在2018年世界衛生組織的統計中，子宮頸癌的好發率位居全球第四位。而在台灣衛福部2017年的統計中，子宮頸癌也位居女性十大癌症死因之中第七位。雖然近幾年因為子宮頸抹片檢測的普及使患者能提早診斷與接受治療，以及子宮頸癌疫苗的廣泛施打有效預防人類乳突病毒的感染，使子宮頸癌的發生率和死亡率逐年降低。然而，在許多開發中國家因為資源的匱乏而缺乏完善的檢測制度，造成子宮頸癌治療與診斷的延誤。當患者被診斷為後期或轉移型的子宮頸癌時，其五年存活率將大福下降且伴隨著不佳的預後。因此，探討子宮頸癌轉移的相關機制以及尋找有效的治療標的是非常重要的議題。
在本實驗中，藉由二維差異電泳和基質輔助雷射脫附游離飛行時間質譜儀分析兩株有著相同基因背景但卻具有不同轉移能力的子宮頸癌細胞株HeLa和高轉移性的HeLa-I5之間蛋白質表現的差異。在68個表現差異的蛋白質中，挑選其中一個在HeLa-I5細胞大量表現的蛋白Progesterone receptor membrane component 1 (PGRMC1) 並針對此蛋白進行更深入的探討。利用核糖核酸干技術使PGRMC1基因表現沈默，發現此蛋白的表現會影響癌細胞的移動與增生能力。此外，也利用CaSki和ME-180兩株子宮頸癌細胞株進行更進一步驗證，皆觀察到相似的結果。期待本實驗透過蛋白質體學的分析能夠提供子宮頸癌轉移相關的診斷標記與具有潛力的治療標的。

Cervical cancer is the fourth most frequently occurring cancer among females around the world in 2018 and it is also the tenth leading cause of death in cancer in Taiwan in 2017. Although the morbidity rate and mortality rate of cervical cancer are declined due to the widespread applications of Pap smear tests and vaccination. However, the morbidity and the mortality rates of cervical cancer remain high in some developing countries because of the lack of well-organized cervical cancer screening programs to detect cervical cancer at early stage which results in delayed treatment and the lack of HPV vaccination among female population also cause greater cervical cancer prevalence. Once patients were diagnosed with metastatic cervical cancer, it usually came with poor prognosis. As a result, it is critical to investigate the mechanism of cervical cancer metastasis and discover therapeutic targets for effective treatment.
In this study, we performed 2D-DIGE and MALDI-TOF/TOF MS to analyze the differentially expressed proteins in HeLa and invasive HeLa-I5 cells. According to our proteomic results, there were 68 differentially expressed proteins between the HeLa and HeLa-I5 cells. One of these differentially expressed proteins called Progesterone receptor membrane component 1 (PGRMC1) was selected as a candidate for further studies. We used small interfering RNA (siRNA) to knockdown PGRMC1 expression in cervical cancer cells, finding that PGRMC1 was correlated with cell migration and progression. A similar phenomenon was also observed in CaSki and ME-180 cells. Except to identify diagnostic markers correlated with cervical cancer metastasis and to discover therapeutic targets for the treatment of cervical cancer via proteomic analysis.

中文摘要……………………………………………………………………................1
Abstract……………………………………………………………………................2
Acknowledgement…………………………………………………………………….3
Content………………………………………………………………………………...5
Abbreviations………………………………………………………………………….7
Introduction……………………………………………………………………………8
1-1 Cervical cancer…………………………………………………………………….9
1-2 Types of cervical cancer…………………………………………………………10
1-3 Risk factors for cervical cancer…………………………………………………..10
1-4 The morbidity and mortality rates of cervical cancer…………………………….10
1-5 Cancer metastasis………………………………………………………………...12
1-6 Cervical cancer metastasis……………………………………………………….13
1-7 Progesterone-receptor membrane component 1(PGRMC1) …………...………14
1-8 Aim of this study…………………………………………………………………15
Materials and Methods……………………………………………………………….17
2-1 Cell lines and cell culture………………………………………………………...17
2-2 Western blot analysis…………………………………………………………….18
2-3 Transwell migration assay……………………………………………………….20
2-4 Flow cytometry in analysis of cell cycle…………………………………………20
2-5 MTT cell viability assay…………………………………………………………20
2-6 siRNA knockdown of targeted protein…………………………………..............21
Results…………………………………………………………………………..........22
3-1 Proteomic analysis of invasive cervical cancer cells compared to parental cervical
cancer cells…………………………………………………………………..…...22
3-2 Establishment of invasive cervical cancer cell lines………………………..........23
3-3 Identify the expression level of EMT markers and PGRMC1 in invasive cervical cancer cell lines…………………………………………………..........................25
3-4 Examine the proliferation ability of invasive cervical cancer cell lines compared to the parental cervical cancer cell lines…..................................................................27
3-5 Reveal cell cycle regulation in invasive cervical cancer cell lines………………29
3-6 The expression of PGRMC1 differed between cervical cancer grade…………...32
3-7 siRNA-mediated PGRMC1 knockdown inhibit the migration ability in cervical
cancer cell lines…………………………………………………..........…………34
3-8 siRNA-mediated PGRMC1 knockdown reduce cell proliferation ability in
cervical cancer cell lines…………………………………………………………37
3-9 Investigate cell cycle regulation in siRNA-mediated PGRMC1 knockdown cell
lines………………………………………………………………………………........39
Discussion…………………………………………………………………………...…42
References……………………………………………………………………………48
Supplementary figures……………………………………………………………….52
Supplementary tables………………………………………………………………...56

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