Inhibition of lung tumor growth in nude mice by siRNACD31 targeting PECAM-1

Small interfering RNA (siRNA) provides a promising therapeutic approach in the silencing of disease-causing genes. In the present study, the use of 2′-O-methyl-modified siRNA-cluster of differentiation 31 (siRNACD31), with cationic liposome RNA interference (RNAi)-mate as a carrier, effectively silenced the platelet endothelial cell molecule 1 (PECAM-1) gene of murine hemangioendothelioma cells in vitro. In vivo, 2′-O-methyl-modified siRNACD31 carried by RNAi-mate was successfully delivered, targeting the PECAM-1 gene in the vasculature of nude mouse lung carcinoma xenografts. The growth of the lung carcinoma xenografts was inhibited by the 2′-O-methyl-modified siRNACD31 and RNAi-mate complexes, and the expression of the PECAM-1 protein was downregulated, with a simultaneous decrease in vascular endothelial growth factor (VEGF) protein in the lung carcinoma xenografts. 2′-O-methyl-modified siRNACD31-RNAi-mate complexes may provide a potential therapeutic strategy in lung carcinoma treatment. The effect of PECAM-1 on VEGF expression may possibly be attributed to the function of PECAM-1 signal transduction.


Introduction
Lung cancer is a leading cause of mortality globally, with the most frequent type being adenocarcinoma. Although platinum-based traditional chemotherapy is currently the first-line therapy for advanced lung cancer, due to its clinical benefits, its use is limited due to significant associated toxicities. In an effort to overcome these limitations, targeted therapies are currently an area of research focus due to our progressive understanding of tumor molecular biology and the tumor microenvironment (TME), including medications targeting the epidermal growth factor receptor, such as gefitinib (1,2) and erlotinib (1,3), and those targeting the vascular endothelial growth factor (VEGF) signaling pathways, such as bevacizumab (4). However, only a small proportion of specific patients benefit from the current targeting agents, with inevitable resistance. The identification of alternative promising molecular targets would be a rational consideration for individual patients with lung cancer.
Tumor growth and invasion is closely associated with the TME. The main components of TME angiogenic endothelial cells are regulated by various bio-mediators, including platelet endothelial cell molecule 1 [PECAM-1; namely cluster of differentiation 31 (CD31)] (5,6) and VEGF (7)(8)(9). PECAM-1 is a biomarker of endothelial cells (10)(11)(12). Experimental studies have indicated that PECAM-1 regulates endothelial cell motility and angiogenesis (13) and is a potential target on TME endothelial cells (14,15). Although it has been shown that the vascular inhibitor that targets the VEGF of the TME can be used an efficacious therapy (4,9,(16)(17)(18)(19), it remains uncertain whether PECAM-1 could be used as an angiogenic inhibitor on the TME. In addition, the delivery system targeting PECAM-1 in vivo requires further exploration.
RNA interference (RNAi) technology shows considerable promise as a nucleic acid-based therapy (20). Small interfering RNA (siRNA) consists of 19-to 23-nucleotide double-stranded RNA duplexes via the formation of an RNA-induced silencing complex (RISC). RISCs specifically identify homologous gene mRNA and induce sequence-specific mRNA degradation leading to silencing of target gene expression. The performance of siRNA-targeted therapy requires a suitable and effective carrier delivery system. Cationic liposomes have been used as effective siRNA carriers in vitro and in vivo (21,22). Achieving systemic RNAi in vivo requires that the siRNA possesses the properties of stability, cellular delivery and tissue bioavailability. Aside from siRNA alone (naked), 2'-O-methyl-modified siRNA CD31 has the strongest resistance towards degradation by exo-and endonucleases in the serum and tissue homogenates (20,23), leading to more effective therapeutic RNAi in vivo.
With respect to previous discussions regarding siRNA delivery systems, the use of 2'-O-methyl-modified siRNA CD31, with cationic liposomes as carriers, would be an attractive candidate technology for systemic delivery of PECAM-1 in vivo (20,22,23). In the present study, the effects of the systemic delivery of siRNA CD31 on the growth of lung adenocarcinoma xenografts were investigated with the application of 2'-O-methyl-modified siRNA CD31 -cationic liposome complexes to silence PECAM-1.

Materials and methods
siRNA and RNAi-mate. The 2'-O-methyl-modified siRNA CD31 molecules used in the present study are described in Table I. siRNA CD31 , 3'-fluorescein amidite (FAM) fluorescence-labeled siRNA CD31 (siRNA CD31 -FAM; described in Table I), stable negative control RNA (SNC; described in Table I) and RNAi-mate were all synthesized by GenePharma Co., Ltd. (Shanghai, China). The primers of PECAM-1 mRNA for reverse transcription polymerase chain reaction (RT-PCR) were also synthesized by GenePharma Co., Ltd. (Table II).
Cell lines and cell treatment. EOMA cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in endothelial growth MED-0002 media (PriCells Biomedical Technology Co., Ltd, Wuhan, China) containing 10% fetal bovine serum (Gibco, Invitrogen Life Technologies, Carlsbad, CA, USA), in 6-well plates at 37˚C, in a 100% humidity cell incubator containing 5% CO 2 , and identified with human anti-factor VIII antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The cells cultured were harvested for assays during the exponential growth phase. The exponential growth EOMA cells (5x10 4 /well) were seeded in 24-well plates containing various agents for 24 h as follows: Naked siRNA CD31 -RNAi-mate (siRNA CD31 group), FAM-labeled siRNA CD31 -RNAi-mate (siRNA CD31 -FAM group), SNC-RNAi-mate (SNC group) and Opti-minimum essential medium (MEM; reduced-serum cell culture medium; Gibco) as a blank control (control group). In brief, the siRNA CD31 -RNAi-mate transfection procedures were as follows: Firstly, 50 µl Opti-MEM and 20 pmol siRNA CD31 (or siRNA CD31 -FAM, SNC or Opti-MEM) were completely mixed, then 50 µl Opti-MEM diluted with 2 µl RNAi-mate reagent was added and the mixture was kept at room temperature for 5 min. Secondly, the diluted siRNA CD31 (or siRNA CD31 -FAM or SNC) and RNAi-mate reagent were mixed gently to form siRNA-lipoplexes at room temperature for 20 min. Finally, 100 µl complexes involving siRNA CD31 -RNAi-mate or siRNA CD31 -FAM-RNAi-mate were respectively added to each well containing the cells and the medium used for transfection, and 100 µl SNC-RNAi-mate and Opti-MEM medium were added respectively to the SNC and control wells. The FAM-fluorescence detection was performed with a confocal microscope (excitation wavelength of 495 nm, emission wavelength of 525 nm; Leica, Mannheim, Germany) after transfection efficiency had been reached for 6 h at 37˚C in a CO 2 incubator. The cell transfection rate was ~80%, and the transfection process continued for 48 h. Assessment of the various specimens were carried out for RT-PCR and western blot analysis, and the MTT assay of the cell proliferation rate was performed as previously described (24). Each assay was performed in triplicate and independently repeated three times. The rabbit anti-PECAM-1 and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GADPH) antibodies were obtained from Santa Cruz Biotechnology, Inc., and the goat anti-rabbit immunoglobulin G monoclonal antibody was purchased from Maixin Technology Co., Ltd., (Shenzhen, Guangdong, China) for the western blot assay. The primer sequences of PECAM-1 used for amplification in the RT-PCR are listed in Table II. The inhibition rate of EOMA cell proliferation was calculated as follows: Inhibition rate of proliferation (%) = [1 -optical density (OD) experimental wells / OD control wells] x 100. The wells containing Opti-MEM were used as the control.
The human lung adenocarcinoma (HLAC) A549 cell line was obtained from the Chinese Academy of Sciences Type Culture Collection (CASTCC; Shanghai, China) and cultivated according to the CASTCC recommendations. The cultured cells were harvested for treatment in vivo during the exponential growth phase.

siRNA CD31 is transfected effectively into EOMA cells in vitro.
The results of FAM-fluorescence detection by confocal fluorescence microscopy showed that siRNA CD31 , with RNAi-mate as a carrier, was successfully transfected into the EOMA cells. The bright fluorescence was emitted from the EOMA cells transfected by the fluorescence FAM-labeled siRNA CD31 (Fig. 1A). Fig. 1B shows the same cells observed by optical microscopy (Olympus BX-51; Olympus Tokyo, Japan).
In vitro siRNA CD31 inhibits the proliferation of EOMA cells.
The results of the MTT assay for the inhibition rates of EOMA proliferation showed that the inhibition rates of the siRNA CD31  The expression of PECAM-1 was observed in the vasculature of various tissues. The results of the immunohistochemical examination indicated that PECAM-1 expression was observed in the vasculature of the lung adenocarcinoma xenograft and the lung, liver, heart, brain and kidney tissues (Fig. 4A-F).
In vivo siRNA CD31 inhibits tumor growth. The volumes of the tumor xenografts in the nude mice treated by siRNA CD31 , with RNAi-mate (the siRNA CD31 group) as the carrier, were smaller than those in the control nude mice (Table III) (P day 10 <0.05 vs. control; P deviation of tumor xenograft volume (DV) <0.01 vs. control). The growth of the tumor xenograft in the nude mice of the siRNA CD31 group was slower than in the control group from day 4, and the DV increased in the latter days (Fig. 5). These results indicated that siRNA CD31 , with RNAi-mate as a carrier, may effectively inhibit the growth of lung adenocarcinoma in vivo.
In vivo siRNA CD31 downregulates PECAM-1 and VEGF expression in tumor xenografts. The results of the PECAM-1 protein expression analysis with ELISA indicated that the measured values (MVs) and BCA correction values of PECAM-1 in the tumor xenografts of the nude mice treated with siRNA CD31 -RNAi-mate complexes (the siRNA CD31 group) were decreased compared with the values of the tumor xenografts of the nude mice treated with saline (control group) (all P<0.01; Fig. 6). However, the MVs and BCA correction values of PECAM-1 of the other tissues (lung, liver, brain, heart and kidney) in the nude mice treated with siRNA CD31 -RNAi-mate complexes were not significantly different compared with the values from the control nude mice treated with saline (all P>0.05; Fig. 6). The VEGF ELISA assay achieved similar results to those of PECAM-1 (Fig. 7).

Discussion
RNAi is a promising therapeutic approach to silencing disease-causing genes, and is usually mediated by siRNA consisting of 19-to 23-nucleotide double-stranded RNA duplexes. The delivery system is the main complication in achieving gene silencing by siRNA technologies in vivo. Previous studies have reported that 2'-O-methyl-modified siRNA possess a strong resistance to the degradation by nuclease in the serum and tissues (20,23). The administration of cationic lipids has been applied for siRNA delivery in vivo (22). The endothelial specific marker, PECAM-1 (i.e., CD31) (10,12,26), is closely associated with angiogenesis (27), and the vasculature of the TME plays a significant role in the proliferation and invasion of tumor cells. PECAM-1 could be used as a potential therapeutic target on the TME with respect to its activity in the pathogenesis of tumors, including lung cancer (28,29).
In the present study, the targeted deliver y of 2'-O-methyl-modified siRNA CD31 and cationic liposome RNAi-mate complexes on endothelial PECAM-1 in vitro and in vivo were investigated. Three important findings were noted in the present study. The first was that the 2'-O-methyl-modified siRNA CD31 -lipoplexes effectively silenced the target gene, PECAM-1, in vitro and in vivo. 2'-O-methyl-modified siRNA CD31 successfully downregulated the PECAM-1 mRNA and protein expression of the EOMA cells using RNAi-mate as a carrier in vitro (Fig. 3), and the expression of PECAM-1 was detected by immunohistochemical examinations in the vasculature of the lung adenocarcinoma xenografts (Fig. 4A) and in the vascular tissues of the lung, liver, heart, brain and kidney in vivo (Fig. 4B-F). These results provided a molecular and cellular basis for targeted treatment with 2'-O-methyl-modified siRNA CD31 and RNAi-mate complexes in vivo. In the in vivo study, the growth of the lung adenocarcinoma xenografts was effectively inhibited by injecting the complexes of 2'-O-methyl-modified siRNA and RNAi-mate via the tail veins of the nude mice (Table III and Fig. 5). Although the expression of the PECAM-1 protein in the lung, liver, heart, brain and kidney tissues was not decreased (Fig. 6), a decrease in PECAM-1 expression was obtained in the lung adenocarcinoma xenografts (Fig. 6). These findings indicated that the 2'-O-methyl-modified siRNA-lipoplexes achieved the targeted silencing of the PECAM-1 gene in the vasculature of the lung adenocarcinoma xenografts in vivo. The achievement of specific targeted silencing of the PECAM-1 gene in tumor xenografts is possibly due to the strong bioavailability of the siRNA CD31 -lipoplexes of the neovascular cells in the TME (22,30). Cationic liposome RNAi-mate may act as a candidate carrier for the systemic administration of siRNA CD31 to other liposomes (21,30). Lung adenocarcinoma with abundant vasculature is the most common pathological type of lung cancer. With respect to the limit and toxicity of traditional chemotherapy on lung cancer, target medications have created a promising research area for the bio-therapy of lung cancer (1)(2)(3)(4). Besides VEGF (4), PECAM-1 would also be a potential target on the vasculature of the TME (14,15,28), as it plays a significant role in angiogenesis (13,27). Although PECAM-1 activity on the modulation of endothelial cells and its effects in tumor angiogenesis are known (14,15), the effects of targeted delivery of PECAM-1 on the growth of lung cancer requires further investigation. It has been demonstrated that 2'-O-methyl-modified siRNA and cationic lipids could be applied as an effective targeted delivery for silencing a target gene (22). On the basis of the previous discussions regarding siRNA delivery systems, the present study possibly provides an important target strategy involving 2'-O-methyl-modified siRNA targeting PECAM-1 using cationic lipids RNAi-mate against the proliferation of lung carcinoma cells (20,22,23). The second important finding in the present study was that the proliferation of the EOMA cells was inhibited by 2'-O-methyl-modified siRNA CD31 using RNAi-mate as a carrier (Fig. 2). PECAM-1 is a membrane protein with signal transduction (10,31,32) occurring via the initiation of downstream signaling pathways, including mitogen-activated protein kinase (33), Erk (34,35) and PI-3/Akt (36). This has significant implications in the regulation of endothelial apoptosis (37,38). Therefore, it is speculated that this finding possibly contributes to the initiation of signal transduction by PECAM-1 and to the induction of EOMA cell apoptosis, leading to the inhibition of the proliferation of EOMA cells by 2'-O-methyl-modified siRNA CD31 .
The third significant finding was that a simultaneous decrease in PECAM-1 (Fig. 6) and VEGF (Fig. 7) was observed when 2'-O-methyl-modified siRNA CD31 downregulated PECAM-1 expression. It is well known that PECAM-1 initiates signal transduction to activate the downstream signaling pathway (31,(33)(34)(35)(36)39) and regulate the generation and release of bio-mediators (27,32,40,41). On the basis of the aforementioned studies and the results of the present study, we believe that the downregulation of PECAM-1 expression using siRNA CD31 silencing of the PECAM-1 gene possibly executed an effect on the signal transduction of PECAM-1, leading to the decrease in VEGF protein expression.
In summary, the present study demonstrated that 2'-O-methyl-modified siRNA CD31 and RNAi-mate complexes may effectively silence the PECAM-1 gene in vitro and in vivo, and downregulate the expression of PECAM-1 and VEGF proteins. siRNA CD31 targeting of PECAM-1 in the TME may be a potential gene therapy for tumors. PECAM-1 regulated the generation of VEGF possibly through the signaling pathway involving PECAM-1. However, the improvement of the delivery system of siRNA CD31 for achieving complete dissolution of the tumor xenografts with siRNA CD31 mediated by lipoplexes and the regulation mechanism of PECAM-1 on VEGF requires further exploration. The combination of various cytokines, including PECAM-1, VEGF, transforming GF and fibroblast GF, contributing to tumor angiogenesis would be a possible candidate for future study.