Henan Provincial People's Hospital/People's Hospital of Zhengzhou University, 7 Weiwu Road, Jinshui District, 450000, Zhengzhou, China.
College of Life Sciences, Henan Agricultural University, 450002, Zhengzhou, China.
Protein J. 2022 Apr;41(2):337-344. doi: 10.1007/s10930-022-10047-8. Epub 2022 May 7.
C-C motif chemokine ligand 5 (CCL5) is crucial in the tumor microenvironment. It has been previously reported to act as a key role in tumor invasion and metastasis. However, the function of exogenous CCL5 in ovarian cancer has not been well-characterized. The present study attempted to express and purify recombinant CCL5 protein and investigate the exogenous CCL5 in ovarian cancer cell proliferation. The human CCL5 was amplified and inserted into the pET-30a vectors for prokaryotic expression in Escherichia coli BL21. Soluble His-CCL5 was successfully expressed with 0.1 mmol/L of isopropyl-β-D-1-tiogalactopiranoside at 25 ℃ and purified by affinity chromatography. Additionally, methyl thiazolyl tetrazolium (MTT) assay demonstrated that CCL5 promotes ovarian cancer cell proliferation; increases the phosphorylation levels of extracellular-signal-regulated kinase and mitogen-activated protein kinase/ERK kinase, and increases the mRNA levels of Jun, NF-κB2, Nras, Relb, and Traf2. Furthermore, treatment with the MEK inhibitor reduced the Jun, NF-κB2, and Traf2 mRNA levels, indicating that exogenous CCL5 increased ovarian cancer cell proliferation, through MEK/ERK pathway activation, and Jun, NF-κB2, and Traf2 expression. The present study provided primary data for further studies to discover more CCL5 functions in ovarian cancer.
细胞因子 C-C 基序趋化因子配体 5（CCL5）在肿瘤微环境中起着至关重要的作用。先前已有研究报道，CCL5 在肿瘤侵袭和转移中起关键作用。然而，外源性 CCL5 在卵巢癌中的功能尚未得到很好的描述。本研究试图表达和纯化重组 CCL5 蛋白，并研究外源性 CCL5 在卵巢癌细胞增殖中的作用。扩增人 CCL5 并将其插入 pET-30a 载体中，在大肠杆菌 BL21 中进行原核表达。在 25℃下，用 0.1mmol/L 的异丙基-β-D-1-硫代半乳糖苷成功表达了可溶性 His-CCL5，并通过亲和层析进行纯化。此外，甲基噻唑基四唑（MTT）测定表明 CCL5 促进卵巢癌细胞增殖；增加细胞外信号调节激酶和丝裂原活化蛋白激酶/ERK 激酶的磷酸化水平，并增加 Jun、NF-κB2、Nras、Relb 和 Traf2 的 mRNA 水平。此外，用 MEK 抑制剂处理可降低 Jun、NF-κB2 和 Traf2 的 mRNA 水平，表明外源性 CCL5 通过 MEK/ERK 通路的激活和 Jun、NF-κB2 和 Traf2 的表达增加了卵巢癌细胞的增殖。本研究为进一步研究 CCL5 在卵巢癌中的更多功能提供了初步数据。
