臺灣博碩士論文加值系統

背景: 絲胺酸蛋白酶23是屬於肽酶S1家族之新穎蛋白。先前研究指出絲胺酸蛋白酶23在乳癌、甲狀腺癌、前列腺癌等癌症中有高度表現。然而，絲胺酸蛋白酶23在這些癌症之分子機制仍待證實。本實驗室先前的研究結果顯示，在表現雌激素受體甲型之乳癌細胞株(MCF-7)中，絲胺酸蛋白酶23會受到雌激素調控並且影響細胞之增生。另外，利用免疫組織染色法發現，絲胺酸蛋白酶23亦會表現於第一型子宮內膜腺癌中。因此，本研究乃探討子宮內膜癌細胞株中，絲胺酸蛋白酶23是否亦受到雌激素調控且影響其細胞之增生。

結果: 本研究發現絲胺酸蛋白酶23亦會表現於雌激素受體甲型的人類子宮內膜癌細胞株- Ishikawa。進一步以核酸干擾系統之方式，評估Ishikawa細胞中的絲胺酸蛋白酶23之功能。然而，此方法則無法有效運用以研究絲胺酸蛋白酶23之功能。另外，MCF-7細胞在雌激素作用下，絲胺酸蛋白酶23之表現量會上升。這樣的現象在Ishikawa細胞，其基因表現量則無明顯改變。另一方面，MCF-7和Ishikawa細胞在TPA作用下，絲胺酸蛋白酶23之基因表現量皆會上升，此結果暗示著絲胺酸蛋白酶23可能會受到AP-1轉錄因子的調控。本研究進一步利用生物資訊學方法分析絲胺酸蛋白酶23之上游啟動子區域，發現該區域含有轉錄因子AP-1轉錄因子結合區域 (TPA-response element; TRE)。綜合上述結果，在MCF-7細胞中，絲胺酸蛋白酶23活化可能會透過雌激素受體甲型之訊息傳遞路徑。然而，Ishikawa細胞中，絲胺酸蛋白酶23則可能是雌激素非依賴性且經由AP-1轉錄因子所活化。本篇論文研究結果顯示，在乳癌與子宮內膜癌細胞株中，雌激素之於絲胺酸蛋白酶23的轉錄調控是具有組織特異性。

Background: Serine protease 23 (PRSS23) is a novel serine protease belong to the peptidase S1 family. Previous studies indicated that PRSS23 could be highly expressed in various cancers such as breast, thyroid and prostate tumors. However, the underlying mechanism of PRSS23 in tumor progression remains unclear. Our previous study indicated that PRSS23 is a downstream estrogen effector gene and mediates the proliferation of estrogen receptor positive breast cancer cells. We found that PRSS23 was also expressed in endometrioid adenocarcinoma by immunohistochemistry staining. This thesis study thus aims to investigate whether PRSS23 could be regulated by estrogen in endometrial cancer and affects its cell proliferation.

Results: I found that PRSS23 was expressed in estrogen receptor positive human endometrial cancer cell line - Ishikawa. I further investigated the function of PRSS23 in Ishikawa cells by RNA inference systems. However, the RNA inference systems failed to generate PRSS23 knockdown Ishikawa cells. The gene expression of PRSS23 could be up-regulated under estrogen stimulation in MCF-7 cells, but not in Ishikawa cells. On the other hand, PRSS23 could be up-regulated in MCF-7 and Ishikawa cells under TPA (12-O-tetradecanoylphorbol-13-acetate)-induced AP-1(Activator protein-1) trans-activation. This is supported by the finding that the upstream promoter region of PRSS23 contains AP-1 protein binding site (TPA-response element; TRE) by bioinformatic prediction. Taken together, the possible transcriptional regulation of PRSS23 in MCF-7 cells might be regulated via ER? dependent pathways. On the other hand, PRSS23 in Ishikawa cells might be regulated through estrogen but AP-1 dependent pathways. This study reveals the striking tissue specificity with distinct transcriptional regulation of PRSS23 by estrogen signaling in breast and endometrial cancer.

List of Contents:
中文摘要 I
Abstract II
誌謝 III
1. Introduction 1
1.1 Preliminary outline 1
1.2 Estrogen, estrogen receptor and its mechanism 1
1.3 Estrogen related female cancer- breast cancer and endometrial cancer 3
1.4 The findings of PRSS23 in previous study and breast cancer 4
1.5 The hypothesis and objective of this study 5
2. Material and Methods 6
2.1 Cell cultures of MCF-7, Ishikawa, HEC-1B and TLA-HEK293T 6
2.2 Cell culture in hormone deprived medium 6
2.3 RNA isolation, First strand cDNA synthesis and Real-Time quantitative PCR 7
2.4 Western blotting 8
2.5 Small interfering RNAs (siRNAs) 9
2.6 Lentiviral short hairpin RNA 10
2.7 BrdU cell proliferation assay 10
2.8 Statistical analysis 11
3. Results 12
3.1 PRSS23 expressed in ER? positive endometrial cancer line 12
3.2 The knockdown system of PRSS23 in Ishikawa cells cannot be established to investigate its function on cell proliferation 13
3.3 PRSS23 might be regulated by steroid hormones in Ishikawa cells 14
3.4 E2 stimulates PRSS23 expression in MCF-7, but not in Ishikawa cells 14
3.5 PRSS23 was up-regulated under TPA induction in MCF-7 and Ishikawa cells 15
4. Discussion 16
4.1 Experimental approach to study the function of PRSS23 in Ishikawa cells 16
4.2 The limitation of BrdU cell proliferation assay 17
4.3 Differential transcriptional regulation of PRSS23 in breast cancer and endometrial cancer 18
5. References 20
List of Table:
Table 1: Real-Time quantitative PCR primer sequence of human genes 25
Table 2: Antibodies list 26
Table 3: The predicted transcription factor binding sites in PRSS23 upstream region 27
List of Figure:
Figure 1: Survey of PRSS23 and ER? expression in endometrial cancer lines 28
Figure 2: PRSS23 siRNA has no effect on Ishikawa cell proliferation 29
Figure 3: MCF-7 and Ishikawa cells were transduced by the medium containing lentiviral particle 30
Figure 4: The protein expression level of PRSS23 was declined in hormone deprived medium in Ishikawa cells 31
Figure 5: Effects of E2 treatment on PRSS23 expression of MCF-7 and Ishikawa cells 32
Figure 6: Effects of tamoxifen treatment on PRSS23 expression of MCF-7 and Ishikawa cells 33
Figure 7: The gene expression of EGR3 in Ishikawa cells 34
Figure 8: Effects of TPA treatment on PRSS23 expression of MCF-7 and Ishikawa cells 35
Figure 9: E2 had no stimulatory effect on the cell proliferation of Ishikawa 36
List of Supplementary Figure:
Supplementary Figure 1: Immunohistochemical detection of PRSS23 in endometrioid adenocarcinoma 37
Supplementary Figure 2: Knockdown of PRSS23 led to reduced cell proliferation in MCF-7 cells 38
Supplementary Figure 3: The possible roles of PRSS23 in breast and endometrial cancer 39

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