摘要
目的探索Sec62对结直肠癌细胞的增殖作用及其潜在机制。方法应用Western blotting技术在结直肠癌细胞和永生化正常结肠上皮细胞中检测Sec62蛋白的表达。依据结直肠癌细胞中Sec62蛋白的表达情况,选取两株结直肠癌细胞分别构建Sec62过表达稳定转染细胞及其对照(NC:过表达阴性对照组,以过表达组阴性对照病毒转染细胞;OE:过表达组,以过表达慢病毒转染细胞)和Sec62敲减稳定转染细胞及其对照(shNC:敲减阴性对照组,以敲减组阴性对照病毒转染细胞;KD-1:敲减组1,以敲减慢病毒1转染细胞;KD-2:敲减组2,以敲减慢病毒2转染细胞)。采用CCK-8、平板克隆形成等增殖相关体外实验检测稳定转染组及其对照组细胞的增殖能力。构建裸鼠皮下移植瘤模型评估Sec62对结直肠癌细胞体内增殖能力的影响,将24只雄性BALB/c裸鼠随机分为NC、OE、shNC、KD-14组,每组6只,将稳定转染组及其对照组细胞分别接种于裸鼠腹股沟处,5周后处死裸鼠,观察各组裸鼠皮下成瘤形成情况。采用流式细胞术检测稳定转染组及其对照组细胞周期变化。利用Western blotting检测稳定转染组及其对照组细胞周期相关蛋白Cyclin D1、CDK4和PCNA的表达。结果Western blotting发现Sec62在结直肠癌细胞中的表达显著高于正常结肠上皮细胞(P<0.01)。CCK-8和平板克隆实验结果均表明与NC细胞相比,OE细胞的增殖能力增强(P<0.05),而与shNC相比,KD-1和KD-2细胞的增殖能力明显降低(P<0.05)。裸鼠皮下移植瘤模型显示Sec62过表达细胞种植后形成的皮下肿瘤质量显著高于NC(P<0.01),而Sec62敲减(KD-1)细胞种植后裸鼠皮下成瘤的质量低于shNC(P<0.01)。流式细胞术检测细胞周期的结果显示,Sec62表达升高后细胞周期进程加快,而Sec62表达被抑制后细胞周期停滞。Western blotting结果表明,与NC细胞相比,OE细胞中Cyclin D1、CDK4和PCNA蛋白表达水平升高,而与shNC细胞相比,KD-1�
Objective To explore the effect and potential mechanism of Sec62 in colorectal cancer(CRC)cell proliferation.Methods The expression of Sec62 was identified in CRC cell lines and immortalized normal colon epithelial cells(FHC)by conducting Western blotting assay.Based on the protein level of Sec62 in various CRC cell lines,two CRC cell lines were selected to construct Sec62-overexpressed stably transfected cell line and control(NC:Sec62-overexpressed negative control group,transfecting cells with Sec62-overexpressed negative virus vector;OE:Sec62-overexpressed group,transfecting cells with Sec62-overexpressed lentivirus)and Sec62-knockdown stably transfected cell line and control(shNC:Sec62-knockdown negative control group,transfecting cells with Sec62-knockdown negative virus vector;KD-1:Sec62-knockdown group 1,transfecting cells with Sec62-knockdown lentivirus 1;KD-2:Sec62-knockdown group 2,transfecting cells with Sec62-knockdown lentivirus 2).The proliferative effects of Sec62 in vitro were identified by CCK-8 and plate clone formation assays.The cell proliferation ability of constructed CRC cells in vivo was evaluated using subcutaneous tumorigenesis assay.Specifically,24 male BALB/c nude mice were randomly divided into 4 groups of 6 mice each,including NC,OE,shNC,and KD-1.Then the constructed cell lines were injected into the flank of the nude mice.The nude mice were sacrificed after 5 weeks to observe the formation of subcutaneous tumors in each group.The role of Sec62 in CRC cell cycle was confirmed by flow cytometry.Moreover,the expressions of Cyclin D1,CDK4 and PCNA in the constructed cell lines were evaluated by Western blotting.Results Western blotting exhibited that the expression of Sec62 in CRC cell lines was significantly higher than that in FHC cells(P<0.01).Both the CCK-8 and plate cloning assays demonstrated that overexpressed Sec62(LV-Sec62)promoted the proliferative ability of CRC cells compared with NC(P<0.05),whereas inhibited Sec62(KD-1,-2)significantly impaired CRC proliferation compared wi
作者
金熠蓉
储屹
吴开春
梁洁
JIN Yirong;CHU Yi;WU Kaichun;LIANG Jie(State Key Laboratory of Cancer Biology&National Clinical Research Center for Digestive Diseases,Department of Gastroenterology,Xijing Hospital,Air Force Medical University,Xi'an 710032,China)
出处
《空军军医大学学报》
CAS
2022年第3期309-314,共6页
Journal of Air Force Medical University
基金
国家自然科学基金(81772650,81421003,81572302)
国家重点实验室专项基金(CBSKL2015Z12)。
