Objective To investigate the effects of a proliferation-inducing ligand (APRIL) on colorectal cancer (CRC) cell growth and migration,and to observe the role of APRIL in CRC biological behavior.Methods The siRNA plasmid vector targeting APRIL gene (APRIL-siRNA) was transfected into human colorectal cancer SW480 cells and recombinant human APRIL (rhAPRIL) was used to stimulate human colorectal cancer HCT-116 cells.Cell proliferation activity was analyzed using cell counting kit-8 (CCK-8),cell cycle was detected by flow cytometry,and the protein expression of cyclin D1,p21 and Bcl-2 was detected by Western blot analysis.Tumor cell migration and invasion were measured by Transwell chambers.RT-PCR was applied to examine the mRNA expression level of MMP-2 and MMP-9.APRIL-siRNA was used to transfect directly SW480 cells,which were injected subcutaneously into nude mice,then the tumor growth and metastasis were observed.Results Cell proliferation ability of APRIL-siRNA-transfected SW480 cells was drastically repressed,and the percentage of G0/G1 phase cells was significantly increased (t =4.12,P ＜ 0.05),accompanied with depressed cyclin D1,Bcl-2 expression and elevated p21 expression.Cell proliferation ability of rhAPRIL-stimulated HCT-116 cells was promoted with a decreased G0/G1 phase ratio (t =3.31,P ＜ 0.05).cyclin D1 and Bcl-2 protein expression was up-regulated while p21 was down-regulated by rhAPRIL stimulation.Metastatic and invasive capacities of APRIL-siRNA-transfected SW480 cells were significantly inhibited compared with their respective controls (both P ＜ 0.05),accompanied with the deregulated MMP-2 and MMP-9 mRNA expression.Metastatic and invasive capacities of rhAPRIL-stimulated HCT-116 cells were promoted with up-regulated MMP-2 and MMP-9 mRNA expression(both P ＜ 0.05).Tumor growth in the group transfected with APRIL-siRNA appeared to be slower than that in the control groups and the expression of MMP-2,MMP-9 in tumor tissues was depressed in the APRIL-siRNA group.Conclusions APRIL facilitates tumor growth and metastasis,and is associated with carcinogenesis and prognosis.Our findings suggest that APRIL might be used as a novel target for the intervention and therapy of colorectal cancer.%目的 探讨增殖诱导配体(APRIL)对结直肠癌细胞生长和转移的影响,以进一步明确APRIL在结直肠癌生物学行为中的作用.方法 APRIL基因的小干扰RNA质粒载体(APRIL-siRNA)转染人结直肠癌细胞SW480,重组人APRIL蛋白(rhAPRIL)刺激结直肠癌细胞HCT-116,采用CCK-8法检测细胞的增殖能力,流式细胞术检测细胞周期变化,Western blot检测细胞周期和凋亡调控蛋白cyclin D1、p21和Bcl-2的表达水平,Transwell小室侵袭和转移试验分析细胞侵袭及转移能力,逆转录聚合酶链反应检测基质金属蛋白酶2(MMP-2)和MMP-9 mRNA的表达变化.APRIL-siRNA转染的结直肠癌细胞SW480接种裸鼠,分析APRIL-siRNA对移植瘤生长和MMP表达的影响.结果 APRIL-siRNA组G0/G1期细胞比例(73.5％)高于非特异性序列转染组(53.9％)和未转染组(51.1％,均P＜0.05).刺激48 h后,400 ng/ml rhAPRIL组和800 ng/ml rhAPRIL组HCT-116细胞G0/G1期细胞比例(分别为34.9％和22.7％)低于未刺激组(45.1％,均P＜0.05).转染48 h后,与非特异性序列转染组相比,APRIL-siRNA组SW480细胞cyclin D1和Bcl-2蛋白表达下降,p21蛋白表达增高.刺激48 h后,与未刺激组相比,400 ng/ml rhAPRILHCT-116细胞组cyclin D1和Bcl-2蛋白表达升高,p21蛋白表达下降.APRIL-siRNA转染SW480细胞后,APRIL-siRNA组Transwell小室滤膜上每低倍视野侵袭和转移细胞数低于非特异性序列转染组.rhAPRIL(100 ng/ml)刺激HCT-116细胞后,侵袭和转移细胞数均高于未刺激组.APRIL-siRNA组SW480细胞中MMP-2和MMP-9 mRNA相对表达水平低于非特异性序列转染组.rhAPRIL刺激48 h后,HCT-116细胞中MMP-2和MMP-9 mRNA相对表达水平明显高于未刺激组.与非特异性序列转染组和未转染组相比,APRIL-siRNA组移植瘤的生长速度较缓慢,APRIL-siRNA组移植瘤组织中APRIL、MMP-2和MMP-9蛋白表达量明显降低.结论 APRIL可促进结直肠癌细胞的生长及转移,与移植瘤的形成有关.
展开▼
