Ultrasound-targeted Microbubble Destruction-mediated Downregulation Of CD133 Inhibits Stemness, Migratory Ability And Epithelial-mesenchymal Transition Of CD133+ Liver Cancer Stem Cells

Background and ObjectiveHepatocellular carcinoma(HCC) is one of most prevalent cancer worldwide, which is an aggressive disease with a poor outcome due to the high incidence of metastasis. Accumulating scientific evidence indicates that tumor formation is driven by a subpopulation of self-renewing cells known as cancer stem cells(CSCs). CSCs have been shown to be responsible for tumor initiation, metastasis, recurrence and chemoresistance.CD133(also known as AC133 or prominin-1), a 5 transmembrane cell surface glycoprotein, has been used to extract a subset of putative stem cells in HCC. CD133+ liver cancer cells possess many stem cell properties,including self-renewal, high proliferation, differentiation and have greater tumorigenicity and chemoresistance. As a liver CSC marker, CD133 also serves as an important indicator for tumor malignant progression, patientsurvival and recurrence rates.The epithelial-mesenchymal transition(EMT) is a transdifferentiation process that converts adherent epithelial cells into migratory mesenchymal cells. Activation of the EMT program is considered an important step in the embryonic development, tumorigenic progression and cancer metastasis.Previous studies have also linked EMT with the properties of CSCs. Cancer stem cells(CSCs) have been identified to be generated by epithelial-mesenchymal transition(EMT) characteristics.UTMD(ultrasound-targeted microbubble destruction), as a promising method for gene and drug delivery, may be combined with RNAi technique successfully. The UTMD system is the combination of ultrasound and microbubbles, which is safer and more effective compared with other methods. UTMD may be a powerful and effective tool for the transfection of specific genes and functional analysis of genes, which may be explored as a useful therapeutic option for liver cancer therapy.The aim of the present study was to evaluate the possibility of shRNA vector transfection mediated by UTMD in LCSCs. We also addressed the issue of whether the UTMD-based shRNA delivery system facilitated gene delivery in liver cancer stem cells(LCSCs); to investigate the change of proliferation, colony formation ability, invasiveness and tumorigenicity of CD133+ liver cancer cells with ultrasound-targeted microbubble destruction-mediated downregulation of CD133; to elucidate the regulatorymechanism of CD133 and EMT in LCSCs.Methods1. Human hepatoma carcinoma SMMC-7721 cell line was cultured and undergone CD133 fluorescence activated cell sorting(FACS), then collected and suspension cultured in the serum-free culture media. Flow cytometry was used to detect the expression of CD133 before and after cells isolation. Spheres-forming and CCK-8 assays in vitro and the nude mice transplantation tumor experiments in vivo were performed to validate the cancer stem-like properties of sorted CD133+ cells.2. The sorted CD133+SMMC-7721 cells were divided into the following three groups: the control group(Control); CD133-shRNA plasmid + Lipofectamine 2000 group(P+L); CD133-shRNA plasmid +ultrasound exposure + microbubbles group(P+UTMD). The transfection efficiency of the cells was detected by inverted fluorescence microscope and flow cytometry. RT-PCR and Western blot were used to detect the gene and protein expression of CD133.3. The stem cell marker, CD44、CD90、Oct4 and Sox2 were analyzed by RT-PCR. The self-renewal capacity was determined by tumor sphere formation assay. CCK-8 and colony formation assays were used to detect cellular proliferation activity. The cell apoptosis of cells was detected by flow cytometry. Transwell chambers cell invasion and migration assays evaluated the invasion and migration ability of cells. In order to constructtransplantation tumor model in nude mice, the cells in three groups respectively inoculated into nude mice subcutaneously. The growth of transplantation tumors of nude mice was observed every other day.4. Epithelial-mesenchymal transition markers, E-cadherin, N-cadherin and vimentin were analyzed by RT-PCR, Western Blot and immunohistochemical staining. The NF-κB signaling pathway was detected to elucidate the underlying mechanism of CD133 regulation of EMT traits in CD133+ liver cancer stem cells.Results1. We sorted out the CD133+ liver CSCs from the HCC cell line SMMC-7721 by FACS. Following sorting, CD133+ SMMC-7721 cells were analyzed by flow cytometry, resulting in a considerable enrichment in the CD133+ cell population(purity>90%) compared to the unsorted SMMC-7721 cells(purity 0.2-2%). We performed the tumorsphere formation assay by culturing the CD133+ cells in serum-free culture medium. Within 14 d of culture, we obtained liver cancer spheroids in CD133+ cells which grew in aggregate clusters and increased in size and amount as time passed. The hepatospheres exhibited high proliferative potential, self-renewal and highly tumorigenic capability.2. The effects of the transfection were assessed with fluorescence,flow cytometry, qRT-PCR and western blotting analysis. We could observe the expression of GFP under an inverse fluorescence microscope. The GFPof the P+UTMD group was stronger than the P+L group. The results of flow cytometry showed that the transfection efficiency of the P+UTMD group was higher than the P+L group(P<0.05). The CD133 expression levels of the groups were determined by qRT-PCR and western blotting analysis. We observed that the relative mRNA and protein levels of CD133 and in the UTMD group of cells were significantly decreased compared to the Lipofectamine or the control group cells(P<0.05).3. The mRNAs of several stem cell associated genes, including CD44,CD90, Oct4 and Sox2, were all down-regulated in the P+UTMD group compared to the P+L group or the control group cells, which was confirmed by qRT-PCR. We also examined the effect of down-regulation of CD133 on self-renewal and sphere-forming ability. The UTMD transfected CD133+ SMMC-7721 cells formed much smaller and less spheroids than cells transduced with Lipofectamine or the control group. CCK-8 assays showed that CD133+ SMMC-7721 cells transfected with CD133-shRNA mediated by UTMD(P+UTMD group) displayed a significantly reduced proliferation rate, compared to the cells transfected with CD133-shRNA mediated by Lipofectamine(P+L) group and the control group(P<0.05). In colony forming assays, the P+UTMD group presented a significant decrease in the number and size of colonies(P<0.05). Next, we detected the cell apoptosis using flow cytometry. LCSCs transfected with CD133-shRNA mediated by UTMD significantly increased the percentageof total apoptotic cells(early apoptotic + late apoptotic). We next performed the transwell migration assay and Boyden chamber assay. The number of LCSCs in the CD133 transfected by UTMD group migrating or invading through the Boyden chamber pores decreased significantly compared with P+L group and the control groups. We next elucidated the effect of CD133 down-regulation on tumorigenic ability of LCSCs using a nude mice xenograft model. The weights and volumes of tumors in P+UTMD group were significantly lower than those of P+L group and control group(P<0.05).4. The expression levels of EMT markers were detected by qRT-PCR and western blotting. The result revealed that E-Cadherin was enhanced but N-Cadherin and vimentin was decreased in CD133 down-regulated LCSCs compared with the CD133+SMMC-7721 cells. The expression of N-Cadherin and vimentin protein in the UTMD transfected group was lower than that of Lipofectamine transfected group and the control group,while the expression level of E-Cadherin in UTMD transfected group was increased significantly. Xenograft tissue samples were analyzed by immunohistochemical staining for the EMT-related proteins E-cadherin,N-cadherin, vimentin and the result was consistent with our previous studies in vitro. To further investigate whether reversion of EMT is associated with the NF-κB signaling pathway in CD133+ liver cancer stem cells, we examined the expression of IκB kinase β(IKKβ), inhibitornuclear factor-kappa B alpha(IκBα), and nuclear factor κB(NF-κB) RelA using Western blot. By shRNA mediated knockdown of CD133, decreased expression of the classical NF-κB signal pathway(IKKβ-IκBα-RelA) was observed in the UTMD and Lipofectamine transfected groups, as compared with the control group.Conclusion1. CD133+ SMMC-7721 liver cancer cells demonstrated many stem cell properties, including self-renewal, highly proliferation, and have greater tumorigenicity, which could be considered as CSC-like subsets of liver cancer cells.2. UTMD exerted a more significant knockdown effect on gene transfection in CD133+SMMC-7721 cells than the Lipofectamine transfected group. Down-regulation of CD133 mediated by UTMD resulted in a reduction of proliferation, malignant properties and tumorigenic ability in CD133+ LCSCs.3. Down-regulation of CD133 mediated by UTMD reversed the EMT process and that EMT mediated by CD133 could be a mechanism of regulation LCSCs initiation, invasion and migration. NF-κB pathway may play an important part in mediating the role of CD133 in regulating EMT phenotype.4. CD133 plays a function rule in regulating proliferative, migratory behaviors, tumorigenesis and EMT in LCSCs.5. UTMD could be a powerful and effective tool for transfection of specific genes and functional analysis of genes, which may be explored as a useful therapeutic option for liver cancer therapy.