Experimental Study Of RBAP48as A Potential Cisplatin Resistance Target For Lung Cancer

BackgroundAccording to the report about the third cause of death throughout the world published by the national ministry of health, Lung cancer has replaced liver cancer to become the first cause of death in our country (about22.7of all malignant tumor death).In the world, the morbidity and mortality of lung cancer is among the top of the various kinds of cancer. According to the statistics reported by38th clinical oncology conference of American, lung cancer killed about1.4million people worldwide each year and the number is rising year by year.Lung cancer has no obvious symptoms in early stage, especially in non-small cell lung cancer by80%of lung cancer.65-80%of patients were belong to middle-late stage when confirmed and had the focal shift in the distance and finally lost the opportunity of surgery and radiotherapy. Therefore, the effect of drug on the treatment of lung cancer is particularly important. Regretfully, at present, there is still lack of a long-lasting and effective drug for lung cancer. The molecular targeted therapeutic drugs, such as Tyrosine kinase signaling pathway inhibitors, has advantages of specific anticancer effects and less toxicity. Even then, there are not candidates for most patients because of limited indications.The combined chemotherapy based on cisplatin recommended by international cancer organization has become a line of NSCLC standard chemotherapy regimens and obtained a certain effect. A number of randomized clinical trials and meta-analyses showed that the effect of different of platinum-based is approximation and at present there is no overall curative effect which is better than that of platinum-based treatment plan. With the widely application of the platinum drugs, the tumor cells has inevitably caused resistance to platinum which make the chemotherapy effect significantly reduced. According to statistics,70-80%of the patients in early period of chemotherapy can alleviate temporarily,but the long-term using will be resistant to platimum drugs which will lead to the high recurrence rate of above60%.However,the drug resistance of the recurrent lung cancer increased significantly while the response rate of chemotherapy drugs was less than30%.It has become a difficulty for lung cancer treatment at present.Platinum is one of the most widely used chemotherapeutic drugs at present, it is the first-line treatment drug for lung cancer, carcinoma of testis, ovarian cancer, head-neck carcinoma, liver cancer, intestinal cancer, breast cancer and so on and also is used in the therapy of osteosarcoma and leukemic. The study found that the formation of platinum drug resistance to tumor cells was unconcerned to multi-drug resistance protein. Although many factors such as copper ion receptors, ATP7B glutathione, DNA repair protein has been thought to play an important role in the formation of platimum resistance, and because of this, there is no suitable target to reverse the platinum resistance of cells. However, once the cisplatin resistance to cancer cells occurred, it will form multi-drug resistance in many chemotherapeutic drugs such as adriamycin, vinblastine, fluorouracil, VP-16mitomycin and so on, so the hazard is particularly serious. Therefore, iucubrating the mechanism of platinum resistance of lung cancer, looking for the specific and platinum resistance related molecular target and developing the drug or therapy which was aimed at platinum resistance of lung cancer has not only become a problem urgently to be solved in clinical treatment of lung cancer, but also a focus of research both inland and abroad.Some research suggests that the DNA methyltransferases inhibitor and Histone deacetylase inhibitors could reverse the down regulation of chemotherapy sensitivity genes caused by DNA methylation and histone acetylation and induce its expression and at last to recover the cisplatin sensibility of cancer cell.The drugs of this type made reversing drug-resistance possible for non-small cell lung cancer treatment. So far, numerous inhibitor of DNMTs or HDACs had been developed and authorized for clinical evaluation. Since the treatment could made the DNA demethylation and histone deacetylation of genome-wide, resulting in the genome unstable and risks of tumorigenesis or growth defects.The treatment of DNA demethylation increase of risks of multidrug resistance and metastasis. Some genes are highly expressed because of DNA demethylation in malignant tumor.Therefore, it is of great significance for seeking more specific epigenetic targets to reverse cancer resistance and as a result to improve the sensitivity of chemotherapy.RBAP48belongs to polycomb group, it is the first identified in the height of the protein isolated from the collection, assembling of chromatin and nucleosome modifi ed compounds. RBAP48as the component of ATP dependent on chromosome recomb ination of complex includes nuclear chromatin remodeling and deacetylase, histone d eacetylase complex Sin3, histone acetylation enzyme complex HDAC, compounds su ch as Lin and CAF-1and adjust the function of them. RBAP48directly affected on R b protein or participate in DNA methylation, histone methylation and acetylation med iated by PRC2and other apparent genetic regulation and played an important role in c ell cycle, cell senescence, cell proliferation and differentiation, gene imprinting and a series of chromatin activities. Several studies have proved that RBAP48has a high expression in cervical cancer, thyroid carcinoma, primary liver cancer, acute myelogenous leukemia and other malignant tumor and is closely related to the development of tumor and chemotherapy sensitivity.RBAP48has been thought to be one of the effective index to judge the recurrence and prognosis of tumor. The research in earlier stage about trichostatin A in vitro has confirmed that the cisplatin resistance of lung cancer cell could be reversed after using HDACi and the expression of core subunit in polycomb group could be restrained on dose dependent, which remind that the epigenetic regulation mediated by PcG may closely associated with cisplatin resistance of lung cancer.However,it still lacks of the systemic research about the expression of RBAP48in lung cancer and its role in cisplatin resistance of cells. Objective:This study intends to lung cancer cells as the main research object,using western blot to confirm the high expression of RBAP48in lung cancer cell and analysis the relation between RBAP48and cisplatin resistance.Using A549cell and A549/DDP cell as the main research object to observe the effect of RBAP48recombinant plasmid and RBAP48gene sileneing by small interfering RNA on cell proliferation and cisplatin sensibility.To research the preliminary mechanism of the formation of cispaltin resistance in lung cancer cell which was regulated by RBAP48.It provides theoretical and experimental basis to further analyze if RBAP48can be the molecular targets to reverse lung cancer cisplatin resistance.This study was divided into three parts.Part1The expression of RBAP48in lung cancer cells and the experimental research about its correlation with cell cisplatin resistance.Objective:Compare the difference expression of RBAP48gene in hunman lung normal cell, lung cancer cell and lung cancer cell resistance to cisplatin and detect the effect of cisplatin on expression of RBAP48in lung cancer cell. Methods:The different expression of RBAP48gene in L132, NHBE,16HBE, A549and A549/DDP cell line was detected with Western blot.The sensibility to cisplatin of A549and A549/DDP was examined by MTT after they were disposed by cisplatin of gradient concentration.The expression of RBAP48in A549/DDP cell line was measured by western blot after it was conducted by cisplatin of different dose.The different expression of RBAP48in A549cell line with the different treatment was detected by western blot. Result:Expression of RBAP48protein were overexpressed by (66.67±28.87),(4.95±2.38),(6.39±2.10),(2.18±0.28) times in A549/DDP cell compared with A549, NHBE,16HBE and L132cells(P<0.01).MTT revealed that the inhibition rate of A549and A549/DDP cells are rising with the increase concentration of cisplatin.The IC50of A549and A549/DDP to cisplatin are7.20±0.87μmol/L,38.10±4.33μmol/L (P<0.01).Western blot revealed that the expression of RBAP48protein in A549/DDP cell treated by cisplatin is rising with the dose and time dependent while the expression of RBAP48in A549cell induced long-term by cisplatin of low dose is up-regulation with the increase of time and dose.Conclusion: RBAP48was high expression in lung cancer cell and its expression was related to the formation of cisplatin resistance of lung cancer cell.As a result, RBAP48promises to be the new target to reverse the cisplatin resistance of lung cancer.Part2To study the role of RBAP48gene in regulating cell proliferation and cisplatin resistance of human lung cancer cells.Objective:Adjust the expression of RBAP48and explore the effect of its expression on proliferation and cisplatin resistance of lung cancer cell. Methods:The pairs of RBAP48-siRNA were designed according to the small interfering RNA. The RBAP48-pEGFP-N1recombinant plasmids was constructed by recombinant DNA technology. Seventy-two hours after lipofectin reagent transfection,the effect of gene regulation was measured by western blot.The proliferation and cisplatin sensibility of lung cancer cell was detected by MTT.The cell cycle status and apoptosis of lung cancer cell were examined by flow cytometry(FCM).The senescence of lung cancer cell was examined by β-Galactosidase dyeing assay. Results:Compared with the untreated group and negative control group,the expression of RBAP48in RBAP48-siRNA group was decreased obviously while in RBAP48-pEGFP-N1group was increased obviously. MTT revealed that the proliferation was restrained notably in RBAP48-siRNA group compared with the untreated group while the cisplatin sensibility increased compared with the untreated group.FCM revealed that the percentage of apoptotic cells in untreated group and RBAP48-siRNA group have no significant change.FCM revealed that the percentage of G1stage cells in RBAP48-siRNA group was increased (24.05±2.50)%compared with the untreated group.These data showed that RBAP48-siRNA restrained the conversion of G1stage. P-Galactosidase dyeing assay revealed that RBAP-siRNA group has the senescence obviously compared with the untreated group and Ctrl-siRNA group.The expression of RBAP48was increased after RBAP-pEGFP-Nl transfection. MTT revealed that the proliferation was enhanced notably in RBAP48-pEGFP-N1group compared with the untreated group while the cisplatin tolerance increased in A549cell. FCM revealed that the percentage of apoptotic cells in untreated group and RBAP48- pEGFP-N1group have no significant change.FCM revealed that the cell cycle distribution in RBAP48-pEGFP-N1group has no significant change compared with untreated group while it can relieve the S stage retardation which was caused by cisplatin. β-Galactosidase dyeing assay revealed that RBAP48-pEGFP-N1group has no the senescence obviously compared with the untreated group and it can reverse the senescence induction which caused by cisplatin. Conclusion:The cisplatin sensibility to A549/DDP cell was enhanced while cell proliferation was suppressed after silence the RBAP48.The cisplatin tolerance to A549cell was enhanced while cell proliferation was promoted.RBAP48plays an important roles in the proliferation and the formation of cisplatin tolerance in cisplatin tolerance.It is the potential target in the gene therapy of lung cancer and adjust the expression of RBAP48in lung cancer cell may be the new way to reverse the cisplatin tolerance of lung cancer.Part3To explore the mechanism of regulation of RBAP48gene expression on cell proliferation and cisplatin resistance in human lung cancer cells.Objective:To detect the effect of regulating of RBAP48on core subunit of PcG of lung cancer cell, histone coordination factor, the expression of the related protein about cell cycle and senescence and explore the molecular mechanism of proliferation in lung cancer cell affected by the expression of RBAP48and cisplatin resistance. Methods:The expression of RBAP48in A549cell was regulated by RBAP48-pEGFP-N1transfection while in A549/DDP cell the expression of RBAP48was regulated by RBAP48-siRNA transfection.Seventy-two hours after transfection, western blot assay was used to determine the change of level of protein of EZH2, Bmi-1, H3K27me3, Acety-H3K27, ubi-H2AK119, P14, P16, Rb, P53, P21, P27, CyclinD1, CDK2, CDK4. Results:Western blot assay revealed that RBAP48-siRNA transfection in A549/DDP cell down-regulated the level of expression of EZH2, Bmi-1, H3K27me3, ubi-H2AK119, cyclinDl, CDK2, CDK4and E2F1, up-regulated the level of expression of Acety-H3K27, P21, P27, P16, P14, Rb and P53.Western blot assay revealed that RBAP48-pEGFP-N1transfection in A549cell down-regulated the level of expression of Acety-H3K27, P21, P27, P16, P14, Rb, P53, up-regulated the level of expression of EZH2, Bmi-1, H3K27me3, ubi-H2AK119, CyclinDl,CDK2,CDK4and E2F1. Conclusion:The proliferation and cisplatin resistance of lung cancer cell regulated by RBAP48may through the histone methylation and ubiquitin catalyzed by PcG and the gene silencing of target mediated by PcG. Silence the expression of RBAP48inhibits the epxression of PcG and bolcks the histoneepigenetic regulation while activates the expression of cell cycle regulatory elements, restrains the cell cycle and induces cell senescence.As a result, it retrains cell proliferation and reverses the cisplatin resistance of cancer cells.