title: Knockdown of PIK3R1 by shRNA inhibits the activity of the splenic macrophages associated with hypersplenism due to portal hypertension.
abstract: Phosphatidylinositol 3-kinase (PI3K) plays a central role in the metabolic actions of insulin. One 85 kDa regulatory subunit of PIK3 is encoded by phosphoinositide-3-kinase, the regulatory subunit 1 (PIK3R1). Our previous study has demonstrated that PIK3R1 was up-regulated significantly in the splenic macrophage (MΦ) of portal hypertensive spleen. In the present study, RNA interference specific to PIK3R1 was employed to investigate its inhibitive effects on the activity of MΦ associated with hypersplenism due to portal hypertension (HS-PHT). The expression of PIK3R1 in the spleen was detected by immunohistochemical staining. Plasmid vector pGenesil-1 expressing specific small hairpin RNA (shRNA) against PIK3R1 and the scrambled shRNA control was constructed. MΦ were isolated and purified by anchored cultivation from patients with HS-PHT (HS-PHT-MΦ) and traumatic rupture of the spleen (Con-MΦ). After transfection into MΦ, PIK3R1 expression at both the mRNA and the protein level was examined by real-time polymerase chain reaction and Western blot. The activities of MΦ were determined, and the expression and activity of NF-κB were also detected. Immunohistochemistry revealed expression and cellular distribution of PIK3R1 in the spleen. The PIK3R1-shRNA was successfully synthesized and cloned into the plasmid vector pGenesil-1, and specifically suppressed PIK3R1 expression at both the mRNA and the protein level. After transfection into HS-PHT-MΦ and Con-MΦ, PIK3R1 knockdown inhibited the viability of MΦ, reduced the phagocytic rate, the rate of antigen-presenting positive cells, the metabolic rate, and the secretion of IL-1β and TNF-α (all p<0.05), and decreased the expression and activity of NF-κB. Our data showed that the knocking down of PIK3R1 with shRNA produced by pGenesil-1 led to inhibition of viability and to decreased activity of MΦ associated with HS-PHT in vitro. Therefore, it is tempting to speculate that PIK3R1 might play a considerable role in the pathogenesis of HS-PHT, and inhibition of PIK3R1 expression might be a novel therapeutic strategy for HS-PHT.
authors.affiliation: Department of Surgery, The Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710004, Shanxi Province, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Shu
authors.initials: S
authors.lastname: Zhang
authors.forename: Zong-Fang
authors.initials: ZF
authors.lastname: Li
authors.forename: Chen
authors.initials: C
authors.lastname: Huang
authors.forename: Song
authors.initials: S
authors.lastname: Ren
authors.forename: Rui
authors.initials: R
authors.lastname: Zhou
authors.forename: An
authors.initials: A
authors.lastname: Jiang
authors.forename: An-Ning
authors.initials: AN
authors.lastname: Yang
mesh_terms: D000328:Adult; D000368:Aged; D002470:Cell Survival; D002478:Cells, Cultured; D015536:Down-Regulation; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D006971:Hypersplenism / Q000209:etiology / Q000235:genetics / Q000473:pathology*; D006975:Hypertension, Portal / Q000150:complications / Q000235:genetics / Q000473:pathology*; D008264:Macrophages / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D058539:Phosphatidylinositol 3-Kinase / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D013154:Spleen / Q000378:metabolism / Q000473:pathology
chemical_list: D034741:RNA, Small Interfering; D058539:Phosphatidylinositol 3-Kinase