| Background:Hepatocellular carcinoma (HCC) is one of the most common liver malignancies and the second most frequent cause of cancer death. Metastasis is always the primary reason for patients’death. Therefore, it is important to explore the mechanism of HCC metastasis.The EMT is a process that changes the cell phenotype between epithelial and mesenchymal states. Studies indicate that EMT plays a critical role in cancer metastatic progression. Therefore, the study of liver cancer EMT provides a new train of thought to explore liver cancer metastasis.CSCs, also known as cancer-initiating cells, are defined as a population of cells that possess the properties of self-renewal, differentiation and tumorigenesis. Therefore, CSCs are hypothesized to be responsible for therapy resistance, recurrence, relapse and metastasis. Therefore, to explore the mechanism of CSCs in the process of tumorigenesis may find a potential target for future liver cancer therapy.MicroRNAs (miRNAs) are short,-22 nt noncoding RNAs that can play important regulatory roles in life processes. It is generally accepted that miRNAs play an important role in cancer pathogenesis, and can act as either oncogenes or tumour suppressors. Many miRNAs regulate HCC development and several miRNAs are associated with EMT or CSCs, including miR-155. Therefore, miRNAs may be important targets for the treatment of liver cancer metastasis via the regulation of the EMT and cancer stem cell phenotype.PartⅠEffects of miR-155 on liver cancer EMT and CSCs acquisitionObjective:1. To obtain cancer stem-like cells by cultured MHCC-97H cells in serum-free culture medium and compare miR-155 expression between spheres and parental cells.2. To explore whether ectopic miR-155 expression in MHCC-97H cells significantly affected liver cancer EMT and CSCs acquisition.Methods:1. To obtain cancer stem-like cells, we cultured MHCC-97H cells in serum-free culture medium; Flow cytometry analysis, qPCR were used to detect the expressions of stem cell markers CD90, CD 133 and Oct4 between spheres and parental cells. Then, the expression of miR-155 was detected with qPCR.2. To investigate the role of miR-155 on CSC properties, we established a lentivirus-mediated stable miR-155 or miR-NC expression cell line using MHCC-97H. We used qPCR and western blot to evaluate the expression of EMT markers E-cadherin, N-cadherin and vimentin, Transwell migration assay and Matrigel invasion assay were used to evaluate the capacity of migration and invasion; Flow cytometry analysis, qPCR were used to detect the expressions of stem cell markers CD90, CD 133 and Oct4, and the capacity of sphere-forming also be evaluated.Results:1. The results indicated that the mRNA and protein levels of E-cadherin were significantly decreased. Conversely, N-cadherin and vimentin were significantly increased. The cell numbers in the invasion and migration assays were significantly increased compared to the miR-NC group cells. We also found that the percentage of CD90+or CD 133+cells in the MHCC-miR-155 group is higher than that in the MHCC-miR-NC group. The enhanced expression of CD90 and CD133 was verified by qRT-PCR, and the mRNA levels of Oct4 were also increased compared to the MHCC-miR-NC group. The number of tumour-spheres (diameter>50 μm) in the MHCC-miR-155 group cells was significantly higher than that in the MHCC-miR-NC group.Conclusion:We demonstrated that miR-155 is responsible for liver cancer cell EMT and CSCs acquisition.Part ⅡMiR-155 targets TP53INP1 to regulate liver cancer stem cell acquisition and self-renewalObjective:1. To determine whether miR-155 can regulate TP53INP1 in liver cancer cells.2. To determine the effects of TP53INP1 on EMT and CSCs acquisition.3. To determine whether TP53INP1 is involved in miR-155-promoted EMT and CSCs acquisition.Methods:1. MiR-155 was transiently up-regulated by the transfection of mimics or NC in both HepG2 and MHCC-97H cell lines, the expression of TP53INP1 was detected by qPCR and western blot.2. We first transfected MHCC-97H and HepG2 cells with TP53INP1 siRNA or NC, then used qPCR and western blot to evaluate the expression of EMT markers E-cadherin, N-cadherin; Flow cytometry analysis, qPCR were used to detect the expressions of stem cell markers CD90, CD133 and Oct4.3. To determine whether TP53INP1 is involved in miR-155-promoted EMT and CSCs acquisition. We divided cells into four groups and transfected cells according to the following combination:① miR-155-inhibitor-NC+TP53INP1-siRNA-NC ② miR-155-inhibitor+ TP53INP1-siRNA NC ② miR-155-inhibitor-NC+TP53INP1-siRNA ④ miR-155-inhibitor+TP53INP1-siRNA. qPCR and western blot were used to detect the expressions of EMT markers and stem cell marker Oct4.Results:1. Both the mRNA and protein expression of TP53INP1 were significantly down-regulated in miR-155 overexpressing MHCC-97H and HepG2 cells compared to miR-NC.2. The result showed that knockdown of TP53INP1 by siRNA was concurrent with E-cadherin suppression and N-cadherin induction. For the effects of TP53INP1 on stem-like traits, we demonstrated in MHCC-97H cells that the CD 133+or CD90+cell population and the mRNA expression of CD133, CD90 and Oct4 were significantly increased by TP53INP1 down-regulation.3. Cells co-transfected TP53INP1 siRNA and miR-155 inhibitors rescued the suppression of Oct4 by miR-155 inhibitors. Additionally, the inhibition of EMT was rescued by TP53INP1 siRNA.Conclusion:These results indicate that miR-155 specifically regulates EMT and stem-like properties via TP53INP1.Part IIITGF-β1 acts through miR-155 to down-regulate TP53INP1 in promoting epithelial-mesenchymal transition and cancer stem cell phenotypes acquisitionObjective:1. To determine the effects of TGF-β1 on EMT and CSCs acquisition.2. To determine TGF-β1 whether elevate the expression of miR-155 in liver cancer cells and suppress the expression of TP53INP1.3. To determine that TGF-β1 indirectly regulates TP53INP1 expression via miR-155 in liver cancer cells.Methods:1. To observe morphological changes after the cells treated with TGF-β1, we used qPCR and western blot to evaluate the expression of EMT markers E-cadherin, N-cadherin; qPCR were used to detect the expression of stem cell markers CD90, CD 133 and Oct4.2. The expression of miR-155 and TP53INP1 were evaluated by qPCR or western blot.3. To determine whether TGF-β1 indirectly regulates TP53INP1 expression via miR-155 in liver cancer cells, we divided cells into three groups and treated cells according to the following combination:① TGF-β1+miR-155-inhibitor ② TGF-β1+miR-155-inhibitor-NC ③ NC. qPCR and western blot were used to detected the expression of EMT markers and stem cell marker Oct4.Results:1. We exposed MHCC-97H cells to TGF-β1 for 48 h, and morphological changes were then observed, western blot assay showed a significant decrease in the expression of E-cadherin and a significant increase in the expression of N-cadherin in TGF-β1-treated cells compared to untreated cells. We also found that the sternness markers CD90, CD133 and Oct4 were increased in TGF-β1-treated cells using qPCR.2. The expression of miR-155 in TGF-β1 treated cells was significantly increased compared to untreated cells, and the expression of TP53INP1 was the opposite.3. The results showed that the miR-155 inhibitors not only rescued the inhibition of TP53INP1 by TGF-β1 but also inhibited the EMT induced by TGF-β1. Additionally, the mRNA levels of Oct4 indicated that the function of TGF-β1 on stemness was also inhibited by miR-155 inhibitors.Conclusion:TGF-β1 indirectly regulates TP53INP1 expression via miR-155 in liver cancer cells. |
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