| Part one:The effect and expression of miR-184 in RB cellsObjective:Detection of miR-184 expression in the normal retina and RB tissues.Then,to investigate the influence of miR-184 to RB proliferation,apoptosis,cell cycle,invasion and migration by using miR-184 inhibitor and mimic.Methods:Trizol method was used to extract total RNA from retinal tissues of RB patients and normal healthy donors and RB cell lines(Y79 cells and WERI-RB-1),expression difference of miR-184 was validated with real-time quantitative polymerase chain reaction(qRT-PCR)technique.Good condition of logarithmic phase WERI-RB-1 cell was divided into six groups:normal control group,blank control group,miR-184 mimic liposome NC group,miR-184 mimic,miR-184 inhibitor NC group and miR-184 inhibitor group.The effects of miR-184 mimic and inhibitor on the expression of miR-184 was validated by qRT-PCR.The effects of miR-184 on RB cell proliferation,apoptosis,cell cycle,migration and invasion were detected by CCK-8 method,Annexin V and PI double staining and flow cytometry,and transwell assay,respectively.Results:Compared with normal tissues,expression of miR-184 was significantly downregulated in RB tissues and cell lines.miR-184 mimic and inhibitor signifcantly upregulated and downregulated miR-184 expression in WERI-RB-1 cells,respectively.miR-184 mainly retarted WERI-RB-1 cells in G0/G1 phase,percentage of G0/G1 phase of RB cells treated with miR-184 mimic was significantly increased and S phase cells was significantly decreased compared with other groups,and miR-184 inhibitor showed a contrary effect with mimic,G2/M phase was not influenced either treated with miR-184 mimic or inhibitor.miR-184 mainly induced early apoptosis of WERI-RB-1 cells and showed no obvious effects on late apoptosis and necrosis.The positive rate was 17.26%in miR-184 mimic group,significantly higher than the blank control group(6.50%),empty liposomes group(7.37%),miR-184 mimics NC control group(7.37%),miR184 inhibitor NC control group(6.62%),and significantly decreased in miR-184 inhibitor transfection group(3.40%)(p<0.05).Transwell assay also confirmed that miR-184 significantly inhibited migration and invasion of WERI-RB-1 cells.Conclusion:miR-184 significantly decreased in RB cells and tissues.Molecular study further confirmed that miR-184 significantly inhibited proliferation,cell cycle progression,migration,invasion,and promoted early apopotisis of WERI-RB-1 cells.Thus,miR-184 plays a suppressive role and may provide a therapeutic agent in RB.Part two:miR-184 target SLC7A5 to regulate biological behaviors of retinoblastoma cellsObjective:To explore the molecular mechanisms of miR-184 in regulating biological behaviors of RB cells,and confirm the role of SLC7A5 in miR-184-mediated suppressive effects.Methods:miRNA Database and Wayne cross analysis to serch the potential gene tagets of miR-184.By using qRT-PCR and western blotting method to detect the effects of miR-184 on potential genes expression,and then confirm the most obvious potential gene.WERI-RB-1 cells were divided into six groups:blank control,liposome NC,miR-184 mimic NC,miR-184 mimic,miR-184 inhibitor NC,and miR-184 inhibitor groups.Construct wildtype pMIR-REPORT-SLC7A5 3’UTR and miR-184 targeted sites(mutl,mut2,mut 3)mutated luciferase plasmid,then useing Luciferase activity assay to confirm the target site of miR-184 on SLC7A5,difference among groups of was compared using single factor analysis of variance(ANOVA).miR-184 and SLC7A5 overexpression plasmid(pcMV6-SLC7A5)were cotransfected into WERI-RB-1 cells,and divided into four groups:miR-184 mimic NC,miR-184 mimic,miR-184 mimic + pcMV6,miR-184 mimic + pcMV6-SLC7A5.CCK8 method and flow cytometry were used to detect whether miR-184 depended on targeting SLC7A5 to suppress WERI-RB-1 cell proliferation and apoptosis,transwell assay was used to evaluate migration and invasion of WERI-RB-1 cells.Results:By using miRNA databse and Wayne cross analysis,we slected two potential targets of miR-184,namely SLC7A5 and TNP02.miR-184 inhibited mRNA expression of both SLC7A5 and TNP02,but more obvious for SLC7A5(p<0.05).Luciferase activity assay confirmed that miR184 mainly targeted position 2494-2513 of 3’UTR(sitel)to inhibit SLC7A5 expression.Cell proliferation analysis showed that the inhibitive rate in miR-184 mimic NC is 2.1%,miR-184 mimic is 16.2%,miR-184 mimic + pcMV6 is 14.8%,miR184 mimic + pcMV6-SLC7A5 is 2.4%,overall,SLC7A5 overexpression significantly rescued the inhibitive effect of miR-184 on RB cell proliferation.Apoptosis analysis showed that the early apoptosis rate in miR-184 mimic NC is 7.8%,miR-184 mimic is 17.3%,miR-184 mimic +pcMV6 is 16.9%,miR-184 mimic + pcMV6-SLC7A5 is 8.2%,overall,SLC7A5 overexpression significantly rescued the promotive effect of miR-184 on RB cell apoptosis.In transwell assay,overexpression of SLC7A5 significantly rescued the suppressive effects of miR-184 on migration and invasion of RB cells.Compared with miR-184 mimic NC group,the migrative rate in miR-184 mimic group is 57.9%and 27.3%,61.8%and 33.3%in miR-184 mimic + pcMV6 group,95.6%and 93.1%in miR-184 mimic + pcMV6-SLC7A5.Conclusion:miR-184 targetes SLC7A5 3’UTR 2494-2513 position to inhibit SLC7A5 expression.As an oncogene,SLC7A5 rescues the suppressive effects of miR-184,and exhibits promotive effects on proliferation,migration,invasion,and inhibitory effect on apoptosis of RB cells.Thus,miR-184 mainly depends on targeting SLC7A5 to inhibit RB progression.Part three:Analysis of PAX6 gene mutation and clinical characteristics in a Chinese Tujia family affected with congenitalaniridiaObjective:To observe the clinical characteristics and identify potential mutation of the PAX6 gene responsible for congenital aniridia in a Chinese Tujia family in central China.Methods:A detailed family history and ophthalmologic examinations data were collected.Genomic DNA was extracted from peripheral blood of seven family members and 100 healthy individuals(50 Tujia nationality,50 Han nationality).The coding regions and flanking sequence of the PAX6 gene of the propositus were amplified by PCR and subjected to DNA sequencing.The genetic sequence of the mutant site of the unaffected family members and control group’s individuals were verified.Results:The clinical characteristics of the affected family members were iris hypoplasia accompanied with orwithout cataract,nystagmus,the foveal dysplasia and keratopathy.cataract and keratopathy were aggravated with age.A heterozygous mutation(c.357+1G>T)was identified in the junction of exon 3 and intron 3 in four patients but not in unaffected family members and 100 healthy individuals.Conclusion:The manifestations were almost consistent in the family,but the degree of iris defect was different.357+1G>A heterozygous mutation of PAX6 had been found to underlie the aniridia,which showed an autosomal dominant inheritance pattern in this western Chinese Tujia family. |
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