干扰素γ诱导蛋白16(IFI16)在多发性骨髓瘤中的功能研究

Functional Study of Interferon Gamma Inducible Protein 16(IFI16)in Multiple Myeloma

目的:探究干扰素γ诱导蛋白16(IFI16)在多发性骨髓瘤(MM)中的生物学功能。方法:将人多发性骨髓瘤细胞(RPMI-8266)分为Control组(未转染的细胞)、NC-sh组(转染阴性对照shRNA慢病毒的细胞)、IFI16-sh组(转染IFI16 shRNA慢病毒的细胞)、NC-OE组(转染阴性对照过表达慢病毒的细胞)和IFI16-OE组(转染IFI16过表达慢病毒的细胞)。使用Lipofectamine 2000进行细胞转染,培养48 h后收集细胞。通过MTT法和集落形成实验检测细胞增殖。通过Annexin V-FITC和PI染色法检测细胞凋亡。通过Transwell检测细胞侵袭。通过qRT-PCR检测IFI16、Bcl-2、Bax、成纤维细胞生长因子(FGF)1、FGF2的m RNA水平。通过Western blot检测IFI16的蛋白表达水平。将裸鼠随机分为NC-sh组和IFI16-sh组,每组6只。NC-sh组和IFI16-sh组裸鼠分别皮下注射转染NC-sh和IFI16-sh的RPMI-8266细胞悬液。35 d后测量肿瘤体积和肿瘤重量。采用ELISA法检测Th1细胞因子[干扰素-γ(IFN-γ)、白介素(IL)-12]、Th2细胞因子(IL-4)和促炎因子[IL-1β、IL-6、肿瘤坏死因子-α(TNF-α)]的水平。结果:与Control组和NC-sh组比较,IFI16-sh组RPMI-8266细胞的IFI16 m RNA和蛋白相对表达量降低(P<0.05),相对细胞活力、集落数量和侵袭细胞数量均降低(P<0.05),细胞凋亡率和Bax m RNA相对表达量均升高(P<0.05),Bcl-2、FGF1和FGF2的m RNA相对表达量均降低(P<0.05)。与Control组和NC-OE组比较,IFI16-OE组RPMI-8266细胞的IFI16 m RNA和蛋白相对表达量升高(P<0.05),相对细胞活力、集落数量和侵袭细胞数量均升高(P<0.05),细胞凋亡率和Bax m RNA相对表达量均降低(P<0.05),Bcl-2、FGF1和FGF2的m RNA相对表达量均升高(P<0.05)。与NC-sh组比较,IFI16-sh组裸鼠的肿瘤体积、肿瘤重量以及肿瘤组织中IFI16的m RNA和蛋白相对表达量均降低(P<0.05),外周血IFN-γ和IL-12水平均升高(P<0.05),IL-4、IL-1β、IL-6和TNF-α水平均降低(P<0.05)。结论:IFI16是MM中的一种致癌基因,下调IFI16可抑制MM细胞的生长和侵袭,其机制与纠正Th1/Th2细胞平衡和抑制促炎因子活性有关。

Objective:To reveal the function of interferon gamma inducible protein 16(IFI16)in multiple myeloma(MM).Methods:Human multiple myeloma cells(RPMI-8266)were divided into Control group(untransfected cells),NC-sh group(cells transfected with negative control shRNA lentivirus),IFI16-sh group(cells transfected with IFI16 shRNA lentivirus),NC-OE group(cells transfected with negative control overexpressing lentivirus)and IFI16-OE group(cells transfected with IFI16 overexpression lentivirus).Lipofectamine 2000 was used for transfection and cells were collected after 48 h culture.Cell proliferation was detected by MTT assay and colony formation test.Apoptosis was detected by Annexin V-FITC and PI staining.Cell invasion was detected by Transwell.The mRNA levels of IFI16,Bcl-2,Bax,fibroblast growth factor(FGF)1 and FGF2 were detected by qRT-PCR.The protein expression level of IFI16 was detected by Western blot.Nude mice were randomly divided into NC-sh group and IFI16-sh group(n=6).Nude mice in NC-sh group and IFI16-sh group were subcutaneously injected with RPMI-8266 cell suspension transfected with NC-sh and IFI16-sh,respectively.After 35 days,the tumor volume was measured and the tumor weight was weighed.The levels of Th1 cytokines[interferon-γ(IFN-γ),interleukin(IL)-12],Th2 cytokines(IL-4)and proinflammatory factors[IL-1β,IL-6,tumor necrosis factor-α(TNF-α)]were detected by ELISA method.Results:Compared with Control group and NC-sh group,the relative expression of IFI16 mRNA and protein of RPMI-8266 cells in IFI16-sh group decreased(P<0.05),relative cell viability and number of colony and invasive cells decreased(P<0.05),the apoptosis rate and relative expression of Bax mRNA increased(P<0.05),the relative mRNA expression level of Bcl-2,FGF1 and FGF2 decreased(P<0.05).Compared with Control group and NC-OE group,the relative expression of IFI16 mRNA and protein of RPMI-8266 cells in IFI16-OE group increased(P<0.05),relative cell viability and number of colony invasive cells increased(P<0.05),apoptosis rate and relative expression of Bax mRNA decreased(P<0.05),the relative mRNA expression level of Bcl-2,FGF1 and FGF2 increased(P<0.05).Compared with NC-sh group,the tumor volume,tumor weight,the relative expression of IFI16 mRNA and protein in tumor tissue of IFI16-sh group decreased(P<0.05),the levels of IFN-γand IL-12 in peripheral blood increased(P<0.05),the level of IL-4,IL-1β,IL-6 and TNF-αin peripheral blood decreased(P<0.05).Conclusion:IFI16 is a kind of oncogene in MM.Down-regulation of IFI16 can inhibit the growth and invasion of MM cells.The mechanism is related to correcting the balance of Th1/Th2 cells and inhibiting the activity of pro-inflammatory factors.