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目的探讨衰老关键蛋白3(fibulin 3,FBLN3)在人结直肠癌SW480细胞增殖及转移中的作用。方法人结直肠癌SW480细胞株分为空白组(培养液2mL)、空载组(FBLN3阴性慢病毒+培养液2mL)和FBLN3过表达组(FBLN3过表达慢病毒+培养液2 mL);采用MTT法观察FBLN3对SW480细胞增殖的影响;流式细胞技术检测FBLN3对SW480细胞周期的影响;Transwell实验观察FBLN3对SW480细胞侵袭、转移的影响;Western blot法检测FBLN3对相关蛋白表达的影响。结果 FBLN3过表达组转染后24h(0.386±0.008)、48h(0.535±0.011)、72h(0.467±0.012)吸光度值均低于空白组(0.460±0.011、0.787±0.009、1.031±0.005)和空载组(0.445±0.009、0.750±0.004、0.949±0.014)(P<0.05),空白组与空载组比较差异无统计学意义(P>0.05);转染48h后FBLN3过表达组G1期细胞比率[(65.02±1.05)%]高于空白组[(47.01±0.90)%]和空载组[(48.25±1.41)%],S期细胞比例[(15.41±1.70)%]低于空白组[(34.90±1.28)%]和空载组[(28.82±2.40)%](P<0.05);转染48h后FBLN3过表达组侵袭细胞计数(47±5)和迁移细胞计数(64±10)均低于空白组(106±7、144±9)及空载组(105±12、145±6)(P<0.05),空白组与空载组比较差异无统计学意义(P>0.05);FBLN3过表达组p-AKT、p-哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、基质金属蛋白酶(matrix metalloproteinase,MMP)-2、MMP-9、Bcl-2及细胞周期蛋白D1(cyclinD1)蛋白表达水平均低于空白组和空载组,凋亡诱导分子Bcl-2相关X蛋白(Bcl-2associated X protein,Bax)和细胞周期蛋白依赖性激酶抑制剂P21蛋白表达均高于空白组和空载组(P<0.05)。结论 FBLN3过表达可抑制AKT/mTOR信号通路活化,抑制侵袭转移因子MMP-2、-9,凋亡抑制因子Bcl-2及细胞周期蛋白cyclinD1表达,促进凋亡诱导分子Bax和细胞周期蛋白依赖性激酶抑制剂P21蛋白的表达,导致肿瘤细胞周期G_1期阻滞,抑制SW480细胞的增殖、侵袭及转移,从而抑制结直肠癌发生、发展。
Objective To investigate the role of fibronectin 3 (FBLN3) in proliferation and metastasis of human colorectal cancer SW480 cells. Methods Human colorectal cancer cell line SW480 was divided into blank group (2 mL culture medium), empty group (FBLN3 negative lentivirus + culture medium 2 mL) and FBLN3 overexpression group (FBLN3 overexpression lentivirus + culture medium 2 mL) The effect of FBLN3 on SW480 cell cycle was observed by MTT assay. The effect of FBLN3 on SW480 cell cycle was detected by flow cytometry. The effect of FBLN3 on invasion and metastasis of SW480 cells was observed by Transwell assay. The effect of FBLN3 on the expression of related proteins was detected by Western blot. Results Absorbance values of FBLN3 overexpression group at 24h (0.386 ± 0.008), 48h (0.535 ± 0.011) and 72h (0.467 ± 0.012) were lower than that of the blank group (0.460 ± 0.011,0.787 ± 0.009,1.031 ± 0.005) (0.445 ± 0.009,0.750 ± 0.004,0.949 ± 0.014) (P <0.05), but there was no significant difference between the blank group and the no-load group (P> 0.05). After transfected with FBLN3 for 48h, (65.02 ± 1.05)%] was higher than that in the blank group [(47.01 ± 0.90)%] and the no-load group [(48.25 ± 1.41)%] (34.90 ± 1.28)%] and no-load group (28.82 ± 2.40)%] (P <0.05). The number of invasive cells and the number of migrating cells in the FBLN3 overexpression group were 47 ± 5 and 64 ± 10 ) Were lower than those in the blank group (106 ± 7, 144 ± 9) and no-load group (105 ± 12, 145 ± 6) (P <0.05), but there was no significant difference between the blank group and the no-load group ), P-AKT in overexpression group FBLN3, mammalian target of rapamycin (mTOR), matrix metalloproteinase (MMP) -2, MMP-9, Bcl- CyclinD1 protein expression levels were lower than the blank group and no-load group, apoptosis-inducing molecules Bcl- The expression of Bcl-2associated X protein (Bax) and cyclin-dependent kinase inhibitor P21were higher than that of blank group and no-load group (P <0.05). Conclusion Overexpression of FBLN3 can inhibit the activation of AKT / mTOR signaling pathway, inhibit the expression of invasion and metastasis factors MMP-2, -9, Bcl-2 and cyclinD1 and promote the apoptosis-inducing molecule Bax and cyclin-dependent The expression of P21, a kinase inhibitor, leads to cell cycle G 1 arrest, inhibits the proliferation, invasion and metastasis of SW480 cells and thus inhibits the development and progression of colorectal cancer.