摘要
肿瘤已成为老年动物常见疾病。多种恶性肿瘤高表达血清和糖皮质激素诱导的蛋白激酶(SGK),但其对肿瘤生长的作用机理还未完全解析。为了探究SGK对肿瘤生长的影响及作用机制,本研究利用人乳腺癌细胞系(MDA-MB-231),通过小干扰RNA(siRNA)敲低SGK,使用二甲氧唑黄(XTT)、溴脱氧尿苷(Brdu)和软琼脂克隆形成试验检测SGK对MDA-MB-231细胞增殖和转化的影响;利用过表达系统构建小鼠胚胎成纤维细胞系NIH3T3/HER2/SGK和NIH3T3/HER2/SGK/p27,通过XTT、Brdu、流式细胞术和软琼脂克隆形成试验探究SGK是否通过抑癌基因p27蛋白调控肿瘤细胞的增殖转化和细胞周期;使用蛋白免疫印迹(WB)和免疫荧光(IF)技术分析SGK调控p27的机制;利用裸鼠构建体内肿瘤模型,通过计算形成肿瘤的体积和免疫组织化学(IHC)技术分析SGK调控肿瘤生长的机制。结果显示,敲低SGK能够显著下调肿瘤细胞的增殖和转化(P<0.05);过表达p27能够明显降低肿瘤细胞的增殖和转化(P<0.05),且G0-G1期的细胞数量增多,而过表达SGK能够逆转这些现象;此外,过表达SGK能够诱导p27定位于细胞质。在小鼠肿瘤模型中也显示,SGK能够降低p27蛋白水平,促进肿瘤生长。结果表明,无论在体外还是体内,SGK均能够通过抑制p27促进肿瘤细胞生长,提示SGK可以作为动物肿瘤疾病诊断和预后的指标,是肿瘤相关药物研发的潜在靶点。
Tumor has become a common disease in elderly animals.Serum and glucocorticoid-induced protein kinase(SGK)is highly expressed in various malignant tumors,but its mechanism in tumor growth has not been fully elucidated.To investigate the impact and mechanism of SGK on tumor growth,this study used the human breast cancer cell line(MDA-MB-231)and knocked down SGK using small interfering RNA(siRNA).The effects of SGK on MDA-MB-231 cell proliferation and transformation were assessed using XTT,bromodeoxyuridine(BrdU),and soft agar colony formation assays.Mouse embryonic fibroblast cell lines NIH3T3/HER2/SGK and NIH3T3/HER2/SGK/p27 were constructed using an overexpression system.XTT,BrdU,flow cytometry,and soft agar colony formation assays were employed to investigate whether SGK regulates tumor cell proliferation,transformation,and cell cycle through the anti-cancer gene p27 protein.The mechanism of SGK regulation of p27 was analyzed using Western blot(WB)and immunofluorescence(IF)techniques.A mouse xenograft tumor model was established,and tumor volume calculation and immunohistochemistry(IHC)were used to analyze the mechanism of SGK regulation of tumor growth.The results showed that knocking down SGK significantly reduced tumor cell proliferation and transformation(P<0.05).Overexpression of p27 markedly decreased tumor cell proliferation and transformation(P<0.05),and the number of cells in the G0-G1 phase increased,which was reversed by SGK overexpression.Additionally,SGK overexpression induced the cytoplasmic localization of p27.In the mouse tumor model,SGK decreased p27 protein levels and promoted tumor growth.These findings suggest that SGK can promote tumor cell growth by inhibiting p27 both in vitro and in vivo,indicating SGK as a potential diagnostic and prognostic indicator for animal tumor diseases and a promising target for cancer drug development.
作者
安一娜
谭姝瑜
刘天龙
许建海
董彦君
AN Yi-na;TAN Shu-yu;LIU Tian-long;XU Jian-hai;DONG Yan-jun(National Key Laboratory of Veterinary Public Health Security,College of Veterinary Medicine,China Agricultural University,Beijing 100193,China;Laboratory of Veterinary Pathology and Nanopathology,College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)
出处
《中国兽医杂志》
CAS
北大核心
2023年第11期1-10,共10页
Chinese Journal of Veterinary Medicine
基金
国家自然科学基金项目(3177130338)。
