MiR-221 has been reported to be overexpressed in human HCC tissues, compared with normal liver tissues or adjacent benign liver tissues [37, 40, 42–48]. Four groups studied the miR-221 expression with fresh frozen samples using miRNA array, Northern blot or RT-qPCR assays [44, 45, 47, 48]. FFPE tissues were used by only two groups to study the miR-221 expression in HCC. Fu et al. used in situ hybridization (ISH) and real time RT-qPCR to detect the miR-221 level. Most recently, Karakatsanis et al.  also investigated the miR-221 expression with real time RT-qPCR in HCC FFPE tissues. In the current study, the result from real time RT-qPCR in 76 cases of HCC samples confirmed the previous reports, which showed that HCC had higher miR-221 level than other liver tissues. The overexpression of miR-221 in HCC indicates that miR-221 plays a critical role in the hepatocarcinogenesis, as an oncogenic miRNA.
Concerning the relationship between miR-221 expression and clinicopathological parameters, several groups reported that miR-221 level is related to the tumor TNM stages [42, 43, 45]. In the present study, miR-221 expression in stage III and IV was found to be significantly higher than that in stage I and II, which was in line with previous reports [42, 43, 45]. In vivo data also supported that miR-221 can promote tumor progression . Meanwhile, consistent with Fu et al. , miR-221 expression was significantly upregulated in metastatic group compared to that in the nonmetastatic group. Additionally, miR-221 expression was correlated with the status of tumor capsular infiltration. In the group of tumor capsular infiltration with cancer cells or tumor with no capsular, miR-221 was significantly higher than that in the group of tumor without capsular infiltration. Generally, the status of tumor capsular infiltration reflects tumor invasion and metastasis. Thus, the result in current study reveals an obvious relation between miR-221 and the infiltration of tumor cells, migration, invasion and metastasis of HCC. The recurrence occurred quicker in the patients with high expression of miR-221 than those with low expression of miR-221. The difference was not significant, however there was a trend that high expression of miR-221 might lead to HCC recurrence. Hence it may be valuable to examine miR-221 expression for the clinical prediction of metastasis of HCC. The mechanisms of miR-221 promoting metastasis could be related to different targets. Garofalo et al.  showed that miR-221, by targeting PTEN and TIMP3 tumor suppressors, induced TRAIL resistance and enhance cellular migration through the activation of the AKT pathway and metallopeptidases. Besides the relationship between miR-221 and clinical stages, metastasis and capsular infiltration, Gramantieri et al.  reported that higher miR-221 levels were observed in multifocal HCCs versus unifocal tumors; miR-221 was also found to be corrected with tumor size and cirrhosis . Different from these reports, miR-221 expression in the current study was not correlated with the number of tumor nodes, tumor diameter or cirrhosis. The number of samples involved and different methods to detect miR-221 could partially explain the discrepancies. Additionally, miR-221 level was neither related to other clinical features, such as: age, gender, differentiation, AFP level or portal vein tumor embolus.
Recently, some miRNAs were also identified in serum and plasma in a remarkably stable form that is protected from endogenous RNase activity [49, 50]. Li et al.  investigated the serum miR-221 expression in HCC. Similarly to HCC tissues, miR-221 was found to be differentially overexpressed in HCC serum samples, and high level of miR-221 expression was correlated with tumor size, cirrhosis and tumor stage. In addition, Kaplan–Meier survival analysis showed that the overall survival rate of the high miR-221 expression group (27.6%) was significantly lower than that of the low miR-221 expression group. Thus serum miR-221 could act as a noninvasive prognostic biomarker for HCC.
MiR-221 was also studied functionally in vitro and in vivo. Pineau et al.  investigated the role of miR-221 in vitro. After transfection of miR-221 precursor, they observed that most HCC cell lines, including HepG2 used also in our study, formed larger colonies than controls. When miR-221 antagomiR was transfected into HLE and Malhavu cells, the cell growth was drastically inhibited. Whereas, the same treatment led to no change in cell proliferation in PLC/PRF5 and Huh6 cell lines. In the current study, we transfected miR-221 inhibitor and mimic by combiMAGnetofection into different HCC cell lines (HepB3, HepG2 and SNU449). The cell growth was monitored by three independent assays: CellTiter96 AQueous One Solution Cell Proliferation Assay, CellTiter-Blue Cell Viability Assay and Hoechst 33342/PI double fluorescent chromatin staining. The results of the three methods were in agreement with each other and enhanced the observation of Pineau et al. . With miR-221 inhibitor, the cell growth was inhibited in all the HCC cell lines tested. By contract, with miR-221 mimic, the cell growth was moderately accelerated in HepB3 and HepG2 cells. However, the effect of miR-221 mimic to enhance the cell growth in SNU449 cells was mild and without significantly difference when compared to the negative controls. Cell cycle was also detected by flow cytometry. When HepB3 cells were transfected with miR-221 mimic post 96 hrs, a 1.8 fold increase in the S-phase cell population was observed, which explained the character of miR-221 accelerating HCC cell growth. Yuan et al.  reported that miR-221 enhances proliferation of in vitro cultivated primary hepatocytes and adeno-associated virus mediated overexpression of miR-221 in the mouse liver also accelerates hepatocyte proliferation in vivo. Furthermore, miR-221 overexpression leads to rapid S-phase entry of hepatocytes during liver regeneration. These findings help explain the mechanism of the relationship between miR-221 and HCC cell proliferation. Fornari et al. reported that cyclin-dependent kinase inhibitor (CDKN) 1C/p57 and CDKN1B/p27 are target genes of miR-221, and Yuan et al.  reported that Aryl hydrocarbon nuclear translocator (Arnt) mRNA can be a novel target of miR-221. Thus, overexpression of miR-221 can promote cell cycle progression, by affecting both CDKN1C/p57, CDKN1B/p27 and Arnt proteins.
There were contradictory reports on the relationship between miR-221 and apoptosis in HCC. Dai et al.  reported in HCC cells, endoplasmic reticulum (ER) stress-induced apoptosis is enhanced by miR-221 mimic and attenuated by miR-221 inhibitor. MiR-221 promoted-apoptosis under ER stress is associated with p27(Kip1)- and MEK/ERK-mediated cell cycle regulation. Thus, they concluded that suppression of miR-221 plays a crucial role in the protection against apoptosis induced by ER stress in HCC cells. On the contrary, Gramantieri et al. [37, 45] found that the apoptosis of HCC cell line SNU449 was induced with knock-down of miR-221. Meanwhile, with a luciferase reporter assay, Bmf,a proapoptotic BH3-only protein , and DNA damage-inducible transcript 4 (DDIT4) , a modulator of the mTOR pathway, were confirmed to be targets of miR-221. Moreover, the analysis of HCC tissues revealed an inverse correlation between miR-221 and activated caspase-3, as a marker of apoptosis . This explains that miR-221 can inhibit apoptosis by targeting Bmf and or/DDIT4. Furthermore, miR-221 has been identified as a potent posttranscriptional regulator of FAS-induced apoptosis  and necrosis factor related apoptosis-inducing ligand related apoptosis . In the current study, Hoechst 33342/PI double fluerenscent staining observed by microscope and APC Annexin V/7-AAD staining by flow cytometry were performed to test the effect of miR-221 on apoptosis in HCC cells. The result of Gramantieri et al.  could be repeated in the present study. More than that, we also found that the apoptosis of HCC cell lines HepB3 and HepG2 was significantly increased with miR-221 inhibitor. However, miR-221 mimic did not succeed in reducing the apoptosis even with the concentration of miR-221 increasing to 300 nmol/L, suggesting that a saturation threshold was reached in these cell lines by a single miRNA mimic. To verify the data of apoptosis, we further detected the caspase-3/7 activity. The result of caspase-3/7 activity was in line with apoptosis. Hence, miR-221 could inhibit the apoptosis of HCC cells.
In vivo test has also been reported using the anti-miR-221 oligonucleotides as a potential therapeutic for HCC in mice . Park et al.  showed that anti-miR-221 oligonucleotides could accumulate in the HCC tumors, reduce endogenous miR-221 oligonucleotides, modulate miR-221-related protein levels, and enhance the survival of tumor-bearing mice.