探討Irisin對人類神經膠質瘤細胞株U-87 MG之機制影響

Abstract

規律運動能降低罹癌風險與延緩癌症進程。目前已知許多運動誘導產生的因子可能是預防癌症的重要因素，而由運動誘導分泌的irisin為一種肌肉激素，作用於白色脂肪細胞並促進其分化成類棕色脂肪細胞，此過程稱為褐化作用。另外，其他研究顯示，irisin顯著降低乳癌、前列腺癌與肺癌細胞株的存活能力 (cell viability)，而我們先前的研究也發現irisin會抑制人類神經膠質瘤細胞株U-87 MG的侵襲能力 (cell invasion)。然而，irisin如何影響癌細胞的分子機制仍未闡明。 本論文中，以E. coli系統表現及純化人類irisin重組蛋白，並探討irisin對人類神經膠質瘤細胞株U-87 MG之影響。在體外實驗結果顯示，irisin使細胞進入休息狀態的G0期來抑制U-87 MG增生，但並不影響細胞凋亡。接著，以轉錄體學分析來研究irisin如何影響U-87 MG的生理功能，觀察到irisin會提高SerpinB2及TFPI-2的mRNA及蛋白質表現，這兩者皆參與細胞侵襲的過程，是多種絲氨酸蛋白酶 (serine proteases) 的抑制物。因此，我們藉由抑制SerpinB2或TFPI-2的基因表現，來評估irisin是否誘導其表現來抑制細胞侵襲。由實驗結果發現抑制SerpinB2表現會降低細胞增生、遷移 (cell migration) 及侵襲能力，但irisin對抑制SerpinB2表現的細胞沒有影響力。此外，抑制TFPI-2表現不影響細胞增生，但會促進細胞遷移及侵襲，且irisin抑制侵襲的能力被抵銷。最後，體內實驗發現，irisin能延緩小鼠皮下異體移植的U-87 MG細胞生長。 由以上實驗結果了解irisin藉由增加TFPI-2的表現而抑制U-87 MG細胞侵襲，然而irisin如何抑制U-87 MG細胞增生的機制須有更進ㄧ步的研究，因此，本論文提議irisin在運動延緩癌症進程中扮演重要的角色，並且具有潛力幫助癌症治療。

Regular exercise can prevent cancer and delay cancer progression. It is known that several exercise-induced factors could be the important factors for cancer prevention. Irisin was identified as one myokine induced by exercise. Irisin targets to white adipocytes and induces white adipocytes differentiating into brown-like adipocytes, and the process is called browning. Other studies revealed that irisin significantly decreases cell viability in breast, prostate and lung cancer cells. Moreover, our previous studies found that irisin inhibits cell invasion in the human glioblastoma cell line, U-87 MG. However, the molecular mechanism of how irisin affects cancer cells remains unclear. In this research, recombinant human irisin protein was produced in E.coli and purified by the Ni-NTA column. Next, we investigated the effects of irisin on U-87 MG. In vitro studies indicated that irisin made cells enter G0 phase, resulting in reduced cell proliferation of U-87 MG, but no effect in apoptosis. Next, transcriptome NGS analysis was performed to further clarify how irisin regulated biological functions of U-87 MG. Both mRNA and protein levels of SerpinB2 and TFPI-2 were up-regulated by irisin. SerpinB2 and TFPI-2 are inhibitors for several serine proteases important for cell invasion. Thus, we knocked down SerpinB2 or TFPI-2 expression to assess if irisin inhibited cell invasion by inducing expression of these two genes. SerpinB2 knockdown cells reduced cell proliferation, migration and invasion, and irisin had no further effects on these cells. Moreover, knockdown of TFPI-2 did not affect cell proliferation, but increased cell invasion and migration. In addition, the inhibition of cell invasion ability by irisin was rescued in TFPI-2 knockdown cells. Furthermore, in vivo studies found that irisin retarded tumor growth in subcutaneous U-87 MG xenograft tumor models. Our results indicated that irisin inhibited U-87 MG cell invasion by up-regulating TFPI-2 expression. However, the mechanism of how irisin reduced cell proliferation will need further investigation. Therefore, we proposed that irisin plays an important role in exercise delaying cancer progression and irisin was a potential target for cancer treatment.