舌鳞癌细胞Tca8113中蛋白激酶C和丝裂原活化蛋白激酶激酶/细胞外调节蛋白激酶通路对诱导型一氧化氮合酶表达的影响

doi:10.7518/hxkq.2018.02.004

摘要/Abstract

摘要：

目的在舌鳞癌细胞Tca8113中,探索与细胞增殖密切相关的诱导型一氧化氮合酶（NOS-2）的表达调控机制。方法采用RNAi技术沉默Tca8113细胞中NOS-2、蛋白激酶C（PKC）-α、PKC-β和PKC-δ的基因;Griess Reagent法检测NOS-2基因沉默后一氧化氮（NO）生成量;CCK8法测定细胞增殖活性;实时定量荧光聚合酶链反应（q-PCR）技术检测各种方法处理后NOS-2、PKC-α、PKC-β和PKC-δ的基因表达;Western blotting技术测定丙二醇甲醚醋酸酯（PMA）作用细胞后的细胞外调节蛋白激酶（ERK）1/2磷酸化程度。结果利用NOS-2的siRNA处理后,Tca8113细胞的增殖能力明显降低（P<0.01）;PKC的活性与NOS-2的基因表达成负相关（P<0.05）;PKC亚型PKC-α、PKC-β和PKC-δ共同参与NOS-2的基因调控（P<0.01）;丝裂原活化蛋白激酶激酶（MEK）/ERK通路负调控NOS-2的基因表达（P<0.05）;PKC通过MEK/ERK通路负调控NOS-2的基因表达（P<0.05）。结论在Tca8113细胞中,PKC通过MEK/ERK通路负调控与细胞增殖相关的NOS-2的基因表达。

关键词: 诱导型一氧化氮合酶, 蛋白激酶C, 丝裂原活化蛋白激酶激酶/细胞外调节蛋白激酶通路, 细胞增殖

Abstract:

Objective To explore the regulatory mechanism of inducible nitric oxide synthase (NOS-2) expression related to proliferation of Tca8113 cells. Methods RNAi mediated by short hairpin RNAs was utilized to knock down NOS-2, protein kinase C (PKC)-α, PKC-β and PKC-δ. Griess Reagent played a significant role on the detection of NO product after NOS-2 silence. The cell proliferation was determined by CCK8 method. Quantitative real-time polymerase chain reaction (q-PCR) was recruited to check the mRNA level of NOS-2, PKC-α, PKC-β and PKC-δ after treated by a variety of ways. Eventually, the measure of phosphorylation of extracellular regulated protein kinases (ERK)1/2 was performed by Western blotting in PMA-treated Tca8113 cells. Results The cell viability of Tca8113 decreased obviously after transfected with NOS-2 siRNA (P<0.01). PKC reduced the expression level of NOS-2 mRNA (P<0.05). PKC-α, PKC-β and PKC-δ worked together to regulate the level of NOS-2 mRNA (P<0.01). Motigen-activated protein kinase kinase (MEK)/ERK signaling pathway regulated the level of NOS-2 mRNA negatively (P<0.05). PKC down regulated the level of NOS-2 mRNA through MEK/ERK signaling pathway (P<0.05). Conclusion PKC regulates the mRNA level of NOS-2 related to proliferation through MEK/ERK signaling pathway in Tca8113 cells..

Key words: inducible nitric oxide synthase, protein kinase C, motigen-activated protein kinase kinase/extracellular regulated protein kinases signaling pathway, cell proliferation

中图分类号:

Q257

高雪峰, 焦海斌, 叶昌成, 刘英群. 舌鳞癌细胞Tca8113中蛋白激酶C和丝裂原活化蛋白激酶激酶/细胞外调节蛋白激酶通路对诱导型一氧化氮合酶表达的影响[J]. 华西口腔医学杂志, 2018, 36(2): 133-139.

Xuefeng Gao, Haibin Jiao, Changcheng Ye, Yingqun. Liu. Effects of protein kinase C and motigen-activated protein kinase kinase/extracellular regulated protein kinases signaling pathway on mRNA level of inducible nitric oxide synthase in Tca8113 cells[J]. West China Journal of Stomatology, 2018, 36(2): 133-139.

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目的基因	上游引物	下游引物
GAPDH	5’-CCTGGACCACCCAGCCAGCA-3’	5’-TGTTATGGGGTCTGGGATA-3’
NOS-2	5’-CAGCGGGATGACTTTCCAAG-3’	5’-AGGCAAGATTTGGACCTGCA-3’
PKC	5’-TCCAAATGGGCTTTCGGAT-3’	5’-TGGATCGGATTTTGGTT-3’
PKC-α	5’-CGGGGACCTCATGTACCACATT-3’	5’-CCCTTCTGAGTCCAGCATGACA-3’
PKC-β	5’-GGGATGAAATGCGACACCTGTA-3’	5’-GACAAGCCATTGGGGTCCATAG-3’
PKC-δ	5’-ATGCCAGACAAAGGTTGCCAAT-3’	5’-GAAATGCCCTGGGGTTCTCTTT-3’

目的基因	上游引物	下游引物
NC	5’-UUCUCCGAACGUGUCACGUTT-3’	5’-ACGUGACACGUUCGGAGAATT-3’
NOS-2	5’-ACAACAGGAACCUACCAGCTT-3’	5’-GCUGGUAGGUUCCUGUUGUTT-3’
PKC-α	5’-AGGAGCCACAAGCAGUAUUTT-3’	5’-AAUACUGCUUGUGGCUCCUTT-3’
PKC-β	5’-ACAAGAACCUCAUCGACUCTT-3’	5’-GAGUCGAUGAGGUUCUUGUTT-3’
PKC-δ	5’-GUCCACAAGCGAUGUCAUGTT-3’	5’-CAUGACAUCGCUUGUGGACTT-3’