Nuclear Proteome Study And MicroRNA Analysis In Response To Cisplatin-induced DNA Damage

BackgroudAs a classic clinical anti-cancer drug, cisplatin has been used for over 30 years in the treatment of various cancers including ovary cancer, testis cancer, bladder cancer, head-and-neck cancer, cervical cancer, and lung cancer. Cisplatin targets DNA and induces crosslinks. Due to the altered structure of DNA, replication cannot proceed, and eventually cell death is triggered. This DNA-Pt adduct is regarded as the main cause of the cytotoxicity of cisplatin.Although cisplatin is still widely used, drug-resistance and side-effect have also been noticed for its clinical application. Through detailed mechanistic study, second generation, such as Oxaliplatin, and third generation drugs, such as Satraplatin, are designed in an effort to replace cisplatin. This suggests that only by better understand the underlying mechanisms for the toxic effects of cisplatin, as well as the mechanisms for drug-resistance, new strategies could be developed for drug discovery. Currently, although there have been a lot studies investigating the effects of cisplatin, many questions remain unanswered. Systems biology approaches, as high-throughput technology, can reveal certain unknown or unexpected associations. Therefore, in the present study, we first applied a proteomic approach to analyze the cellular response to cisplatin.Considering the main target of cisplatin is DNA, and repair also occurs in the nuceleus, we were interested to examine the changes of nuclear proteome upon cisplatin treatment. Thus, nuclear proteins were extracted and analyzed by 2-Dimensional poly Acrylmide Gel Electrophoresis (2D-PAGE), and proteins with changed expression were further identified by mass spcetrometry (MS).mciroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression in eukaryotes. Over 60% of mammal RNAs can be regulated by miRNAs. It has been shown that the oncogen p53 can regulate certain miRNAs, while itself can also be regulated by other miRNAs. Becuase it is well known that p53 is associated with cellular response to DNA damage, it is possible that miRNAs might also be involved in cisplatin-induced DNA damage response. Therefore, in the second part of this study, we utilized another high-throughput technology—microRNA chips, to investigate the possible roles of miRNAs in cisplatin-induced DNA damage response.Part I.Nuclear proteome study for cisplatin-induced DNA damage response GoalUsing 2D-PAGE to search for those nuclear proteins with changed expression after cisplatin treatment, in order to identify those involved in DNA damge response.Methods1. Measurement of cell survival and DNA damage: Trypan blue exclusion, MTT and immunofluorescent microscopy2. Nuclear protein extraction, ultrafiltraton and concentrate of extracted proteins, determine protein concentration by BCA and Bradford method3.2D-PAGE combined with MS identification4. Verification by Western blot and real-time PCR; RNAi for fuctional study; and flow cytometry to evaluate apoptosis and DNA damageResults1. With longer exposure time and higher concentration, the cytotoxicity of cisplatin increased, as well as the formation of yH2AX foci, indicator for DNA double strand breaks.2. Cisplatin treatment (5?M) induced cell cycle arrest. After 24 h, cells arrested in S phase increased from 35% in control to 86% in treatment group; while cells in G1 phase decreased from 54% to 2%. For cells in G2 phase, there was only a moderate decrease from 19% to 12%. However, apoptotic cells increased from 5.7% in control to 17.5% after cisplatin treatment.3. After cisplatin treatment, there were about 98 protein spots showed significant changes at different time points by 2D-PAGE analysis. 28 proteins were identified by MS. Among them, 6 were common to all different treatment groups. Of the rest 22 proteins, 12 were upregulated, and 10 were downregulated by cisplatin treatment.4. Three identified proteins, namely Annexin A1 (ANXA1), Lamin B1 and Hsp27, were verified by quantitative PCR, Western blot, and immunofluorescent microscopy.5. Functional study was further conducted for ANXA1. First the effect on yH2AX foci formation was observed by blocking ANXA1 expression using RNA interference (RNAi). It was found that after 5?M cisplatin treatment, the fluorescent intensity was significantly increased in RNAi group compared to control. Similar results were also obtained by using Western blot and flow cytometry.6. It was shown that apoptotic cell ratio already increased from 23.7% in control to 37.9% by blocking ANXA1 expression only. Further cisplatin exposure increased apoptotic cell ratio in both groups, but no significant difference were observed.Conclusion:1. There exist time- and dose-dependent effects for the cytotoxicity and yH2AX foci formation for cisplatin.2.28 nuclear proteins were identified, among which are involved in biogenesis of microRNA, the regulation of transcription, transport, splicing, cell cycle and division, chorosome separation, cell stress response, and apoptosis.3. After Annexin A1 knockdown, apoptosis increased significantly, and DNA damage induced by cisplatin was enhanced. Part?.microRNAs analysis in response to cisplatin-induced DNA damage GoalTo obtain the miRNA expression profiles after cisplatin-induced DNA damage, in order to understand the effects and underlying mechanisms for cisplatin from the post-transcription regulation level.Methods Total RNA (including miRNAs) were extracted, purity was analyzed using Bionanalyzer, and concentration was determined by Nanodrop. After miRNA chip analysis, selected miRNAs were subjected to real time PCR verificaiton. One of the selected miRNAs, miR-191, was further studied for its relationship with DNA damage using different methods, including transfection, immunofluorescent microscopy, flow cytometry, and Western blot.Results1. miRNAs chip analysis identified 40 miRNAs showed significant changes after cisplatin treatment. 13 of them were verified by real time PCR.2. One of the identified miRNAs, miR-191, was furter evaluated. It was found that cells transfected with miR-191mimic showed decreased cell survival after cisplatin treatment, indicating that miR-191 rendered cells more sensitive to cisplatin.3. By transfecting cells with miR-191 mimic alone induced more yH2AX foci formation compared to control. Cisplatin treatment further increased yH2AX foci formation in transfected cells. Detailed analysis showed that the average fluorescent intensity was significantly increased in miR-191 mimic transfected cells after cisplatin treatment.4. Flow cytometry analysis revealed that cisplatin increased the yH2AX-positive cells from 10.5% in control to 25.1% in miR-191mimic transfected cells, a 3-fold increase. Similar trend was also obtained by Western blot. Conclusion1. A group of miRNAs that showed changed expression by cisplatin treatment were identified.2. A relationship between cisplatin-induced DNA damage and miR-191 was established.