ABSTRACT
Esophageal cancer (EC) incidence remains to be on a global rise supported by an unchanged recurrence and 5-year survival rate owing to the development of chemoresistance. Resistance to cisplatin, one of the majorly used chemotherapeutic drugs in EC, is a major nuisance. This study sheds light on miRNA dysregulation and its inverse relation with dysregulated mRNAs to guide pathways into the manifestation of cisplatin resistance in EC. A cisplatin-resistant version of an EC cell line was established and comparative profiling by NGS with the parental cell line was employed to identify dysregulation in miRNA and mRNA levels. Protein-protein interaction network analysis was done using Cytoscape, followed by Funrich pathway analysis. Furthermore, selective significant miRNAs were validated using qRT-PCR. miRNA-mRNA integrated analysis was carried out using the Ingenuity Pathway Analysis (IPA) tool. Expression of various established resistance markers supported the successful establishment of cisplatin-resistant cell line. Whole-cell small RNA sequencing and transcriptome sequencing identified 261 miRNAs and 1892 genes to be significantly differentially expressed (DE), respectively. Pathway analysis indicated enrichment of EMT signaling, supported by NOTCH, mTOR, TNF receptor, and PI3K-mediated AKT signaling pathways, in chemoresistant cells. Validation by qRT-PCR confirmed upregulation of miR-10a-5p, miR-618, miR-99a-5p, and miR-935 and downregulation of miR-335-3p, miR-205-5p, miR-944, miR-130a-3p, and miR-429 in resistant cells. Pathway analysis that followed IPA analysis indicated that the dysregulation of these miRNAs and their target genes may be instrumental in the development and regulation of chemoresistance via p53 signaling, xenobiotic metabolism, and NRF2-mediated oxidative stress. This study concludes the interplay between miRNA and mRNA as an important aspect and occurrence in guiding the regulation, acquisition, and maintenance of chemoresistance in esophageal cancer in vitro.
Subject(s)
Esophageal Neoplasms , MicroRNAs , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Cisplatin/pharmacology , Cisplatin/metabolism , Cisplatin/therapeutic use , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/genetics , Cell Line , RNA, Messenger/genetics , Gene Expression Profiling , Gene Expression Regulation, NeoplasticABSTRACT
BACKGROUND: Esophageal cancer is an aggressive malignancy. miR-335-5p is reported to possess both tumour suppressor and tumour promoter activities in different cancers. OBJECTIVES: We investigated the role of miR-335-5p in esophageal cancer by expression and functional studies. MATERIALS AND METHODS: The role of miR-335-5p in ESCC was evaluated using MTT assay, cell cycle analysis, colony formation assay, scratch assay, matrigel invasion, and migration assay. RESULTS: Our expression studies showed a significantly decreased expression of tissue and circulating miR-335-5p in esophageal cancer. Our results herein report a key tumour suppressive role of miR-335-5p in esophageal carcinogenesis by inhibiting proliferation, migration, and invasion in ESCC cells. Using RNA-seq and Insilico analysis we found TTK to be a newly identified direct target and confirmed it by using luciferase assay. CONCLUSION: Overall, our expression and functional analysis results demonstrated herein point towards the potential role of miR-335-5p in esophageal tumorigenesis. Moreover, this is the first report showing TTK as a downstream target of miR-335-5p.
Subject(s)
Cell Cycle Proteins , Esophageal Squamous Cell Carcinoma , MicroRNAs , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Esophageal Neoplasms/genetics , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/metabolism , Esophageal Squamous Cell Carcinoma/pathology , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Neoplasm Invasiveness , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolismABSTRACT
Geminiviral replication initiator protein (Rep) is a key player in geminiviral rolling circle mode of replication. However, the virus exploits various host cellular machineries for its replication. Study of these host factors is important to understand the geminiviral DNA replication in greater details. With this view, we screened for the peptides interacting with the Rep protein of a representative of geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), employing phage display technique. Through this screen, we have identified a host transcription factor, NAC083, as a potential MYMIV-Rep-binding partner. In silico docking studies also suggested possible binding of NAC083 peptide to MYMIV-Rep. We validated the interaction between MYMIV-Rep and Arabidopsis thaliana full-length NAC083 protein using in vitro pull-down assay and yeast two-hybrid analysis. NAC proteins are well-known transcription factors belonging to the largest gene families in plants. This study demonstrates for the first time the interaction of NAC083, a member of NAC transcription factor family, with MYMIV-Rep protein thereby indicating its possible role in MYMIV DNA replication.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Arabidopsis/virology , Begomovirus/metabolism , DNA Helicases/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Begomovirus/genetics , DNA Helicases/genetics , Models, Molecular , Molecular Sequence Data , Protein Binding , Sequence Alignment , Trans-Activators/genetics , Transcription Factors/chemistry , Transcription Factors/genetics , Viral Proteins/geneticsABSTRACT
Geminiviruses replicate their single-stranded genomes with the help of only a few viral factors and various host cellular proteins primarily by rolling-circle replication (RCR) and/or recombination-dependent replication. AtRAD51 has been identified, using the phage display technique, as a host factor that potentially interacts with the Rep protein of mungbean yellow mosaic India virus (MYMIV), a member of the genus Begomovirus. In this study, we demonstrate the interaction between MYMIV Rep and a host factor, AtRAD51, using yeast two-hybrid and ß-galactosidase assays, and this interaction was confirmed using a co-immunoprecipitation assay. The AtRAD51 protein complemented the rad51∆ mutation of Saccharomyces cerevisiae in an ex vivo yeast-based geminivirus DNA replication restoration assay. The semiquantitative RT-PCR and northern hybridization data revealed a higher level of expression of the Rad51 transcript in MYMIV-infected mungbean than in uninfected, healthy plants. Our findings provide evidence for a possible cross-talk between RAD51 and MYMIV Rep, which essentially controls viral DNA replication in plants, presumably in conjunction with other host factors. The present study demonstrates for the first time the involvement of a eukaryotic RAD51 protein in MYMIV replication, and this is expected to shed light on the machinery involved in begomovirus DNA replication.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/virology , Begomovirus/metabolism , DNA Replication , Host-Pathogen Interactions , Rad51 Recombinase/metabolism , Amino Acid Sequence , Animals , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Begomovirus/genetics , DNA Helicases/genetics , DNA Helicases/metabolism , DNA, Viral/biosynthesis , DNA, Viral/genetics , DNA, Viral/metabolism , Immunoprecipitation , Models, Molecular , Molecular Sequence Data , Rad51 Recombinase/chemistry , Rad51 Recombinase/genetics , Sequence Alignment , Sequence Analysis, DNA , Two-Hybrid System Techniques , Viral Proteins/genetics , Viral Proteins/metabolism , Virus ReplicationABSTRACT
Geminiviruses are plant pathogens with single-stranded (ss) DNA genomes of about 2.7 kb in size. They replicate primarily via rolling-circle replication (RCR) with the help of a few virally encoded factors and various host-cell machineries. The virally encoded replication initiator protein (Rep) is essential for geminivirus replication. In this study, by interaction screening of an Arabidopsis thaliana cDNA library, we have identified a host factor, MCM2, that interacts with the Rep protein of the geminivirus mungbean yellow mosaic India virus (MYMIV). Using yeast two-hybrid, ß-galactosidase and co-immunoprecipitation assays, we demonstrated an interaction between MYMIV-Rep and the host factor AtMCM2. We investigated the possible role of AtMCM2 in geminiviral replication using a yeast-based geminivirus DNA replication restoration assay and observed that the AtMCM2 protein complemented the mcm2∆ mutation of S. cerevisiae. Our data suggest the involvement of AtMCM2 in the replication of MYMIV ex vivo. The role of MCM2 in replication was confirmed in planta by a transient replication assay in both wild-type and mutant Arabidopsis plants through agroinoculation. Our data provide evidence for the involvement of AtMCM2 in geminiviral DNA replication, presumably in conjunction with other host factors, and suggest its importance in MYMIV DNA replication.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/virology , Begomovirus/physiology , DNA Helicases/metabolism , DNA Replication , Host-Pathogen Interactions , Trans-Activators/metabolism , Arabidopsis Proteins/genetics , Begomovirus/pathogenicity , Gene Deletion , Genetic Complementation Test , Immunoprecipitation , Protein Interaction Mapping , Saccharomyces cerevisiae/enzymology , Two-Hybrid System Techniques , beta-Galactosidase/analysisABSTRACT
Geminiviruses primarily encode only few factors, such as replication initiator protein (Rep), and need various host cellular machineries for rolling-circle replication (RCR) and/or recombination-dependent replication (RDR). We have identified a host factor, RAD54, in a screen for Rep-interacting partners and observed its role in DNA replication of the geminivirus mungbean yellow mosaic India virus (MYMIV). We identified the interacting domains ScRAD54 and MYMIV-Rep and observed that ScRAD54 enhanced MYMIV-Rep nicking, ATPase, and helicase activities. An in vitro replication assay demonstrated that the geminiviral DNA replication reaction depends on the viral Rep protein, viral origin of replication sequences, and host cell-cycle proteins. Rad54-deficient yeast nuclear extract did not support in vitro viral DNA replication, while exogenous addition of the purified ScRAD54 protein enhanced replication. The role of RAD54 in in planta replication was confirmed by the transient replication assay; i.e., agroinoculation studies. RAD54 is a well-known recombination/repair protein that uses its DNA-dependent ATPase activity in conjunction with several other host factors. However, this study demonstrates for the first time that the eukaryotic rolling-circle replicon depends on the RAD54 protein.
