The Mechanism Of Mir-145 In The Proliferation And Apoptosis Of Retinoblastoma Cell

Retinoblastoma,which incidence is 1 in 20000 in the world, is the most common intraocular malignancy in children and one of the very few life-threatening ophthalmic conditions. There are approximately 1000 new cases occurring each year in our country, accounting for 20% of the world (Danian Chen,2007) Children with retinoblastoma are diagnosed before 3 years of age. The diagnosis of retinoblastoma in older is extremely rare. Patients present with the pupil color change and strabismus. The cause of tumor is the imbalance between cell proliferation, differentiation and apoptosis. miRNA is an abundant class of small, roughly 20-24 nucleotide (nt)-long, noncoding RNA,and widely expressed in tissues and cells. Studies have showed that miRNA was associated with tumor occurrence and development. This study used liposome-mediated gene transfer technology, examined the effect of miRNA on retinoblastoma cell proliferation and apoptosis, and provided experimental evidence for the new treatment of retinoblastoma tumor. Expression of IGF-1R on retinoblastoma was estimated by immunohistochemistry,and cultured human retinoblastoma cells Y79 cells.We explored the possibility of a new target treatment of IGF-1R on retinoblastoma. Furthermore,we studied regulation mechanism of miR-145 and IGF-1R on the human retinoblastoma cells. Our study included the following three parts:Part 1. Influence of MicroRNA 145 on the proliferation and apoptosis in retinoblastoma Y79 cellsObject:To observe the expression of MicroRNA 145 (miR-145) in human retinoblastoma Y79 cells and explore the effect of miR-145 on the proliferation and apoptosis of Y79 cells.Methods:Human retinoblastoma cells, Y79 cells were divided into four groups: miR-145 intervention group,control miRNA intervention group,empty liposome group and blank control group. miR-145 was transfected into Y79 cells by lipofection. The expression of miR-145 was detected at 48h after transfection by RT-PCR..Cell proliferation inhibition was measured by Cell Counting Kit-8 (CCK-8) assay. Flow cytometery was used to examine cell cycles.The apoptosis was detected by Annexin/PI double immunofluorescence and flow cytometer. One-way ANOVA was used to analyze the overall data of every group.Results:The Y79 cells were transfected by miR-145 successfully with lipofection. RT-PCR analysis showed that the expression of miR-145 in the miR-145 intervention group was significantly increased as compared to the control miRNA intervetion group,empty liposome group and blank control group(79.06±3.45 vs 1.06±0.03,0.93 ±0.02 and 1.00±0.02, P<0.05).The proliferation inhibition rate in the miR-145 intervention group was highest in in the miR-145 intervention group,Compared to the the control miRNA intervetion group(2.57%),empty liposome group (3.97%). Cell cycle was depressed in G1 cycle. The results of immunofluorescence and flow cytometer showed that ratioes of Annexin or Annexin/PI double positive were highest in those in the control miRNA intervention group, empty liposome group and blank control group, the difference was significant(P<0.05).Conclusion:miR-145 can inhibited the proliferation and induced the apoptosis in Y79 cells.Part 2. Expression and founction of IGF-1R in retinoblastoma Y79 cellsObject:To observe the expression of IGF-1R in human retinoblastoma Y79 cells and explore the effect of IGF-1R on the proliferation and apoptosis of human retinoblastoma Y79 cells.Methods:Cultured human retinoblastoma cells Y79 cells were divided into three groups:IGF-1R siRNA intervention group, negative siRNA group, and blank control group. IGF-1R siRNA was transfected into Y79 cells by lipofection. The mRNA level of IGF-1R was detected at 24h after transfection by RT-PCR.. The level of IGF-1R protein was tested by Western blotting. Proliferation inhibition was measured by Cell Counting Kit-8 (CCK-8) assay. Flow cytometery was used to examine cell cycles. The apoptosis was detected by Annexin/PI double immunofluorescence and flow cytometer. One-way ANOVA was used to analyze the overall data of every group.Results:Immunohistochemistry confirmed the expression of IGF-1R in human retinoblastoma tissue. IGF-1R siRNA specially deter the expression of IGF-1R in Y79 cells. RT-PCR analysis showed that the expression of IGF-1R mRNA in the siRNA group(0.426) was significantly increased as compared to the scrambled siRNA group (1.000) and blank control group(P<0.01). Western blotting demonstrated the level of IGF-1R protein of siRNA group was 22.4% of scrambled siRNA group and 24.0% of blank control group. The result of the proliferation inhibition test demonstrated that the A value(OD) in the siRNA group (0.30) was lower than scrambled siRNA group(0.40) and blank control group(0.42), the difference was significant(P<0.05).The results of immunofluorescence and flow cytometer showed that ratioes of Annexin were higher in the siRNA group(28.9%) than the scrambled siRNA group(6.4%)and blank control group (6.6%). The difference was significant(P<0.01).Conclusion:IGF-1R were expression in human retinoblastoma cells, and specially deterring the expression of IGF-1R inhibited the proliferation of Y79 cells.Part3. miR-145 Regulates Proliferation and Apoptosis of Human Retinoblastoma Y79 cells via Down regulation of IGF-1R ExpressionObject:To explore the mechanism of IGF-1R on the proliferation and apoptosis of human retinoblastoma Y79 cells.Methods:Cultured human retinoblastoma cells Y79 cells were divided into three groups:miR-145 mimics group, negative miR group, and blank control group. The mRNA level of IGF-1R was detected at 24h after transfection by RT-PCR. The level of IGF-1R protein was tested by Western blotting. A luciferase reporter plasmids pMIR-REPORT-IGF-1R 3'-UTR was constructed.luciferase activity measurement test the interaction between miR-145 and IGF-1R. One-way ANOVA was used to analyze the overall data of every group.Results:RT-PCR analysis showed that the expression of IGF-1R mRNA in the miR-145 mimics group(0.498) was significantly increased as compared to the negative miR group(0.951) and blank control group (1.000) (P0.05).Western blotting demonstrated the reduced level of IGF-1R protein of miR-145 mimics group(P<0.01),which was 37.5% of negative miR group and 36.9% of blank control group.The results of luciferase activity measurement displayed reduced activity of miR-145mimics group (P<0.01), demonstrate interaction between miR-145 and IGF-1R.Conclusion:miR-145 can restrain proliferation of human retinoblastoma via downregulation of IGF-1R expression...