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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2010, Vol. 4 Issue (3) : 336-341     DOI: 10.1007/s11684-010-0096-4
RESEARCH ARTICLE |
Autoimmune regulator regulates autophagy in THP-1 human monocytes
Liang SHI, Li-Hua HU(), Yi-Rong LI
Clinical Laboratory Medicine Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
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Abstract  

The autoimmune regulator (AIRE) is a crucial factor for the induction of central tolerance, and mutations in this gene lead to abnormal immune responses. However, the role of AIRE in autophagy in immune cells, especially in monocytes, is obscure. In the present study, we found that overexpression of AIRE in THP-1 human monocytes resulted in increased endogenous light chain 3 (LC3)-II level and elevated LC3 positive vesicles. Moreover, an autophagy inhibitor or knockdown of AIRE by small interference RNA attenuated these effects. In contrast, the expression of p62/SQSTM1 remained unchanged in THP-1 cells after the corresponding treatment. Our findings indicate that AIRE plays a role in the regulation of autophagy in THP-1 human monocytes.

Keywords autoimmune regulator      autophagy      monocytes      light chain 3 (LC3)     
Corresponding Authors: HU Li-Hua,Email:sliang@smail.hust.edu.cn   
Issue Date: 05 September 2010
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-010-0096-4     OR     http://academic.hep.com.cn/fmd/EN/Y2010/V4/I3/336
Fig.1  Expression of AIRE in THP-1 cells. (a) The expression of AIRE mRNA in blood monocytes (Mono) and THP-1 cells were assayed by RT-PCR. G3PDH served as an endogenous reference gene. (b) the protein expression of AIRE was analyzed by Western blotting in AIRE siRNA and/or plasmids transfected THP-1 cells. Actin was designated as a loading control. Lane 1: blood monocytes; lane 2: THP-1 cells; lane 3: mock transfection; lane 4: AIRE plasmids; lane 5: AIRE siRNA and plasmids; lane 6: control siRNA and plasmids. (c) the transfection efficiency of plasmid DNA or siRNA was indicated by fluorescent microscopy. AIRE-GFP fusion proteins were located in discrete dot like structures in AIRE plasmids (AIRE) or AIRE siRNA with plasmids (si AIRE) or control siRNA with plasmids (si control) transfected cells, while mock transfection (C3) showed homogeneous distribution of green fluorescence in whole cell body (× 10). (d) the transcription of AIRE was assayed by RT-PCR in THP-1 cells after 48 h of mock transfection (lane 1) or AIRE plasmids transfection (lane 2). Likewise, AIRE mRNA of cells that were treated by AIRE siRNA (lane 3) or control siRNA (lane 4) was detected. AIRE: autoimmune regulator; RT-PCR: reverse transcription-polymerase chain reaction; G3PDH: glyceraldehyde 3 phosphate dehydrogenase; GFP: green fluorescent protein.
Fig.2  The effect of AIRE on autophagy. (a) Immunoblot analysis of endogenous LC3-II conversion in mock (C3) or AIRE plasmids (AIRE) transfected cells was performed after 48 h of transfection in the absence (left) or presence (right) of wortmannin (100 nmol/L). Cells (control), which were cultured in Earle’s balanced salt solution (EBSS) for 2 h, served as positive control. (b) autophagosome formation of the three groups of cells described above was visualized by immunofluorescent staining with anti-LC3 and TRITC conjugated second antibody (× 40). (c) the number of LC3 positive dots or vacuoles per cell from different groups was quantified by using five random fields of fluorescent microscopy followed by inclusion of counting a minimum of 100 cells per field. Data represented the mean of three independent experiments. *: <0.05, between AIRE plasmids transfection (AIRE) or positive control (control) and mock transfection (C3). DAPI: 4′,6-diamidino-2-phenylindole; AIRE: autoimmune regulator; LC3: light chain 3.
Fig.3  Negative regulation of AIRE siRNA in autophagy. (a) The level of endogenous LC3-II protein was analyzed by Western blotting after 48 h of cotransfection with AIRE plasmids and AIRE siRNA (si AIRE) or control siRNA (si control). Before lysis of cells, cells were starved for 2 h and then proteins were extracted. (b) immunofluorescent staining of autophagic vacuoles was performed as described in Fig. 2b (× 40). (c) the method of counting LC3 positive dots was the same as that described in Fig. 2c. *: <0.05 between si AIRE group and the control group. AIRE: autoimmune regulator; LC3: Light chain 3; DAPI: 4′,6-diamidino-2-phenylindole.
Fig.4  AIRE independent expression of p62/SQSTM1 in autophagy. After 48 h of transfection with plasmids (AIRE) or AIRE siRNA (si AIRE), the protein level of p62/SQSTM1 was analyzed by Western blotting and compared with respective control group (C3 or si-control). Starved cells were used as positive control (control), and actin was designated as a loading control. AIRE: autoimmune regulator.
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