ABSTRACT
BACKGROUND: Hepatocellular carcinoma (HCC) is an extremely aggressive malignant tumor associated with high migratory and invasive potential. The present study intends to explore regulatory mechanism of p53/microRNA (miR)-29c-3p/A disintegrin and metalloproteinase 12 (ADAM12) axis in HCC based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. METHODS: Putative miR-29c-3p binding sites on ADAM12 3'UTR were verified by a luciferase assay. The binding affinity of p53 to miR-29c-3p was assessed based on CRISPR/Cas9 technology to construct a p53 knockout (p53-/-) HCCLM3 cell line. Furthermore, the effect of p53/miR-29c-3p/ADAM12 was assessed on maligant phenotypes in vitro and tumor formation and metastasis in nude mice. RESULTS: ADAM12 was highly expressed but miR-29c-3p was poorly expressed in HCC. miR-29c-3p inhibited migratory and invasive abilities of HCC cells by targeting ADAM12 expression. p53 was found to target and upregulate miR-29c-3p, thus downregulating ADAM12 and conferring inhibitory effect on HCC cell activities. Moreover, ADAM12 knockout or p53 overexpression reduced HCC tumor formation and metastasis, which were reversed by further silencing of miR-29c-3p. CONCLUSION: The identification of the p53/miR-29c-3p/ADAM12 axis in migration and invasion of HCC may potentially further our understanding of mechanisms underpinning HCC, and also bear translational value as novel molecular targets.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , MicroRNAs , Animals , Mice , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/pathology , Mice, Nude , MicroRNAs/genetics , Tumor Suppressor Protein p53/genetics , ADAM12 Protein/metabolismABSTRACT
Histone deacetylases (HDACs) are entwined with the pathogenesis of various cancers and potentially serve as promising therapeutic targets. Herein, we intend to explore the potential role of HDAC1 inhibitor (JSL-1) in the tumorigenesis and metastasis of cholangiocarcinoma (CC) and to highlight the molecular basis of its function. As shown by bioinformatics analysis and immunohistochemical detection, high HDAC1 expression was witnessed in CC tissues relative to matched controls from patients with cholecystitis. The molecular network that HDAC1 silencing reduced the enrichment of HDAC1 and Snail on the TPX2 promoter was identified using immunoprecipitation and chromatin immunoprecipitation assays. Both short hairpin RNA (shRNA)-mediated knockdown of HDAC1 and JSL-1 treatment exhibited anti-proliferative, anti-migration and anti-invasion effects on CC cells through downregulation of TPX2. The in vivo xenograft model was developed in nude mice. Consistently, the anti-tumorigenic and anti-metastatic properties of shRNA against HDAC1 and HDAC1 inhibitor were validated in the in vivo settings. Taken together, our data supported the notion that HDAC1 inhibitor retards the initiation and development of CC via mediating the TPX2/Snail axis, highlighting the anti-tumor molecular network functioned in CC.
Subject(s)
Cholangiocarcinoma , Histone Deacetylase 1 , Histone Deacetylase Inhibitors , Animals , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cholangiocarcinoma/genetics , Histone Deacetylase 1/antagonists & inhibitors , Histone Deacetylase 1/genetics , Histone Deacetylase Inhibitors/pharmacology , Humans , Mice , Mice, Nude , Microtubule-Associated Proteins/genetics , Neoplasm Metastasis , RNA, Small Interfering , Snail Family Transcription Factors/geneticsABSTRACT
Gallbladder cancer is a malignant tumor with a high mortality rate. Accumulating evidence supports that lncRNA MEG3 may halt the progression of gallbladder cancer, while the downstream mechanism is rarely studied. Thus, we aim to investigate the molecular basis of the tumor-suppressing role of lncRNA MEG3 in gallbladder cancer. The expression of lncRNA MEG3 and CXCL3 was measured in patient serum and cell lines of gallbladder cancer. The viability, apoptosis, migration, and invasion of gallbladder cancer cells were assessed following ectopic MEG3 expression, as detected by CCK-8, flow cytometry, and Transwell assays. The interaction among lncRNA MEG3, EZH2, and CXCL3 was explored through ChIP, RNA pull-down, and RIP assays. The effects of lncRNA MEG3 and CXCL3 on tumor growth were evaluated by a mouse xenograft model. lncRNA MEG3 was expressed at a low level in gallbladder cancer patient serum and cell lines, while CXCL3 was highly expressed. MEG3 overexpression repressed the malignant behaviors of gallbladder cancer cells and promoted their apoptosis. MEG3 was mainly localized in the nucleus. MEG3 bound to EZH2, and EZH2 catalyzed the H3K27 trimethylation of the CXCL3 promoter region. MEG3 downregulated CXCL3 by activating EZH2-mediated H3K27 trimethylation of CXCL3; MEG3 overexpression attenuated cancer cell malignant behaviors in vitro and suppressed tumor growth in vivo in gallbladder cancer by inhibiting CXCL3 expression. Altogether, our results indicate that lncRNA MEG3 impedes gallbladder cancer development via the EZH2-CXCL3 axis, offering potential biomarkers for gallbladder cancer management.
Subject(s)
Chemokines, CXC , Enhancer of Zeste Homolog 2 Protein , Gallbladder Neoplasms , RNA, Long Noncoding , Animals , Apoptosis/physiology , Cell Line, Tumor , Cell Proliferation/physiology , Chemokines, CXC/genetics , Chemokines, CXC/metabolism , DNA Methylation , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Gallbladder Neoplasms/genetics , Gallbladder Neoplasms/metabolism , Gallbladder Neoplasms/pathology , Heterografts , Humans , Methylation , Mice , Promoter Regions, Genetic , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolismABSTRACT
BACKGROUND: Recent studies have emphasized determining the ability of microRNAs (miRNAs) as crucial regulators in the occurrence and development of pancreatic cancer (PC), which continues to be one of the deadliest malignancies with few effective therapies. The study aimed to investigate the functional role of miR-135b and its associated mechanism to unravel the biological characteristics of tumor growth in pancreatic cancer stem cells (PCSCs). METHODS: Microarray analyses were initially performed to identify the PC-related miRNAs and genes. The expression of miR-135b and PCSC markers in PC tissues and cells was determined by RT-qPCR and western blot analysis, respectively. The potential gene (JADE-1) that could bind to miR-135b was confirmed by the dual-luciferase reporter assay. To investigate the tumorigenicity, migration, invasion, and stemness of PC cells, several gain-of-function and loss-of-function genetic experiments were conducted. Finally, tumor formation in nude mice was conducted to confirm the results in vivo. RESULTS: miR-135b was highly-expressed in PC tissues and PCSCs, which was identified to specifically target JADE-1. The overexpression of miR-135b promoted proliferation, migration, and invasion of PCSC, inhibited cell apoptosis and increased the expression of stemness-related factors (Sox-2, Oct-4, Nanog, Aldh1, and Slug). Moreover, miR-135b could promote the expression of phosphorylated AKT and phosphorylated mTOR in the AKT/mTOR pathway. Additionally, miR-135b overexpression accelerated tumor growth in nude mice. CONCLUSIONS: Taken together, the silencing of miR-135b promotes the JADE-1 expression, which inactivates the AKT/mTOR pathway and ultimately results in inhibition of self-renewal and tumor growth of PCSCs. Hence, this study contributes to understanding the role of miR-135 in PCSCs and its underlying molecular mechanisms to aid in the development of effective PC therapeutics.
