lncRNA AK054386 Functions as a ceRNA to Sequester miR-199 and Induce Sustained Endoplasmic Reticulum Stress in Hepatic Reperfusion Injury

Figure 3

AK054386 functions as a ceRNA and interacts with miR-199. Subcellular localization of AK054386 by qRT-PCR of the subcellular fractionations of BNL-CL2 cells. The in situ expression of AK054386 was analyzed by FISH using a FITC-AK054386 RNA probe or a FITC-scrambled sequence RNA probe (green), and the nuclear DNA was counterstained with DAPI (blue). (b) The relative levels of AK054386 were analyzed by qRT-PCR after miR-199 overexpression or inhibition. Data are shown as the of 6 independent experiments. ANOVA, . (c) Target validation using luciferase reporters in HEK293 cells. Mut: mutant; NC: scrambled RNA; Luc: luciferase. ANOVA, . (d) RIP experimental results in BNL-CL2 cells using the MS2-MS2-BP system. The enrichment of miRNAs was measured by qRT-PCR. (e) Changes in the previously reported targets of miR-199 after AK054386 overexpression or knockdown as analyzed by qRT-PCR. Data are shown as the of 6 independent experiments. ANOVA, and . (f, g) The levels of miR-199 and its target genes after transfection of wild-type AK054386 or mut-AK054386 expression vectors. The RNA levels were tested by qRT-PCR. Data are shown as the of 6 independent experiments. ANOVA, and . (h) AK054386 overexpression can directly compete with GRP78, ATF6, and IRE1a for miR-199 binding. Student’s -test, . (i) GRP78, ATF6, and IRE1a protein levels were affected by the changes in wild-type AK054386 as measured by Western blot. (j) ChIP results show the binding of NF-κB to the AK054386 promoter. For all qRT-PCR analyses in this figure, GAPDH or U6 was used as an endogenous control. Data are shown as the The results are statistics from three independent experiments unless otherwise indicated. Student’s -test, . All the experiments were done in BNL-CL2 cells except for the luciferase reporter assays in (h), which were done in 293 cells. (k) Model for the AK054386-related regulatory loop in the modulation of hepatocyte ERS in hepatic I/R. In hepatic IRI, overactivated UPR causes sustained ERS, which induces the activation of nuclear factor-κB (NF-κB1). NF-κB can bind to the AK054386 promoter and induce its transcription. This lncRNA then sequesters miR-199, resulting in the upregulation of GRP78, ATF6, and IRE1a, which promotes aggravated and sustained ER stress. This positive feedback response causes hepatocyte apoptosis and cell death.

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