缺氧誘導之環形核糖核酸circSFMBT2抑制乳癌細胞中之腫瘤惡性發展

Abstract

乳癌是女性中最常見的癌症，也是全球女性癌症相關死亡的第二大原因。在腫瘤形成的過程中，會產生缺氧的腫瘤微環境，從而導致腫瘤更加的惡性並產生治療抗性。而近期有許多報導指出非編碼核糖核酸 (non-coding RNAs)，例如：環形核糖核酸 (circular RNAs, circRNAs)，在癌細胞的分子調控路徑和機制中扮演著腫瘤抑制或是致癌基因的角色，進而影響細胞功能以及癌症的進程。並且，環型核糖核酸的表現量會在缺氧的環境下產生變化。然而，缺氧誘導的環形核糖核酸在乳癌中的調控和功能仍不清楚。因此，本研究的目的是探討缺氧誘導的環形核糖核酸circSFMBT2在乳癌細胞MCF-7中的調控機制及其功能。我們從先前實驗室的乳癌細胞次世代定序(next generation sequencing)資料中篩選出缺氧環境下表現量顯著上升的環狀核糖核酸circSFMBT2。接著確認其環形構造，並驗證在缺氧環境中的不同乳癌細胞株間表現量皆顯著上升。大量表現circSFMBT2 抑制了細胞增殖、遷移、侵襲和上皮間質轉換。接下來，為了研究 circSFMBT2 的調控機制，我們利用細胞核質分離的技術，顯示circSFMBT2主要位於細胞質，表明circSFMBT2的調控機制可能是通過與microRNA或RNA結合蛋白（RNA binding protein, RBP）的相互作用。首先我們利用生物信息學工具 RBPDB、ATtRACT 和 RBPmap 來預測候選 RBP，並通過 RNA pulldown技術進行驗證。 RNA結合蛋白PABPC1被證明與circSFMBT2結合。另一方面，我們用ENCORI 和 miRDB 預測了潛在的結合microRNA，然而qRT-PCR實驗中顯示沒有 microRNA 與 circSFMBT2 結合。最後，通過使用Affymetrix微陣列分析circSFMBT2下游基因。在qRT-PCR，在大量表現 circSFMBT2 的細胞中沒有基因顯示出顯著變化，這意味著 circSFMBT2 的調控機轉可能不在轉錄。目前，為了探索circSFMBT2的轉錄後調控，我們進行質譜分析以分析過表達circSFMBT2的MCF-7細胞中蛋白質的差異量。總之，我們的結果表明，circSFMBT2 能夠抑制乳腺癌的細胞功能並與 PABPC1 結合。隨著對circSFMBT2調控機制的深入了解，我們希望開發一種新的乳腺癌治療方案。

Breast cancer is the most prevalent cancer among women, and the second major cause of cancer-related deaths in women worldwide. The hypoxic microenvironment of solid tumor is formed in the progress of carcinogenesis, which lead to more malignant and treatment resistance. Recently, circular RNAs (circRNA), a kind of noncoding RNAs, were discovered to influence the progress of tumorigenesis, and were induced under hypoxia. However, the regulation and functions of hypoxia-induced circRNA in breast cancer remains unclear. Therefore, the purpose of this study is to identify the regulatory mechanism and its function of a hypoxia-induced circRNAs, circSFMBT2, in breast cancer MCF-7 cells. Firstly, hypoxia-responsive circRNA candidates in breast cancer MCF-7 cells under different oxygen concentrations were identified by RNA-sequencing with the criteria of fold change ≥1.5X. Among the differentially expressed circRNAs, circSFMBT2 was chosen for further experiments. The up-regulation of circSFMBT2 under hypoxia was validated by quantitative RT-PCR. The functional assays showed that overexpression of circSFMBT2 inhibited cell proliferation, migration, invasion and epithelial–mesenchymal transition. Next, in order to investigate the regulatory mechanisms of circSFMBT2, nuclear and cytoplasm fractionation were performed. The results showed circSFMBT2 predominantly localized in the cytoplasm, indicating that the regulatory mechanism of circSFMBT2 might be via interaction with microRNA or RNA binding proteins (RBP). In order to identify the RBPs, bioinformatic tools, RBPDB, ATtRACT, and RBPmap, were first used to predict the candidate RBPs and validated by RNA pulldown assays. The RNA binding protein PABPC1 was proved to bind with circSFMBT2. On the other hand, the potential sponging-microRNAs were predicted by ENCORI and miRDB, followed by qRT-PCR. No miRNAs were shown to bind with circSFMBT2. Lastly, the functions of circSFMBT2 were explored by identifying its down-stream genes using Affymetrix microarrays with the criteria of fold change ≥1.5X and P value <0.05. The top 10 of 96 differentially expressed genes were chosen for validation by quantitative RT-PCR. However, no genes showed significantly changes in cells overexpressing circSFMBT2, implying the regulation of circSFMBT2 might not at the transcriptional level. In summary, circSFMBT2 was able to inhibit cellular function of breast cancer and bind to PABPC1. With deeper understanding of the regulatory mechanisms of circSFMBT2, I hope to develop a novel treatment regime for breast cancer.