LncRNA expression profile during autophagy and Malat1 function in macrophages

Abstract

Long noncoding RNAs (lncRNAs) are a class of functional non-coding transcripts that are longer than 200 nt and regulate gene expression via diverse mechanisms in eukaryotes. In fact, they have emerged as critical epigenetic and transcriptional regulators of autophagy in mammals in response to various stressors. Autophagy not only plays a crucial role in maintaining cellular homeostasis, but it is also essential to immunity, targets intracellular pathogens for degradation, modulates inflammation, and participates in adaptive immune responses. However, the expression profile of lncRNA and its role in regulating autophagy in macrophages have been poorly defined. Here, we used transcriptomic and bioinformatics to analysis LncRNA expression profile during autophagy and functional studies to evaluate the function of the metastasis-associated lung adenocarcinoma transcript-1 (Malat1) lncRNA in macrophages. A total of 1112 putative lncRNAs (240 novel lncRNAs) were identified, including 831 large intergenic, 129 intronic, and 152 anti-sense lncRNA, of which 59 differentially expressed transcripts exhibited a greater than 1.5-fold change under different conditions. The interaction of Malat1 lncRNA with microRNA (mir)-23-3p and lysosomal-associated membrane protein 1 (Lamp1) was found, Malat1 releases inhibition of Lamp1 expression in macrophages through competitive adsorption of mir-23-3p. The results of this study provide a better understanding of lncRNA function in macrophages and a basis for further investigation into the roles and mechanisms of ncRNA in immunology, particularly the functions of Malat1 and mir-23-3p in the pathogenesis of macrophages.

Fig 1. An integrative techflow and computational pipeline for the systematic identification of LncRNAs in Raw264.7.

(A) Informatics pipeline for the identification of LncRNAs in Raw264.7. (B) Venn chart showing the numbers of candidate LncRNAs filtered by the CNCI, CPC (Coding Potential Calculator), CPAT, and PFAM with the default parameters. (C) Box plots of log10 maximum expression values (FPKM+1) for protein-coding (Blue) and lncRNA (red) genes. Boxes represent first and third quartiles. Whiskers are 1.5-times the interquartile range. (D) Average score of Noncoding and Coding sequences calculated with CPC and CPAT.

Fig 2. Characteristics of Raw264.7 lncRNAs.

(A) Numbers of lincRNAs, intronic lncRNAs and antisense; ncRNAs in Raw264.7. (B) Transcript length distribution of lincRNAs, intronic lncRNAs and antisense lncRNAs. (C) The number of exons per transcript for LncRNAs. (D) Distribution of lincRNAs, intronic lncRNAs and anti-sense along each chromosome.

Fig 3. LncRNA expression file.

(A) The numbers of DEL under each condition. (B) Two expression clusters containing 59 DELs. (C-E) Different type and chromosome distribution of DEL in each group (C: 50 nM Rapa, D: 3 mM 3-MA, E: STV).

Fig 4. RT-qPCR validation of eight LncRNA relative expression level upon 50 nM Rapa stimulation 2 hours in Raw264.7 cell lines (Mean±SD, n = 3).

Fig 5. Effects of Malat1 on autophagy in macrophages.

(A) RNA-FISH was performed to confirm sub cellular distribution of Malat1 in Raw264.7 cells (Scale bar = 10 μm, n = 3). (B) Confocal microscope was used to check the autophagy flux in different groups (Scale bar = 10 μm, n = 3). (C) The expression of Malat1 in Raw264.7 cells infected with PCDH-Duo-Malat1 (PD-Malat1) or vector (PCDH-Duo) Lentivirus was assessed by RT-qPCR (Mean±SD; n = 3). GAPDH was used as the endogenous control. (D) The expression of Malat1 was assessed by RT-qPCR in Raw264.7 cells transfected with Malat1-SiRNA and ASO mix (Mean±SD; n = 3). (E) Fluorescent LC3 dots per cell was quantified by Image J software, the data represent the Mean±SD of three independent experiments. (F and H) Autophagy marker P62 and LC3 were assessed by Western blot in PD-Vector or PD-Malat1 group. The expression of autophagy marker protein includes P62, LC3 was normalized against β-actin. (G and J) Autophagy marker P62, ATG5 and LC3 were assessed by Western blot in the Malat1-siRNA or Si-NC groups. The expression of autophagy marker protein includes ATG5, P62 and LC3 was normalized against GAPDH. *P<0.05 or ** P<0.01 in experiments vs. the corresponding control group.

Fig 6. Lamp1 is a target of Mir-23b-3p in macrophage.

(A) Four different databases were used to cross-predict mir-23b-3p targets and 26 potential targets presented in Venn. (B) The bioinformatics prediction of mir-23a/b-3p MRE in Lamp1. (C) Luciferase reporter assay was performed in 293T cells co-transfected with pmirGLO-Lamp1-Wt or Mut and mimics or mimics-NC in Raw264.7 cells. (D) RT-qPCR was performed to examine the expression of Lamp1 in Raw264.7 cells transfected with mimics-NC, mimics, inhibitor-NC, or inhibitor.

Fig 7. mir-23b-3p inhibits autophagy in macrophage.

(A) Immunofluorescence of endogenous LC3 was checked in Raw264.7 cells under different treatment conditions (Scale bar = 10 μm). (B) GFP-LC3 puncta per cell was quantified by Image J software, the data represent the Mean±SD of three independent experiments. (C) Autophagy flux analysis was conducted in Raw264.7 cells in Rapa+NC, Rapa+mimics, or Rapa + Inhibitor group to confirm mir-23-3p function (Scale bar = 10 μm). (D) Fluorescent LC3 dots per cell was quantified by Image J software, the data represent the Mean±SD of three independent experiments. (E-H) Western blot was used to detect the protein levels of Lamp1, ATG5, P62, LC3 I/II in Raw264.7 cells treated with concentration gradients of (E) mimics (0–200 nM) or (G) inhibitors (0–400 nM). Normalized gray ratio was also compared, the expression of protein was normalized against β-actin (Mean±SD; n = 3).

Fig 8. Malat1 functions as CeRNA to regulate Lamp1 expression by sponging mir-23-3p in macrophage cells.

(A) RT-qPCR was performed to evaluate the expression levels of Malat1, mir-23b-3p, and Lamp1 in Raw264.7 cells respective treatment with 50 nM Rapa 2h or 3 mM 3-MA 12h (Mean±SD; n = 3). (B) RT-qPCR was used to test mir-23b-3p expression in the different groups, including NC, Si-NC, Malat1-siRNA, Malat1-siRNA+inhibitor-NC (INC), Malat1-siRNA+inhibitor, or inhibitor (Mean±SD; n = 3). (C) The bioinformatics predicted MRE of mir-23b-3p on Malat1. (D) Luciferase reporter assay was performed in 293T cells co-transfected with pmirGLO-Malat1-Wt or Mut and mimics or mimics-NC (Mean±SD; n = 3). (E) Luciferase reporter rescue experiment was conducted to confirm co-transfected pmirGLO-Malat1-Wt, pmirGLO-Lamp1-Wt and mir-23b-3p mimics effect the relative luciferase activity in 293T cells (Mean±SD; n = 3). (F) WES was used to confirm over-expression Malat1 can promote Lamp1 expression in Raw264.7 cells. (G) Graphic summary of Malat1 function in this work.