Mir-155 Targets TP53INP1 to Regulate Liver Cancer Stem Cell Acquisition and Self-Renewal ⇑ Fengchao Liu, Xin Kong, Lin Lv, Jian Gao

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FEBS Letters 589 (2015) 500–506

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MiR-155 targets TP53INP1 to regulate liver cancer stem cell acquisition and self-renewal ⇑ Fengchao Liu, Xin Kong, Lin Lv, Jian Gao

Department of Gastroenterology, Second Afﬁliated Hospital, Chongqing Medical University, Chongqing, China

article info abstract

Article history: In liver cancer, miR-155 up-regulation can regulate cancer-cell invasion. However, whether miR-155 Received 29 October 2014 expression is associated with liver cancer stem cells (CSCs) remains unknown. Here, we show that Revised 5 January 2015 miR-155 expression is up-regulated in tumor spheres. Knock-down of miR-155 resulted in suppres- Accepted 9 January 2015 sion of tumor sphere formation, through a decrease in the proportion of CD90+ and CD133+ CSCs and Available online 17 January 2015 in the expression of Oct4, whereas miR-155 overexpression had the opposite effect. TP53INP1 was Edited by Quan Chen determined to be involved in the CSCs-like properties that were regulated by miR-155. Thus, miR- 155 may play an important role in promoting the generation of stem cell-like cells and their self- renewal by targeting the gene TP53INP1. Keywords: Liver cancer stem cell Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. MiR-155 Tumor Protein 53-Induced Nuclear Protein 1 Tumor sphere Liver cancer

1. Introduction plants [10]. Recently, increasing studies have revealed that many miRNAs play crucial roles in tumorigenesis [11,12], including CSCs Hepatocellular carcinoma (HCC) is the sixth most prevalent [13,14]. Recent data suggest that there are miRNAs regulate stem- cancer and the third most frequent cause of cancer-related death like traits in liver cancer cells, including miR-214 [15], miR-148a [1]. Although surgical resection have significantly improved sur- [16], miR-612 [17]. It has been reported that miR-155 was up-reg- vival in patients at very early stage, however, long-term survival ulated in many cancers and plays an important role in tumorigen- remains unsatisfactory and development of tumor recurrence is esis [18,19], including HCC [20]. However, its molecular roles in the main cause of long-term death [2]. Therefore, we urgently need stem-like traits remain largely unknown. to understand the molecular mechanisms of the occurrence of liver Herein, we report that miR-155 play an important role in the cancer. acquisition of CSC phenotype. We demonstrated that the down- Cancer stem cells (CSCs), also termed as cancer initiating cells, is regulation of miR-155 can decreased the subpopulation of CD90+ a small set of self-renewing cells are believed to initiate and main- and CD133+ cells, the formation of spheres, and suppressed the tain cancers [3]. Liver CSCs dictate a hierarchical organization that expression of Oct4. Additionally, we also found that TP53INP1 is shared in both organogenesis and tumorigenesis [4]. CSCs in liver (Tumor Protein 53-Induced Nuclear Protein 1) could regulated cancer cells can be enriched by sorting cells with specific surface the CSC phenotype, which was regulated by miR-155. These find- markers such as CD90 [5], CD133 [6] or detection of side popula- ings suggest that miR-155 regulates the acquisition of stemness tion (SP) phenotype by Hoechst33342 exclusion [7] or by sphere in liver cancer cells and it may serve as a potential target in future formation using serum-free culture medium [8]. We have recently liver cancer therapy. reported that spheres cultured in serum-free culture medium (SFM) can up-regulated the mRNA and protein levels of CD90, 2. Methods CD133 and the stem cell-specific transcription factor Oct4 [9]. MicroRNAs (miRNAs) are a class of endogenous 22 nt non- 2.1. Cell lines and culture coding RNAs can play important regulatory roles by targeting mRNAs for cleavage or translational repression in animals and The human liver cancer cell line MHCC-97H was purchased from the Liver Cancer Institute, Zhongshan Hospital, Fudan Univer- ⇑ Corresponding author. Fax: +86 23 63711527. sity, Shanghai, China. The human liver cancer cell line, HepG2, was E-mail address: [email protected] (J. Gao).

http://dx.doi.org/10.1016/j.febslet.2015.01.009 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. F. Liu et al. / FEBS Letters 589 (2015) 500–506 501 purchased from the Chinese Academy of Sciences Cell Bank, Shang- cytometer (BD Biosciences). Isotype-matched mouse antibodies hai, China. The above cell lines were maintained in Dulbecco’s served as controls. modiﬁed Eagle medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 units/ml penicillin and 2.6. Cell sphere formation assay 100 lg/ml streptomycin, and the cells were maintained at 37 °C 3 in 5% CO2. CSCs sphere formation were performed in serum-free Single cells (1 Â 10 ) were plated in a 24-well ultra-low attach- culture medium (SFM), SFM was composed of DMEM/F12 (Gibco), ment plate (Corning, Corning, NY) in the serum-free culture med- 10 ng/ml basic ﬁbroblast growth factor (bFGF; PeproTech), 20 ng/ ium (SFM) described above. After 14 days of culture, the number ml EGF (PeproTech) and 20 ll/ml B27 supplement (Life of tumor spheres formed (diameter > 50 lm) was counted under Technologies). an inverted microscope.

2.2. RNA extraction and real-time RT–PCR 2.7. Protein extraction and Western blotting

Total RNA was extracted from the cultured cells using Trizol Total protein was extracted from the cells using RIPA lysis buf- (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruc- fer and the protein concentration was determined using a BCA Kit tions. For miRNA analysis, real-time PCR was performed using the (Pierce). The proteins were separated by sodium dodecyl sulfate– Hairpin-it™ miRNAs RT-PCR Quantitation Kit (GenePharma, polyacrylamide gel electrophoresis and transferred to polyvinyli- Shanghai, China). All miRNA data are expressed relative to the U6 dene difluoride membranes (Millipore). The membranes were small nuclear RNA and analyzed using the 2ÀDDCt method. For incubated overnight at 4 °C with primary antibodies after blocked the analysis of mRNA expression, complementary cDNA synthesis with 5% defatted milk. The membranes were subsequently washed was performed using the PrimeScript™ RT Reagent Kit with gDNA using Tris-buffered saline with 0.1% Tween (TBST) and incubated Eraser (Takara), Real-time PCR was conducted using SYBR Premix with horseradish peroxidase-linked secondary antibody for 1 h. ExTaq (Takara) under standard conditions according to the manu- Blots were visualized using enhanced chemiluminescence reagent facturer’s instructions. Real-time PCR was performed on the CFX96 (Engreen). The following antibodies were used: b-actin (YT0099, Real-Time PCR Detection System (Bio-Rad, USA). GAPDH was the Immunoway), anti-TP53INP1 (AB62004a; SangonBiotech, Shang- internal control, and analyzed using the 2ÀDDCt method. The prim- hai, China). ers are listed in Supplementary Data Table S2. 2.8. Statistical analysis 2.3. Cell transfection The SPSS 21 software was used for statistical analysis. All data MiR-155 agomir, small interfering RNAs (siRNA) and negative were acquired from at least three independent experiments and control (NC) were designed and synthesized by GenePharma are presented as the mean ± S.D. The Student’s t-test was used (Shanghai, China). The cells were transfected using siRNA-Mate™ for comparisons between two independent groups. P < 0.05 was (GenePharma) according to the manufacturer’s protocol. Briefly, considered statistically significant. the cells were seeded at 2–3 Â 105 cells per well and grown to 30–50% confluence. Transfection complexes were prepared accord- 3. Results ing to the instructions and were added directly to the cells. The miR-155 agomir and negative control were used at a final concen- 3.1. Up-regulation of miR-155 is associated with the population of tration of 50 nM, and the TP53INP1 siRNA and negative control CSCs were used at a final concentration of 100 nM. RT-PCR was used to assess the miR-155 levels after incubation for 24–48 h, and Wes- Many studies confirmed that the spheres cultured in serum-free tern blot was used to determine the level of protein expression culture medium (SFM) are highly enriched for cancer stem cells. To after incubation for 48 h. The sequence of the agomir and small investigate whether miR-155 is associated with the phenotype of siRNA are listed in Supplementary Data Table S3. CSCs, we cultured MHCC-97H cells in SFM. After 2 weeks, spheres formation was observed under an inverted microscope (Fig. 1A). It 2.4. Establishment of the miR-155 stable knock-down cell line has been reported that the CD90+ cells, from MHCC-97L/H cell lines displayed CSC phenotype [5]. Colony formation assays, and in vivo To establish stable transduction, we purchased a lentiviral vec- SCID mice tumorigenesis experiments were used to characterize tor containing the anti-sense of miR-155 or miR-control sequence CSCs in CD133+ MHCC-97H cells. Tumor nodules from the from GeneChem (Shanghai, China). Transfection was performed CD133+ cells isolated from MHCC97H cells appeared after 7 weeks using polybrene (GeneChem) according to the manufacturer’s from 1 Â 104 inoculated cells, While tumor nodules from the 5 instruction. Briefly, the cells were seeded at 2 Â 10 cells per well CD133À cells appeared after 7 weeks need from 2 Â 105 inoculated and grown to 20–40% confluence, then the cells were transfected cells (Supplementary Data Fig. A and Table S1). Additionally, the at a multiplicity of infection (MOI) of 40. After 72–96 h, the cells CD133+ cells showed a higher clonogenic capacity than CD133À were verified by fluorescence microscopy and qRT-PCR. cells (Supplementary Data Fig. B). So in the subsequent experiments we chose CD90 and CD133 as the surface markers of CSCs. 2.5. Flow cytometric analysis The population of CD90+ and CD133+ cells were measured by flow cytometric analysis, and we found that the percentage of CD90+ 6 Up to 1 Â 10 cells were resuspended in 100 ll PBS containing and CD133+ cells were increased in spheres compared to the 20 ll FcR Blocking Reagent (Miltenyi Biotec) and placed on ice parental cells (Fig. 1B). The mRNA levels of CD90 and CD133 were for 10 min to block the Fc receptors. After incubating with the also increased in sphere cells, detected by qRT-PCR (Fig. 1C). It has anti-human antibodies, PE-CD90 and PE-CD133 (Miltenyi Biotec), been reported that transcription factor Oct4 is a critical player in for 30 min on ice in the dark, the cells were washed with 1 ml maintaining the self-renewal state of liver cancer stem cells [21], ice-cold PBS for two times. The collected cells were resuspended and we found that the expression of Oct4 mRNA was significantly in 300 ll PBS and were detected using a FACSCanto II flow higher in spheres than in parental cells (Fig. 1D). These data 502 F. Liu et al. / FEBS Letters 589 (2015) 500–506

Fig. 1. Up-regulation of miR-155 is associated with CSCs. (A) Representative image of tumor sphere generated after 14 days of single-cell cultures in SFM. Scale bar: 50 lm. (B) A ﬂow cytometry histogram analysis shows the enrichment of CD90 and CD133 expression in spheres as compared to the adherent parental cells, P < 0.05. qPCR analysis was performed for CD90, CD133 (C) and stem cell-associated gene Oct4 (D) in spheres versus adherent parental cells. (E) The expression of miR-155 was higher in spheres compared with parental cells, detected by qPCR, ⁄P < 0.05.

Fig. 2. Inhibition of stem-like cell phenotypes by stable knockdown of miR-155 in MHCC-97H cells. (A) qPCR analysis of miR-155 expression in MHCC-97H cells lacking miR- 155. (B) Flow cytometry analysis of CD133+ and CD90+ cells distribution in anti-NC and anti-miR-155 cells, P < 0.05. (C and D) The relative mRNA levels of CD90, CD133 and Oct4 were detected by qPCR assay. (E) anti-miR-155 cells were able to generate a decreased number of spheres as compared with anti-NC cells. Scale bar: 100 lm, ⁄P < 0.05. demonstrated that SFM (serum-free culture medium) could enrich 3.2. Silencing of miR-155 decreased population of CSCs for CSCs by spheres formation. We furthered our study to test the expression of miR-155 in To examine the effects of miR-155 on CSC phenotype in liver sphere cells and parental cells by qPCR. The result demonstrated cancer cells, we established a lentivirus-mediated stable miR-155 that miR-155 expression was enriched in the sphere cells as com- down-regulation cell line (anti-miR-155) or negative control cell pared with the parental cells (Fig. 1E). The result prompted us to line (anti-NC) using MHCC-97H, and the inhibition of miR-155 study whether miR-155 play an important role in the stem-like was detected by qPCR (Fig. 2A). We first measured the proportion phenotype acquisition and self-renewal. of CD90+ or CD133+ cells in anti-miR-155 cells and negative control F. Liu et al. / FEBS Letters 589 (2015) 500–506 503 cells. The percentage of cells with CD90+ or CD133+ in anti-miR- cells by qPCR, and found that these cells showed increased CSC 155 cells was significantly less than in negative control group markers expression compared to the miR-155-NC-HepG2 and (Fig. 2B). Moreover, the relative mRNA expression of CD90 and miR-155-NC-MHCC-97H cells, respectively (Fig. 4C and D), sug- CD133 in anti-miR-155 cells and anti-NC cells were also measured. gesting that the over-expression of miR-155 increased the cancer The relative mRNA levels of CD90 and CD133 in anti-miR-155 cells stem-like cell stem-ness. were significantly decreased as compared to control groups, assessed by real time RT-PCR analysis (Fig. 2C). Additionally, the 3.4. Down-regulation of TP53INP1 promotes the acquisition of CSC-like mRNA level of Oct4 was also decreased in anti-miR-155 cells com- phenotypes pared to the anti-NC cells (Fig. 2D). To further demonstrate the results above, we performed the sphere-formation assay, and the It has been reported that TP53INP1 is a cancer suppressor, result indicated that the sphere number in anti-miR-155 cells which is associated with an oncogenic microRNA miR-155 was significantly smaller than anti-NC cells (Fig. 2E). These results [22,23]. And repressing TP53INP1 significantly enhanced the abil- suggested that suppression of miR-155 could decrease the CSC pro- ity of the CD133À cells to initiate spheroid formation [24], but portion and weaken CSCs self-renewal in liver cancer cells. whether silencing TP53INP1 could increase the population of To further confirm the role of miR-155 in regulating CSC pheno- CD133+ cells is unknown. To investigate the effect of silencing type, miR-155 agomir or negative control was transfected into TP53INP1 on the CD133+ cancer cell population, we firstly transfec- established anti-miR-155 cells (anti-miR-155-agomir or anti- ted MHCC-97H cells with siRNA of TP53INP1 or negative control miR-155-NC), and miR-155 re-expression was detected by qPCR (siTP53INP1 or siNC), and the suppression ratio was verified by (Fig. 3A). As compared to the negative control groups, forced re- qRT-PCR and Western blot (Fig. 5A). Flow cytometric analysis expression of miR-155 increased the proportion of CD90+ and showed that knocked-down of TP53INP1 can increased CD133+ CD133+ cells (Fig. 3B), and the mRNA expression of CD90, CD133 and CD90+ cells (Fig. 5C). To further indicate the role of TP53INP1 and Oct4 (Fig. 3C). Additionally, the sphere-formation ability was on CSC phenotype, we determined the expression of CD90, CD133 increased in anti-miR-155-agomir group compared to the anti- and Oct4 in siTP53INP1 and siNC cells used qPCR. The result miR-155-NC group (Fig. 3D). showed that the mRNA level of CD90, CD133 and Oct4 was significantly increased in siTP53INP1 cells compared to the siNC cells 3.3. Overexpression of miR-155 increased CD90/CD133/Oct4 (Fig. 5B). These results suggest that the down-regulation of expression TP53INP1 could promote acquisition of CSC-like phenotypes.

To further examine the role of miR-155 in regulating CSC phe- 3.5. MiR-155 regulate CSC phenotype through targeting TP53INP1 notype, miR-155 was transiently up-regulated by transfection of agomir or NC in both HepG2 and MHCC-97H cells, and the overex- Our results showed that down-regulation of miR-155 could pression of miR-155 was conﬁrmed by qPCR (Fig. 4A). We exam- increase the protein expression of TP53INP1 and forced re-expres- ined liver cancer stem cell markers, CD90, CD133 and Oct4 in sion of miR-155 yielded an opposing effect (Fig. 6A). Additionally, both miR-155-agomir-HepG2 and miR-155-agomir-MHCC-97H the expression of TP53INP1 was signiﬁcantly down-regulated in

Fig. 3. Induction stem-like cell phenotypes by re-expression of miR-155 in anti-miR-155 cells. (A) miR-155 expression was up-regulated in anti-miR-155 cells after transfected anti-miR-155 cells with miR-155 agomir (anti-miR-155-agomir), detected by qPCR. (B) Flow cytometry analysis showed the increase of CD90+ and CD133+ cells after transfected anti-miR-155 cells with miR-155 agomir, P < 0.05. (C) The expression levels of CD90, CD133 and Oct4 in anti-miR-155-agomir and anti-miR-155-NC cells. (D) anti-miR-155-agomir cells generated a greater number of spheres as compared with anti-miR-155-NC cells. Scale bar: 200 lm, ⁄P < 0.05. 504 F. Liu et al. / FEBS Letters 589 (2015) 500–506

Fig. 4. Overexpression of miR-155 increased CD90, CD133 and Oct4 expression. (A) The levels of miR-155 in MHCC-97H and HepG2 cells transfected with miR-155 agomir or NC were analyzed by qPCR. (B) The levels of TP53INP1 protein in MHCC-97H and HepG2 cells transfected with miR-155 agomir or NC were analyzed by Western blotting. The expression of stem-cell markers CD90, CD133 and Oct4 in MHCC-97H and HepG2 cells transfected with miR-155 agomir or NC (C) and (D), ⁄P < 0.05.

anti-miR-155 MHCC-97H cells signiﬁcantly rescued miR-155- inhibited stem markers CD90, CD133 and Oct4 expression (Fig. 6B and C), which further suggesting that targeting TP53INP1 is a mechanism by which miR-155 exerts its function.

4. Discussion

Recent ﬁndings support the concept that cancer stem cells (CSCs) are integral to the development and perpetuation of several forms of human cancer [25], and accumulating evidence has demonstrated the existence of cancer stem cells in liver cancer. It has been reported that, in HCC, CSC markers include epithelial cell adhesion molecule (EpCAM) [26], CD133 [27], CD90 [5], CD24 [28]. However, the critical step was to characterize the underlying molecular and biological mechanisms by which these cells can acquire their speciﬁc-phenotype. The deregulation of miR-155 has been implicated in various human tumors including human hepatocellular carcinoma. Never- theless, the role of miR-155 in liver cancer CSCs phenotype remains largely unknown. Accumulating evidence demonstrated that there are relationships between the EMT and CSCs, through the EMT pro- gram cells can gain CSC characteristics [29]. It has been reported that miR-155 plays an important role in the regulation of EMT Fig. 5. The roles of TP53INP1 on stem-like cell phenotypes. (A) The levels of [18]. So we hypothesized that miR-155 may also play an important TP53INP1 mRNA or protein in MHCC-97H cells transfected with siRNA were role in the CSCs-like properties in HCC cells. In the current study, analyzed by qPCR and Western blotting. (B) The expression of stem-cell markers we demonstrated that miR-155 functions to promote the acquisi- CD90, CD133 and Oct4 in siTP53INP1 cells and siNC cells. (C) Changes in CD90+ and + tion of stem cell phenotype. It has been reported that miR-155 CD133 cell population after transfection with siTP53INP1 as compared with siNC, + + P < 0.05, ⁄P < 0.05. expression in the CD133 /CD338 cells (lung cancer stem cell) was higher than that in the lung cancer A549 cells [30]. However, in ovarian cancer-initiating cells miR-155 was reported as a sup- miR-155 overexpressing MHCC-97H and HepG2 cells compared to pressor [31]. All these results indicated that miR-155 does play miR-NC (Fig. 4B), which further demonstrated the results of previ- an important role in the process of cancer stem cell generation, ous studies that TP53INP1 is a target gene of miR-155. Based on the but may play different roles in different tumors cells. above conclusions we concluded that miR-155 may regulate CSC It has been reported that serum-free culture medium (SFM) is phenotype by targeting TP53INP1. To investigate whether miR- increasingly used as an effective method to enrich and identify 155 exerts its function by targeting TP53INP1, we then performed CSCs [32,33]. Therefore, we used this method to enrich for liver a rescue experiment. As expected, transfection of si-TP53INP1 into cancer stem cells. The expression of liver cancer stem cell markers, F. Liu et al. / FEBS Letters 589 (2015) 500–506 505

Fig. 6. MiR-155 regulated CSC phenotype through target TP53INP1. (A) The expression levels of TP53INP1 in anti-miR-155 cells (left) or anti-miR-155 cells transfected with miR-155 agomir (right), as determined by Western blotting. (B and C) The effect of anti-miR-155 on stem phenotype markers were partially attenuated by silencing of TP53INP1, ⁄P < 0.05. such as CD90, CD133 and Oct4 were significantly increased in 155 exerts due to down-regulation of the tumor suppressor spheres compared with parental cells. These results indicated that TP53INP1. The present study implies the intriguing possibility of spheres cultured in SFM containing CSCs, we used spheres as a miR-155 may be used as a potential therapeutic target for the liver CSC model to explore the relationship between CSCs and treatment of patients with HCC. miR-155. We found that the expression of miR-155 in spheres was significantly increased compared to parental cells, which sug- 5. Conflict of interest gests that miR-155 plays an important role in regulating the stem phenotype in liver cancer cells. Next, using a lentiviral vectors No potential conflicts of interest were disclosed. approach, we transfected MHCC-97H cells with miR-155 inhibitor lentiviral vectors and stably silenced miR-155 in MHCC-97H cells. Acknowledgments We found that MHCC-97H cells stably lacking miR-155 decreased CD90+ and CD133+ cell population, Oct4 expression and weakened This work was supported by Key project of Chongqing Medical sphere-forming capacity. Science Foundation (2011-1-055); National Natural Science Foun- It is well known that TP53INP1 is a tumor suppressor gene dation of China (81101827); Science and technology research pro- [22,34], is a p53 target gene that induces cell growth arrest and ject by Chongqing Municipal Education Commission (KJ1400214); apoptosis by modulating p53 transcriptional activity. Our current Science and technology planning project by Yuzhong District of observation together with what has been reported recently sug- Chongqing (20140102). gests that TP53INP1 is a direct target gene of miR-155. It has been reported that miR-130b promotes CD133+ liver tumor-initiating Appendix A. Supplementary data cell growth and self-renewal via TP53INP1 [24], so we concluded that miR-155 may regulates stem phenotype by targeting Supplementary data associated with this article can be found, in TP53INP1 in liver cancer cells. To determine the hypothesis, we the online version, at http://dx.doi.org/10.1016/j.febslet.2015.01. firstly knock-down TP53INP1 using siRNA. The results showed that 009. TP53INP1 blockage increased CD133+ and CD90+ cells, and up-reg- References ulated Oct4. Expectedly, we also observed that CD90, CD133 and Oct4 attenuated by anti-miR-155 rescued by knockdown of [1] Forner, A., Llovet, J.M. and Bruix, J. (2012) Hepatocellular carcinoma. Lancet TP53INP1. Taken together, the biological effects of miR-155 on 379 (9822), 1245–1255. CSCs phenotype are probably due to the repression of TP53INP1. [2] Pang, R.W., Joh, J.W., Johnson, P.J., Monden, M., Pawlik, T.M. and Poon, R.T. (2008) Biology of hepatocellular carcinoma. Ann. Surg. Oncol. 15 (4), 962–971. In conclusion, the results of this study revealed up-regulation of [3] Pardal, R., Clarke, M.F. and Morrison, S.J. (2003) Applying the principles of miR-155 in CSCs cells and showed that miR-155 regulates the pro- stem-cell biology to cancer. Nat. Rev. Cancer 3 (12), 895–902. portion of CD90+ or CD133+ CSCs, the expression of stem cell-asso- [4] Yamashita, T. and Wang, X.W. (2013) Cancer stem cells in the development of ciated gene Oct4 and the capacity of spheres-formation, which liver cancer. J. Clin. Investig. 123 (5), 1911–1918. [5] Yang, Z.F., Ho, D.W., Ng, M.N., Lau, C.K., Yu, W.C., Ngai, P., Chu, P.W., Lam, C.T., indicated that miR-155 may play an important role in the CSCs Poon, R.T. and Fan, S.T. (2008) Significance of CD90+ cancer stem cells in acquisition and self-renewal. Importantly, the function of miR- human liver cancer. Cancer Cell 13 (2), 153–166. 506 F. Liu et al. / FEBS Letters 589 (2015) 500–506

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