For studying the physiological properties of stem like cells in HCC, a large number of stem-like cells were needed. As a result, sphere forming assay was performed with the protocol of neuron sphere (Singh et al., 2003). After culturing 6 days, two different kinds of HCC cell lines, Huh7 and Hep3B could both form sphere cells on ultralow-attachment dish (Figure 1).
3.2 YB-1 was up-regulated during sphere forming
To analyze the changes of genes expressing pattern between normal and sphere cultures, I performed quantitative PCR test. Some CSCs related genes, CD133, CD90 and EpCAM, were up-regulated in sphere cells (Marquardt et al., 2010). YB-1 mRNA was also up-regulated during sphere forming (Figure 2).
3.3 YB-1 translocated into nucleus in sphere cells
The previous studies have shown that the different subcellular localization of YB-1 may indicate its different regulation functions, such as tumour recurrence or drug resistance (Stein et al., 2001). Compared with normal HCC cells, YB-1 would translocate into nucleus in immunofluorescent staining of sphere HCC cells (Figure 3 A-B).
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3.4 The self-renewal ability of YB-1 KD cells decreased
Self-renewal ability leaded CSCs to maintain their cells pool in tumor tissues.
Self-renewal ability of CSCs could be evaluated by sphere and colony formation (Hu and Smyth, 2009; Oppitz et al., 2002; Singh et al., 2003). To investigate whether YB-1 affects the self-renewal ability of HCC CSCs, YB-1 KD Huh7 cells were established (Figure 4A). In Figure 4B and 4C, YB-1 KD Huh7 cells formed fewer colonies in colony formation assay and revealed lower stem cell frequency in extreme limiting dilution assay (Table 1 and Figure 5).
3.5 The stem cell population would be regulated by YB-1
Owing to functions of YB-1 in liver development and regeneration, I wanted to know the relation between YB-1 and stem cells in HCC. First, I performed side population flow cytometry, which could distinguish the stem-like cells from heterogeneous cells by the ability to pump out hoechst 33342 dyes (Zhou et al., 2001).
While YB-1 was knocked down, the ratio of stem-like cells was decreasing from 0.596% to 0.31%. On the contrary, the ratio of stem-like cells in sphere cells would increase from 0.596% to 1.65% with YB-1 up-regulation (Figure 6). Next, I analyzed the liver stem/progenitor cell surface marker, EPCAM, which had been reported also expressing in HCC cancer stem cells (Yamashita et al., 2009) by flow cytometry. The
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ratio of EPCAM+ cells in shYB1 cells would decrease with YB-1 down-regulation (Figure 7).
3.6 The pluripotent genes were downregulated in YB-1 KD cells
Because knock-down YB-1 would decrease stem-like cells in HCC, I would like to know how YB-1 mediates self-renewal ability of HCC cells. First, I measure the typical stemness gene related to self-renewal, Oct4, Nanog and Sox2, which was highly expressed in embryonic stem cells. My results showed that those genes were down regulated in YB-1 KD HCC cells, so did the well-known cancer stem cell marker as well as hepatic lineage stem cell marker, AFP (Figure 8A). On the contrary, the liver lineage differentiated marker, albumin, was up-regulated (Figure 8B). I also tried to overexpress YB-1 and test Nanog transcription activity but the result is negative.
(Supplementary figure 1)
3.7 The pluripotent genes were up-regulated in sphere cells
Beside the YB-1 KD HCC cells, I also investigate some pluripotent genes, such as OCT4, cMYC and NANOG in sphere cells. Those genes are up-regulated in sphere cells (Figure 8C). In the mean while, the mRNA expression of CSCs marker genes, BMI1, CD44, EPCAM and CD133 are also up-regulated in sphere cells (Figure 9).
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3.8 EMT related genes were altered in different YB-1 expression pattern
Epithelial-mesenchymal transition (EMT) and cancer metastasis are considered as the properties of CSCs in tumor progression.(Zubeldia et al., 2013). From my previous data, YB-1 was up-regulated in sphere cells, and stemness genes were down-regulated in YB-1 KD cells. Thus, YB-1 may also affect EMT and metastasis-related genes. In YB-1 KD cells, I found BMI1, Vimentin and SNAIL1, which have been known to participate in cancer metastasis and EMT were also down-regulated by qPCR analysis.
(Figure 10).
3.9 KD YB-1 cells had low ability of drug resistance
Many studies suggest that cancer stem cells had stronger ability of drug resistance and YB-1 seemed to play important role in cancer stem cells (Singh and Settleman, 2010) so I want to investigate the relationship with YB-1 and drug resistance in Huh7.
After treated with traditional anti HCC drug, doxorubicin (0.15 μg/ml) or sorafenib (0.15mM) for 3 days, cell viability of YB-1 KD cells was decreased compared to shLac cells (Figure 11).
3.10 Increase the proapoptotic genes in YB-1 KD cells
Some studies suggested that repression of proapoptotic gene might enhance the survival rate of cells under a stress condition (Bertrand et al., 2009). In order to know
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whether YB-1 affects the anti-stress ability in HCC cells, proapoptotic genes, E2F1 and BAX were evaluated in normal and YB-1 KD HCC cells. As shown in figure 12, mRNA expression level of E2F1 and BAX were up-regulated in mitomicyin C treated YB-1 KD cells.
3.11 Wnt/β-catenin pathway might be regulated by YB-1
From my results, YB-1 may alter the expression of genes related to self-renewal, CSCs markers, EMT and apoptosis in HCC CSCs. How YB-1 modulates these properties was still unclear. Recently, a ChIP-on-Chip bioinformatics data indicated YB-1 may affect Wnt/β-catenin pathway, which regulates several key stemness properties of normal and tumor stem cells in breast cancer (Finkbeiner et al., 2009). In HCC, whether YB-1 alters Wnt signaling needs to further investigate. By immunofluorescence staining, active Wnt signaling led β-catenin accumulation and translocation in the cell nucleus of HCC sphere cells (Figure 13A-B). Furthermore, the expression of Wnt signaling target genes, AXIN1 and AXIN2, as well as important signal transducer, β-catenin, were down-regulated in YB-1 KD cells (Figure 14A-B).
On the contrary, β-catenin protein would express more in sphere cells (Figure 14B).
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3.12 Epigenetic modulators were affected after Knock down of YB-1
There was an article reporting that YB-1 would play important role in mechanism of controlling Wnt/β-catenin pathway (Finkbeiner et al., 2009). In this article, the author also shown that some epigenetic modulators were regulated by YB-1. In the other hand, stem-like properties acquisition also had a relationship with epigenetic regulation (Apostolou and Hochedlinger, 2013). As a result, I combined their results in table 2 and believed that some of epigenetic modulators would be regulated by YB-1. After knock down YB-1 with siRNA-oligo, the mRNA level of SOX2 decreased while the genes related to DNA methylation pattern change, DNMT1 and DNMT3A, as well as histone modification protein, HDAC1, were up-regulated (Figure 15).
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