| Hepatocellular carcinoma (HCC), most of primary liver cancer, is the fifth most frequent malignant tumors and the third most common cause of cancer mortality. Nowadays, though surgical resection is valid to provide long-term survival for HCC patients, the high postoperative recurrence and metastasis rate is a major problem making the patients undergoing curative resection poor outcome. Consequently, to explore effective biomarkers to predict outcome and adjuvant treatments is crucial to prevent and treat postoperative recurrence and improve the patient’s life span.Several studies have demonstrated that microRNAs (miRNAs) modulate cellular gene expression through the RNA interference (RNAi) pathway. Half of all miRNAs are located at fragile chromosomal sites and in regions commonly deleted or amplified in various human cancers, suggesting that miRNAs may be important for carcinogenesis and tumor progression. These miRNAs are called oncomiRs. It is difficult to inhibit multiple oncomiRs in cancer cells because each oncomiR may independently contribute to tumor development, although repressing oncomiRs during carcinogenesis and tumor progression may be a novel cancer therapy. The AGO family is the essential protein component of the RNAi machinery, and it can regulate the biogenesis of multiple miRNAs. AGO family members are present in all RNA-induced silencing complexes (RISCs) and are required for the production of mature miRNAs. AGO1is a major component of RISCs and is a well-studied AGO in mammals. The relationship between AGO1and cancer has been reported in several recent investigations. However, AGO1was just reported to overexpress in intratumoral tissues and there are limited data on the role of AGO1in HCC. Therefore, exploring the relationship between AGO1and HCC to identify an effective biomarker may be more promising for treatment compared to suppressing miRNAs.AGO1expression was measured in HCC cell lines with different metastatic potential (HCCLM3, MHCC97L, and HepG2) by RT-PCR and Western Blot. It was also measured in HCC tissue specimens by RT-PCR. We found that both mRNA and protein levels of AGO1expression increased in parallel with the metastatic potential of HCC cell lines (P=0.014). In HCC tissue specimens, the mRNA level of AGO1 was significantly higher in intratumoral tissues than in peritumoral tissues (P<0.001).We further depleted AGO1in HCCLM3and HepG2cells using AGO1-targeting siRNAs with different suppressive effects (control siRNA, AGO1-2427, and AGO1-2235). The suppressive effects of the siRNAs were validated at24h by RT-PCR and Western blot. AGO1-2235and AGO1-2427demonstrated significant suppression effects compared with control siRNA sequences and AGO1expression was significantly lower after AGO1-2235transfection than after AGO1-2427transfection (P=0.003and P <0.001, respectively). In HCCLM3and HepG2cell lines, down-regulation of AGO1caused a significant decrease in cell proliferation at48h following transfection (P=0.039and P=0.014, respectively). Decreased AGO1expression was accompanied by a reduction in HCC cell invasion, as measured by the Matrigel Transwell assay (both P <0.001). Microscopic examination revealed a significant decrease in the wound closure rate in cells transfected with AGO1siRNA compared to the control cells at24h, as determined by the scratch wound assay (both P <0.001). Depletion of AGO1resulted in a significant decrease in P53and vascular endothelial growth factor (VEGF) expression. This effect was first observed at24h, but the P value was not significant. P53expression was decreased most dramatically at48h following transfection with AGO1-2235or AGO1-2427compared to the control siRNA. The variations in VEGF expression were similar to P53, with the most dramatic changes occurring at72h.Basing on the previous results, we constructed HCC tissue microarrays (TMAs; n=200) and measured AGO1and CD31expression in TMAs by immunohistochemistry. We counted MVD (microvessel density) through CD31staining and assessed its relationship with AGO1. Meanwhile, the relationship between AGO1and clinicopathological features and its prognostic value was also invastigated. AGO1staining was most prominent in the cytoplasm of intratumoral HCC cells and adjacent liver cells. Nuclear staining was also detected in several sections. The positive rates of AGO1expression in intratumoral and peritumoral tissues were45.0%and27.0%, respectively. Patients with positive intratumoral AGO1expression had a higher incidence of young age (P=0.039), high serum AFP (P=0.002), HBeAg-positive status (P=0.049), liver cirrhosis (P=0.045), intrahepatic or extrahepatic recurrence (P <0.001) and high MVD (P <0.001). Positive intratumoral AGO1was an independent risk factor for overall survival (P= 0.008) and recurrence-free survival (P <0.001). It was also associated with a poor early (within24months) and late phase (after24months) RFS (P=0.020and P <0.001, respectively). Furthermore, the predictive value of intratumoral AGO1expression within subgroups:AFP≤20ng/ml and>20ng/ml (P=0.001and P=0.008, respectively); tumor size≤5cm and>5cm (P=0.002and P=0.007, respectively); HBsAg status positive and negative (P=0.005and P=0.002, respectively) was investigated and the prognostic significance of AGO1was retained.In conclusion, AGO1may regulate the proliferation, migration, and invasion of HCC cells and angiogenesis in HCC tissues through the P53and VEGF pathways. It is also associated with HBV viremia and some other adverse clinicopathologic features. AGO1may be a novel prognostic factor and theraputic target. |
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