Roles Of MicroRNA-25 In Liver Cancer Stem Cells And Its Underlying Mechanisms

Background:Hepatocellular carcinoma(HCC)represents as the fifth most commonly diagnosed malignancy and the third leading cause of cancer-related death worldwide.Due to the extensive research efforts,understanding of the molecular mechanisms involved in the tumorigenic transformation of HCC has been clarified gradually.However,the molecular targets for HCC therapy remain insufficient.Recent researches suggest that human cancer can be considered as a stem cell disorder.According to these studies,tumors may be viewed as abnormal growth which is driven by a small population of cancer cells called cancer stem cells(CSCs)that are endowed with the ability of self-renewal.Previous studies have identified the CD133+ phenotype as the molecular marker of liver cancer stem cells(LCSCs).The LCSCs have been found to show a greater colony-forming efficiency and greater ability to form tumor in vivo.In addition,the LCSCs are believed to be responsible for the high resistance to chemotherapeutic drugs.Therefore,to treat the HCC more effectively,it is imperative that LCSCs be specifically targeted.Tumor necrosis factor(TNF)-related apoptosis inducing ligand(TRAIL)is a cytokine,which belongs to the TNF superfamily.As the TRAIL triggers apoptosis selectively in tumor cells without damaging the normal tissue cells,it has been tested in clinical trials for cancer therapy.However,unfortunately,some cancer cells(especially CSCs)are resistant to TRAIL and the mechanism of this resistance is not fully understood.Therefore,it's urgent to identify the mechanisms and take strategies to decrease the resistance of CSCs to TRAIL.MicroRNAs(miRNAs),small functional RNAs of 19-25 nt,are a class of short non-coding RNA molecules and play important regulatory roles by binding to the target mRNAs at the 3'untranslated region(3' UTR).Increasing evidence showed that miRNAs have significant roles in a number of biological processes such as proliferation,differentiation,metabolism and apoptosis.Aberrantly expression of miRNAs is associated with multiple human diseases including cancer.In the present study,we found the miR-25 was dysregulated in LCSCs.We therefore investigated the relationship between the miR-25 and the treatment of TRAIL in LCSCs,which might serve as a potential target for HCC therapy.Aims:The aim of this study is to observe the influence of miR-25 expression on the malignancy of LCSCs,to find out the signal transduction of miR-25 in LCSCs,and to investigate the strategies to increase the sensitivity of LCSCs to TRAIL treatment.Methods:Cell samples of HepG2,Huh7,PLC and L-02 were stained with anti-CD 133-FITC,and then analyzed and sorted as the LCSCs using the fluorescent-activated cell sorting equipment.Total RNA was extracted from the cells.The expression level of miR-25 was measured by reverse transcription and Quantitative Real-Time Polymerase ChainReaction.Cell-based experiments were carried out by transfection of RNA oligonucleotides including miR-25 mimics,miR-25 antisense-oligonucleotides(anti-miR-25),control RNA oligonucleotides(miR-NC)and small interfering RNA of PTEN(PTEN siRNA).Cell lines were treated with TRAIL.MiR-25 expression and cell viability were then analyzed to find out the influence of miR-25 level on LCSCs malignancy.The target of miR-25 gene is predicted using three different public databases.Dual-luciferase report,co-immunoprecipitation and western blot were then conducted to detect the generation of apoptosis-related cytochrome C,apoptogenic compounds and ROS.Results:In the present study,we observed significant up-regulation of miR-25 in LCSCs compared with the non-CSCs.Furthermore,we found that knockdown of miR-25 by its antisense oligonucleotide(anti-miR-25)significantly increased the sensitivity of LCSCs to TRAIL-induced apoptosis.The gene of PTEN,which is a natural inhibitor of PI3K,was found to be directly regulated by miR-25 in HepG2-CSCs.We demonstrated that knockdown of miR-25 increased the expression of PTEN.Mechanistically,Inhibition of Bad phosphorylation,which is regulated by the PTEN/PI3K/AKT pathway,is essential for the functional roles of anti-miR-25 in HepG2-CSCs.Conclusions:In summary,we have provided several pieces of evidence to prove that the miR-25 is associated with the sensitivity of liver cancer stem cells to TRAIL-induced apoptosis.Mechanically,we demonstrate that the knockdown of miR-25 promotes the TRAIL-induced apoptosis by inhibiting the PI3K/Akt/Bad signaling pathway through the miR-25/PTEN axis.The combination with anti-miR-25 and TRAIL may represent a novel strategy for the treatment of LCSCs.