ABSTRACT
Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism and involved in many diseases, including cancer. CFIm25, a subunit of the cleavage factor I encoded by NUDT21, is required for 3'RNA cleavage and polyadenylation. Although it has been recently reported to be involved in glioblastoma tumor suppression, its roles and the underlying functional mechanism remain unclear in other types of cancer. In this study, we characterized NUDT21 in hepatocellular carcinoma (HCC). Reduced expression of NUDT21 was observed in HCC tissue compared to adjacent non-tumorous compartment. HCC patients with lower NUDT21 expression have shorter overall and disease-free survival times than those with higher NUDT21 expression after surgery. Knockdown of NUDT21 promotes HCC cell proliferation, metastasis, and tumorigenesis, whereas forced expression of NUDT21 exhibits the opposite effects. We then performed global APA site profiling analysis in HCC cells and identified considerable number of genes with shortened 3'UTRs upon the modulation of NUDT21 expression. In particular, we further characterized the NUDT21-regulated genes PSMB2 and CXXC5. We found NUDT21 knockdown increases usage of the proximal polyadenylation site in the PSMB2 and CXXC5 3' UTRs, resulting in marked increase in the expression of PSMB2 and CXXC5. Moreover, knockdown of PSMB2 or CXXC5 suppresses HCC cell proliferation and invasion. Taken together, our study demonstrated that NUDT21 inhibits HCC proliferation, metastasis and tumorigenesis, at least in part, by suppressing PSMB2 and CXXC5, and thereby provided a new insight into understanding the connection of HCC suppression and APA machinery.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Carrier Proteins/genetics , Cleavage And Polyadenylation Specificity Factor/genetics , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Proteasome Endopeptidase Complex/genetics , 3' Untranslated Regions/genetics , Animals , Carcinogenesis/genetics , Cell Line, Tumor , Cell Proliferation/genetics , DNA-Binding Proteins , Disease-Free Survival , Gene Expression Regulation, Neoplastic/genetics , Humans , Male , Mice , Mice, Nude , Polyadenylation/genetics , Transcription FactorsABSTRACT
Tamoxifen-resistant (TAMR) estrogen receptor-positive (ER+) breast cancer is characterized by elevated Erb-B2 receptor tyrosine kinase 2 (ERBB2) expression. However, the underlying mechanisms responsible for the increased ERBB2 expression in the TAMR cells remain poorly understood. Herein, we reported that the ERBB2 expression is regulated at the post-transcriptional level by miR26a/b and the RNA-binding protein human antigen R (HuR), both of which associate with the 3'-UTR of the ERBB2 transcripts. We demonstrated that miR26a/b inhibits the translation of ERBB2 mRNA, whereas HuR enhances the stability of the ERBB2 mRNA. In TAMR ER+ breast cancer cells with elevated ERBB2 expression, we observed a decrease in the level of miR26a/b and an increase in the level of HuR. The forced expression of miR26a/b or the depletion of HuR decreased ERBB2 expression in the TAMR cells, resulting in the reversal of tamoxifen resistance. In contrast, the inactivation of miR26a/b or forced expression of HuR decreased tamoxifen responsiveness of the parental ER+ breast cancer cells. We further showed that the increase in HuR expression in the TAMR ER+ breast cancer cells is attributable to an increase in the HuR mRNA isoform with shortened 3'-UTR, which exhibits increased translational activity. This shortening of the HuR mRNA 3'-UTR via alternative polyadenylation (APA) was observed to be dependent on cleavage stimulation factor subunit 2 (CSTF2/CstF-64), which is up-regulated in the TAMR breast cancer cells. Taken together, we have characterized a model in which the interplay between miR26a/b and HuR post-transcriptionally up-regulates ERBB2 expression in TAMR ER+ breast cancer cells.
Subject(s)
3' Untranslated Regions/drug effects , Breast Neoplasms/drug therapy , Drug Resistance, Neoplasm , ELAV-Like Protein 1/metabolism , MicroRNAs/metabolism , Receptor, ErbB-2/metabolism , Tamoxifen/pharmacology , Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cleavage Stimulation Factor , Female , Humans , MicroRNAs/antagonists & inhibitors , Mutation , Neoplasm Proteins/agonists , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Polyadenylation/drug effects , RNA Interference , RNA Stability/drug effects , RNA, Messenger/agonists , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Neoplasm/agonists , RNA, Neoplasm/antagonists & inhibitors , RNA, Neoplasm/chemistry , RNA, Neoplasm/metabolism , RNA-Binding Proteins/agonists , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptor, ErbB-2/agonists , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-2/genetics , Response Elements/drug effects , Up-Regulation/drug effectsABSTRACT
We analyzed the anti-inflammatory and antinociceptive activities of total flavonoids (TF) found in black mulberry fruits. The TF content was 20.9 mg/g (dry weight). Two anthocyanins, cyanidin-3-O-glucoside (8.3 mg/g) and cyanidin-3-O-rutinoside (2.9 mg/g), were identified in the fruits by UPLC. The TF of black mulberry fruits had significant reducing power and radical (OH(-), O2(·-), DPPH and ABTS) scavenging activities that was demonstrated in a dose-response curve. The TF had inhibitory activities on xylene-induced ear edema and carrageenan-induced paw edema in mice. In addition, TF had antinociceptive activities in the two nociceptive phases of formalin test. We used ELISA to detect the pro-inflammatory cytokines IL-1ß, TNF-α, IFN-γ, and NO in the serum of mice. These cytokines were significantly inhibited or scavenged by TF (50 and 100 mg/kg). The results demonstrated that TF of black mulberry possess anti-inflammatory and analgesic effects that might correlate to its antioxidant activities and inhibition of pro-inflammatory cytokines.
