Micrornas Expression Profiling In Er Stress Response And Their Target Genes Verification

Approximately one third of proteins in eukaryotic cells enter Endoplasmic reticulum lumen to complete their folding process and undergo some posttranslational modifications. If the influx of nascent, unfolded polypeptides exceeds the folding and/or processing capacity of the ER, the normal physiological state of the ER is perturbed, signaling pathways, termed the unfolded protein response (UPR), are activated to adjust the balance between the efficiency and fidelity of protein folding and ER protein load. Endoplasmic reticulum stress (ER Stress) related with the etiology of numerous disease states, including metabolic disease, atherosclerosis, and neurodegenerative disease.MicroRNAs are small endogenous RNAs of about22nucleotides in length that posttranscriptionally regulate gene expression by forming imperfect hybrids with sequences in the3’untranslated region. MicroRNAs have been linked to various cellular stresses, among them processes that impinge on the function of the ER, e.g., hypoxia, insulin secretion, and B cell differentiation. In this study, attempts were made to uncover microRNAs that participate in the process of ER Stress, and to gain some insight about the potential biological functions of these microRNAs. Following study was carried out:1. Expression profiling of microRNAs involved in ER stress responseER Stress inducer Tunicamycin (Tm) treated HEK293cell for8h, then profiled microRNAs expression levels by microRNAs microarrays. The expression levels of four microRNAs were changed under statistical significant circumstance (P<0.05). miR-26a was down-regulated, whereas miR-148a, miR-181b and miR-30d up-regulated, although the change was moderate.2. Establishment of the real time RT-PCR analysis method of microRNA, and verification of the microarrays profiling result.We established the real time RT-PCR analysis method of microRNA. Analysis of of melting curve and the slope of the replication curve indicated the method was reliable. Then assessed the kinetics of miR-26a, miR-148a, miR-181b and miR-30d within24h by real time RT-PCR analysis. The result showed that only miR-148a was up-regulated, and its expression level reached2fold after24h of Tm treatment. Different dose of Tm treatment show that the intensity of miR-148a’s induction was positively co-related with the intensity ER Stress.3. miR-148a directly regulate UPR related gene PDIA3and CNXSeven genes, including GRP78, GRP94, HRD1, OS9, EDEM1, PDIA3and CNX, were predicted by the online software to be miR-148a’s potential target, Construct the3’UTR luciferase reporter plasmid of each gene. Co-transfection of luciferase reporter plasmid with miR-148a mimics showed miR-148a mimics significantly down-regulated the relative luciferase unit of PDIA3and CNX. Correspondently, co-transfection of luciferase reporter plasmid with miR-148a inhibitor significantly up-regulated the relative luciferase unit of PDIA3and CNX. And the mutation of the seed of region on the3’UTR luciferase reporter plasmid of PDIA3made the former regulative ability of miR-148a mimics inert. Together, these results show miR-148a directly regulate UPR related gene PDIA3and CNX.4. Kinetics of induction for miR-148a、PDIA3and CNX in ER Stress responseHEK293cells were treated with Tm for24h and assess the kinetics of induction for miR-148a、PDIA3and CNX in ER Stress response by real time RT-PCR. The result shows miR-148a%PDIA3and CNX were all moderately but steadily up-regulated in ER Stress response. PDIA3was induced earlier than other two, and its induction level was consistently but moderately higher than miR-148a. Whereas the induction level of CNX and miR-148a imitate one another.