Chen Qi, He Quan, Zhuang Lingling, Wang Kunya, Yin Chunhua, He Linsheng
Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, People's Republic of China.
Department of Obstetrics and Gynecology, Jiangxi Provincial People's Hospital, Nanchang, Jiangxi 330006, People's Republic of China.
Onco Targets Ther. 2019 Nov 14;12:9697-9706. doi: 10.2147/OTT.S209757. eCollection 2019.
This study aimed to explore the effects of interferon-γ inducible protein 10 (IP10) and complementarity-determining region 3 (CDR3) of T cells receptor on ovarian cancer cells and the involved mechanisms.
IP10 and CDR3 were linked with single-chain antibody (scfv) and exotoxin gene muton of (PE40) to construct IP10-CDR3scfv and IP10-CDR3-PE40scfv. Then, we constructed pcDNA3.1-IP10-CDR3scfv and pcDNA3.1-IP10-CDR3-PE40scfv plasmids which were proved by HindIII/EcoRI digestion. SKOV3 cells and HOSEpiC cells were incubated with fluorescein isothiocyanate (FITC) labeled IP10-CDR3scfv and IP10-CDR3-PE40scfv proteins and protein levels were examined by flow cytometry. After gene transfection, SKOV3 cells were divided into four groups: Control, pcDNA3.1(+) negative control (NC) (pcDNA3.1(+) NC transfection), IP10-CDR3scfv (IP10-CDR3scfv transfection) and IP10-CDR3-PE40scfv (IP10-CDR3-PE40scfv transfection). Levels of IP10, CDR3, Caspase-3, cleaved Caspase-3 and Bcl-2 were determined by RT-PCR and Western blot. Cell viability and apoptosis were investigated by CCK-8 assay and Annexin V-FITC/PI assay, respectively.
The levels of FITC-labeled IP10-CDR3scfv and IP10-CDR3-PE40scfv proteins in the SKOV3+IP10-CDR3scfv group and the SKOV3+IP10-CDR3-PE40scfv group were remarkably higher than that in the SKOV3 group (P<0.05). So was the HOSEpiC related groups. There was no obvious difference in the levels of IP10, CDR3, Caspase-3, cleaved Caspase-3 and Bcl-2 between the control group and the pcDNA3.1(+) NC group. However, compared with the control group, the levels of Caspase-3 and Bcl-2 were reduced notably and the levels of IP10, CDR3 and cleaved Caspase-3 were elevated sharply in the IP10-CDR3scfv and IP10-CDR3-PE40scfv groups (P<0.05). The control group and the pcDNA3.1(+) NC group demonstrated similar cell viability and apoptosis. However, compared with the control group, cell viability in the IP10-CDR3scfv and IP10-CDR3-PE40scfv groups decreased significantly and cell apoptosis increased (P<0.05).
IP10-CDR3 could reduce the viability and induce the apoptosis of ovarian cancer cells by down-regulating the expression of Bcl-2 and Caspase-3.
本研究旨在探讨干扰素-γ诱导蛋白10（IP10）及T细胞受体互补决定区3（CDR3）对卵巢癌细胞的影响及其作用机制。
将IP10和CDR3与单链抗体（scfv）及绿脓杆菌外毒素A基因（PE40）突变体连接，构建IP10-CDR3scfv和IP10-CDR3-PE40scfv。然后构建pcDNA3.1-IP10-CDR3scfv和pcDNA3.1-IP10-CDR3-PE40scfv质粒，经HindIII/EcoRI酶切鉴定。用异硫氰酸荧光素（FITC）标记的IP10-CDR3scfv和IP10-CDR3-PE40scfv蛋白孵育SKOV3细胞和人卵巢上皮细胞（HOSEpiC），通过流式细胞术检测蛋白水平。基因转染后，将SKOV3细胞分为四组：对照组、pcDNA3.1(+)阴性对照组（pcDNA3.1(+) NC转染组）、IP10-CDR3scfv组（IP10-CDR3scfv转染组）和IP10-CDR3-PE40scfv组（IP10-CDR3-PE40scfv转染组）。通过RT-PCR和蛋白质印迹法检测IP10、CDR3、半胱天冬酶-3（Caspase-3）、裂解的Caspase-3和Bcl-2的水平。分别采用CCK-8法和Annexin V-FITC/PI法检测细胞活力和凋亡情况。
SKOV3+IP10-CDR3scfv组和SKOV3+IP10-CDR3-PE40scfv组中FITC标记的IP10-CDR3scfv和IP10-CDR3-PE40scfv蛋白水平显著高于SKOV3组（P<0.05）。HOSEpiC相关组亦是如此。对照组和pcDNA3.1(+) NC组之间IP10、CDR3、Caspase-3、裂解的Caspase-3和Bcl-2水平无明显差异。然而，与对照组相比，IP10-CDR3scfv组和IP10-CDR3-PE40scfv组中Caspase-3和Bcl-2水平显著降低，IP10、CDR3和裂解的Caspase-3水平急剧升高（P<0.05）。对照组和pcDNA3.1(+) NC组表现出相似的细胞活力和凋亡情况。然而，与对照组相比，IP10-CDR3scfv组和IP10-CDR3-PE40scfv组细胞活力显著降低，细胞凋亡增加（P<0.05）。
IP10-CDR3可通过下调Bcl-2和Caspase-3的表达降低卵巢癌细胞活力并诱导其凋亡。
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