shRNA介导沉默肺腺癌转移相关转录本1表达对人卵巢癌细胞株OVCAR3侵袭与转移的影响  

Effects of shRNA-mediated silence of lung adenocarcinoma transcript 1 on the invasion and metastasis of ovarian carcinoma cell line OVCAR3

作　　者：周艳清[1] 李娟[1] 谭琳玉 徐霞英 吕华兵[1] 温启荣[1] 生秀杰[1] 

机构地区：[1]广州医科大学附属第三医院妇产科广东省产科重大疾病重点实验室,510150

出　　处：《肿瘤研究与临床》2015年第10期664-668,672,共6页Cancer Research and Clinic

基　　金：广东省科技计划（20138021800307）

摘　　要：目的检测肺腺癌转移相关转录本1（MALAT-1）在人卵巢癌细胞株中的表达，并探讨shRNA介导沉默MALAT-1表达对卵巢癌细胞OVCAR3生物学功能的影响。方法采用实时荧光定量聚合酶链反应（qRT-PCR）检测ES-2、SKOV3、OVCAR3、A27804株卵巢癌细胞中MALAT-1mRNA水平的表达差异，设计构建4条特异性干扰MALAT-1表达的shRNA—pGPU6／GFP／Neo表达载体和-条不干扰任何基因表达的空载体作为阴性对照，转染高表达MALAT-1的OVCAR3细胞，qRT-PCR筛选并鉴定有效沉默MALAT-1的shRNA序列。应用CCK-8比色法、体外黏附与划痕实验、Transwell迁移及侵袭实验检测MALAT-1沉默后OVCAR3细胞生长增殖、黏附、划痕修复、迁移及侵袭能力的改变。结果筛选出高表达MALAT-1的OVCAR3细胞株，有效沉默MALA-1基因后，OVCAR3细胞株的增殖、划痕修复能力明显抑制，黏附能力有不同程度降低。Transwell迁移实验中，干扰组转入底层膜细胞数量为（52．17±4．48）个，低于阴性对照组的。（286．50±12．23）个和空白对照组的（295．67±6．96）个；在Transwell侵袭实验中，干扰组转入底层膜细胞数为（37．33±2．40）个，低于阴性对照组的（239．00±15．72）个和空白对照组的（222．67±20．85）个，干扰组与对照组比较差异有统计学意义（P〈0．001）。结论利用shRNA介导沉默MALAT-1的表达可以有效抑制卵巢癌细胞株OVCAR3的增殖、黏附、划痕修复以及迁移、侵袭能力，MALAT-1有望成为卵巢癌治疗的有效靶基因。Objective To evaluate the expression of metastasis-associated lung adenocarcinoma transcript 1 （MALAT-1） in ovarian cancer cell lines, and to investigate the biological effects of down-regulated MALAT-1 on OVCAR3 cells. Methods qRT-PCR analysis was used to examine the expression level of MALAT-1 gene in ovarian cancer cells, including ES-2, A2780, SKOV3 and OVCAR3 cell lines. For functional research, four shRNA oligos specially targeting MALAT-1 and a empty vector were designed and constructed into pGPU6/GFP/Neo, then transfected into OVCAR3 cells, qRT-PCR was used to confirm the effective suppression of MALAT-1. Changes of proliferation and adhesion of cells were analyzed by CCK-8 and adhesion assays. Wound-healing, transwell migration and invasion assays were used to examine migration and invasion of MALAT-l-silencing cells in vitro. Results The expression of MALAT-1 gene in OVCAR3 cells was high, and qRT-PCR results confirmed successfully the knockdown of MALAT-1 after transient transfection. After successful suppression of MALAT-1, the proliferation, wound-healing and adhesion ability in vitro were inhibited to some degree. In transwell migration assay, the number of migration cells in MALAT- 1-silencing group was 52.17±4.48, which is much less than that in the negative and control groups （286.50± 12.23 and 295.67±6.96, respectively）. In invasion assay, the number of invasion cells passing the transwell membrane in MALAT-l-silencing group （37.33±2.40） was also decreased significantly, compared to that in the negative and control groups （239.00±15.72 and 222.67±20.85, P 〈 0.05）. Conelusions shRNA-mediatedsilence of MALAT-1 can effectively inhibit the proliferation, adhesion, migration and invasion abilities of ovarian cancer cell line OVCAR3 in vitro, indicating MALAT-1 is expected to be a target gene for the treatment of ovarian cancer.

关 键 词：卵巢肿瘤 MALAT-1 肿瘤浸润 肿瘤转移 RNA干扰 

分 类 号：R737.31[医药卫生—肿瘤]