Study of inhibition of hepatocellular carcinoma proliferation after down-regulating Claspin

Abstract

Objective To explore the relationship between the expression of Claspin in liver cancer and the clinical features of patients and the roles of Claspin in the proliferation of hepatocellular carcinoma (HCC). Methods Using the TCGA public database, analyze the expression level of Claspin mRNA in 374 cases hepatocarcinoma tissues and 50 cases adjacent tissues, and its survival and clinical features. Real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were used to detect the expression of Claspin mRNA and protein in hepatoma cell lines Hep3B cells. Methyl thiazol tetrazolium (MTT) method and colony formation assay were used to understand the changes of proliferation of Hep3B cells after Claspin down-regulation. The stable cell line screened out by G418 was inoculate to establish the subcutaneous xenograft tumor in nude mice, 3 groups and 6 mice each group. The tumor growth curve was plotting and histological examination was performed. Results Claspin mRNA expression was higher in HCC tissues (5.643±1.599) than in normal liver tissues (2.752±1.674) (t=11.941, P=0.000), the OS time and PFS time of the low expression Claspin mRNA patients was high than high expression patients [ (3.659±0.355) years vs (2.289±0.357) years, Log Rank=15.993, P=0.000; (6.072±0.426) years vs (4.771±0.462) years, Log Rank=11.830, P=0.001]. The level of Claspin mRNA expression was related to the pathological TNM stage (χ2=6.149, P=0.013)、grade of liver cancer (χ2=8.563, P=0.003) and AFP level (χ2=11.202, P=0.001), and was also an independent predictor of prognosis (R=0.579, P=0.009). Compared with the blank control group, the down-regulation of Claspin group decreased the proliferation ability at 72 h [(1.316±2.471)%, t=4.823, P=0.000] and the relative colony formation cells [(28.000±3.152)%, t=39.540, P=0.000]. As for the subcutaneous hepatoma xenograft in nude mice, the down-regulation group had significantly slower tumor growth than the blank group and negative control group[(144.300±15.340) mm3 vs. (377.000±20.280) mm3/(363.300±26.980) mm3,t=22.410 and 17.280, both P=0.000], the down-regulation group had significantly lower Claspin expression than he blank group (t=8.367, P=0.000) and negative control group (t=9.303, P=0.000). Conclusion The expression of Claspin is significant high in HCC and also an independent predictor of poor prognosis, down-regulation of Claspin can inhibit the proliferation of liver cancer cells and xenograft in nude mice. Key words: Claspin; Hepatocellular carcinoma; Gene expression; Proliferation