ABSTRACT
Long non-coding RNA HOX Transcript Antisense RNA (HOTAIR) is overexpressed in multiple cancers with diverse genetic profiles. Importantly, since HOTAIR heavily contributes to cancer progression by promoting tumor growth and metastasis, HOTAIR becomes a potential target for cancer therapy. However, the underlying mechanism leading to HOTAIR deregulation is largely unexplored. Here, we performed a pan-cancer analysis using more than 4,200 samples and found that intragenic exon CpG island (Ex-CGI) was hypermethylated and was positively correlated to HOTAIR expression. Also, we revealed that Ex-CGI methylation promotes HOTAIR expression through enhancing the transcription elongation process. Furthermore, we linked up the aberrant intragenic tri-methylation on H3 at lysine 4 (H3K4me3) and Ex-CGI DNA methylation in promoting transcription elongation of HOTAIR. Targeting the oncogenic CDK7-CDK9-H3K4me3 axis downregulated HOTAIR expression and inhibited cell growth in many cancers. To our knowledge, this is the first time that a positive feedback loop that involved CDK9-mediated phosphorylation of RNA Polymerase II Serine 2 (RNA PolII Ser2), H3K4me3, and intragenic DNA methylation, which induced robust transcriptional elongation and heavily contributed to the upregulation of oncogenic lncRNA in cancer has been demonstrated. Targeting the oncogenic CDK7-CDK9-H3K4me3 axis could be a novel therapy in many cancers through inhibiting the HOTAIR expression.
Subject(s)
Cyclin-Dependent Kinase 9 , Histones , Neoplasms , RNA Polymerase III , RNA, Long Noncoding , Cell Line, Tumor , Cyclin-Dependent Kinase 9/metabolism , DNA Methylation , Feedback, Physiological/physiology , Gene Expression Regulation, Neoplastic , Histones/metabolism , Humans , Neoplasms/genetics , Neoplasms/metabolism , RNA Polymerase III/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolismABSTRACT
BACKGROUND: Circular RNAs (circRNAs) play important roles in many biological processes. However, the detailed mechanism underlying the critical roles of circRNAs in cancer remains largely unexplored. We aim to explore the molecular mechanisms of circRTN4 with critical roles in pancreatic ductal adenocarcinoma (PDAC). METHODS: CircRTN4 expression level was examined in PDAC primary tumors. The oncogenic roles of circRTN4 in PDAC tumor growth and metastasis were studied in mouse tumor models. Bioinformatics analysis, luciferase assay and miRNA pulldown assay were performed to study the novel circRTN4-miRNA-lncRNA pathway. To identify circRTN4-interacting proteins, we performed circRNA-pulldown and mass spectrometry in PDAC cells. Protein stability assay and 3-Dimensional structure modeling were performed to reveal the role of circRTN4 in stabilizing RAB11FIP1. RESULTS: CircRTN4 was significantly upregulated in primary tumors from PDAC patients. In vitro and in vivo functional studies revealed that circRTN4 promoted PDAC tumor growth and liver metastasis. Mechanistically, circRTN4 interacted with tumor suppressor miR-497-5p in PDAC cells. CircRTN4 knockdown upregulated miR-497-5p to inhibit the oncogenic lncRNA HOTTIP expression. Furthermore, we identified critical circRTN4-intercting proteins by circRNA-pulldown in PDAC cells. CircRTN4 interacted with important epithelial-mesenchymal transition (EMT)- driver RAB11FIP1 to block its ubiquitination site. We found that circRTN4 knockdown promoted the degradation of RAB11FIP1 by increasing its ubiquitination. Also, circRTN4 knockdown inhibited the expression of RAB11FIP1-regulating EMT-markers Slug, Snai1, Twist, Zeb1 and N-cadherin in PDAC. CONCLUSION: The upregulated circRTN4 promotes tumor growth and liver metastasis in PDAC through the novel circRTN4-miR-497-5p-HOTTIP pathway. Also, circRTN4 stabilizes RAB11FIP1 to contribute EMT.
Subject(s)
Epithelial-Mesenchymal Transition , MicroRNAs/genetics , Nogo Proteins/genetics , Pancreatic Neoplasms/etiology , Pancreatic Neoplasms/metabolism , RNA, Circular , RNA, Long Noncoding/genetics , Adult , Aged , Animals , Biomarkers, Tumor , Cell Line, Tumor , Disease Models, Animal , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Middle Aged , Neoplasm Grading , Neoplasm Staging , Pancreatic Neoplasms/pathology , RNA InterferenceABSTRACT
The detailed biological functions of circular RNA (circRNA) are largely unexplored. Using circRNA sequencing, we identified 169 differentially expressed circRNA in pancreatic ductal adenocarcinoma (PDAC) cells compared with nontumor human pancreatic ductal epithelial cells. Among them, circFOXK2 was validated with significant upregulation in PDAC cells and 63% of primary tumors (53 of 84). circFOXK2 promoted cell growth, migration, and invasion and was involved in cell-cycle progression and apoptosis. circFOXK2 contained multiple miRNA binding sites, functioning as a sponge for miR-942, which in turn promoted expression of ANK1, GDNF, and PAX6. A novel and highly specific circRNA-pulldown followed by mass spectrometry analysis identified 94 circFOXK2-interacting proteins, which were involved in cell adhesion, mRNA splicing, and structural molecule activity. Of these, circFOKX2 interactions with YBX1 and hnRNPK enhanced expression of oncogenes NUF2 and PDXK. Knockdown of circFOXK2 reduced binding of YBX1 and hnRNPK to NUF2 and PDXK, in turn decreasing their expression. Collectively, our findings demonstrate that circFOXK2 in complex with YBX1 and hnRNPK promotes expression of oncogenic proteins that contribute to PDAC progression. SIGNIFICANCE: This study reveals a prominent role for the circRNA circFOXK2 in PDAC progression, suggesting that circFOXK2 might be a novel diagnostic marker for PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal/metabolism , Forkhead Transcription Factors/genetics , MicroRNAs/metabolism , Pancreatic Neoplasms/metabolism , RNA, Circular/metabolism , RNA-Binding Proteins/metabolism , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Growth Processes/physiology , Cell Line, Tumor , HEK293 Cells , Heterografts , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , Neoplasm Metastasis , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , RNA, Circular/genetics , RNA-Binding Proteins/geneticsABSTRACT
A colorimetric assay is developed for detection of lncRNA HOTTIP by one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) coupled with positively-charged gold nanoparticles ((+)AuNP) for diagnosis of pancreatic cancer. This assay allows simple, rapid, and sensitive quantification of lncRNA down to 50 copies.
