Study On The Effects Of SMYD3 Silencing By MicroRNA On Biological Behavior And The Mechanism Of SMYD3 Regulating C-Myc In MCF-7 Cells

Objectives 1. To construct eukaryotic expression plasmids of microRNA targeting SMYD3(SET and MYND domaineontaining protein3)gene, and stably transfecting MCF-7 cells to detect their gene-silencing effects. 2. To investigate the effects of SMYD3 silence on the biological characteristics of human breast cancer MCF-7 cells. 3. To study on the effects and possible mechanism of SMYD3 silence on WNT10B and c-Myc gene expressions in human breast cancer MCF-7 cells.Methods 1. Four eukaryotic expression plasmids targeting SMYD3 and one negative control plasmid of microRNA were constructed and stably transfected into MCF-7 cells. Meanwhile, a blank control group was established. The transfection efficiency of each group was observed under a fluorescence microscopy. The inhibitory effects of SMYD3 were identified by RT-PCR and Western Blot analysis after blasticidin screening. 2. The cellular proliferation and apoptosis activities of MCF-7 cells were analyzed respectively by MTT assay. The plate clone formation experiment and Matrigel invasion assay were used to detect the proliferation and invasiveness activities of the MCF-7 cells in vitro. 3. SMYD3, WNT10B and c-Myc mRNA expression changes was assayed by RT-PCR.Results 1. DNA sequencing results indicated all of the microRNA sequences that inserted into pcDNA6.2-GW/EmGFPmiR eukaryotic expression vectors completely meet the design. The results from RT-PCR and Western Blot showed an inhibition of expression of SMYD3 at different levels in the four interfered groups and two interfered groups (pcDNA-SMYD3mi-3 and pcDNA-SMYD3mi-4) of highly inhibitory effects were selected. 2. Results from MTT assay showed the cellular proliferation activities of pcDNA-SMYD3mi-3 and pcDNA-SMYD3mi-4 were markedly decreased compared with that of the control group(P<0.05). The plate clone formation rates of the two interference groups (15.1%) (18.7%) were obviously decreased in comparison with the control group (65.4%) (p<0.05), and there was no markedly difference between that of the negative control group (63.2%) and the blank control group (p>0.05). The results of FCM demonstrated early apoptosis rates of pcDNA-SMYD3mi-3 (19.3±1.61)% and pcDNA-SMYD3mi-4 (17.6±0.45)%, respectively were significantly different to that of control group (P<0.05). The matrigel in vitro invasion assay revealed that the mean cell invasion percentages of the two interference groups (8.4±1.2) (7.6±1.4) were significantly lower than that of the blank control group (53.4±3.2) and the negative control group (46.8±4.6) (P<0.05). 3. The relative mRNA expressions of the two interference groups separately: WNT10B (0.012±0.001), (0.013±0.001) and c-Myc (0.054±0.005), (0.055±0.002), were all respectively down-regulated compared with the negative control group and the blank control group (P<0.05).Conclusions 1. The eukaryotic expression plasmids of microRNA we constructed could respectively inhibit the expression of SMYD3 in the breast cancer MCF-7 cells which suggestes the plasmids for a further application. 2. SMYD3 silence does inhibit the proliferation, promote the apoptosis and suppress the invasion of the breast cancer MCF-7 cells. 3. It may be due to its upgrading WNT10B and c-Myc expressions that SMYD3 causes proliferation promoting and apoptosis inhibiting of the breast cancer MCF-7 cells. It may be related to the over-expression of SMYD3 activated its downstream gene WNT10B expression to an abnormal level and then made epithelial-mesenchymal transition happened that the over-expression of SMYD3 can markedly upgrade the invasion and cause the transfer of the breast cancer MCF-7 cells.