膀胱癌相关蛋白基因对HeLa细胞增殖的抑制作用

Inhibitory Effect of Bladder Cancer Related Protein Gene on Hela Cell Ploliferation

背景与目的:通过癌基因与抑癌基因的基因分类表达芯片筛选发现,在宫颈癌组织中,膀胱癌相关蛋白基因(bladdercancerrelatedprotein,BLCAP)表达降低最为明显。提示该基因可能与宫颈癌发生相关。本研究旨在探讨该基因对宫颈癌细胞增殖的抑制作用。方法:用RT-PCR法检测54例宫颈癌组织和25正常宫颈组织中BLCAP基因的表达情况。制备BLCAP全长cDNA并克隆到真核表达载体pLXSN上,稳定转染至宫颈癌细胞系HeLa中,细胞计数方法和克隆形成实验观察稳定转染BLCAP基因的HeLa细胞的细胞形态、细胞增殖及集落形成能力;利用DNAladder检测BLCAP基因引起HeLa细胞凋亡情况;裸鼠体内抑瘤实验观察BLCAP基因的抑瘤作用。结果:BLCAP在宫颈癌组织中不表达或低表达,与正常宫颈组织相比明显降低;转染BLCAP基因的HeLa细胞倍增时间为69.4h,较未转染HeLa细胞(27.5h)和转染空载体的HeLa细胞(30.2h)明显延长,差异有显著性(P﹤0.05);细胞同步对比实验显示,转染BLCAP基因的HeLa细胞的克隆形成率明显低于未转染HeLa细胞(t=5.98,P<0.01)和转染空载体的HeLa细胞(t=4.69,P﹤0.01);HeLa细胞在转染BLCAP基因后出现了DNA梯形条带。将转染BLCAP基因的HeLa细胞、转染空载体的HeLa细胞和未转染的HeLa细胞分别注射入BALB/c裸鼠体内,30天后处死动物,剥离肿瘤组织并称重,各组平均瘤重分别为1.015g、1.612g和1.530g,转染BLCAP基因的细胞抑瘤能力与两对照组相比,差异有显著性(P<0.05)。肿瘤病理切片结果显示,稳定转染BLCAP基因的HeLa细胞所成肿瘤组织,癌组织被纤维组织包裹,边缘整齐,未侵犯肌层和脂肪组织,部分癌巢边缘癌细胞可见散在类似凋亡细胞的细胞群。而未转染质粒组及转染空载体pLXSN质粒组所成肿瘤组织癌组织突破肌层呈浸润性生长,癌巢边缘癌细胞病理性核分裂像多见。结论:BLCAP基因在宫颈癌组织中表达下调,对HeLa细胞的增殖具有明显抑制作用,为宫颈癌相关候选抑癌基因。

BACKGROUND ＆ OBJECTIVE： Bladder cancer related protein （BLCAP） gene was found downregulated most in primary cervical cancer tissues using oncogene/tumor suppressor gene microarray screening in our previous study, therefore this study was to explore the possible correlation between BLCAP gene and cervical cancer. METHODS： BLCAP expression was investigated in 54 cervical cancer and 25 normal tissues by reverse transcription polymerase chain reaction （RT-PCR）. Full length of BLCAP cDNA was cloned into pLXSN expression vector and stably transfected into cervical cancer cell line HeLa cells. Cell proliferation, colony formation ability and apoptosis were determined by cell number counting, colony formation and DNA ladder assay. Nude mice were used to study the anti-tumor effect of BLCAP gene in vivo. RESULTS： BLCAP gene expression was significantly down-regulated or even not observed in human cervical cancer tissues compared to the normal ones. The cell doubling time of HeLa cells transfected with BLCAP was significantly elevated to 69.4 hrs （P〈 0.05）, compared to that of the parental cells （27.5 hrs） or cells transfected with empty vectors （30.2 hrs）. Moreover, in comparison with control cells, the colony formation efficiency of cells transfected with BLCAP gene was significantly lower （t=5.98, P〈0.01）. BLCAP expression also sensitized Hela cells to apoptosis induced by serum deprivation. In vivo, smaller size of tumors were formed in mice after the injection of cells transfected with BLCAP for 30 days compared to those injected with parental cells or cells transfected with empty vector （tumor wet weights were 1.015 g, 1.612 g, and 1.530 g, respectively, P〈0.05）. Furthermore, under pathological examinations, tumor tissues formed by cells transfected with BLCAP gene displayed less invasive potential with integral vascular fibrous capsules; muscle or adipocyte tissue invasion was not observed. In comparison, necropsy revealed that the tumors formed from the control cells were attached to the underlying muscles; histologically, the neoplastic cells were locally invasive and associated with fibrous connective tissues. These cells exhibited severer cytoplasmic and nuclear pleomorphism compared to cells transfected with BLCAP. CONCLUSION： BLCAP gene is down-regulated in cervical carcinoma tissues and could suppress tumorigenic ability and growth of HeLa cells, thus it may be a potential suppressor candidate gene of cervical carcinoma.