研究癌細胞中細胞訊息傳遞路徑之調控：探討CD36在肺癌的機制以及miR-548ba在食道癌的機制

Abstract

根據政府歷年統計資料顯示，從70年代開始至今癌症是台灣十大死因之首且癌症發生數連年增加，因此目前癌症發生最新統計資料顯示，和西元2018年相比，西元2019年台灣罹癌個數增加最多的是肺癌，且不論在台灣男性以及在台灣女性肺癌癌症發生數位居前三，此外根據最新癌症死亡數統計指出在西元2021年死亡率最高的癌症是肺癌。然而有些癌症好發於台灣男性，在西元2019年十大癌症發生率性別比差異最大的癌症為食道癌，男性罹患食道癌的機率為女性的15倍。雖然已經有許多研究探討肺癌以及食道癌發生之相關細胞訊號途徑，但是癌症細胞訊號途徑調控很複雜，所以本研究會分別探討CD36以及miR-548ba對肺癌以及食道癌細胞癌化之機制。 實驗室先前已經針對肺癌患者進行研究指出相較於正常肺細胞，CD36低表現量可能在肺癌中扮演重要角色，因此本研究探討CD36低表現量於肺癌生長之影響及機制。首先我們發現CD36基因靜默(Silencing)會抑制CL1-0增生能力(Proliferation)，然而CD36大量表現(Overexpression)不會改變CL1-5的增生能力，但CD36大量表現會抑制H1299的增生能力。在細胞遷移能力實驗當中，使用Transwell發現CD36基因靜默會促進CL1-0遷移能力(Migration)，然而CD36大量表現不會改變CL1-5以及H1299的遷移能力。在細胞凋亡實驗(Cell Apoptosis Assay)當中，CD36基因靜默會降低CL1-0死亡速率，然而CD36大量表現不會改變CL1-5的死亡速率。最後CD36基因靜默會減少CL1-0吞噬之葡萄糖量以及抑制醣解作用(Glycolysis)活性。 除了基因之外，許多研究也指出小分子核醣核酸(microRNA, miRNA)在各種人類癌症中也扮演重要的角色。因此實驗室先前透過次世代定序(Next-Generation Sequencing, NGS)以及即時定量聚合酶連鎖反應(Real-Time Quantitative Polymerase Chain Reaction, QPCR)驗證後發現相較於正常食道細胞，miR-548ba高表現量可能在食道癌中扮演重要角色，因此本研究探討miR-548ba高表現量於食道癌生長之影響及機制。由實驗結果發現miR-548ba大量表現會促進CE81T的增生能力以及增加CE81T細胞群落數目，但不會改變CE81T遷移能力。接著使用預測miRNA目標基因的軟體以及即時定量聚合酶連鎖反應驗證的發現，MOB1B可能是miR-548ba的目標基因，然而MOB1B是否是miR-548ba的直接目標基因仍需後續實驗驗證。 綜合來看，首先CD36在不同肺癌細胞中所造成之細胞生理功能的影響也不同，此外miR-548ba會促進CE81T細胞增生能力，期望透過本研究能提供後人新的研究方向以找出新的肺癌以及食道癌致癌機制。

According to the statistics released by government in the past few years, cancer is the majority of dead reason from 1980s to present in Taiwan. Moreover, the cancer incidence is increasing year after year. Based on the latest statistics of cancer incidence and cancer death released by the Health Promotion Administration in 2019 and 2021 respectively, lung cancer ranked the top three cancer incidence and cancer death in both gender in Taiwan. Additionally, the esophageal cancer incidence in male was fifteen times as much as the esophageal cancer incidence in female. Although most of cancer research focus on lung and esophageal carcinogenesis related cell signaling, the regulation of cancer cell signaling is complex. Thus, in this study, I would focus on the function of CD36 and miR-548ba in lung and esophageal carcinogenesis respectively. In our lab, previous study found that compared with normal lung cells, CD36 low expression may play an important role in lung cancer. Therefore, we investigated the regulatory mechanism of CD36 low expression in lung cancer. First, we found that CD36 silencing inhibited CL1-0 proliferation, whereas CD36 overexpression did not alter CL1-5 proliferation, but CD36 overexpression inhibited H1299 proliferation. Using Transwell, we found that CD36 silencing promoted CL1-0 migration, but CD36 overexpression did not change CL1-5 and H1299 migration. In cell apoptosis assays, CD36 silencing reduced the death rate of CL1-0, whereas CD36 overexpression did not alter the death rate of CL1-5. Finally, CD36 silencing reduced the amount of glucose consumption and inhibited glycolysis activity in CL1-0. Except for genes, many studies had also found that microRNAs (miRNAs) also play important roles in various human cancers. Therefore, in our lab previously found that the high expression of miR-548ba may play an important role in esophageal cancer compared with normal esophageal cells by next-generation sequencing and real-time quantitative polymerase chain reaction. Therefore, we investigated the regulatory mechanism of miR-548ba high expression in esophageal cancer. First, it was found that miR-548ba overexpression promoted CE81T proliferation and increases CE81T colony number, but did not change CE81T migration. Then, using the software for predicting miRNA target genes and the verification by real-time quantitative polymerase chain reaction and found that MOB1B may be the target gene of miR-548ba, but whether MOB1B is the direct target gene of miR-548ba still needs to be verified by subsequent experiments. Overall, my study indicated that CD36 had diverse effects on the physiological functions in different lung cancer cells, and miR-548ba could promote CE81T proliferation. Therefore, it is hoped that it can provide new aspects for researchers to find new lung and esophageal carcinogenesis mechanism in this study.