ABSTRACT
Hepatocellular carcinoma (HCC) poses an increasing threat to humans, due to its poor prognosis. Nuclearenriched abundant transcript 1 (NEAT1), a type of long noncoding (lnc)RNA, has been found to function in a variety of cancer types. However, the role of NEAT1 in HCC is poorly understood. Reverse transcriptionquantitative PCR was used to detect the expression levels of NEAT1, microRNA (miR)503 and Smoothened (SMO) mRNA in HCC tissues and cells. MTT and flow cytometry assays were used to investigate cell viability and apoptosis, respectively, while Transwell assays were performed to investigate cell invasion and migration. StarBase and TargetScan were utilized to predict the target sequence between miR503 and NEAT1 or SMO, the results of which were verified using a dualluciferase reporter assay. The protein expression level of SMO was measured using western blot. The RNA expression level of NEAT1 and SMO was significantly elevated in HCC tissues and cells compared with that in the corresponding healthy tissues and cells, which was contrary to miR503 expression level. NEAT1 silencing was found to restrict the viability, migration and invasion of the cells, while simultaneously induced apoptosis in the HCC cell line. Further studies found that miR503 expression was negatively correlated with NEAT1 or SMO. It was also confirmed that NEAT1 directly interacted with miR503 and miR503 could bind to the 3'untranslated region of SMO. Furthermore, overexpression of NEAT1 or SMO could reverse the effects of miR503mediated inhibition on cell viability, invasion, migration and promotion of apoptosis in the HCC cell lines. These results demonstrated that downregulation of NEAT1 impeded the viability, migration, invasion and induced apoptosis through the NEAT1/miR503/SMO axis in the HCC cell line.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Down-Regulation , Gene Expression Regulation, Neoplastic , Gene Silencing , Liver Neoplasms/metabolism , MicroRNAs/biosynthesis , Neoplasm Proteins/biosynthesis , RNA, Long Noncoding/biosynthesis , RNA, Neoplasm/biosynthesis , Smoothened Receptor/biosynthesis , Adult , Aged , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Female , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Male , MicroRNAs/genetics , Middle Aged , Neoplasm Proteins/genetics , RNA, Long Noncoding/genetics , RNA, Neoplasm/genetics , Smoothened Receptor/geneticsABSTRACT
Osteosarcoma (OS) is a malignant bone cancer lacking of effective treatment target when the metastasis occurred. This study investigated the implication of MicroRNA-326 in OS proliferation and metastasis to provide the clue for the treatment of metastatic OS. This study knocked down SP1 in MG63 and 143B cells and then performed Microarray assay to find the expression of miRNAs that were influenced by SP1. MTT, EdU, wound-healing and cell invasion assays were performed to evaluated cell proliferation and invasion. OS metastasis to lung was detected in a nude mice model. ChIP assay and DAPA were applied to determine the regulatory effect of SP1 and histone deacetylase 1 (HDAC) complex on miR-326 expression. Human OS tissues showed lowly expressed miR-326 but highly expressed Sp1 and HDAC. Sp1 recruited HDAC1 to miR-326 gene promoter, which caused the histone deacetylation and subsequent transcriptional inhibition of miR-326 gene. miR-326 deficiency induced the stimulation of SMO/Hedgehog pathway and promoted the proliferation and invasion of 143B and MG63 cells as well as the growth and metastasis in nude mice. SP1/HDAC1 caused the transcriptional inhibition of miR-326 gene by promoting histone deacetylation; miR-326 deficiency conversely stimulated SMO/Hedgehog pathway that was responsible for the proliferation and metastasis of OS.
Subject(s)
Bone Neoplasms/pathology , Histone Deacetylase 1/physiology , MicroRNAs/antagonists & inhibitors , Neoplasm Metastasis/physiopathology , Neoplasm Proteins/physiology , Osteosarcoma/pathology , RNA, Neoplasm/antagonists & inhibitors , Smoothened Receptor/biosynthesis , Sp1 Transcription Factor/physiology , Adolescent , Adult , Animals , Bone Neoplasms/genetics , Cell Division/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Female , Gene Knockdown Techniques , Histone Deacetylase 1/antagonists & inhibitors , Histone Deacetylase 1/genetics , Humans , Male , Matrix Metalloproteinase 9/physiology , Mice , Mice, Nude , MicroRNAs/biosynthesis , MicroRNAs/genetics , Neoplasm Metastasis/genetics , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Osteosarcoma/genetics , Osteosarcoma/secondary , RNA Interference , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Smoothened Receptor/genetics , Sp1 Transcription Factor/antagonists & inhibitors , Sp1 Transcription Factor/genetics , Xenograft Model Antitumor Assays , Young Adult , Zinc Finger Protein GLI1/physiologyABSTRACT
Previous studies report aberrant activation of the hedgehog signaling pathway in the progression of various cancers. This study aimed to investigate the expressions of smoothened and downstream glioma-associated oncogene homology-1 in gastric cancer and the underlying molecular mechanisms. Here, we first detected the expression in 58 cases of primary gastric cancer tissue and matched normal tissue specimens by western blot analysis and quantitative reverse transcription polymerase chain reaction. Cell proliferation and cycle were assayed in gastric cancer cells after blocking the hedgehog pathway by lentiviral-short hairpin RNA knockdown. In vitro inhibition of hedgehog pathway resulted in decreased cell proliferation and migration. Our studies demonstrate an important role for smoothened and glioma-associated oncogene homology-1 in gastric cancer and suggest inhibition of hedgehog pathway as a novel and potent strategy to treat gastric cancer patients.
Subject(s)
Smoothened Receptor/genetics , Stomach Neoplasms/genetics , Zinc Finger Protein GLI1/genetics , Cell Cycle/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Hedgehog Proteins/genetics , Humans , Lentivirus/genetics , Male , Signal Transduction , Smoothened Receptor/biosynthesis , Stomach Neoplasms/pathology , Zinc Finger Protein GLI1/biosynthesisABSTRACT
Accumulating evidence indicates that hedgehog signaling plays a pivotal role in pathological angiogenesis and is involved in wound-healing responses in a number of adult tissues, including the liver. We previously demonstrated that hedgehog signaling promoted proliferation and inhibited apoptosis in hepatic stellate cells. This study was aimed to evaluate the effect of tetramethylpyrazine (TMP) on hedgehog signaling and to further examine the molecular mechanisms of TMP-induced antiangiogenesic effects in liver fibrosis. We found that TMP ameliorated the expression of proangiogenic markers vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor 2 (VEGF-R2), platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor-ß receptor (PDGF-ßR) and hypoxia inducible factor 1α (HIF-1α), concomitant with reduced abundance of endothelial markers platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), CD34 and von willebrand factor in vivo and in vitro. Interestingly, TMP attenuated the abundance of sonic hedgehog, smoothened (Smo) and glioblastoma but increased the expression of hedgehog-interacting protein in liver sinusoidal endothelial cells, which was underlying mechanism for the antiangiogenesic activity of TMP. Downregulation of Smo activity, using selective Smo inhibitor cyclopamine, lead to a synergistic effect with TMP, whereas Smo overexpression plasmid impaired the induction of antiangiogenesic effects of TMP. Overall, these results provide novel implications to reveal the molecular mechanism of TMP-inhibited liver sinusoidal angiogenesis, by which points to the possibility of using TMP-based antiangiogenic drugs for the treatment of liver fibrosis. © 2017 IUBMB Life, 69(2):115-127, 2017.
