down-regulation was negatively correlated with prognosis. Overexpression of lncRNA-199 could inhibit the growth and metastasis of HCC, while knockdown lncRNA-199 had the opposite effect. Down-regulated lncRNA-199 also could induce epithelial-mesenchymal transformation (EMT). Luciferase reporter assay demonstrated lncRNA-199 could modulate NRIP1 by sponge miR-576-5p. Conclusion: LncRNA-199 inhibited growth and metastasis through miR-576-5p/NRIP1 axis in HCC. This study revealed a novel unknown lncRNA function in malignant tumors, especially in HCC. At the same time, the regulatory mechanism of lncRNA-199 in HCC was clarified.

2018 ◽  
Vol 49 (4) ◽  
pp. 1403-1419 ◽  
Yunxiuxiu Xu ◽  
Xinxi Luo ◽  
Wenguang He ◽  
Guangcheng Chen ◽  
Yanshan Li ◽  

Background/Aims: To investigate the biological roles and underlying molecular mechanisms of long non-coding RNA (lncRNA) PVT1 in Hepatocellular carcinoma (HCC). Methods: qRT-PCR was performed to measure the expression of miRNA and mRNA. Western blot was performed to measure the protein expression. CCK-8 assay was performed to determine cell proliferation. Flow cytometry was performed to detect cell apoptosis. Wounding-healing assay and Transwell assay was performed to detect cell migration and invasion. Dual luciferase reporter assay was performed to verify the target relationship. Quantichrom iron assay was performed to check uptake level of cellular iron. Results: PVT1 expression was up-regulated in HCC tissues and cell lines. Function studies revealed that PVT1 knockdown significantly suppressed cell proliferation, migration and invasion, and induced cell apoptosis in vitro. Furthermore, PVT1 could directly bind to microRNA (miR)-150 and down-regulate miR-150 expression. Hypoxia-inducible protein 2 (HIG2) was found to be one target gene of miR-150, and PVT1 knockdown could inhibit the expression of HIG2 through up-regulating miR-150 expression. In addition, the expression of miR-150 was down-regulated, while the expression of HIG2 was up-regulated in HCC tissues and cell lines. Moreover, inhibition of miR-150 could partly reverse the biological effects of PVT1 knockdown on proliferation, motility, apoptosis and iron metabolism in vitro, which might be associated with dysregulation of HIG2. In vivo results showed that PVT1 knockdown suppressed tumorigenesis and iron metabolism disorder by regulating the expression of miR-150 and HIG2. Conclusion: Taken together, the present study demonstrates that PVT1/miR-150/HIG2 axis may lead to a better understanding of HCC pathogenesis and provide potential therapeutic targets for HCC.

2021 ◽  
Vol 19 (1) ◽  
Shuai Xue ◽  
Fengqin Lu ◽  
Chunhui Sun ◽  
Jingjing Zhao ◽  
Honghua Zhen ◽  

Abstract Background It has been reported that long-chain non-coding RNA (lncRNA) zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) is an oncogene in various cancers, including hepatocellular carcinoma (HCC). We investigated the role and mechanism of ZEB1-AS1 as a competitive endogenous RNA (ceRNA) combined with miR-23c in HCC cell proliferation and invasion. Methods QRT-PCR was used to detect ZEB1-AS1 and miR-23c expressions in HCC tissues and cells. The dual luciferase reporter assay detected the targeted regulation of miR-23c and ZEB1-AS1. We also performed the correlation analysis of their expression in HCC tissues by the Spearman’s correlation analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of hepatoma cells. Cell invasion was assessed by the Transwell assay. Results QRT-PCR results indicated ZEB1-AS1 was upregulated and miR-23c was downregulated in HCC tissues and cell lines. ZEB1-AS1 knockdown hampered the proliferation and invasion of HCC cells. Dual luciferase reporter assay showed that miR-23c is a target of ZEB1-AS1, and ZEB1-AS1 was significantly negatively correlated with the miR-23c expression in HCC tissues. The results of MTT and Transwell assay showed that miR-23c inhibition restored the inhibitory effect of ZEB1-AS1 knockdown on HCC cells proliferation and invasion. Conclusions As a ceRNA, lncRNA ZEB1-AS1 may play a vital role in inhibiting HCC progression through miR-23c, which will provide new clues and theoretical basis for the HCC diagnosis and treatment.

2020 ◽  
Pengcheng Li ◽  
Junhui Xing ◽  
Jianwu Jiang ◽  
Xinyu Tian ◽  
Xuemeng Liu ◽  

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.

2020 ◽  
Vol 19 (1) ◽  
pp. 39-44
Bangming Pu ◽  
Yong Cao ◽  
Yan Li ◽  
Li Tang ◽  
Jiyi Xia ◽  

Purpose: To explore the molecular function of miR-196b-5p in hepatocellular carcinoma (HCC).Methods: MiR-196b-5p expression levels in HCC tissue samples were assessed by qRT-PCR. MiR-196b-5p was knocked-down or over-expressed in HepG2 cells by transfecting the cells with plasmids expressing either a miR-196b-5p inhibitor or mimic, respectively, while cell proliferation was  assessed by MTT assay. The interaction of miR-196b-5p with target molecules was confirmed using luciferase reporter assay. Cell cycle was investigated by flow cytometry, while NFκBIA expression was assessed by western blotting.Results: MiR-196b-5p was over-expressed in HCC, and miR-196b-5p expression levels in patients with HCC were related to tumor grade. MiR-196b-5p over-expression promoted cell proliferation and colony formation and suppressed cell cycle arrest and apoptosis. The results of luciferase reporter assay showed that miR-196b-5p reduced NFκBIA expression in HepG2 cells by binding to a response element in the 3′ UTR of NFκBIA. Further investigation showed that NFκBIA interacts with NFκB1 and reduces the concentration of NFκB1 in HepG2 cells. The promoter of ATP-binding cassette sub-family B member 1 (ABCB1) was also targeted and bound by NFκB1, which altered the expression of ABCB1 in HepG2 cells.Conclusion: MiR-196b-5p regulates cell proliferation in drug-resistant HCC cell lines via activation of the NFκB/ABCB1 signaling pathway. Keywords: Hepatocellular carcinoma, miR-196b-5p, NFκBIA, NFκB1, ABCB1

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242179
Yichao Zhao ◽  
Chaoqian Zhu ◽  
Qing Chang ◽  
Peng Peng ◽  
Jie Yang ◽  

Objective This study aims to explore the mechanism of the miR-424-5p/E2F7 axis in hepatocellular carcinoma (HCC) and provide new ideas for targeted therapy of HCC. Methods Bioinformatics analysis was used to identify the target differentially expressed miRNA in HCC and predict its target gene. qRT-PCR was employed to verify the expression of miR-424-5p and E2F7 mRNA in HCC cells. Western blot was performed to detect the effect of miR-424-5p ectopic expression on the protein expression of E2F7. CCK-8 was used to detect proliferative activity of HCC cells and flow cytometry was carried out for analyzing cell cycle distribution. Dual luciferase reporter assay was conducted to verify the direct targeting relationship between miR-424-5p and E2F7. Results We observed that miR-424-5p was down-regulated in HCC cells. CCK-8 showed that overexpression of miR-424-5p inhibited cell proliferation, and flow cytometry showed that miR-424-5p could block cells in G0/G1 phase. E2F7 was up-regulated in HCC cells, and E2F7 overexpression could facilitate the proliferative ability of HCC cells and promote the cell cycle progressing from G0/G1 to S phase. Furthermore, dual-luciferase reporter assay indicated that miR-424-5p could directly down-regulate E2F7 expression. Analysis on cell function demonstrated that miR-424-5p inhibited the proliferation of HCC cells and blocked cell cycle at G0/G1 phase by targeting E2F7. Conclusion Our results proved that E2F7 was a direct target of miR-424-5p, and miR-424-5p could regulate cell cycle and further inhibit the proliferation of HCC cells by targeting E2F7.

2020 ◽  
Liu Yang ◽  
Yinan Zhang ◽  
Jun Bao ◽  
Ji-Feng Feng

Abstract Background: It has been well documented that long non-coding RNAs (lncRNAs) regulate numerous characteristics of cancer, including proliferation, migration, metastasis, apoptosis, even metabolism. LncRNA BCYRN1 (BCYRN1) is a newly identified brain cytoplasmic lncRNA with 200-nucleotide, which was discovered highly expressed in tumor tissues of cancer, including hepatocellular carcinoma, gastric cancer and lung cancer. However, the roles of BCYRN1 in colorectal cancer (CRC) remain obscure. This study was designed to reveal the acts of BCYRN1 in the occurrence and progression of CRC.Methods: RT-PCR was used to detect the expression level of BCYRN1 in tumor tissues and CRC cell lines. Knock down BCYRN1 in CRC cells, evaluate cell proliferation changes by CCK-8 test, EdU test, and Ki-67 and PCNA expression; evaluate cell migration and invasion changes by wound healing assay, Transwell assay and invasion-related protein expression . Through flow cytometry analysis to assess whether BCYRN1 regulates apoptosis of CRC cells. The dual luciferase reporter gene detects the competitive binding of BCYRN1 to miR-204-3p. In vivo experiments to evaluate the effect of BCYRN1 on tumor development. TargetScan analysis and dual luciferase reporter gene detect the target gene of miR-204-3p. Rescue experiments verified the effect of BCYRN1 on CRC by regulating the effect of miR-204-3p on KRAS.Results: We found that compared with normal tissues and human intestinal epithelial cells (HIECs), BCYRN1 levels were significantly increased in tumor tissues and cell lines of CRC. We further determined that knockdown of BCYRN1 inhibited proliferation, migration, invasion, and promoted apoptosis of CRC cells. In addition, bioinformatics analysis and dual luciferase reporter assay showed that BCYRN1 served as a competitive endogenous RNA (ceRNA) to regulate the development of CRC through competitively binding to miR-204-3p. Further studies proved that overexpression of miR-204-3p reversed the effects of BCYRN1 on CRC. Next, TargetScan analysis and dual luciferase reporter assay indicated that KRAS was a target gene of miR-204-3p and negatively regulated by miR-204-3p. A series of rescue experiments showed that BCYRN1 affected the occurrence and development of CRC by regulating the effects of miR-204-3p on KRAS. In addition, tumorigenic experiments in CRC model mice confirmed that down-regulated BCYRN1 effectively inhibited tumor growth. Conclusions: Our findings suggested that BCYRN1 plays a carcinogenic role in CRC by regulating the miR-204-3p/KRAS axis.

2021 ◽  
Vol 21 (1) ◽  
Qingyun Pan ◽  
Ying Huang ◽  
Yirui Wang ◽  
Deke Li ◽  
Changjiang Lei

Abstract Background Long non-coding RNA is considered to be essential to modulate the development and progression of human malignant cancers. And long non-coding RNA can act as crucial modulators by sponging the corresponding microRNA in tumorigenesis. We aimed to elucidate the function of ACTA2-AS1 and its molecular mechanism in colon adenocarcinoma. Materials and methods The expression of ACTA2-AS1, miR-4428 and BCL2L11 in colon adenocarcinoma tissues were detected via qRT-PCR. SW480 and HT29 cells were transfected with shRNA ACTA2-AS1, OE ACTA2-AS1, miRNA mimics of miR-4428, miR-4428 inhibitor, si-BCL2L11 and over-expression of si-BCL2L11. Cell proliferation, colony formation and apoptosis were respectively assessed using CCK-8 assay, colony assay and flow cytometry. Luciferase reporter assay was performed to verify the targets of ACTA2-AS1 and miR-4428. Tumor subcutaneous xenograft mode was constructed to explore tumor growth in vivo. Results ACTA2-AS1 was obviously downregulated in human colon adenocarcinoma tissues and colon adenocarcinoma cell lines. Silence or over-expression of ACTA2-AS1 promoted or inhibited cell proliferation and colony formation abilities, and regulated apoptosis. The silence of ACTA2-AS1 resulted in the decrease of Bax and increase of Bal2, while restored in OE ACTA2-AS1 group when compared with the control transfected cells. In addition, luciferase reporter assay revealed that ACTA2-AS1 interacted with miR-4428 and suppressed its expression. miR-4428 could bind to 3ʹ untranslated region of BCL2L11 and modulated the expression of BCL2L11 negatively. Knockdown of ACTA2-AS1 and over-expression of BCL2L11 reversed the biological function that ACTA2-AS1 mediated by knockdown ACTA2-AS1 alone. Conclusion Our data demonstrated that ACTA2-AS1 could suppress colon adenocarcinoma progression via sponging miR-4428 to regulate BCL2L11 expression.

2016 ◽  
Vol 38 (6) ◽  
pp. 2500-2508 ◽  
Yang Liu ◽  
Yi Chai ◽  
Jian Zhang ◽  
Junwei Tang

Backgroud/Aims: Previous studies have shown that miR-501 is involved in the development of hepatocellular carcinoma (HCC) by promoting cell proliferation through CYLD. From the published MirSNP database that enrolls all single nucleotide polymorphisms(SNPs) of microRNA (miRNA), we found an interesting SNP (rs112489955, G>A) located in the mature region of miR-501. Methods: We performed a case-control study focusing on the predicted SNP located in miRNA-501 to investigate the further relationship of the SNPs with miRNAs among HCC patients. Genotyping, real time PCR assay, cell transfection and the dual luciferase reporter assay were used in our study. Results: Bioinformatic analysis indicated that this SNP would inhibit the binding of miR-501 to CYLD. In a case-control study, subjects with the variant genotypes (AG, GG) showed a significantly increased risk of HCC relative to AA carriers. A significant association of miR-501 variant genotypes with enhanced tumor growth was also observed. Further functional analyses indicated that patients with the AA genotype might attenuate the level of CYLD compared to that regulated by miR-501 with the GG genotype. A dual luciferase reporter assay also confirmed that miR-501 with the A allele had reduced binding to CYLD. We further confirmed a suppression of cell proliferation and promotion of apoptosis in SMMC-7721 and Hep3B cell lines treated with the AA genotype. Conclusions: We identified a novel SNP located in miR-501 acting as an important factor of the HCC susceptibility by modulating miR-501 and CYLD levels.

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