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目的通过慢病毒载体沉默乳腺癌细胞系SK-BR-3中血小板衍生生长因子D(PDGF-D)基因的表达,探讨PDGF-D基因沉默后对乳腺癌细胞增殖、凋亡及迁移的调控作用。方法应用RT-PCR及蛋白质印迹方法检测5种乳腺癌细胞系中PDGF-D的表达状况;设计人PDGF-D基因的shRNA慢病毒载体,在293T细胞中包装病毒并感染PDGF-D高表达的乳腺癌细胞系,运用蛋白质免疫印迹的方法鉴定其对PDGF-D基因的沉默效果;PDGF-D基因沉默后通过细胞增殖实验和软琼脂克隆形成实验检测其对细胞增殖能力的影响,并应用流式细胞技术检测细胞凋亡;运用Transwell迁移实验检测细胞的体外侵袭迁移能力。结果 PDGF-D在5种细胞系中存在差异性表达,其中具有低转移潜能的HT-29和SK-BR-3细胞与具有高转移潜能的LOVO、RKO和SW620细胞相比,具有更高的PDGFD表达水平(t=3.880,P<0.05)。选取低转移细胞株中PDGF-D表达相对较高的SK-BR-3作为研究对象,构建PDGF-D shRNA慢病毒载体沉默SK-BR-3细胞中PDGF-D的表达,蛋白质免疫印迹结果提示,PDGF-D稳定沉默的SK-BR-3细胞系构建成功;在稳定沉默PDGF-D基因后,MTT细胞增殖实验和软琼脂克隆形成实验表明SK-BR-3细胞增殖能力增强(F=75.23,P<0.001);流式细胞技术检测显示PDGF-D基因沉默后可抑制SK-BR-3细胞凋亡(F=84.44,P<0.001);细胞小室实验结果显示,PDGF-D沉默后SK-BR-3迁移能力增强(F=155.9,P<0.001)。结论 PDGF-D基因可能与乳腺癌的细胞增殖、凋亡与转移有关,进而参与了乳腺癌的发生、发展。
Objective To investigate the regulatory effect of PDGF-D gene on the proliferation, apoptosis and migration of breast cancer cells by lentiviral vector silencing the expression of PDGF-D gene in breast cancer cell line SK-BR-3 . Methods The expression of PDGF-D in five breast cancer cell lines was detected by RT-PCR and Western blotting. ShRNA lentiviral vector with human PDGF-D gene was designed and packaged in 293T cells to infect 293T cells with high expression of PDGF-D The effect of PDGF-D gene silencing on PDGF-D gene expression was evaluated by Western blotting. The effect of PDGF-D gene silencing on cell proliferation was tested by cell proliferation assay and soft agar colony formation assay Cell apoptosis was detected by cell-based assay. Transwell migration assay was used to detect the invasion and migration of cells in vitro. Results PDGF-D was differentially expressed in five cell lines, in which HT-29 and SK-BR-3 cells with low metastatic potential had higher (P <0.05) higher than LOVO, RKO and SW620 cells with high metastatic potential PDGFD expression level (t = 3.880, P <0.05). Select SK-BR-3 with relatively high expression of PDGF-D in low-metastatic cell line as study object, and to construct PDGF-D shRNA lentiviral vector to silence PDGF-D expression in SK-BR-3 cells. Western blot results suggest , SK-BR-3 cell line stably silenced by PDGF-D was successfully constructed. After stable silencing of PDGF-D gene, MTT cell proliferation assay and soft agar colony formation assay showed that the proliferation ability of SK-BR-3 cells was enhanced , P <0.001). Flow cytometry showed that PDGF-D gene silencing could inhibit the apoptosis of SK-BR-3 cells (F = 84.44, P <0.001) Migration of -BR-3 was enhanced (F = 155.9, P <0.001). Conclusion The PDGF-D gene may be involved in the proliferation, apoptosis and metastasis of breast cancer cells, which may be involved in the occurrence and development of breast cancer.