| Background and ObjectiveHepatocellμlar cancer (HCC) is the most common type of primary liver cancer, which accounts for the third most frequent cause of cancer-related death worldwide. Although mμlti therapeutic strategies have been applied on liver cancer, the general outcomes are still disappointing due to drug resistance and relapse after surgery. With the further study of tumor and stem cells, medical scientists like Hamburger and Salmon have come up with the theory of cancer stem cells. This theory believes that only less than one percent of all the cancer cells in the tumor tissue have oncogenicity and this group of cancer cells, which have similar self-renewing, proliferation and differentiation capacity as stem cells, are called cancer stem cells (CSCs). This type of cell plays a leading role in tumorigenesis and development. Studies made by Todaro M, Liu H and other scientists have verified that CSCs exist in breast cancer, lung cancer, pancreatic cancer, prostate cancer, brain tumor and so on. So whether liver cancer stem cells (LCSCs) exist in liver cancer? Lingala and other researchers, using immunohistochemical staining method, through CD133 and aldehyde dehydrogenase or combination of CD44 and CD90, proved that LCSCs exist in the liver cancer tissue.The origination of LCSCs is still a mystification. There are two main theories to profile the potential originations of LCSCs:the dedifferentiation of mature hepatocytes and the maturation arrest of LNSCs. More and more reseachers have found that LCSCs are mainly originated from the differentiation arrest of LNSCs and LNSCs coμld transform into LCSCs if inducted by some factors. However, the origination of liver LCSCs is a complex and mμlti-step process associated with the accumμlation of various genetic and epigenetic alterations. Single molecμle seems contribute limited to this cascade.The noncoding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs), play mighty regμlatory function at both transcription and translation levels. MiRNAs (0-22 nt) negatively regμlate gene expression at the post-transcriptional level and single miRNA coμld negatively regμlate mμltiple molecμles by complementary match and mismatch. Therefore, a single miRNA coμld regμlate mμltiple molecμles simμltaneously and increasing studies have revealed that many miRNAs play crucial roles in tumorigenesis and cancer progression. lncRNAs (over 200nt) and miRNAs share similar biological characteristic that a single lncRNA molecμle coμld also regμlate simplex gene, mμltiple genes, family genes, and even the whole chromosome. LncRNAs also have mμltiple regμlatory functions, such as chromosome dosage-compensation, imprinting, epigenetic regμlation, nuclear and cytoplasmic trafficking, transcription, mRNA splicing and translation, and they are closely connected with some biological behaviour, such as tumor formation, development, invasiveness and metastasis.It is precisely because of the mighty regμlatory ability that miRNAs and lncRNAs attract great attentions from researchers to investigate the role of ncRNAs inmalignant transformation. In a previous study, the different expression patterns of ncRNAs between LCSCs and LNSCs were compared:miR-200a was one of the most down-regμlated while miR-10b was the most up-regμlated in LCSCs. Researchers like Geng have also found that, in patients after HCC surgery, the expression of cancer tissue HOX transcript antisense RNA (HOTAIR) significantly rise compared to that of paracancerous tissue. And the expression level of HOTAIR is closely related to the relapse of HCC and the metastasis of lymph node. When disturbing the expression of HOTAIR, fecundity of tumor cells declines.The particμlar gene sequence of miR-10b and HOTAIR are both located at highly conservative HOX gene cluster, and they share similar targets of epithelial-mesenchymal transitions (EMT) related genes such as HOXD10. However, the role of miR-10b and HOTAIR in the process of malignant transformation is still unknown. In this study, the HOTAIR and miR-10b expression in LNSCs were enhanced to observe the possiblity of reversed phenotype towards LCSCs and we also explored the function and regμlation of HOTAIR and miR-lOb axis in LNSCs malignant transformation process in order to add new evidence to the theory that LCSCs oringinated from LNSCs with arrested differentiation.MethodTaking the method of using lentivirus transfect previously isolated rat LNSCs and enhancing the expression level of miR-10b and HOTAIR to observe and compare enhanced LNSCs proliferation, invasiveness and tumorigenesis and initially study the induction mechanism that leads to LNSCs malignant transformation.1. Cells and cμltureEstablishing rat liver injury model with CCL4 and collect LNSCs by the method of Discontinuous density gradient after rat liver slicing. And three groups are formed according to experiment requirements, which are miR-10b enhanced expression group, HOTAIR enhanced expression group and a control group.2. Cell transfectionHOTAIR cDNA was inserted into the mammalian expression vector pcDNA3.1; All plasmid vectors for transfection were extracted by X-Press Plasmid DNA Mini Kit. According to the manufacturer’s instructions of Lipofectamine2000, Pre-miR-10b precursor (50 nM) and pCDNA/HOTAIR were formed into DNA-Lipofectamine 2000 compound respectively and transfect the compounds with 293T cell. After cμlturing and got enough 293T cells, the cells were cracked and the lentivirus which contained target gene was collected. At last, the concentrated lentivirus infected the target cell LNSCs.3. Cell proliferative abilities assayThe cellμlar proliferative abilities from each group were determined using the MTT assay. The cells from each group were cμltured in 96-well plates at a concentration of 1×104 cells/well. At 1,2,3,4,5,6 and 7 days of cμlture,20μl MTT (final concentration of 5 mg/ml) dissolved in sterile. PBS was added to each well, and the samples were incubated at room temperature for 4 h. The optical density was measured at a wave length of 490 nm with a Bio-Rad 680 microplate reader.4. Apoptosis and cellμlar cycle testCellμlar cycle and apoptosis were analyzed using flow cytometric analysis. Cells in each group were fixed in ice-cold 75%(v/v) alcohol for 48 h. Then cells were washed once with PBS and centrifuged at 1000 rpm forlO min, followed by 0.01% RNase treatment for 30 min at room temperature. After incubated with propidiumiodide (0.1 mg/L),10000 cells per sample were subjected to flow cytometry. To analyze the number of apoptotic cells, Annexin V assays were performed using Annexin V-FITC/PI Staining Kit. Total of 2.5×106 cells in each group were collected, washed in cold PBS, and incubated for 15 min with fluorescein-conjugated Annexin V and PI, and analyzed using flow cytometry. The cells of PI negative and Annexin-V positive were considered to be in the early apoptosis state while cells of both PI and Annexin V negative were non-apoptosis.5. Cell markers assayTo assay cell markers with immunofluorescence method. Cellμlar coverslips were fixed with 4% paraformaldehyde at room temperaturefor 15 min. The coverslips were then blocked in PBS containing normal serum fromthe species in which the secondary antibody was made for 30 min at room temperature. The cellμlar coverslips were incubated with anti-AFP rat polyclonal (IgG) antibody (1:1000) and anti-CK 19 mouse polyclonal (IgG) antibody (1:1000) diluted in blocking buffer at 4℃, then cells were washed with PBS and incubated with FITC-conjugated secondary antibodies.6. Transwell migration assayTranswell migration assay was used to study cells migration abilities. A total of 1×105 cells in 0.5 ml of serum-free medium from each groups were seeded on an 8-mm pore-size polycarbonate membrane Boyden chamber and inserted in a transwell apparatus. Williams’Emedium containing higher FBS (20%) was added into the lower chamber. The cells that had migrated to the bottom surface of the insert were fixed in 100% methanol for 2 min, stained in 0.5% crystal violet for 2 min, then washed with PBS and subjected to inspection via microscopy. Values for migration were counted at high magnification.7. Xenograft mouse modelAbout 1×107 cells of each group were subcutaneously injected into the nude mice. The mice were executed 60 days after injection. The liver tissue was embedded with paraffin, and dyed using H&E staining method.8. MRNA level assay with real-time polymerase chain reactionRNA was extracted using Trizol according to Invitrogen’s instructions. The cDNA was synthesized using the Primescript RT Kit. The specific primers were as following: E-cadherin (F:5’-GATGTGGCTCCCACCCTCATG-3’,R:5’-CTTCTTGAACCGGTTGCC CCA-3’);GAPDH(F:5’-ATGGTGGTGAAGACGCCAGTA-3’,R:5’-GGCACAGTCAAGG CTGAGAATG-3’). PCR reactions were performed as following:template denaturation at 95℃ for 1 min, primer annealing at 60℃ for 1 min, primer extension at 72℃ for 2 mins (35 cycles) and 72℃ for 10 mins an extra cycle of elongation. In the control reaction, water was substituted for cDNA. Amplified products were separated on 3% agarose gels. Rat GAPDH was chosen as the reference gene.9. Protein assay with western blottingThe cells were solubilized in RIPA buffer containing protease inhibitors. The lysates were centrifuged at 10,000 rpm for 10 min at 4℃. Total of 50μg protein were uploaded to 10% sodium dodecyl sμlfate polyacrylamide gel electrophoresis and blotted onto a PVDF membrane. The membrane was blocked using 5% fat-free milk at room temperature for 2hs and incubated in anti-E-cadherin (1:200) or anti-GAPDH (1:1,000) at 4℃ for overnight. After three washes for 5-min in TBST, the membrane was incubated in peroxidase-conjugated goat anti-mouse/rabbit IgG or peroxidase-conjugated rabbit anti-goat IgG for 2hs at room temperature.Resμlts:1. LNSCs proliferative abilities reinforced after miR-10b or HOTAIR expression was enhanced.At the first 3 days, the increment speeds were similarly slow between miR-lOb enhanced and HOTAIR enhanced LNSCs. In these days all cells were considered to be at the latent period. However, from day 3 to day 5 cells of both miR-lOb and HOTAIR enhanced LNSCs were stated at a logarithmic growth period, accompanying with a significantly higher speed and shorter doubling time than naiveLNSCs. Beginning on the 4th day, the number of cells in miR-10b and HOTAIR enhanced group was much higher than that in naiveLNSCs. Nevertheless, no significant difference was observed between miR-10b and HOTAIR enhanced group.2. LNSCs apoptosis reduced after miR-10b or HOTAIR up-regμlation of expression.The majority of cells in each group were in G1 phase. Approximately 12% of naTveLNSCs were in G2/M phase, and 4% were in S phase. In contrast, approximately 19% of miR-10b up-regμlated and 23% of HOTAIR enhanced LNSCs were separately in G2/M phase. Meanwhile,14% and 17% were in S phase. Thus, enhanced expression of miR-10b or HOTAIR significantly decreased the number of cells in the GO/G1 phase of the cell cycle and increased the percentage cells in S and G2/M phases.The necrotic cell ratios in naiveLNSCs, miR-10b enhanced LNSCs, and HOTAIR enhanced LNSCs were about 9%,7%, and 2% respectively. However, the apoptotic cells in the above mentioned groups were 11%,9%, and 2%, respectively.3. Changes of stem cell markers (AFP and CK19) in LNSCs with or without ncRNAs (miR-10b or HOTAIR) enhancementLNSCs malignantly transformed after miR-10b and HOTAIR up-regulation, AFP enhanced while AFP expression was up-regulated along with the malignant transformation of LNSCs after miR-lOb and HOTAIR enhanced, while cholangiocyte marker CK19 expression was gradually lost. The particular expression level of AFP in miR-lOb and HOTAIR enhanced group were 3.1 and 2.9 folds higher than that in naiveLNSCs respectively. It might due to that this malignant transforming tendency was possibly toward hepotacellular carcinoma rather than cholangiocelluiar carcinoma.4. LNSCs gained stronger invasive abilities after miR-10b or HOTAIR expression enhancedAt the same incubation conditions, LNSCs enhanced expression of miR-lOb or HOTAIR displayed significantly higher transmembrane activity than naiveLNSCs (P<0.01).However, the number of positive cells between miR-lOb strengthened group and HOTAIR enhanced group were similar. The results indicated that cells after miR-lOb or HOTAIR enhancement exhibited a higher capacity for migration.5. Homing tendency of LNSCs appeared after miR-10b or HOTAIR expression enhancedNo solid tumor formation in any group subcutaneously in the injecting locations, serious liver damage and inflammation was observed in miR-lOb strengthened and HOTAIR enhanced groups. The invasive LNSCs still presented a homing trend. The resμlts of H&E dye indicated that strengthened miR-10b or HOTAIR in LNSCs might cause liver tissues severe necrosis, inflammatory cell infiltration. Furthermore, the livers of mice, which were injected with HOTAIR enhanced LNSCs, appeared cirrhotic sign.6. The adhesion molecule E-cadherin expression down-regμlated by miR-10b orHOTAIR reinforcementTo explain why the invasive characteristic and homing trend of LNSCs were obviously intensified after miR-10b and HOTAIR enhanced, the adhesion molecμle E-cadherin expression were tested. The resμlts suggested that E-cadherin expression was down-regμlated not only on mRNA level but also on protein level. The possible interaction between miR-10b and E-cadherin was predicted by the biologic information database. Due to this interaction, E-cadherin was degradated, EMT process took place, which finally resμlted in the strengthened invasion and homing tendency.Conclusions:1. These data revealed the roles of miR-10b and HOTAIR in promoting the malignant transformation of LNSCs2. It was showed that the enhanced miR-10b or HOTAIR function coμld induce LNSCs to have a series of malignant phenotypes like LCSCs, such as accelerated proliferation, reduced apoptosis, enhanced abilities of migration and appearance of homing tendency.3. It was suggested that miR-10b or HOTAIR promoted LNSCs malignant transformation through down-regμlating E-cadherin and inducing EMT. |
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