| Objective To construct a retroviral vector for microRNA-21 (miR-21) up-regulation. MHCC-97H was transformed with retroviral vector to establish a cell line stably expressing miR-21. To uncover its function and mechanism of miR-21 with liver cancer stem cells (LCSCs) and determine its effect on clone formation, invasion, migration and nude mouse transplantation tumor.Methods 1. We design the following primers, which are complementary to the nucleotide sequences that flanked the miR-21 gene. The 589 bp of flanking sequence was amplified from liver cell genomic. DNA using by polymerase chain reaction (PCR), and cloned into the BamH1 and Sal1 sites of the pBABE-puro lentiviral vector, and named as pBABE-puro-pre-miR-21. PIK, and negative control (pBABE-puro-negative control) or pBABE-puro-pre-miR-21 were transfected HEK 293T cells When the cells were incubated for 48 hours, the medium containing the lentiviral particles were harvested.2.MHCC-97H cell was into 6-well plates. The cell was setted as follows:(a) negative control (NC) group, in which the cells were subjected to transfection with retroviral particles without miR-21; (b) pre-miR-21 group. the cells were grown in DMEM medium containing puromycin and without antibiotics for 2 months. The level of miR-21 was analyzed by Quantitative Real-Time Reverse Transcription-PCR (RT-PCR) between the two groups.3. The level of LCSCs markers such as OCT4. CD13、Ep-CAM and CD90 were analyzed by Quantitative Real-Time Reverse Transcription-PCR (RT-PCR) and Western blotting(WB) between the two groups.4. Tablet clone formation test and transwell assay miR-21 effects on MHCC-97H cell migration and invasion between the pre-miR-21 group and the NC group.5. NC group and pre-miR-21 group cells were suspended and injected subcutaneously into both side of the male BALB/C nude mouse. Observe the nude mice into tumor and make tumor growth curves Tumor volumes were calculated by using the equation:volume (mm3)=(length ×width2)/2. And after 34 days the mice were killed and tumors were weighted.Result 1. The retroviral vector liquid of pBABE-puro-pre-miR-21 and pBABE-puro-NC were successfully constructed.2. RT-PCR analysis showed that miR-21 expression was 7.78±1.51-fold higher in miR-21 cultures than in NC ones (P<0.05). A cell line stably expressing miR-21 was successfully constructed.3. Expression of the LCSC markers CD 13, Ep-CAM, CD90 and OCT4 was significantly higher in the miR-21 cell line than in the NC cell line, at both the protein level (Figure 2A) and mRNA level (p<0.05)4. CFE was significantly higher in the miR-21 cell line (24.8±3.1%) than in the NC cell line (15.3±3.6%; P=0.026). Further, the relative cell number of migration was significantly higher in the miR-21 cell line (210.3±5.8) than in the NC cell line (104.8±6.5, P< 0.001) and the relative cell number of invasion through an extracellular matrix coating was significantly higher in the miR-21 cell line (107.3±2.3) than in the NC cell line (56.7±2.5, P< 0.001).5. BALB/C nude mice were inoculated with miR-21 or NC cells and tumors were allowed to grow. In both groups of mice, tumors were visible after 5 d. As time went on, miR-21 tumors grew significantly faster. Mean tumor volume at the end of the 34-d observation period was 773.62±163.46 mm3 in the miR-21 group and 502.79±33.94 mm3 in the NC group (P= 0.048), while the corresponding mean weights were 0.422±0.019 g and 0.346±0.006 g (P=0.003). Tumor pathology was verified by histopathology.Conclusion MiRNA-21 strengthens LCSC characteristics, including colony formation, invasion, and migration. This phenotypic reinforcement may result from the regulation by miR-21 of LCSC marker expression. |
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