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Regulation of secreted type Ⅱ cGMP-dependent protein kinase on proliferation-related gene expression of human gastric cancer cell line |
LI Guo-rui, PANG Ji, QIAN Hai, WU Yan, CHEN Yong-chang |
(School of Medicine, Jiangsu University, Zhenjiang Jiangsu 212013, China) |
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Abstract Objective: To explore the effect of secreted type Ⅱ cGMP-dependent protein kinase (PKG Ⅱ) on gene expression in human gastric cancer cell lines HGC-27 and AGS. Methods: Illumina HiSeq 2000 high-throughput sequencing platform was used to sequence the transcriptome of AGS human gastric cancer cells treated with recombinant full-length human PKG Ⅱ which was expressed by baculovirus in Sf9 insect cells using an N-terminal Glutathione S-transferases (GST) tag. Several genes associated with proliferation were selected from differentially expressed genes (DEGs) caused by recombinant PKG Ⅱ treatment, and the effect of recombinant PKG Ⅱ on the transcription level of related genes under the activation of cGMP analog 8-pCPT-cGMP was verified by real-time quantitative PCR. The effect of GST-PKG Ⅱ on the expression of related protein was verified by Western blotting. Results: Transcriptome sequencing results showed a significant difference in expression of 579 genes, of which 267 up-regulated genes and 312 down-regulated genes with the treatment of GST-PKG Ⅱ. Among the 9 cell proliferation related genes selected by RNA-Sequencing, there was a consistent trend in the mRNA levels of EGF, DFNA5, DACT1, and PSCA with the RNA-Sequencing result both in HGC-27 and AGS human gastric cell line under the treatment of GST-PKG Ⅱ and 8-pCPT-cGMP(P<0.05), compared with blank control group. The protein level of EGF was down-regulated with the treatment of GST-PKG Ⅱ and 8-pCPT-cGMP, which was consistent with the results of RNA-Sequencing and qRT-PCR. Conclusion: Secreted PKG Ⅱ could affect the expression of various proliferation-related genes included EGF in AGS and HGC-27 gastric cancer cells from the transcription level, and inhibit the translation of EGF.
[Key words]secreted type Ⅱ cGMP-dependent protein kinase; gastric cancer cells; gene expression; RNA-sequencing; epidermal growth factor
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Received: 31 January 2020
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