结直肠癌细胞增殖诱导配体的异常表达促进肿瘤的生长和转移

Abnormal expression of APRIL in colorectal cancer cells promotes tumor growth and metastasis

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-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，Westernblot检测细胞周期和凋亡调控蛋白cyclinD1、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摹质金属蛋白酶2（MMP-2）和MMP-9mRNA的表达变化。APRIL．siRNA转染的结直肠癌细胞SW480拨种裸鼠，分析APRIL-siRNA对移植瘤生长和MMP表达的影响。结果APRIL—siRNA组G。／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48h后，400ng／mlrhAPRIL组和800ng／mlrhAPRIL组HCT-116细胞Go／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48h后，与非特异性序列转染组相比，APRIL—siRNA组SW480细胞cyclinD1和Bcl-2蛋白表达下降，p21蛋白表达增高。刺激48h后，与未刺激组相比，400ng／mlrhAPRILHCT-116细胞组cyclinD1和Bcl-2蛋白表达升高，p21蛋白表达下降。APRIL—siRNA转染SW480细胞后，APRII，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（100ng／m1）刺激HCT-116细胞后，侵袭和转移细胞数均高于末刺激组。APRIL—siRNA组SW480细胞中MMP-2和MMP-9mRNA相对表达水平低于非特异性序列转染组。rhAPRIL刺激48h后，HCT-116细胞中MMP-2和MMP-9mRNA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，APRll．-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。

Objective To investigate the effects of a proliferation-inducing ligand （APR1L） 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 Transwel] chambers. RT-PCR was applied to examine the mRNA expression level of MMP-2 and MMP-9. APRILsiRNA was used to transfeet directly SW480 cells, which were injected subcutaneously into nude mice, then the tumor growth and metastasis were observed. Results Cell proliferation ability of APRIL-siRNAtransfected SW480 cells was drastically repressed, and the percentage of G0/G1 phase cells was significantlyincreased （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 ）. cyelin D1 and Bcl-2 protein expression was up-regulated while p21 was down-regulated by rhAPR1L 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-I16 cells were promoted with up-regulated MMP-2 and MMP-9 mRNA expression（ both P 〈 0.05 ）. Tumor growth in the group transfeeted 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.