The Effection Of RNA Interference Against Connexin43 On Cultured Human RPE Cells And Its Proteome Analysis

Background and aim It is certified that connexin43 is the primary connexin expressed by RPE. Cx43 participates in the formation of gap junctiongap junction, so that it promotes information exchange among cells, and contributes to the proliferation , differentiation and migration of cells. Normal structure and function of RPE play an important role in sustaining the integrity and physiological function of RPE layer, the abnomality of which is related to many eye diseases. Here we investigate the effect of cx43 on RPE by RNA interference and its proteome analysis, to provide probable path to cure eye disease.Method 1,Primary cultured human RPE cells were identified by ck, S-100 and GFAP through immunocytochemistry method, and the rate of live/dead cells was evaluated by acridine orange/ propidium iodide staining. After the cell growth curves was recorded, RPE cells of the 3-5th passages were utilized. 2,Three different siRNA (siRNAl,siRNA2,siRNA3) targeting against human cx43 gene and one negative control siRNA were designed and transfected into cultured human RPE cells via liposome reagent. The most effective siRNA can be determined by semi-quantitative reverse transcription PCR(RT-PCR. 3,To the most effective siRNA, after transfected into human RPEs with different concentration, the cellular proliferate activities were messured by MTT colorimetry ; the percentages of RPE in different cell circle phase was assayed by FCM; the changes of phenotypical properities were observed with SCM; the protein expression of cx43 was studied through immunocytochemistry stain and Weston blot; the communication intercellular was calculated with FRAP; and the ability of recovery was assessed by using an in vitro wound healing model.4,The total proteins of siRNA1 and RPE were seperated by two-dimensional gel electrophoresis and visualized by silver staining. Proteins with significant expression alterations were selected and their peptide mass fingerprints (PMFs ) were obtained by matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS).The PMFs were used to search NCBInr database by Auto MS-Fit software. The proteins were identity and analysis its biological significenceResults 1,The just digested hRPE cells were polygon , contained large quantity of pigments and easy to passage, but those pigments in the RPE cells decreased along with the cell division. The percentage of cells, with positive of anti-CK,s-100 and negative of anti-GFAP,is up to 94%.according to this, the cultured cells can be identified as highly purified human retinal pigment epithelium cells. The rate of live/dead cell was about 96% through AO/PI staining, the cell grow state in good condition, the cells go into exponential phase of growth after passaged 3-4 days. the cells would be passaged every 6 days. 2,Three pairs of siRNAs were reduced the synthetic of cx43 mRNA in different levels, and the most effective siRNA was siRNA1.the inhibited rate was up to 72% 3,After siRNA1 transfected into human RPE cells, the cellular proliferate activities enhanced significantly in an concentration-dependent manner until it achieved saturate at 1.25ug/ml, and the maximal growth rate was 46%. the cells were passaged every 5 days; the percentate of cells in S phase was increased obviously(p<0.05); the changes of cell phenotype was observed through SCM: the cell volume enlarged and tentacles lie on the surface of cell increased visibly; the protein expression of cx43 was decreased obviously by Immunocytochemistry stain and Weston blot. Cultured RPE cells expressed cx43 in cellular membrane, cytoplasm and nucleus. 24 hours after transfection the protein expression of cx43 was depressed and 48 hours the depression rate was about 76% through image analysis, which was coincidence with the results of Weston blot, its maximal inhibited rate was 62% at 48 hours ; the ability of communication intercellular and recovery form injury were decreased significantly after cx43 gene was interfered. 4,By silver staining, there are 861 protein spots in normal RPE group and 887 protein spots were showed in siRNA1 group. The average matching rate in RPE group and siRNA1 group were 92% and 93% separately. Compared with two groups There were 78 different spots which's volume value changed≥2.0 times. Among them, 16 protein spots increased in abundance and 18 spots showed lower expression. There were 19 protein spots showed in RPE but not in siRNA1 group, and 25 spots showed in siRNA1 but not in RPE group. 10 protein spots were chosen randomly to be performed mass spectrometry analysis, and 7 of them were identified successfully: SP-H antigen, p27BBP protein, voltage-dependent anion channel, mitogen activated protein kinase binding protein 1, heat shock 70kDa protein 8 isoform 1 and inosine monophosphate dehydrogenase 2 were up regulated, while ACTB protein was down regulated.Conclusion RNA interference is a highly specific effective tool for gene research work. Connexin 43 is not only effect the gap junction-mediated intercellular communication but also very important for some cell behaviors: such as cell growth, cell circle and recovery ability from injury. The abnormal of connexin43 can cause many proteins ' disorder through proteome analysis. Connexin43 may play an important role in many ocular diseases...