MicroRNA-150Mediated Regulation Of The Maligant Phenotype Of Liver Cancer Stem Cells

Hepatocellular carcinoma (HCC) is one of the most prevalentaggressive malignancies in the world. In the worldwide, HCC is the fourthmost cause of death from cancer in the world because of its high malignancyand poor prognosis, and the incidence is rising in China possibly due to theincreased HCV infection. Although advance of conventional clinicaltreatment for HCC has been achieved at some centers, the overallfive-year survival rate for the whole HCC population at large is still verypoor over the past several decades, partly due to the tumor cells to thetherapeutic resistance.Recently, specific subpopulations of cell with stem-like properties,termed tumor-initiating calls or cancer stem cells (CSCs), have the abilityto drive and sustain tumor growth. The theory of CSCs provides a newtherapeutic strategie for the anti-tumor therapy. According to this hypothesis,CSCs have the capacity to extensive proliferation, self-renewal anddifferentiation. Several studies have indicated that CSCs are thereal roots of the treatment resistance and recurrence of malignant tumor.Therefore, exploring the mechanisms of CSCs in the occurrence anddevelopment of tumor is therefore important for the development of novel therapeutic strategies.microRNAs (miRNAs) are a novel class of small non-coding RNAs(21~23nucleotides) that negatively regulate gene expression. miRNAs canfunction either as oncogenes or as tumor suppressors. miRNAs have beenimplicated in maintenance of CSCs phenotype via their ability to affectexpression of genes and proteins. Emerging evidences demonstrate animportant role of miRNAs in regulation many cellular processes such ascell differentiation, cell proliferation and tumorigenesis. Whereas theknowledge about miRNAs expression in liver CSCs are still preliminary.Therefore, screening for differences in miRNA expression betweennon-CSCs and CSCs may help to exploring the functional impacts ofmiRNA on malignant phenotype of CSCs.Objective:1. To isolate and identify the CD133~+Liver CSCs from the liver cancercell lines by MACS sorting.2. To analyze the differential miRNAs expressions profiles betweenCD133~+Liver CSCs and CD133-non-CSCs, and then find the commonbioinformatics features of the significantly up-regulation miRNAs innon-CSCs.3. To investigate the effect of miR-150on CD133~+liver CSCsmalignant phenotype, such as cell proliferation, cell differentiation andmigration, and explore the molecular mechanism of miR-150in maintenance of CD133~+liver CSCs phenotype.Methods:1. Magnetic separation was performed to obtain CD133~+CSCs cellsand CD133-cells. The purity of sorted cells was evaluated by FACS with theFACS-Calibur machine and Western blotting.2. The proliferation was examined by means of a cell proliferationassay using Cell Counting Kit-8(CCK-8). The ability of colony forming ofCD133~+cells and CD133-cells were observed by colony formation assay.The CD133~+cells were cultured in serum-free medium to determine themonoclonal formation characteristics. The tumor formation ability of theCD133~+cells was determined by transplantation into NOD/SCID mice.3. For miRNA profiles analysis, total RNA was isolated from theCD133~+fractions and CD133-fractions using TRIZOL. The miRNAsexpression profiles of CD133~+fractions and CD133-fractions werescreened by microarray. The differentially expressed miRNAs were verifiedby Realtime-PCR. And a BLAST search was performed to identify thebiological functions of these significant differentially expression miRNAs,using two commonly used prediction algorithms (TargetSCAN and PicTar).4. The HIV lentiviral system expression miR-150was commerciallysynthesized and transfected into CD133~+cells. To investigate the potentialrole of miR-150in liver cancer stem cells, we examined whetherover-expression of miR-150could inhibit the CD133~+cells and their self-renewal potential. The effects of miR-150over-expression on cellproliferation, tumorsphere formation, cell apoptosis and cell invasion werefurther evaluated. The predicted miR-150binding site in c-Myb3'-untranslated region (UTR) was confirmed by dual-luciferase reportergene assay. The expression of predicted target gene was detected byRealtime-PCR and Western blotting.Results:1. The CD133~+subpopulations in the SMMC-7721cell line wereanalyzed by flow cytometry, which ranged from0.20%to2.16%. Aftersorting, the content of CD133~+subpopulation was enriched up to92.02±5.12%. Differential expression level of CD133protein between theCD133~+SMMC-7721cells and CD133-SMMC-7721cells was confirmedby Western blotting (0.873±0.05vs.0.067±0.015).2. The isolated CD133~+SMMC-7721cultured in a serum-free mediumformed tumorspheres within24～72h, whereas the CD133-SMMC-7721cells did not exhibit tumorspheres formation. Moreover, Spheroids formedfrom CD133~+cells were removed from the serum-free medium and allowedto attach to a substratum. Expanding adherent cells from the spheroids can beobserved at two days of culture.3. The CD133~+cells possessed significantly higher proliferation thanCD133-cells. Remarkably, the proliferation rate of CD133~+cells wassignificantly higher than CD133-cells on the fifth day. In the colony formation assay, we found CD133~+cells possessed higher colony formationefficiency than CD133-cells (271.22±57vs.22.24±6.56).4. CD133~+SMMC-7721cells, but not CD133-SMMC-7721cells,could efficiently initiate tumors in NOD/SCID. As little as1,000CD133~+SMMC-7721cells could initiate tumors in2of5injected mice6weeks afterinoculation. Moreover,10,000CD133~+SMMC-7721cells initiated tumorsin5of5injected mice, whereas no tumors were found in injected of CD133-SMMC-7721cells.5. The miRNA microarray analysis showed that several differentiallyexpressed miRNAs between CD133~+and CD133-cells. Interestingly,several miRNAs were up-regulated in the CD133-HCC fractions, includinghsa-miR-150, hsa-miR-152, hsa-miR-888, hsa-miR-519e, hsa-miR-532-5p.These findings have been confirmed by Real-time PCR assays. To betterunderstand the biological functions of differentially expressed miRNAs,potential targets of these miRNAs were predicted by three commonly usedprediction algorithms(miRanda,TargetSCAN and PicTar. Through onlineforecasting, we focus on miR-150, which have potential target sites ofoncogene c-Myb mRNA. Two miR-150potential target sites reside atnucleotides904-910and nucleotides968-974of the c-Myb3'UTR.6. The3'UTR fragment (nt904to nt910and nt968to nt974) of c-Mybcontaining miR-150and the mutated binding site of c-Myb3'UTRsequence were cloned into the firefly luciferase reporter vector pMIR-REPORT (Ambion). All constructed were verified by sequencing.Cotransfection with c-Myb-3'UTR and miR-150caused a36%decrease inthe luciferase activity compared with the negative control. Luciferaseactivity assays demonstrate a direct targeting of the3'UTR of c-Myb bymiR-150.7. Western blotting results demonstrated that over-expression ofmiR-150in CD133~+cells significantly decreased c-Myb protein expression,while the control vectors or blank control had no effect on expression ofc-Myb protein (0.31±0.07vs.0.80±0.09and0.81±0.08). RT-PCRanalysis demonstrated that miR-150has little if any effect on c-Myb mRNAlevels (0.85±0.04vs.0.81±0.03and0.80±0.09).8. CD133~+cells transfected with miR-150displayed a significantlyreduced proliferation rate, compared to cells transfected with control vectors.And flow cytometry analysis also showed that up to25.21±9.26%ofCD133~+cells became apoptotic on72h after transfected with miR-150,whereas the apoptosis in the control vectors group and blank control groupwere6.39±1.02%and5.87±9.26%, respectively. Furthermore,over-expression of miR-150in CD133~+SMMC-7721cells led to a68%reduction of invasiveness in Transwell assay compared with cells transfectedwith control vectors.Conclusions:We successfully sorted CD133~+cells from SMMC-7721cells, and which demonstrated some CSCs-like properties and abilities. The CD133~+SMMC-7721cells show higher proliferation, colony formation, spheroidformation ability in vitro and higher tumorigenicity ability in vivo. miR-150was significantly lower expression in CD133~+SMMC-7721, which may beinvolved in maintenance of CD133~+liver CSCs phenotype. We also showthat miR-150interacts with the3'UTR of c-Myb mRNA andover-expression of miR-150downregulates c-Myb protein levels.Over-expression of miR-150lead to a decrease in cell proliferation andtumorsphere formation, a decrease in cell survival, a suppress in cellinvasiveness. Therefore, our findings demonstrate for the first time thatmiR-150plays an important role in maintenance the malignant phenotypeof CD133~+liver CSCs, at least partly via the direct modulation of targetgene c-Myb.