宿主微核醣核酸miR-146a抑制沙門氏菌在宿主細胞內的生長與複製

Abstract

微核醣核酸(microRNAs)是21-24nt長度的non-coding RNA，可以調節其他基因的表現。研究指出當病原菌感染宿主時，宿主微核醣核酸可以調控發炎或產生抗病原菌感染的免疫反應來抵禦病原菌。因此微核醣核酸在宿主防禦病原菌上有著重要的角色。沙門氏菌(Salmonella)是革蘭氏陰性菌且可生長於宿主細胞內。沙門氏菌可感染吞噬細胞與非吞噬細胞，當沙門氏菌存活於非吞噬細胞內則可再感染鄰近之細胞，若存活於吞噬細胞內可透過血液或淋巴擴散於全身而造成全身性的感染。然而胞內菌在感染宿主腸道上皮細胞時，宿主微核醣核酸的表現或調控關係仍然未知。 實驗使用人類腸道癌症上皮細胞(HT29)感染鼠傷寒沙門氏菌(Salmonella typhimurium)來探討宿主微核醣核酸之表現與病原菌之關係。利用TLDA array分析在沙門氏菌感染後4小時，宿主微核醣核酸之表現量。TLDA分析後發現宿主細胞之miR-146a在沙門氏菌感後其表現量會增加。為了進一步研究宿主miR-146a在沙門氏菌感染時所扮演的角色，使用miR-146a過表現的細胞株(HT29與HIEC，人類腸道正常上皮細胞)感染沙門氏菌並且觀察其細胞內細菌生長(intracellular growth)之情形。實驗結果顯示在大量表現miR-146a的細胞株其細胞內之細菌生長數目明顯比對照組少。同時，也利用antagomir抑制內源性miR-146a的表現，結果也發現細胞內之細菌數與對照組間無明顯差異。另一方面，利用西方墨點分析也看到miR-146a會促進autophagy之產生。 以上實驗結果得知，宿主miR-146a的表現，會抑制沙門氏菌在宿主細胞內的生長與複製，此抑制作用是透過autophagy的方式來清除細胞內的細菌。

MicroRNA (miRNA) is a large family of short, non-coding RNAs, which have been discovered playing essential roles in many cellular processes. In mammalian, bacteria have been found to regulate miRNAs during infection progression. Salmonella could invade the non-phagocytic cells and then survive. Salmonella may cause systemic diseases via blood dissemination in infected patients. The intestinal epithelial is the first line of host anti-bacterial defense. Here, we found Salmonella typhimurium infection upregulates the miR-146a expression in human colon cancer epithelial cells, HT29, at 4 hours post infection by TLDA array assay. We further investigate the role of miR-146a in Salmonella infection. We used the colony formation assay to measure the intracellular bacterial numbers post infection in HT29 cells and human intestinal epithelial cells, HIEC. Our results found the enforced expression of miR-146a was restricting Salmonella intracellular replication after Salmonella infection. In contrast, neutralizing miR-146a by specific antagomiR restored Salmonella intracellular replication ability in Salmonella-infected cells. Furthermore, we found the enforced expression of miR-146a increased autophagy. The intracellular bacterial growth results suggest that miR-146a might have a role in Salmonella intracellular survival through xenophagy, was able to clean cytosolic pathogens.