N6-Methyladenosine Modification in Helocobacter pylori Infection

Background & Aims: N6-Methyladenosine (m6A) is one of the most abundant messenger RNA (mRNA) internal modifications in eukaryotes and can regulate many aspects of RNA biology, including splicing, localization, stability and translation. m6A has been well-studied in regulating cellular responses to viral infection, but the role of m6A in the interplay between host and bacteria is still less clear. In the present study, I investigated the role of m6A modification in the host response to Helicobacter pylori infection.Methods: Normal gastric epithelial cell line HFE145 was used as cellular host model for H. pylori infection in vitro. C57BL/6 mice gavaged with H. pylori were used for in vivo study. Dot-blot assay and m6A enzyme-linked immunosorbent assay (ELISA) were performed to measure global m6A levels. mRNA and protein levels were determined by reverse transcription-quantitative PCR (qPCR) and Western blots, respectively. H. pylori colonized level was determined by 16S ribosomal DNA quantitative PCR, immunofluorescence and colony formation assay. Small interfering RNA (siRNA) was used to knock down target gene expression. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was performed to depict the global change of m6A patterns on mRNA in H. pylori-infected HFE145 cells. m6A-specific PCR was conducted to validate the results of MeRIP-seq.Results: Co-culturing with H. pylori at a multiplicity of infection (MOI) of 100 increased the protein levels of m6A writers - WTAP, METTL3 and METTL14 in a time-dependent manner in human gastric epithelial cells HFE145. The up-regulation of m6A writers was also observed in the gastric tissues of mice acutely infected with H. pylori. Concordantly, H. pylori-infected HFE145 and mouse tissue exhibited increased global levels of m6A-modified RNA. Knockdown of WTAP, a component of m6A writer complex, effectively reduced total m6A-modified RNA levels in vitro. Functionally, abolition of m6A modification by WTAP knockdown increased the intracellular survival of H. pylori. Mechanically, MeRIP-seq data showed that oxidized low-density lipoprotein receptor 1 (OLR1) is a downstream target of WTAP. Knockdown of WTAP reduced m6A level on 3' untranslated region (UTR) of OLR1, which was further validated by m6A-specific PCR. The reduced m6A level on 3'UTR promoted the decay of OLR1. Silencing of OLR1 decreased intracellular H. pylori abundance and abolished the effect of WTAP knockdown.Conclusion: Gastric epithelial cells respond to acute H. pylori infection by WTAP-dependent m6A RNA modification that promotes OLR1 decay to limit the infection. These findings highlight that blockage of OLR1 is a novel therapeutic target to control H. pylori infection.研究背景及目的: N6-甲基線嘌呤（m6A）是真核生物中最廣泛的一種信使RNA內部修飾， 並且可以多方面地調節RNA的生物學功能， 包括RNA的剪切， 細胞內定位， 穩定性和翻譯。m6A在調節細胞對病毒感染的反應方面已經得到了充分的研究。 但是m6A在宿主和細菌的互作中的作用還不清楚。 本研究的目標旨在探究m6A化學修飾在宿主應對幽門螺桿菌感染中所發揮的作用。方法：正常胃上皮細胞HFE145用作幽門螺桿菌感染宿主的體外模型。C57BL/6小鼠施予幽門螺桿菌灌胃則用於體內研究。點印跡（Dot-blot）和針對m6A的酶聯免疫吸附試驗（ELISA）用於測定細胞和組織樣品中的總體m6A水平。實時螢光定量PCR用於檢測mRNA水平，蛋白印跡技術（Western-blot）則用於檢測蛋白表達量。16S rDNA PCR， 免疫螢光和克隆形成試驗用於測定細胞內的幽門螺桿菌數量。 小干擾RNA（siRNA）用於敲降目的基因的表達。甲基化RNA免疫共沉淀測序（MeRIP-seq）用於描繪HFE145感染幽門螺桿菌後總體mRNA上m6A的變化圖譜。m6A特異性PCR則用於驗證MeRIP-seq的結果。結果：以感染複數100進行幽門螺桿菌和細胞的共孵育可以使胃上皮細胞HFE145中m6A甲基轉移酶-WTAP，METTL3和METTL14的表達雖時間增加。此外， 在幽門螺桿菌灌胃小鼠的組織中也能觀察到m6A甲基轉移酶表達的增加。 相應地， 幽門螺桿菌感染的HFE145和灌胃小鼠組織中都顯示RNA上m6A水平的升高。敲降m6A甲基轉移酶複合物種的組分WTAP可以顯著下降細胞中m6A修飾RNA的水平。 功能上，敲降WTAP導致的m6A水平下降可以增加細胞內的幽門螺桿菌DNA水平，增加幽門螺桿菌螢光信號以及克隆形成能力。機制上， MeRIP-seq分析結果顯示OLR1是WTAP的潛在下游靶點。敲降WTAP可以降低OLR1上3'UTR的m6A修飾水平，該結果也通過m6A特異性PCR進行了驗證。並且3'UTR上m6A水平的下降可能促進了OLR1的降解。當我們使用siRNA沈默OLR1可以降低細胞內幽門螺桿菌的水平並逆轉敲降WTAP導致的細胞內細菌水平的增加。結論：幽門螺桿菌感染引起的WTAP依賴的m6A水平升高可以促進OLR1的降解從而抑制細菌的感染。該發現提示可以通過阻斷OLR1來達到抑制幽門螺桿菌感染的效果。ZENG, Judeng.Ph.D. Chinese University of Hong Kong 2021.Includes bibliographical references (leaves )Abstracts in English and Chinese.Title from PDF title page (viewed on ...