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Title Page

Contents

ABSTRACT 10

INTRODUCTION 12

MATERIALS & METHODS 15

1. Cell culture 15

2. Three-Dimensional culture 15

3. Immunohistochemistry (IHC) 16

4. Transfection of Small interfering RNA (siRNA) 16

5. Transfection of miRNA precursors & inhibitors 17

6. RNA isolation 17

7. MiRNA expression analysis 17

8. Western blotting analysis 18

9. 5-Aza-2'-deoxycytidine treatment 19

10. Purification of genomicDNA and bisulfite treatmentn 19

11. Methylation analysis (Pyrosequencing & MS-HRM assay) 19

12. Lentiviral microRNA systems 21

13. Immunofluorescence 21

14. Statistical analysis 22

RESULTS 23

1. Epithelial-to-mesenchymal transition (EMT) is associated with cyst development in ADPKD 23

2. miR-192 and miR-194 are downregulated in vivo in kidney tissues of both human ADPKD patients and Pkd1 conditional knockout (cKO) mice 29

3. miR-192/215 and miR-194 influence cyst growth 33

4. Epithelial-to-mesenchymal transition (EMT)-associated genes are differentially expressed by miR-192/215 and miR-194 36

5. Downregulation of miR-192 and miR-194 is related to DNA hypermethylation 39

6. Overexpression of miR-192 and miR-194 influences cyst progression in vivo 47

DISCUSSION 55

REFERENCES 58

ABSTRACT IN KOREAN 60

List of Tables

Table 1. Information of antibodies used western blotting 18

Table 2. Primer sequences for methylation-specific high resolution melting (MS-HRM) assay 42

List of Figures

Figure 1. Cyst development of Pkd1 conditional knockout (cKO) mice at all over the age. 25

Figure 2. The differential protein expression levels of epithelial-to-mesenchymal transition (EMT)-associated genes in P13 of Pkd1... 27

Figure 3. Inhibition of cyst growth by knockdown of epithelial-to-mesenchymal transition (EMT)-associated genes. 28

Figure 4. The expression levels of miR-192 and miR-194 in human kidney tissues. 30

Figure 5. The expression levels of miR-192 and miR-194 in Pkd1 conditional knockout (cKO) mice. 31

Figure 6. The expression levels of miR-192 and miR-194 in collecting duct cells isolated from the kidneys of Pkd1 conditional knockout (cKO) mouse. 32

Figure 7. Accelerated cyst growth by inhibition of miR-192/215 and miR-194. 34

Figure 8. Repression of cyst growth by overexpression of miR-192/215 and miR-194. 35

Figure 9. The differential expression of epithelial-to-mesenchymal transition (EMT)-associated genes by miR-192/215 and miR-194 inhibition. 37

Figure 10. The differential protein expression of epithelial-to-mesenchymal transition (EMT)-associated genes by miR-192/215 and miR-194... 38

Figure 11. Bisulfite pyrosequencing analysis of methylation level of hypermethylated MIR-192-194-2 in demethylating agent, 5-aza-dC,... 41

Figure 12. The normalized melting curves of the methylation-sensitive high resolution melting (MS-HRM) analysis of hypermethylated MIR-192-194-... 43

Figure 13. Mature and primary miR-192 and miR-194 expression levels upon 5-aza-dC treatment in WT9-12 cells. 44

Figure 14. The differential protein expression of epithelial-to-mesenchymal transition (EMT)-associated genes in 5-aza-dC treated WT9-12 cells. 45

Figure 15. Proposed schematic diagram on relation among miR-192/215 and miR-194, methylation, epithelial-to-mesenchymal transition (EMT), and... 46

Figure 16. The expression levels of miR-192 and miR-194 in collecting duct cells isolated from the kidneys of Pkd1 conditional knockout (cKO) mice. 49

Figure 17. The expression levels of miR-192 and miR-194 in lentivirus infected mIMCD cells. 50

Figure 18. Injection of lentiviral-overexpressed miR-192 and miR-194 in Pkd1 conditional knockout (cKO) mouse model. 51

Figure 19. The observation of cyst progression in Pkd1 conditional knockout (cKO) mice. 53

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