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SOX12 Upregulation Is Associated with Metastasis of Hepatocellular Carcinoma and Increases CDK4 and IGF2BP1 Expression

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SOX12 Upregulation Is Associated with Metastasis of Hepatocellular Carcinoma and Increases CDK4 and IGF2BP1 Expression European Review for Medical and Pharmacological Sciences 2017; 21: 3821-3826 SOX12 upregulation is associated with metastasis of hepatocellular carcinoma and increases CDK4 and IGF2BP1 expression P. Y UA N1, L. MENG2, N. WANG3 1Department of Interventional Therapy, the People’s Hospital of Jianhu, Jianhu, Jiangsu, China 2Clinical Laboratory, the People’s Hospital of Lanling, Linyi, Shandong, China 3Department of Ultrasound, the First Hospital of Hebei Medical University, Shijiazhuang, Hebei, China Introduction Abstract. – OBJECTIVE: In this study, we studied the expression profile of SOX12 gene in different pathological stages of hepatocellular Hepatocellular carcinoma (HCC) is one of carcinoma (HCC) and explored the possible the most common cancers and is also a leading downstream genes related to its regulation of cause of cancer-related death worldwide1. Local HCC invasion and migration. and distant metastases are indicators of poor MATERIALS AND METHODS: Bioinformatic prognosis and are also the main causes of can- data mining was performed in liver cancer co- cer-related death2,3. Therefore, elucidating the hort in TCGA. HepG2 cells were used as in vitro cell model to assess the effect of SOX12 on molecular mechanisms underlying HCC metas- CDK4 and IGF2BP1 expression. The association tasis is critical for identifying novel therapeutic between the expression of SOX12, CDK4 or IG- targets. F2BP1 and survival time in HCC patients was SYR-related high mobility group (HMG) box also assessed based on the data in TCGA. (SOX) family proteins are a conserved group RESULTS: The regional lymph node metasta- of transcriptional factors widely participate in sis (N1) and distant metastasis (M1) cases had both normal embryonic development and onco- significantly increased SOX12 expression than 4,5 5,6 the lymph node negative (N0) and distant metas- genic processes . Previous studies found that tasis negative (M0) cases respectively. CDK4 the SOX factors have extraordinary regulations and IGF2BP1 were among the top 20 SOX12 on cancer cell proliferation, migration, inva- co-upregulated genes in HCC, which have well sion and tumor metastasis in multiple types of characterized enhancing the effect on HCC cell cancer. SOX12 belongs to the SOXC group of growth, invasion, and metastasis. In HepG2 SOX family, together with SOX4 and SOX117. cells, knockdown of SOX12 reduced IGF2BP1 These three SOX members show overlapping and CDK4 expression, while enforced SOX12 ex- 8 pression significantly enhanced their expres- expression patterns and molecular properties . sion. Low SOX12 or CDK4 expression was asso- SOX12-null mice are viable and do not show ciated with significantly better 3-year survival obvious malformations due to the compensation and better 5-year survival. Low IGF2BP1 expres- of SOX4 and SOX117. The oncogenic effect of sion was associated with significantly better SOXC group in HCC has been reported. Briefly, 3-year survival in HCC patients. CONCLUSIONS: SOX4 contributes to hepato-carcinogenesis via SOX12 upregulation is asso- 9 ciated with regional and distant metastasis of inhibiting p53-mediated apoptosis . SOX12 in- HCC. SOX12 can increase the expression of duces epithelial-mesenchymal transition (EMT) CDK4 and IGF2BP1, which confer malignant by trans-activating Twist1 expression and also phenotypes to HCC. The expression of SOX12, enhances cancer cell invasion and metastasis via IGF2BP1 or CDK4 might be potential clinical increasing the expression of fibroblast growth prognostic markers for HCC. factor binding protein 1 (FGFBP1)10. Among the Key Words 16 SOX genes, SOX12 was identified as one of SOX12, CDK4, IGF2BP1, Metastasis, Hepatocellular the most up-regulated genes in HCC compared carcinoma. to adjacent normal tissues10. Corresponding Author: Na Wang, MD; e-mail: [email protected] 3821 P. Yuan, L. Meng, N. Wang As a transcription factor, the downstream Quantitative Real-Time-PCR (QRT-PCR) regulation of SOX12 in HCC is still not fully 24 h after infections, Total RNA was extracted understood. In this paper, we further studied its from cell samples by using the High Pure RNA expression profile in different pathological stages Isolation Kit (Bio-Rad, Hercules, CA, USA) and of HCC and explored the possible downstream then was reversely transcribed into cDNA using genes related to its regulation of HCC invasion the iScript cDNA Synthesis Kit (Bio-Rad, Hercu- and migration. les, CA, USA). Then, QRT-PCR was performed to detect SOX12 expression using TaqMan Master Mix (Applied Biosystems, Foster City, CA, USA) Materials and Methods and the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The Data Mining in Liver Cancer Cohort relative mRNA expression was normalized to in TCGA GAPDH. The primers for SOX12 were: forward, Gene expression in liver cancer cohort in TC- 5’-CGCGATGGTGCAGCAGCG-3’ and reverse, GA was analyzed using the UCSC Xena browser 5’-GCCACTGGTCCATGATCTTC-3’. Relative (http://xena.ucsc.edu/). The genes co-expressed mRNA expression levels (fold changes) between with SOX12 in liver cancer cohort in TCGA was groups were calculated using the 2-ΔΔCT method. analyzed using the cBioPortal for Cancer Ge- nomics11,12 and further verified using the UCSC Western Blotting Xena. The associations between the expression Total protein were extracted from HepG2 cells of SOX12, CDK4 or IGF2BP1 and survival of 48 h after infection by using a lysis buffer. Then HCC patients were also assessed using the UCSC the samples were subjected a conventional western Xena. blotting as described previously15. The primary antibodies used include anti-SOX12 (SAB1411973, Protein-Protein Interaction (PPI) Sigma-Aldrich, Carlsbad, CA, USA), anti-CDK4 Network (ab137675, Abcam, Cambridge, MA, USA), an- The genes co-upregulated with SOX12 in liv- ti-IGF2BP1 (ab82968, Abcam), and anti-β-actin er cancer cohort in TCGA were loaded into (ab8226, Abcam). After incubation and washing, the Search Tool for the Retrieval of Interacting the membranes were further incubated with HRP Genes (STRING) (http://string-db.org/) database conjugate secondary antibodies. The blot signals for analysis of PPI network. The evidence level were visualized using the ECL Western blotting was set to > 0.7, which indicates experimentally substrate (Promega, Madison, WI, USA). validated interactions. Statistical Analysis Cellular SOX12 Expression Identification Statistical analysis was performed using Graph- Immunostaining of the distribution and ex- Pad Prism 6.0. The difference between groups was pression of SOX12 in HCC cell line HepG2 cells evaluated by unpaired, two-tailed Student’s t-test. were reviewed in Human Protein Atlas, an online The difference between Kaplan-Meier curves were database offering the combination of transcrip- assessed using the log-rank test. The test statistics tomics and antibody-based proteomics for map- (χ2) and p-value (χ2 distribution) were given. p < ping the human proteome down to the single cell 0.05 indicates statistical significance. level13,14. Cell Culture and Transfection Results Human HCC HepG2 cells were purchased from the American Type Culture Collection and SOX12 Upregulation is Associated with were cultured in Dulbecco’s Modified Eagle Me- Regional and Distant Metastasis of HCC dium (DMEM) at 37°C in a 5% CO2 incubator. One recent study reported that SOX12 can pro- The medium was supplemented with 10% FBS, mote metastasis of HCC10. In this study, we further 100 μg/ml penicillin, and 100 μg/ml strepto- characterized the expression profile of SOX12 in mycin. SOX12 lentiviral shRNA and lentiviral different stages of HCC. By using the data from expression particles were obtained from Geneco- liver cancer cohort in TCGA, we compared the poeia. HepG2 were infected with the lentiviral expression profiles of SOX12 in different patho- particles in the presence of Polybrene. logical stages (Figure 1A). The results showed 3822 SOX12 upregulation is associated with metastasis of hepatocellular carcinoma Figure 1. SOX12 upregulation is associated with regional and distant metastasis of HCC. A-D, Heat map (left) and bar chart (right) of SOX12 mRNA expression in different pathological stages (A), different T stages (B), different N stages (C) and different M stag- es (D) of HCC. The number of patients in different stages were listed below the bar chart. Analysis was performed using the UCSC Xena based on liver cancer cohort in TCGA database. that SOX12 expression gradually increased with 2A, red arrows), which have well characterized en- the development of pathological stages (Figure hancing effect on HCC cell growth, invasion and 1A). Then, we compared SOX12 expression in metastasis16-18. Therefore, we decided to further different T stages. The results showed similar investigate whether there is any regulative effect SOX12 expression from T1 to T4 stages (Figure of SOX12 on CDK4 and IGF2BP1. By reviewing 1B). However, by comparing SOX12 expression the heat map and performing regression analy- between the cases with or without regional lymph sis using the UCSC Xena, we further confirmed node metastasis and between the cases with or moderate level of co-upregulation between SOX12 without distant metastasis, we observed that the and CDK4 (Figure 3A-B) and between SOX12 regional lymph node metastasis (N1) and distant and IGF2BP1 (Figure 3A and C). SOX12 immu- metastasis (M1) cases had significantly increased nostaining in Human Protein Atlas showed that SOX12 expression (Figure 1C-D). SOX12 has nucleoplasm expression in HepG2
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