Meng Fanling, Chen Xiuwei, Song Hongtao, Lou Ge, Fu Songbin
Department of Gynecology, The Affiliated Tumor Hospital, Harbin Medical University, Harbin, 150081, China.
Department of Pathology, The Affiliated Tumor Hospital, Harbin Medical University, 150 Ha Ping Road, Nangang District, Harbin, Heilongjiang Province, 150081, China.
Chem Biol Drug Des. 2016 Jan;87(1):121-30. doi: 10.1111/cbdd.12632. Epub 2015 Aug 31.
LAPTM4B (lysosome-associated protein transmembrane 4 beta) play an important role in several human carcinomas. We examines the effects of RNA interference mediated downregulation of human lysosomal-associated protein transmembrane 4 beta expression on the biological behavior of the human serous adenocarcinoma cell line NIH:OVCAR3. This study investigated the expression level of lysosomal-associated protein transmembrane 4 beta in several ovarian cancer cell lines. RNA interference mediated by recombinant lentiviral vectors expressing an artificial lysosomal-associated protein transmembrane 4 beta miRNA was used to induce long-lasting downregulation of lysosomal-associated protein transmembrane 4 beta gene expression in NIH:OVCAR3 cells. Lysosomal-associated protein transmembrane 4 beta expression as well as the motility, migration potential, and proliferation of the tumor cells was measured by flow cytometry, real-time polymerase chain reaction, Western blot analysis, transwell migration assays, wound healing assays, and cell counting kit-8 assays. In addition, the cell cycle analysis utilized fluorescence-activated cell sorting. Four recombinant plasmid expression vectors encoding premiRNAs against lysosomal-associated protein transmembrane 4 beta (pcDNA-LAPTM4B-miR-1, -2, -3, and-4) were constructed and transfected into 293T cells, which overexpress lysosomal-associated protein transmembrane 4 beta. The recombinant lentiviral vector for lysosomal-associated protein transmembrane 4 beta RNA interference was packaged with pcDNA-LAPTM4B-miR-3, which had the highest interfering efficiency, thereby successfully generating stable transfectants. Compared with the control cells, the LAPTM4B-miRNA-transfected NIH:OVCAR3 cells exhibited significant decreases in cell motility and invasion. Furthermore, LAPTM4B depletion resulted in a significant decrease in proliferating cell nuclear antigen, vascular endothelial growth factor, MMP2, MMP9, and CDK12 expression. We propose that lysosomal-associated protein transmembrane 4 beta expression may be an oncogene-inducing feature of invasive ovarian cancer cells and may be a potential therapeutic target for ovarian cancer treatment.
LAPTM4B（溶酶体相关蛋白跨膜4β）在多种人类癌症中发挥重要作用。我们研究了RNA干扰介导的人类溶酶体相关蛋白跨膜4β表达下调对人浆液性腺癌细胞系NIH:OVCAR3生物学行为的影响。本研究调查了几种卵巢癌细胞系中溶酶体相关蛋白跨膜4β的表达水平。使用表达人工溶酶体相关蛋白跨膜4β miRNA的重组慢病毒载体介导的RNA干扰来诱导NIH:OVCAR3细胞中溶酶体相关蛋白跨膜4β基因表达的长期下调。通过流式细胞术、实时聚合酶链反应、蛋白质免疫印迹分析、Transwell迁移试验、伤口愈合试验和细胞计数试剂盒-8试验来检测溶酶体相关蛋白跨膜4β的表达以及肿瘤细胞的运动性、迁移潜能和增殖。此外，利用荧光激活细胞分选进行细胞周期分析。构建了四种编码针对溶酶体相关蛋白跨膜4β的前体miRNA的重组质粒表达载体（pcDNA-LAPTM4B-miR-1、-2、-3和-4），并将其转染到过表达溶酶体相关蛋白跨膜4β的293T细胞中。用干扰效率最高的pcDNA-LAPTM4B-miR-3包装用于溶酶体相关蛋白跨膜4β RNA干扰的重组慢病毒载体，从而成功产生稳定转染子。与对照细胞相比，转染LAPTM4B-miRNA的NIH:OVCAR3细胞的细胞运动性和侵袭能力显著降低。此外，LAPTM4B缺失导致增殖细胞核抗原、血管内皮生长因子、MMP2、MMP9和CDK12表达显著降低。我们认为溶酶体相关蛋白跨膜4β表达可能是侵袭性卵巢癌细胞的一种致癌诱导特征，可能是卵巢癌治疗的潜在靶点。
