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postgraduate thesis: Role of miR-338-5p in targeting Id-1 and modulating esophageal cancer cell invasion and chemoresistance

TitleRole of miR-338-5p in targeting Id-1 and modulating esophageal cancer cell invasion and chemoresistance
Authors
Issue Date2015
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Han, L. [韩亮]. (2015). Role of miR-338-5p in targeting Id-1 and modulating esophageal cancer cell invasion and chemoresistance. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5689261
AbstractEsophageal cancer is the eighth most frequent cancer and the sixth leading cause of cancer mortality in the world. Esophageal squamous cell carcinoma (ESCC) is the predominant histologic subtype of esophageal cancer in Asia. Late diagnosis, metastasis and resistance to chemoradiotherapy all contribute to the low survival rate. Inhibitor of differentiation or DNA binding (Id-1) is overexpressed in esophageal cancer and activates the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway, which plays important roles in regulating a wide range of cellular functions including cell proliferation, survival and angiogenesis. In this study, tumor suppressive microRNAs (miRNAs) targeting Id-1 in esophageal cancer were identified. Ten miRNAs containing seed sequences complementary to the 3'UTR of Id-1 were identified using bioinformatics software. Amongst these miRNAs, western blot analysis and dual luciferase assay showed that miR-29b, miR-29c and miR-338-5p could suppress Id-1 by direct binding to its 3'UTR. MTT cell proliferation, colony formation, wound healing and invasion chamber assays were used to show that miR-29b and miR-29c exerted suppressive effects on ESCC cell proliferation, migration and invasion in vitro. Importantly, qRT-PCR and western blot analyses showed that miR-338-5p, which became the focus of this study, was downregulated whereas Id-1 was upregulated in 5-fluorouracil (5-FU) resistant (FR) esophageal cancer cells. Forced overexpression of miR-338-5p resulted in downregulation of endogenous Id-1 in the FR cells. MTT cell proliferation assay, western blot analysis of cleaved caspase-3 and flow cytometry analysis of sub-G1 apoptotic cell population showed that overexpression of miR-338-5p could sensitize FR esophageal cancer cells to 5-FU treatment in vitro, and that these effects could be partially abolished by overexpression of Id-1. On the other hand, knockdown of miR-338-5p induced 5-FU resistance in the chemosensitive esophageal cell lines in vitro, while knockdown of Id-1 could partly reduce the resistance to 5-FU. In addition, the role of miR-338-5p in chemoresistance was verified in vivo by tumor xenograft study. Furthermore, wound healing and transwell cell migration assays were performed to demonstrate that miR-338-5p reduced the migration and invasion properties of esophageal cancer cells, and inhibition of miR-338-5p showed opposite effects. These anti-metastasis related effects of miR-338-5p were partially mediated through regulating Id-1. Hypermethylation of pri-miR-338 promoter was identified by methylation-specific PCR in the parental ESCC cells and the FR sublines, and treatment with 5-aza-2'-deoxycytidine restored the expression level of miR-338-5p and downregulated Id-1. Analysis of ESCC clinical samples showed that miR-338-5p was significantly downregulated in the ESCC tumors compared with the adjacent non-tumorous esophageal tissue. Moreover, the expression level of miR-338-5p was inversely correlated with Id-1 in ESCC tumor and non-tumorous esophageal tissue. Taken together, these results suggest that miR-338-5p can directly and negatively regulate Id-1 to play important inhibitory roles in invasion and chemoresistance of esophageal cancer. The findings of this study provide new insights into the roles of miRNAs in esophageal cancer, and may also aid the development of miRNA-based therapies for advanced and chemoresistant esophageal cancer.
DegreeDoctor of Philosophy
SubjectDrug resistance in cancer cells
Cancer invasiveness
Small interfering RNA
Esophagus - Cancer - Chemotherapy
Dept/ProgramBiomedical Sciences
Persistent Identifierhttp://hdl.handle.net/10722/235748

 

DC FieldValueLanguage
dc.contributor.authorHan, Liang-
dc.contributor.author韩亮-
dc.date.accessioned2016-10-14T23:25:48Z-
dc.date.available2016-10-14T23:25:48Z-
dc.date.issued2015-
dc.identifier.citationHan, L. [韩亮]. (2015). Role of miR-338-5p in targeting Id-1 and modulating esophageal cancer cell invasion and chemoresistance. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5689261-
dc.identifier.urihttp://hdl.handle.net/10722/235748-
dc.description.abstractEsophageal cancer is the eighth most frequent cancer and the sixth leading cause of cancer mortality in the world. Esophageal squamous cell carcinoma (ESCC) is the predominant histologic subtype of esophageal cancer in Asia. Late diagnosis, metastasis and resistance to chemoradiotherapy all contribute to the low survival rate. Inhibitor of differentiation or DNA binding (Id-1) is overexpressed in esophageal cancer and activates the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway, which plays important roles in regulating a wide range of cellular functions including cell proliferation, survival and angiogenesis. In this study, tumor suppressive microRNAs (miRNAs) targeting Id-1 in esophageal cancer were identified. Ten miRNAs containing seed sequences complementary to the 3'UTR of Id-1 were identified using bioinformatics software. Amongst these miRNAs, western blot analysis and dual luciferase assay showed that miR-29b, miR-29c and miR-338-5p could suppress Id-1 by direct binding to its 3'UTR. MTT cell proliferation, colony formation, wound healing and invasion chamber assays were used to show that miR-29b and miR-29c exerted suppressive effects on ESCC cell proliferation, migration and invasion in vitro. Importantly, qRT-PCR and western blot analyses showed that miR-338-5p, which became the focus of this study, was downregulated whereas Id-1 was upregulated in 5-fluorouracil (5-FU) resistant (FR) esophageal cancer cells. Forced overexpression of miR-338-5p resulted in downregulation of endogenous Id-1 in the FR cells. MTT cell proliferation assay, western blot analysis of cleaved caspase-3 and flow cytometry analysis of sub-G1 apoptotic cell population showed that overexpression of miR-338-5p could sensitize FR esophageal cancer cells to 5-FU treatment in vitro, and that these effects could be partially abolished by overexpression of Id-1. On the other hand, knockdown of miR-338-5p induced 5-FU resistance in the chemosensitive esophageal cell lines in vitro, while knockdown of Id-1 could partly reduce the resistance to 5-FU. In addition, the role of miR-338-5p in chemoresistance was verified in vivo by tumor xenograft study. Furthermore, wound healing and transwell cell migration assays were performed to demonstrate that miR-338-5p reduced the migration and invasion properties of esophageal cancer cells, and inhibition of miR-338-5p showed opposite effects. These anti-metastasis related effects of miR-338-5p were partially mediated through regulating Id-1. Hypermethylation of pri-miR-338 promoter was identified by methylation-specific PCR in the parental ESCC cells and the FR sublines, and treatment with 5-aza-2'-deoxycytidine restored the expression level of miR-338-5p and downregulated Id-1. Analysis of ESCC clinical samples showed that miR-338-5p was significantly downregulated in the ESCC tumors compared with the adjacent non-tumorous esophageal tissue. Moreover, the expression level of miR-338-5p was inversely correlated with Id-1 in ESCC tumor and non-tumorous esophageal tissue. Taken together, these results suggest that miR-338-5p can directly and negatively regulate Id-1 to play important inhibitory roles in invasion and chemoresistance of esophageal cancer. The findings of this study provide new insights into the roles of miRNAs in esophageal cancer, and may also aid the development of miRNA-based therapies for advanced and chemoresistant esophageal cancer.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.lcshDrug resistance in cancer cells-
dc.subject.lcshCancer invasiveness-
dc.subject.lcshSmall interfering RNA-
dc.subject.lcshEsophagus - Cancer - Chemotherapy-
dc.titleRole of miR-338-5p in targeting Id-1 and modulating esophageal cancer cell invasion and chemoresistance-
dc.typePG_Thesis-
dc.identifier.hkulb5689261-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiomedical Sciences-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b5689261-

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