Abstract: Objective To analyze the gene expression of hamster kidney normal BHK21 cells after inhibiting serine
threonine tyrosine kinase 1 (STYK1/NOK) by transcriptome analysis, and analyze its possible signaling pathways. Methods
BHK21 cells were divided into empty vector group (transfected with 6 µg empty vector pBs/U6), low concentration
transfection group (2 µg si-STYK1/NOK plasmid + 4 µg empty vector pBs/U6) and high concentration transfection group ( 6 µg si-STYK1/NOK plasmid). Western blot assay was used to detect STYK1 / NOK protein expression in 48 hours after
transfection. Based on the detection results, the empty RNA group and high transfection concentration group were selected to extract total RNA for the transcriptome sequencing. The sequences obtained by sequencing were filtered and compared to obtain differentially expressed genes. After obtaining the differentially expressed genes, R software was used to analyze the biological functions of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The interactions in the STRING protein interaction database were used to analyze the differential gene-protein interaction networks. QRT-PCR was used to verify the key differentially expressed genes methylenetetrahydrofolate dehydrogenase 2 (Mthfd2), activating
transcription factor 5 (Atf5), phospholipase A2 group Ⅱ A (Pla2g2a) and 6-phosphofructo-2-kinase/fructose-2, 6-
biphosphatase 3 (Pfkfb3). CCK-8 method was used to detect cell proliferation in empty vector group and high transfection
concentration group. Results Western blot results showed that the expression of STYK1/NOK protein was significantly
inhibited in the high-concentration transfection group. Analysis of the transcriptome sequencing results revealed a total of 44
differentially expressed genes, including 19 up-regulated genes and 25 down-regulated genes in the empty vector group and the high-concentration transfection group. GO enrichment analysis found that the differentially expressed genes mainly
affected biological regulation, cellular processes, metabolic regulation, cellular biological processes, cells, cellular parts,
extracellular regions, ligands and catalytic activity. KEGG pathway analysis found that the differentially expressed genes
were mainly concentrated in the immune system, cancer, cell cycle, signal transduction and amino acid metabolism. The
qRT-PCR results showed that the relative expression levels of Mthfd2 and Atf5 were significantly increased in the high
concentration transfection group (P＜0.05), and the relative expression levels of Pfkfb3 and Pla2g2a were significantly
decreased (P＜0.05), which was consistent with the sequencing results. Cell proliferation experiments showed that the cell
proliferation was significantly higher in the high-concentration transfection group than that in the empty vector group (P＜
0.01). Conclusion The inhibition of STYK1 / NOK expression can result in the enhanced expression of proto-oncogenes,
the promoted expression of tumor suppressor genes and the decreased cell proliferation in BHK21 cells.

Key words: transcriptome, gene expression profiling, serine threonine tyrosine kinase 1, BHK21 cell, differentially
expressed genes