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Graduate School of Ajou University Department of Biomedical Sciences 4. Theses(Ph.D)

Epigenetic regulation of BTG2/TIS21/PC3 and its tumor suppressive role in invasive human cancers

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dc.contributor.advisorIn Kyoung Lim-
dc.contributor.authorPreethi Devanand-
dc.date.accessioned2019-10-21T07:30:58Z-
dc.date.available2019-10-21T07:30:58Z-
dc.date.issued2018-02-
dc.identifier.other27461-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/19113-
dc.description학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2018. 2-
dc.description.tableofcontentsABSTRACT ......................................................... i TABLE OF CONTENTS ........................................................iii LIST OF FIGURES ........................................................vi ABBREVIATIONS ......................................................viii I. INTRODUCTION ..........................................................1 II. EXPERIMENTAL METHODS ..........................................................7 A. Tissues and cell cultures ..........................................................7 B. RNA isolation and reverse transcription ..........................................................8 C. Real-time and RT-PCR analyses ..........................................................8 D. Methylation-specific PCR (MSP) and unmethylation-specific PCR (USP) analyses..........................................................................................................9 E. Western blotting ..........................................................9 F. DNMT activity assay ........................................................10 G. Chromatin immunoprecipitation (ChIP) analysis..............................................10 H. Cloning of CpG islands and sequencing analyses.............................................10 I. Immunoprecipitation/immunoblot analysis ........................................................11 J. Regulation of gene expression ........................................................11 K. Tumorigenesis study ........................................................12 L. Invasion assay ........................................................13 M. Immunohistochemistry analysis ........................................................14 N. Preparation of recombinant Tipα protein ........................................................15 O. Subcellular fractionation .......................................................15 P. Statistical analysis .......................................................15 III. RESULTS .......................................................16 A. Downregulation of BTG2 expression in human MIBC by DNA methylation of BTG2 gene........................................................................................................16 B. Inverse correlations between the expressions of BTG2 vs DNMT1 and DNMT3a in MIBC.........................................................................................18 C. Upregulation of BTG2 expression in EJ bladder cancer cells upon Decitabine treatment.............................................................................................22 D. Chromatin remodeling at the promoter and intron of BTG2 gene after decitabine treatment..............................................................................................27 E. Sp1, the transcription factor, for BTG2 gene upon decitabine treatment.........28 F. Induction of BTG2 expression by knockdown of DNMT1 in EJ cells.............32 G. Downregulation of tumorigenesis and cell cycle arrest by BTG2 overexpression .......................................................35 H. Inhibition of tumor invasion by BTG2 via downregulation of DNMT1 expression ......................................................38 I. Direct effect of BTG2/TIS21 expression on the inhibition of cancer invasiveness ......................................................41 J. Immunohistochemical findings ......................................................44 K. Induction of BTG2 expression by decitabine treatment in various cancer cell lines in addition to APRO gene expression analyses...............46 L. Expression of BTG2/TIS21 is increased in mucous epithelium infected with H. pylori, but lost in human gastric adenocarcinoma...................................48 M. Absence of endogenous BTG2/TIS21 expression in the gastric cancer cells ......................................................51 N. Expression of BTG2/TIS21 is epigenetically regulated in gastric adenocarcinoma ......................................................52 O. Inhibition of cancer cell proliferation by BTG2/TIS21 gene..............................54 P. Inhibition of Tipα activity by BTG2/TIS21 in human gastric cancer cells.........55 Q. Inhibition of Tipα activity by downregulation p-ERK1/2 in human gastric cancer cells .....................................................57 R. Downregulation of nucleolin expression by BTG2/TIS21.................................59 S. Expression of nucleolin, Tipα receptor, was reduced by BTG2 expression via inhibiting Sp1binding to the nucleolin promoter.........................61 T. Inverse regulation of Tipα -induced TNFα expression by NCL and BTG2 .....................................................64 U. Reciprocal expression of BTG2/TIS21 and nucleolin expressions in normal and cancer regions…................................................................................68 V. Inverse regulation of overall survival of gastric cancer patients by BTG2/TIS21 and NCL genes..................................................................................71 IV. DISCUSSION .....................................................74 V. SUMMARY .....................................................80 VI. CONCLUSIONS .....................................................82 VII. REFERENCES .....................................................83 국문요약 .................................................95-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.titleEpigenetic regulation of BTG2/TIS21/PC3 and its tumor suppressive role in invasive human cancers-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.department일반대학원 의생명과학과-
dc.date.awarded2018. 2-
dc.description.degreeDoctoral-
dc.identifier.localId800314-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000027461-
dc.description.alternativeAbstractB cell translocation gene 2 (BTG2/TIS21/PC3) belongs to the family of antiproliferative (APRO) genes and reported as a tumor suppressor by our group and others. Expression of BTG2 is significantly reduced in cancers developed in various organs and tissues. EJ (bladder carcinoma cells), MKN-1 (gastric cancer cells) are highly invasive and metastatic cells with very less endogenous BTG2 expression due to epigenetic regulation. Significantly lower endogenous expression of BTG2 was observed in human muscle-invasive bladder cancers (MIBC) than matched normal tissues and non-muscle invasive bladder cancers (NMIBC). BTG2 expression was inversely correlated with increased expression of the DNA methyltransferases DNMT1 and DNMT3a in MIBC, but not NMIBC, suggesting a potential role for BTG2 expression in muscle invasion of bladder cancer. Over 90% of tumor tissues revealed strong methylation at CpG islands of the BTG2 gene, compared with no methylation in the normal tissues, implying epigenetic regulation of BTG2 expression in bladder carcinogenesis. BTG2 is constitutively expressed in mucous epithelium and parietal cells of gastric glands in stomach, and the expression was increased in mucous epithelium with H. pylori infection as opposed to loss in human gastric adenocarcinoma. Indeed, adenoviral transduction of BTG2 significantly inhibited Tipα activity in MKN-1 and MGT-40, human and mouse gastric cancer cells, respectively, thereby downregulated TNFα expression and Erk1/2 phosphorylation via reducing nucleolin, Tipα receptor, expression. Chromatin immunoprecipitation proved that BTG2 inhibited Sp1 expression and it’s binding to the promoter of nucleolin gene. In addition, BTG2 expression significantly reduced membrane localized nucleolin expression in cancer cells and the loss of BTG2/TIS21 expression rather induced cytoplasmic nucleolin availability in gastric cancer tissues, evidenced by immunoblot and immunohistochemistry. The higher expression of BTG2 and the lower nucleolin expression accompanied with the better overall survival of the poorly differentiated gastric cancer patients.-
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