The Role Of SOX18 Induced Proliferation, Migration And Invasion In Hepatocellular Carcinoma Cells

Objective:Hepatocellular carcinoma(HCC) is one of the most common malignancies in the world. Recent studies have demonstrated that SOX18 is highly expressed in various types of cancer. The SOX family consists of a group of transcription factors that are defined by a highly conserved high-mobility group(HMG) DNA-binding domain.Knockout experiments demonstrated that SOX genes have a wide range of roles in cancer development. For instance, SOX7 and SOX17 act as tumor suppressors in various types of cancer through suppres-sion of Wnt signaling. In the case of SOX18,overexpression of SOX18 has been found in gastric cancer, as compared to normal gastric tissues. Furthermore, SOX18 expression was found to be correlated with a poor clinical outcome of patients with ovarian, non-small cell lung cancer or invasive ductal breast carcinoma. However, little is known concerning the expression pattern and biological functions of SOX18 in HCC.Methods:Seventy-five patients admitted to Shanxi Medical University First Hospital were enrolled in the present study. In each case, HCC and adjacent non-tumorous tissues were snap-frozen in liquid nitrogen and stored at-80?C until RNA extraction. Kaplan-Meier survival analysis was used to investigate the clinical outcome of HCC patients with low or high SOX18 expression. We determined the protein and mRNA levels of SOX18 in five HCC cell lines, BEL-7404, MHCC-97 H, MHCC-97 L, HepG2 and SMC-7721, by western blot analysis and real-time PCR, respectively. To observe the effect of migration,invasion proliferation, cell cycle and apoptosis of hepatocellular carcinoma cells after SOX18 expression was knock down. To gain further insight into the biological pathways involved in HCC pathogenesis through the SOX18 pathway, GESA, a method of analyzing and interpreting microarray and the data using biological knowledge.Results:Statistical analysis using the Student’s t-test showed that SOX18 mRNA was significantly overexpressed in the HCC tissues when compared with that in the normal tissues(P<0.001). We reanalyzed high throughput RNA-sequencing data of the HCC cohort of TCGA and also found a significant increase in SOX18 expression in the HCC tissues, and high SOX18 expression was compared with that in the normal tissues. The survival time of the patients with high SOX18 expressing tumors was significantly shorter than that of patients with low SOX18 expressing tumors(P<0.01). Higher protein and mRNA levels of SOX18 were observed in two cell lines, MHCC-97 H and HepG2.Silencing of SOX18 suppresses the proliferation, while induces G1 phase arrest and cell apoptosis in HCC cells. Downregulation of SOX18 inhibits the motility and invasiveness of HCC cells. To probe the SOX18 associated pathways on an unbiased basis, we performed GSEA using high throughput RNA sequencing data of the HCC cohort from The Cancer Genome Atlas project(TCGA). GSEA is designed to detect coordinated differences in expression of predefined sets of functionally related genes. Among all the188 predefined ‘KEGG pathway gene sets, the focal adhesion pathway and chemokine signaling pathways were identified as having a significant association with SOX18 expression in the HCC. SOX18 siRNA regulates the mRNA and protein expression of RhoA, PDGFB, IGF1 R, CCL2, CCL3 and CCL5 in HCC cells.Conclusion:The present study proved for the first time that SOX18 plays a key role in the proliferation and metastasis of HCC cells. Moreover, SOX18 may regulate these biological processes through the focal adhesion and chemokine signaling pathways, thus providing useful information for the targeted therapy of HCC. As the SOX18 expression level is associated with patient survival, inhibition of SOX18 in tumor tissues may provide an effective therapeutic strategy.