Regulation Of Apoptosis Genes By RBM5and The Effect On Chemoresistance Of Lung Adenocarcinoma

Lung cancer is one of the malignant tumors seriously threatening humanhealth and life worldwide. The incidence rate of lung cancer increasedrapidly year by year. Non-small cell lung cancer (NSCLC) accounts forapproximately85%of primary lung cancer cases. Platinum-basedcombination chemotherapy is the standard therapy for NSCLC. Despite therecent introduction of new platinum chemotherapeutic compounds, cisplatinremains widely used for the treatment of lung cancer. Cisplatin exertsanticancer effects via multiple mechanisms, yet its most prominent mode ofaction involves the generation of DNA lesions followed by the activation ofthe DNA damage response and the induction of apoptosis. However, theefficacy of cisplatin treatment is often impaired by the emergence of tumorcell resistance to this drug. As in some clinical settings cisplatin constitutesthe major therapeutic option, the development of chemosensitizationstrategies constitute a goal with important clinical implications. To improve the efficacy of cisplatin in clinical oncology, it is important to elucidate themechanisms by which lung cancer cells acquire the ability to evadecisplatin-induced cell death.Complicated mechanisms account for the cisplatin-resistance of tumorcells. One important mechanism by which tumor cells develop resistance tocisplatin is related to resistance to apoptosis. Molecular changes that have thepotential to cause apoptotic dysregulation, including activation ofantiapoptotic factors, inactivation of pro-apoptotic effectors, and/orreinforcement of survival signals. The development of novel therapeuticagents directed against these apoptosis regulation targets, which have beenshown to demonstrate enhanced apoptotic killing and sensitize resistantcancer cells to antineoplastic agents. Therefore, therapy targeting the specificmolecular alterations underlying the development of platinum resistancerepresents an attractive therapeutic strategy.RNA binding motif protein5(RBM5) is an RNA-binding protein thathas the ability to modulate apoptosis. Further, RBM5resides in the most“sought-after” tumor suppressor locus in lung cancer,3p21.3. Homozygousdeletions at this locus are the earliest and the most frequently genetic alteration in lung cancer. There is a growing body of literature on RBM5suggesting that RBM5is involved in apoptosis, cell proliferation andoncogenesis. Defects in apoptosis underpin both tumorigenesis and drugresistance. However, the role of RBM5in the development of acquiredresistance against chemotherapeutic agents, including cisplatin, in humanlung cancer has yet to be elucidated.In this study, we focused our research specifically on lungadenocarcinoma cells A549and their cisplatin resistant variant A549/DDPcells. We observed the regulation on apoptosis gene of RBM5and the effecton response to cisplatin in vivo and in vitro. This study provided platform forscreening and the discovery of new apoptosis genes related with tumorchemoresistance. These findings suggest that RBM5may act as a biomarkerwith the ability to predict a response to cisplatin, and that it may serve as aprognostic indicator in lung cancer patients, as well as acting as a moleculartarget for enhancing and resensitizing cisplatin-resistant tumors to cisplatin.Methods（1） RBM5mRNA and protein expression in the A549andA549/DDP cells was analyzed by semi-quantitative RT-PCR and Western blot.（2） MTT assays were used to evaluate chemosensitivity tocisplatin. Apoptosis was assessed by DAPI nuclear staining and flowcytometric analysis with an Annexin-V-FITC apoptosis kit. Cytosoliccytochrome c, cleaved caspase-3and cleaved caspase-9were detected byWestern blot.（3） The A549/DDP cells were then transfected with apcDNA3.1-RBM5plasmid using LipofectAMINE2000prior to treatmentwith cisplatin. We performed semi-quantitative RT-PCR and Western blotanalyses to confirm the expression of RBM5mRNA or protein.（4） Small interfering RNA (siRNA) sequences targeting humanRBM5and a non-target sequence were constructed. The RBM5-specificsiRNA was transfected into A549cells. Semi-quantitative RT-PCR andWestern blot analyses were performed to confirm the knockdown of RBM5mRNA or protein.（5） Transplant tumor model was obtained by injecting A549/DPPand A549cells to nude mice for study in vivo. Cisplatin group was givenintraperitoneal injection of2.5mg/kg cisplatin twice a week. Combination therapy group was administered with recombinant attenuated S.Typhimurium for3times and intraperitoneal injection of cisplatin forA549/DDP model. Lipidosome and siRNA were injected intra-tumor andperi-tumor for A549transplanted tumor twice a week. Tumor tissues wereobtained21days after the first treatment. Growth curve, Tumor weight andvolume were compared.（6） The tumors were weighted and performed the TUNEL andimmunohistochemistry (IHC) experiments respectively. The proteinexpression levels of RBM5, PCNA, cleaved-caspase-9andcleaved-caspase-3by Western blot and immunohistochemical method.（7） The apoptosis related genes expression change in theover-expression or down-regulated RBM5cells was detected by PCR arrayscreening in vivo and in vitro.Results（1） The expression of RBM5mRNA and protein wassignificantly reduced in the A549/DDP cells compared with the A549cells.（2） Exogenous expression of RBM5by the pcDNA3.1-RBM5resensitized the response of A549/DDP to cisplatin, resulting in a significant increase in tumor-suppressing activity induced by cisplatin. RBM5-enhancedchemosensitivity was associated with the release of cytochrome c into thecytosol, activation of caspase-9and the downstream marker caspase-3. Theexpression of apoptosis related genes changed to take the pro-apoptoticeffect.（3） The expression of RBM5mRNA and protein increased in thetransplanted human lung adenocarcinoma cells A549/DDP in nude miceconfirmed by RT-PCR, Western blot and IHC. Correlated with the results invitro, the introduction of RBM5enhanced the effect of cisplatin onA549/DDP transplanted tumors and induced the pro-apoptotic trend ofapoptosis genes.（4） After transfected the RBM5-specific siRNA,semi-quantitative RT-PCR and Western blot analyses were performed toconfirm the expression of RBM5mRNA or protein, and knockdown ofRBM5mRNA or protein. Downregulation of RBM5with siRNA in the A549cells inhibited the response of A549to cisplatin, resulting in a significantdecrease in tumor-suppressing activity induced by cisplatin. The expressionof apoptosis related genes changed to take the anti-apoptotic effect. （5） Injection of si-RBM5intra-tumor and peri-tumor reduced theexpression of RBM5mRNA and protein in A549transplanted tumor. Alltumors with A549cells increased in size during the experiment. However,the si-RBM5did not have any influence on the therapeutical effect alone orcombined with cisplatin.Conclusions（1） Cisplatin-resistant A549/DDP cells expressed lower RBM5levels than the parental A549cells. This implies a correlation betweencisplatin resistance and the expression of RBM5.（2） Ectopic expression of RBM5by pcDNA3.1-RBM5inducedcell apoptosis and promoted cisplatin-induced apoptosis in the A549/DDPcells. Cytosolic cytochrome c and the expression of cleaved caspase-3andcleaved caspase-9were clearly enhanced in the A549/DDP cells co-treatedwith pcDNA3.1-RBM5and cisplatin, compared with the negative control.Overexpression of RBM5up-regulated the expression of pro-apoptotic genesand down-regulated the expression of anti-apoptotic genes. These resultssuggest that the upregulated expressions of RBM5might sensitize tumorcells to cisplatin-based chemotherapy through regulation the expression of apoptotic genes.（3） Both mRNA and protein expression of RBM5significantlydecreased in the siRBM5-A549cells in vitro. Knockdown of RBM5conferred resistance to cisplatin-induced apoptosis and down-regulated theexpression of pro-apoptotic genes and up-regulated the expression ofanti-apoptotic genes in A549cells. The si-RBM5injection intra-tumor andperi-tumor indeed reduced the expression of RBM5in tumor tissue. However,we did not observe the influence of si-RBM5on the chemotherapeutic drugsin nude mice bearing human lung adenocarcinoma, which suggested thatthere is a complex regulatory mechanism in the regulation of response tocisplatin in vivo.（4） RBM5relates to cisplatin resistance in human lungadenocarcinoma. Regulation of RBM5might influence the response tocisplatin through modulating the apoptotic genes, and thus RBM5may be apromising target to overcome chemoresistance in lung cancer. Our resultssuggest that RBM5has the potential to act as a biomarker for the predictionof the cisplatin response and thus provide important data on the prognosis ofpatients with lung cancer. This study also further clarified and discovery and reversal of drug resistance related gene of apoptosis.