Mechanism Study Of Roles Of Long Non-coding RNA In The Invasion Of The Central Nervous System By Meningitic Escherichia Coli

Bacterial meningitis is a serious and life-threatening infection of the central nervous system(CNS)caused by infection of the meningeal tissue surrounding the brain.In the animal husbandry industry,bacterial meningitis often causes huge economic losses,it also seriously threatens food safety and public health;in humans,it has high morbidity and mortality,and it is a major cause of death and disease disability worldwide,especially in children.Escherichia coli(E.coli)is the most common gram-negative pathogenic bacterium causing meningitis,and it is also a common model strain for the study of bacterial meningitis.Generally,to cause meningitis,E.coli must survive in the blood stream,interact with and penetrate the blood brain barrier(BBB),and invoke inflammatory responses.Although good results have been achieved for bacterial meningitis treatment by antibiotic therapy,the molecular mechanisms by which bacteria penetrate the BBB,disrupt BBB integrity,and cause neuroinflammatory responses remain largely unclear,so the overall mortality rate of this disease is still high with neurologic sequelae.In the past,long non-coding RNAs(lnc RNAs)were considered to be transcriptional noises that do not function.In recent years,lnc RNAs have been increasingly recognized as regulators of infectious diseases,accompanied by a variety of regulatory mechanisms.However,in bacterial meningitis,especially E.coli meningitis,there are few studies on the function of lnc RNAs.Based on the sequencing results of differentially expressed lnc RNAs in Meningitic E.coli(MNEC)-infected human brain microvascular endothelial cells(h BMECs),this study searched for lnc RNAs with regulatory functions in each step of MNEC breaking through the BBB and analyzed its molecular mechanism.According to the different regulatory mechanisms,the research contents of this study are as follows:1.Lnc C11orf54-1 regulates neuroinflammation by activating(Nuclear factor kappaB,NF-κB)signaling pathway.This study first characterized a significantly down-regulated lnc RNA lnc C11orf54-1in the lnc RNA sequencing results.Northern blot assay confirmed that this lnc RNA was degraded during MNEC infection to generate mg U2-30 and mg U2-19,a process was mediated by translocated RNase Coilin.Functionally,lnc C11orf54-1-originated noncoding RNA mg U2-30 interacted with interleukin-1 receptor-associated kinase 1(IRAK1)to induce its oligomerization and autophosphorylation,thus promoting the activation of NF-κB signaling.Overexpressing/knocking-out of mg U2-30 further confirmed that mg U2-30 could promote the expression of cytokines in MNEC-infected h BMECs and aggravate the neuroinflammatory responses.2.Lnc RSPH9-4 facilitates blood–brain barrier disruption via mi R-17-5p/MMP3 axis.This study found that lnc RSPH9-4 was significantly up-regulated in h BMECs after MNEC infection,and it was found to mediate the disruption of BBB integrity by overexpressing/knocking-down of lnc RSPH9-4 in h BMECs.RNA-sequencing analysis of h BMECs revealed a total of 639 m RNAs and 299 micro RNA(mi RNAs)were significantly changed in response to lnc RSPH9-4 overexpression.Bioinformatics analysis and experimental verification of these data confirmed that lnc RSPH9-4 can regulate the expression of downstream genes through a common lnc RNA regulation mechanism,that is,as a molecular sponge to competitively sponging mi R-17-5p,thereby increasing MMP3 expression.MMP3 was able to target and degrade intercellular tight junctions and disrupting the integrity of h BMECs monolayer.It demonstrated that MNEC-induced lnc RSPH9-4 aggravated disruption of the tight junctions in h BMECs through the mi R-17-5p/MMP3 axis.3.The polypeptide Small regulatory polypeptide of bacterial invasion(SPBI)encoded by lnc MOB3A-2 promotes the invasion of MNEC into the BBB.In addition,this study investigated the coding potential of host lnc RNAs in infectious diseases for the first time,and confirmed that the open reading frame(ORF)of lnc RNA lnc MOB3A-2 could encode a 9.5 k Da polypeptide SPBI in the bacterial meningitis model.This result was also verified by the anti-SPBI antibody we prepared.The adhesion and invasion assay further confirmed that SPBI could promote MNEC to invade h BMECs,and this effect was mediated by the cytoskeleton rearrangement of h BMECs promoted by SPBI.Thus,it might be concluded that lnc MOB3A-2 could encode a polypeptide SPBI,which promotes the invasion of MNEC into the BBB.This study analyzed the molecular mechanism of lnc RNAs regulation of gene expression from three dimensions,that is,lnc RNAs function as molecular scaffold to bind protein,lnc RNAs function as molecular sponge to bind mi RNA and lnc RNAs coding polypeptide.It elucidated the roles of three lnc RNAs(lnc C11orf54-1,lnc RSPH9-4 and lnc MOB3A-2)in regulating each key step of MNEC breaking the BBB and eventually leading to bacterial meningitis.It also provides new insights and therapeutic targets for the development of new drugs for the treatment of bacterial meningitis and its neurological sequelae.