Degradation of SNAP29 in HeLa cells by Acinetobacter baumannii depending on LncRNA-GAS5
DING Wenyi1, AN Zhiyuan2
1. Department of Clinical Laboratory, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China; 2. Medical Research Center, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China
Abstract：The present paper aims to construct the eukaryotic overexpression vector and knockdown vector of LncRNA-GAS5 (long non-coding RNA-growth arrest-specific transcript 5), and to study its role in the expression of SNAP29 in HeLa cells infected by Acinetobacter baumannii (A. baumannii). LncRNA-GAS5 gene was amplified by polymerase chain reaction (PCR) and cloned into pcDNA3.1 (＋) eukaryotic expression vector to form pcDNA3.1-GAS5 eukaryotic expression vector. Through RNA interference technology, the oligonucleotide sequences for knockdown of GAS5 were designed and constructed into pRNAT-U6.1/Neo vector to form pRNAT-U6.1-GAS5 knockdown vector. The expression of GAS5 was detected by real-time PCR to determine whether the overexpression and knockdown were successful or not. Western blotting was used to detect the changes of SNAP29 expression in HeLa cells after overexpression or knockdown of GAS5. In addition, the expression of SNAP29 in A. baumannii-infected HeLa cells at different times and the expression of SNAP29 in A. baumannii-reinfected HeLa cells after knockdown or overexpression of GAS5 were detected by Western blotting. The results showed that pcDNA3.1-GAS5 and pRNAT-U6.1-GAS5 were successfully constructed. pcDNA3.1-GAS5 could induce high expression of LncRNA-GAS5 in HeLa cells. pRNAT-U6.1-GAS5 significantly reduced the expression of GAS5 in HeLa cells. The overexpression of LncRNA-GAS5 decreased the expression of SNAP29 in HeLa cells, while the knockdown of LncRNA-GAS5 increased the expression of SNAP29 in HeLa cells. The expression of SNAP29 decreased progressively with A. baumannii infection time. The knockdown of LncRNA-GAS5 could restore the expression of SNAP29 degraded by A. baumannii, while the overexpression of LncRNA-GAS5 could aggravate the degradation of SNAP29 by A. baumannii. The results suggest that LncRNA-GAS5 has a negatively regulatory effect on SNAP29 expression, and the degradation of SNAP29 by A. baumannii depends on LncRNA-GAS5. It lays a theoretical foundation for further discovering the deep mechanism of autophagy degradation disorder caused by A. baumannii.
丁文一1，安志远2. LncRNA-GAS5对鲍曼不动杆菌降解HeLa细胞中SNAP29的调控作用[J]. 微生物与感染, 2022, 17(4): 220-226.
DING Wenyi1, AN Zhiyuan2. Degradation of SNAP29 in HeLa cells by Acinetobacter baumannii depending on LncRNA-GAS5. JOURNAL OF MICROBES AND INFECTIONS, 2022, 17(4): 220-226.
2022, Vol. 17 
