Study Of Silencing Id-1 On Adenoid Cystic Carcinoma Biological Behaviors

Objective:1. To establish a novel green fluorescent protein (GFP)-expressing nude mice orthotopic model which can be visualized in real time with fluorescence imaging and detected in a noninvasive and continuous way for tumor's development, invasion and metastasis.2. To demonstrate the effectiveness and feasibility of RNAi in the tongue of orthotopic mice model which is mediated by lentivirus injection into the tumor.3. To investigate the inhibition of silencing Id-1 gene by RNAi on adenoid cystic carcinoma (ACCM) in vivo and in vitro.4. To research the effect of Id-1 expression on cell proliferation, invasion, apoptosis, and so on in ACCM and to explore the possibility of Id-1 being a new therapeutic target for ACCM treatment.Methods:1. ACCM cells were cultured in DMEM supplemented with 10%fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin,100 U/ml streptomycin) at 37℃, 5%CO2. After collecting viral supernatant through a 0.45μm celluslose acetate filter, viral titer was determined as 108 cfu/L finally.1×105 ACCM cells in a 6-well plate, cultured for about 12-16h, were exposed to viral supernatant at a multiplicity of infection, in the presence of 8μg/ml polybrene for 24h. Subsequently, the viral supernatant was replaced with fresh medium. ACCM-GFP cells were acquired with 800μg/ml G418 selection for 2 weeks and then were collected, counted. Split one cell per well in 96-well plate. Colonies were grown in DMEM containing FBS and antibiotics at 37℃,5%CO2.2. ACCM-GFP cells were harvested with trypsin and washed with prechilling DMEM for 3 times.2×105 ACCM-GFP cells in 0.1 ml medium were injected submucosally into lingual central part at the cross-point of forward 2/3 and backward 1/3 in 60 BALB/c nu/nu male mice. Injection should be finished in 40 minutes and the medium containing cells can be placed on the ice temporarily. The mice were six weeks old, with a body weight of 20-25g.3.7 days after ACCM-GFP cells injection, mice were imaged at various time points using a cooled CCD camera. To acquire better images, mice were anesthetized, and the tongue was pulled out of the oral cavity. Next, anesthetized mice were pictured by the whole-body optical imaging system. Green fluorescence images were also collected using 60s exposure time with 465nm excitation/535nm emission filters but no binning. The comparison between ACCM-GFP and ACCM cells in morphology, capability of cell growth and invasion, and histology was analyzed. RNA and protein were extracted from ACCM-GFP and ACCM cells in vitro. mRNA and protein expression of Id-1 were evaluated with RT-PCR and Western Blot.4.2 weeks after ACCM-GFP injection,5×107TU in 100μl of Id-1-siRNA-Lentivirus and NC-siRNA-Lentivirus were injected in a multiple intratumoral direction respectively every other day and for 3 times continuously. The multiplicity of infection (MOI) is 50.5. Each tumor was measured every other day by using a caliper. Graphs were plotted to illustrate tumor growth in each group. Mice were photographed for fluorescent images by KODAK Image Station. The mice were scanned with fluorescence with excitation maximum 465nm and 560nm for GFP and RFP respectively. Mice were sacrificed at day 3,4,5,6,7,10 and 14 after RNAi. Total RNA and protein were isolated from the tissue and Id-1 expression in mRNA and protein level were detected by qRT-PCR and Western Blot. Immunohistochemistry analysis of Ki-67 expression was conducted 7 days after RNAi. 6. Infection of ACCM cells by Id-1-siRNA-Lentivirus in vitro was applied to analyze the transduction efficiency. At different time points (24h,48h, and 72h), cells were harvested for FACS analysis to confirm the transduction efficiency (465nm excitation/535nm emission). Meanwhile, ACCM-GFP cells infected with Id-1-siRNA-Lentivirus were observed under the green/red fluorescence to determine the transduction efficiency.2 X 104 ACCM cells were seeded in each well of a 6-well plate. The cells were infected in DMEM containing 10%FBS and 5μg/ml polybrene when the confluence was about 50%-80%and Id-1-siRNA-Lentivirus was introduced at a multiplicity of infection (MOI) of 50 for 24h.4 days later, apoptosis was measured using Hoechst 33258 staining. Cells at a concentration of 103 per well were seeded in the 96-well plate and incubated for 24h and then infected with lentivirus particles at a MOI of 50. MTT assay was performed for each day after RNAi to study the effect of Id-1 siRNA on cell proliferation. Cell invasion was assayed using a Boyden chamber cell with culture-chamber-insert system. On the 5th day of RNAi, ACCM cells infected by Id-1-siRNA-Lentivirus in 6-well plate were collected for cell invasion analysis (Wright's stain).Results:1. ACCM-GFP cells isolated by G418 screening were able to express GFP stably after 2 months'culture and thawed after 6 months'freezing-storage when G418 was already removed. The GFP sign was completely strong. GFP was distributed in the cytoplasm.2. From the viewpoint of histology, there was no significant difference between ACCM and ACCM-GFP cells. Histopathologically, most ACCM-GFP tumors presented solid form. It was composed of solid epithelial islands with central areas of necrosis; the cells were small, basophilic and hyperchromatic with a densely granulated nucleus and easily visualized mitotic figures. The epithelial island was composed of cubic cells with scarce cytoplasm, oval nuclei with eosinophilic content, surrounded by a band of dense muscular fibrous tissue, well vascularized, and moderate diffuse chronic inflammatory infiltration. ACCM-GFP cells showed no significant difference from ACCM cells in morphology, Id-1 protein expression, cell proliferation, and invasion ability.3. ACCM-GFP tumor incidence measured by GFP expression and histological analysis was 100%for all mice in the control and test groups. Transduction efficiency in vitro was evaluated by flow cytometry. With the stable expression of RFP, the high transduction efficiency of 83.5%,68.45%and 24.89%could be detected 72h,48h and 24h after lentiviral infection respectively. Meanwhile, ACCM-GFP cells infected with Id-1-siRNA-Lentivirus were observed with green and red fluorescence respectively and the transduction efficiency of lentivirus was as high as about 80%.4. Detected with green fluorescence, some features of original ACCM tumor, such as notches along the tumor border, cystic cavities inside the tumor, etc. can present in ACCM-GFP tumors. Meanwhile, a metastic lymphatic node invaded by ACCM-GFP cells can be detected by GFP 3 weeks after ACCM-GFP cell injection, although it is not palpable. The fluorescence sign was strong in spite of the limited areas involved by metastasis. However, we did not find metastasis in lung and other tissues. ACCM-GFP tumors treated with RNAi presented weak green fluorescence with excitation of 465nm, while strong red fluorescence with excitation of 560nm.5.12 days after the infection with RNAi-containing lentivirus particles, tumor growth began to decrease. Although the tumor size was still increasing after 12 days of RNAi, the difference of test and controls group was significant. However, the negative controls showed the same tumor growth speed as the blank controls. Tumor growth inhibition efficiency could be 26.14%-39.17%. Immunohisto-chemical staining showed Ki-67 positive cells in tumors treated with NC-siRNA-Lentivirus were similar in quantity to ACCM-GFP tumors, however, cellular proliferation indices of tumors derived from Id-1-siRNA-Lentivirus-treated mice differed significantly from others.6. Until the 3rd day after injection, Id-1-siRNA-Lentivirus-treated tumors, NC- siRNA-Lentivirus-treated tumors and ACCM-GFP tumors showed no reduction in the levels of Id-1; whereas those infected with Id-1-siRNA-Lentivirus for more than 3 days showed significantly reduced levels of mRNA. mRNA level decreased to a relative stable state from the 4th to the 14th day. The expression was markedly downregulated in tumors from the 4th day of infection with Id-1-siRNA-Lentivirus (p<0.01), while it remained similar in blank control, NC-siRNA and Id-1-siRNA group infected for no more than 3 days. Protein level decreased to a relative stable state from the 6th day.7. Cells in culture after 4 days of infection with Id-1-siRNA exhibited lots of apoptosis, which was characterized by pyknotic and fragmented nuclei. Condensed bright apoptotic nuclei were readily observed amidst the transducted cells. In control cultures, round and large nuclei appeared with regular contours and cells with smaller nuclei and condensed chromatin were rarely seen. Repression of Id-1 expression in ACCM-GFP was found to inhibit invasion in vitro. Cells passed PET membrane in test group were significantly less than control groups (p<0.01). MTT assays showed that Id-1-siRNA-Lentivirus-treated tumor cells grew significantly more slowly than others (p<0.01) frome the 5th day after RNAi. Inhibition efficiency in test group was around 40%-50%. No significant difference was detected in cells treated with NC-siRNA-Lentivirus compared with untreated cells.Conclusion:1. ACCM cells can be infected 2 times successively by retrovirus. The insertion of retroviral genome into ACCM's had no influence on the physiological behavior in our study. ACCM-GFP cells displayed the same characteristics as ACCM cells in cell proliferation, invasion ability, Id-1 mRNA and protein expression.2. Our established GFP-expressing ACCM tumor model should be useful for the evaluation of novel treatment strategies for ACCs including neoadjuvant chemotherapy or gene therapy. It is also an important new tool to help us to understand the key events of cancer development and metastasis especially interaction of the metastatic tumor with a wide array of normal organs and tissues. It is one of the best tumor models of adenoid cystic carcinoma.3. Lentiviral vector shows high transfection efficiency, stable expression and reliable safety in this research.4. Specific Id-1-siRNA can downregulate significantly Id-1 gene expression in mRNA and protein level. It can also inhibit tumor development, cell proliferation and cell invasiveness as well. Cell apoptosis can be induced at the same time.5. Id-1 has a positive relationship with cell proliferation, invasiveness and so on in adenoid cystic carcinoma. It is an ideal target gene for adenoid cystic carcinoma treatment.6. Lentivirus-mediated RNAi presents advantage of stability, high efficiency and similar administration to clinic. This effective technique proves to be promising for genetic therapy of adenoid cystic carcinoma.