沉默Gpr48基因表达对皮肤鳞状细胞癌细胞系A431 PKC信号通路活化及细胞侵袭的影响

doi:10.3877/cma.j.issn.2095-1221.2019.04.007

目的

分析沉默G蛋白偶联受体48 （Gpr48）基因表达对皮肤鳞状细胞癌（SCC）蛋白激酶C （PKC）信号通路及细胞侵袭能力的影响。

方法

构建ShRNA感染皮肤SCC细胞株A431，设计干扰Gpr48表达shRNA（shRNA-Gpr48组）及阴性对照shRNA-NC组，采用实时定量反转录聚合酶联反应（RT-PCR）检测Gpr48相对表达量；以蛋白印迹法（Western Blot）测定PKC相关抗体蛋白表达情况，进行Transwell小室实验检测细胞侵袭能力，总结Gpr48缺失对三丙胺（TPA）刺激A431细胞株PKC活化及细胞侵袭能力的影响，为SCC靶向治疗提供参照。组间比较采用独立样本t检验。

结果

qRT-PCR结果：shRNA-Gpr48组Gpr48相对表达量（0.27±0.06）低于shRNA-NC组（1.01±0.12），差异具有统计学意义（t?= 17.441，P?< 0.01），shRNA-Gpr48组p-PKCδ （0.463±0.035）低于shRNA-NC组（0.721±0.074），shRNA-?Gpr48组NF-κB p65 mRNA （0.631±0.079）低于shRNA-NC组（0.834±0.102），差异具有统计学意义（t?= 9.966、4.975，P均< 0.01），Notch1 mRNA（0.981±0.037）高于shRNA-NC组（0.562±0.041），差异具有统计学意义（t?= 23.991，P?< 0.01）；Western Blot结果：shRNA-Gpr48组p-PKCδ（0.221±0.034）低于shRNA-NC组（0.292±0.046）、NF-κB p65蛋白表达（0.512±0.047）低于shRNA-NC组（0.636±0.051），差异具有统计学意义（t?= 3.925，5.653，P均< 0.01），Notch1（0.524±0.063）高于shRNA-NC组（0.421±0.076），差异具有统计学意义（t?= 3.299，P?< 0.01）；shRNA-Gpr48组侵袭细胞率为（35.03±1.54）﹪，低于shRNA-NC组的（51.83±2.76）﹪，差异具有统计学意义（t?= 16.809，P?< 0.01）。

结论

沉默Gpr 48缺失表达可能通过抑制PKC活化，降低p-PKCδ、NF-κB p65表达，上调抑癌基因Notch1表达，抑制癌细胞增殖、侵袭。

关键词: 皮肤, 鳞状细胞癌, G蛋白偶联受体48, 信号通路

Objective

To analyze the effects of silencing G-protein-coupled receptor 48 (Gpr48) gene expression on protein kinase C (PKC) signaling pathway and cell invasion in skin squamous cell carcinoma (SCC) .

Methods

Gpr48 interfering shRNA (shRNA-Gpr48 group) and negative control shRNA were infected into skin SCC cell line A431. And the relative expression of Gpr48 was detected by real-time quantitative reverse transcription polymerase chain reaction (RT-?PCR) . Expressions of PKC-related antibody proteins were determined by Western Blot. Transwell chamber assay was used for cell invasion. Statistical analysis was performed with SPSS 20.0-software. The independent sample t test was used to compare the differences of the above indexes in shRNA-Gpr48 group and shRNA-NC group. The effects of Gpr48 deletion on activation of A431 cell line PKC and and cell invasion stimulated by tripropylamine (TPA) were determined.

Results

qRT-PCR results showed the relative expression of Gpr48 in the shRNA-Gpr48 group was lower than that in the shRNA-NC group (0.27±0.06) vs (1.01±0.12) (t = 17.441, P < 0.001) . The mRNA expression of p-PKCδ (0.463±0.035) vs (0.721±0.074) , and NF-κB p65 was lower in the shRNA-Gpr48 group compared to the shRNA-NC group (0.631±0.079) vs (0.834±0.102) (t?= 9.966, 4.975, all P?< 0.01) while the mRNA expression of Notch1 was higher (0.981±0.037) vs (0.562±0.041) (t?= 23.991, P?< 0.001) in the shRNA-Gpr48 group. Western Blot results showed that the protein expression of p-PKCδ (0.221±0.034) vs (0.292±0.046) , and NF-κB p65 (0.512±0.047) vs (0.636±0.051) (t?=3.925, 5.653, all P?< 0.01) was lower in the shRNA-Gpr48 group compared to the shRNA-NC group while the expression of Notch1 was higher in the shRNA-Gpr48 group (0.524±0.063) vs (0.421±0.076) (t?= 3.299, P?< 0.01). The cell invasion rate in the shRNA-Gpr48 group was lower than that in the shRNA-NC group (35.03±1.54) ﹪ vs (51.83±2.76) ﹪ (t?= 16.809, P?< 0.01) .

Conclusion

Silencing Gpr 48 expression may inhibit cancer cell proliferation and invasion by inhibiting PKC activation, decreasing expressions of p-PKCδ and NF-κB p65 and up-regulating the expression of tumor suppressor gene Notch1.

Key words: Skin, Squamous cell carcinoma, G-protein-coupled receptor 48, Signal pathway

表1 RT-PCR反应各引物序列

基因	引物序列（5'→3'）
β-actin	上游：GGAGACAACCTGGTCCTCAG
?	下游：ACCCAGAAGACTGTGGATGG
PKCδ	上游：CTCGTTTCTTCAAGCAGCCAA
?	下游：GTGAACCACAAAGCTACAGACT
Notch1	上游：GCAACAGCTCCTTCCACTTC
?	下游：GCCTCAGACACTTTGAAGCC
NF-κB p65	上游：GCTGTGCGGCTCTGCTTC
?	下游：GGCTGGGGTCTGCGTAGGGA
Gpr48	上游：ACCTGGAGACCTTAGACTTG
?	下游：CCACGAATGACTGGGAAATG

表1 RT-PCR反应各引物序列

基因	引物序列（5'→3'）
β-actin	上游：GGAGACAACCTGGTCCTCAG
?	下游：ACCCAGAAGACTGTGGATGG
PKCδ	上游：CTCGTTTCTTCAAGCAGCCAA
?	下游：GTGAACCACAAAGCTACAGACT
Notch1	上游：GCAACAGCTCCTTCCACTTC
?	下游：GCCTCAGACACTTTGAAGCC
NF-κB p65	上游：GCTGTGCGGCTCTGCTTC
?	下游：GGCTGGGGTCTGCGTAGGGA
Gpr48	上游：ACCTGGAGACCTTAGACTTG
?	下游：CCACGAATGACTGGGAAATG

图1 SCC A431细胞稳转株Gpr48基因敲出效率

图2 倒置显微镜下观察两组细胞形态（×400）

表2 Gpr48缺失对SCC A431细胞株各基因表达的影响（ ± s）

分组	p-PKCδ	Notch1	NF-κB p65
shRNA-NC组	0.721±0.074	0.562±0.041	0.834±0.102
shRNA-Gpr48组	0.463±0.035	0.981±0.037	0.631±0.079
t值	9.966	23.991	4.975
P值	< 0.01	< 0.01	< 0.01

表2 Gpr48缺失对SCC A431细胞株各基因表达的影响（ ± s）

分组	p-PKCδ	Notch1	NF-κB p65
shRNA-NC组	0.721±0.074	0.562±0.041	0.834±0.102
shRNA-Gpr48组	0.463±0.035	0.981±0.037	0.631±0.079
t值	9.966	23.991	4.975
P值	< 0.01	< 0.01	< 0.01

图3 Western Blot测定A431细胞各蛋白表达

表3 Gpr48缺失对SCC A431细胞株各蛋白表达的影响（ ± s）

分组	p-PKCδ	Notch1	NF-κB p65
shRNA-NC组	0.292±0.046	0.421±0.076	0.636±0.051
shRNA-Gpr48组	0.221±0.034	0.524±0.063	0.512±0.047
t值	3.925	3.299	5.653
P值	< 0.01	< 0.01	< 0.01

表3 Gpr48缺失对SCC A431细胞株各蛋白表达的影响（ ± s）

分组	p-PKCδ	Notch1	NF-κB p65
shRNA-NC组	0.292±0.046	0.421±0.076	0.636±0.051
shRNA-Gpr48组	0.221±0.034	0.524±0.063	0.512±0.047
t值	3.925	3.299	5.653
P值	< 0.01	< 0.01	< 0.01

图4 Gpr48缺失对SCC A431细胞株侵袭能力的影响

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Effects of silencing Gpr48 gene expression on PKC signaling pathway activation and cell invasion in skin squamous cell carcinoma cell line A431

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Effects of silencing Gpr48 gene expression on PKC signaling pathway activation and cell invasion in skin squamous cell carcinoma cell line A431