Title page
ACKNOWLEDGEMENT
CONTENTS
ABBREVIATION 10
ABSTRACT 11
Ⅰ. INTRODUCTION 13
Ⅱ. MATERIALS AND METHODS 15
Ⅱ. 1. Isolation of RNA 15
Ⅱ. 2. Synthesis of cDNA 15
Ⅱ. 3. Suppression Subtraction Hybridization 16
Ⅱ. 4. Analysis of Gene Expression by RT-PCR 17
Ⅱ. 5. Cell Line and Time Course 18
Ⅲ. Results 19
Ⅲ. 1. Identification of Novel Gene Overexpressed in Human Breast cancer using SSH 19
Ⅲ. 2. Differently Expression Gene in Breast Tissues and Cell Lines 25
Ⅲ. 3. Serum Stimulation in Normal WI-38 Fibroblasts 29
Ⅳ. Discussion 31
Ⅴ. Conclusion 33
References 34
Summary 38
Table 1. Identification of Known Tumor Specific Genes from SSH Libraries 21
Table 2. Identification of Unknown Tumor Specific Genes from SSH Libraries 23
Table 3. The Primer used in RT-PCR Analysis 26
Figure. 1. Scheme of the SSH method. Type e molecules are formed only if the sequence is up-regulated in the tester cDNA. Solid lines represent the Rsa I-digested tester or drive cDNA. Solid boxes represent the outer part of the Adaptor1 and 2R longer strands and corresponding PCR primer1 sequence. Clear boxes represent the inner part of Adaptor1 and the corresponding Nested PCR primer 1 sequence; shaded boxes represent the inner part of Adaptor 2R and the corresponding Nested PCR primer 2R sequence 20
Figure. 2. Reduction of GAPDH abundance by PCR-selected subtraction. PCR was performed on unsubtracted (Lane 1-4) or subtracted (Lane 5-8) PCR product with the GAPDH 5 ´and GAPDH 3´ primers included in the kit. Lane 1&5: 18cycles; Lane 2&6:23cycles; Lane 3&7: 28cycles; Lane 4&8: 33cycles. Lane M: marker 24
Figure. 3. Expression analysis of 3 Clones in various breast tissues by RT-PCR. In the five-pair sample, the expression of mRNA of clone is significantly higher than normal. The clones were determined to be highly expressed in mRNA of tumor tissues but only weak or no expression in most of the normal. N: normal breast tissues; T: breast tumor tissues; 18srRNA: control 27
Figure. 4. Expression analysis of 3 clones in various cell lines by RT-PCR. In human cancer cell lines, 3 clones were expressed in the SV40-immortalized (WI-38 VA13) cell line, lung carcinoma cell line (NCI-H596). "Clone 135" was expressed prostate carcinoma cell line (DU-145), and colon carcinoma (KM1214). Lane 1, normal lung (WI-38); lane 2, SV40-immortalizated (WI-38 VA13); lane 3, lung carcinoma ( NCI-H596); lane4, normal colon (CCD-18Co); lane 5, colon carcinoma (KM1214); lane 6, normal prostate (RWP 28
Figure. 5. Induction of expression of 3 clones in WI-38 fibroblast Cell The expression of 3 clones was significantly increased from 0.5-1hr after 10% FBS treatment, afterwards decreased in WI-38 cell of 10% FBS treatment for 2hr 30