| 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 PIWI family is the essential protein component of the RNAi machinery, and it can regulate the biogenesis of multiple miRNAs. PIWI family members are present in all RNA-induced silencing complexes (RISCs) and are required for the production of mature miRNAs. HIWI (also known as PIWIL1) expresses mainly in human germ cells. Express patterns and biologic capacities of HIWI are reported as the key elements in carcinogenesis, tumor development and prognosis of various human cancers in recent studies. However, there are limited data on the role of HIWI in HCC. Therefore, exploring the relationship between HIWI and HCC to identify an effective biomarker may be more promising for treatment compared to suppressing miRNAs.HIWI expression was measured in HCC cell lines with different metastatic potential (HepG2, SMMC7721, MHCC97L, MHCC97H and HCCLM3) 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 HIWI expression increased in parallel with the metastatic potential of HCC cell lines (P<0.001). In HCC tissue specimens, the mRNA level of HIWI was significantly higher in intratumoral tissues than in peritumoral tissues (P<0.001).We further depleted HIWI in HCCLM3, MHCC97H and MHCC97L cells using HIWI-targeting siRNAs with different suppressive effects (control siRNA, HIWI-970and HIWI-1510). The suppressive effects of the siRNAs were validated at24h by RT-PCR and Western blot. HIWI-970and HIWI-1510demonstrated significant suppression effects compared with control siRNA sequences and HIWI expression was significantly lower after HIWI-1510transfection than after HIWI-970transfection (both P<0.001). In HCCLM3, MHCC97H and MHCC97L cell lines, down-regulation of HIWI caused a significant decrease in cell proliferation at72h following transfection (both P<0.001). Decreased HIWI expression was accompanied by a reduction in HCC cell invasion, as measured by the Matrigel Transwell assay (both P<0.001).Basing on the previous results, we constructed HCC tissue microarrays (TMAs; n=168) and measured HIWI expression in TMAs by immunohistochemistry. The relationship between HIWI and clinicopathological features and its prognostic value were also invastigated. HIWI stained the cytoplasm, membrane and nucleus of intratumoral HCC cells and adjacent liver cells. The positive rates of HIWI expression in intratumoral and peritumoral tissues were26.19%and2.98%, respectively (P<0.001). Patients with positive intratumoral HIWI expression had a higher incidence of large tumor size (P=0.047), more intrahepatic metastasis (P=0.027) and more lymph node metastasis (P=0.004). PCNA staining occurred mainly in the nucleus in tumor or paritumoral liver tissue. In positive rate of PCNA expression was97.73%in tissue with positive HIWI expression, whereas it was66.13%in tissue with negative HIWI expression. PCNA expression was positively correlated with HIWI (P<0.001). Positive intratumoral HIWI was an independent risk factor for overall survival (P=0.007) and recurrence-free survival (P=0.036). Furthermore, the predictive value of intratumoral HIWI was a risk factor associated with OS and RFS in the AFP<300ng/ml subgroup (P=0.002and P<0.001, respectively) and Edmondson-Steiner Ⅰ-Ⅱ subgroup (P=0.038and P=0.022, respectively) was investigated respectively, and the prognostic significance of HIWI was retained. However, in AFP>300ng/ml (P=0.463and P=0.987, respectively) and Edmondson-Steiner Ⅲ-Ⅳ subgroups(P0.331and P=0.912, respectively), HIWI was not associated with prognosis of HCC patients. In summary, our data have demonstrated that HIWI may be a useful prognostic factor for HCC patients after curative resection, particularly in conditions of low serum AFP and low Edmondson-Steiner grade. In addition, HIWI probably plays a crucial role in carcinogenesis, proliferation, and metastasis of HCC. |
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