The Effect Of RNA Interference Of HMGA2 Related With Silicosis Fibrosis On The Expression Of The Collagen Type Ⅰand Ⅲ In Human Embryo Lung Fibroblast

Objectives In this study, HMGA2 was silenced in human embryonic lung fibroblast cells MRC-5 with HMGA2-si RNA being transfected into these cells. After stimulating MRC-5 with the alveolar macrophages(AM) supernatant, the expression of collagen type I and III was examined to explorethe role of HMGA2 in the process of silicosis fibrosis.Methods 1 The collection of alveolar macrophages(AM) of the patients with silicosis:The AM were obtained from the alveolar lavage of the patients with silicosis. After being centrifuged, filtrated and washed by PBS, the AM were stained by HE, antacid, Wright’s staining for identification, then were further curthered in DMEM medium with 10% fetal bovine serum(FBS).2 The AM were curthered with non-serum medium containing Si O2 for 18h, then the supernatant was collected filtered and saved.3 The construction of sh RNA expression plasmids: The sh RNA expression plasmids and the corresponding negative control plasmids were constructed into p GPU6/GFP/Neo–sh GAPDH by Shanghai Gemma Company. 4 The function of sh RNA expression plasmids were investigated by real time PCR and western-blot: The sh RNA expression plasmids were transfected into embryonic lung fibroblast cells with LipofectamineTM2000. After 24-48 h,the efficiency of transfection was examined by fluorescence microscope, then the total m RNA and protein of the MRC-5 cells were extracted and were investigated by reverse transcriotion real time PCR(RT-PCR) and western-blot assays.5 MRC-5 cells were divided into four groups: control group(C), in which the MRC-5 cells were curthered with 10% FBS DMEM medium; the stimulation group(S), in which the MRC-5 cells were curthered with supernatant of AM after being stimulated by Si O2; the transfection group(T), in which the MRC-5 cells were transfected with sh RNA expression plasmids and curthered with AM supernatant as stimulation group described; the negative plasmids transfection group(N), in which the MRC-5 cells were transfected with negative plasmids and curthered as the stimulation group described. 6 Immunocytochemistry and Western-blot methods were used to detect the expression of collagen type I and III: The expression of collagen type I and III of MRC-5 cells in each group was detected respectively 12 h, 24 h, 36 h, 48 h and 72 h after stimulation.Results 1 The plasmids were transfected with LipofectamineTM2000 into the human embryo lung fibroblasts MRC-5 cells at ratio plasmids: Lipofectamine TM2000 liposomes= 2μg:3μl. After 24h-48 h, the efficiency of transfection was observed by fluorescence microscope and the result showed that more than 90% MRC-5 cells were transfected with plasmids successfully with green fluorescence expressing.2 The expression of HMGA2 in MRC-5 cells was detected by RT-PCR and Western- blot assays after the transfection of plasmids.The results confirmed that the plasmid p GPU6/GFP/Neo-HMGA2-homo-768 had the highest interferential efficiency. Negative plasmids transfected group had no significant difference with control group.3 The results of immunocytochemistry and Western blot showed that the MRC-5 cells of control group expressed collagen type I and III; the expression of collagen type I and III in MRC-5 cells of stimulation group was upregulated, compared with the cells of control group(P < 0.05); the expression of collagen type I and III in transfected group was doweregulated compared with stimulation group(P < 0.05); however, there was no difference between transfection group and the stimulation group(P>0.05).Conclusions1 The expression of collagen type I and III in MRC-5 cells which were curthered with AM supernatant was inhibited significantly after the silence of HMAG2 by sh RNA plasmids. 2 Downregulation of HMAG2 by sh RNA plasmids provided evidences for silicosis therapy.