| Background:Laryngeal carcinoma is the most common cancer in upper respiratory tract, accounting for approximately25%of Head and Neck Cancer. The classical treatment is surgery or radiotherapy, with chemotherapy or not. The partial or total loss of laryngeal function or serious side effects induced by classical treatments have a great impact on patients’quality of life. Furthermore, some patients suffer from recurrent cancer after treatments. Although there have been great advance in surgery type, radiotherapy method and chemotherapy regime in recent30years, the survival rates are still not obviously improved. For those who have lost the opportunity of radical treatments, palliative chemotherapy is the only choice. The5-year survival rate is around30%.In the past20years, molecular targeted therapy has developed to be a new anti-tumor method. For Head and Neck Squamous Cell Carcinoma (HNSCC), the most popular molecular targeted therapy are monoclonal antibodies and low molecular tyrosine kinase inhibitors that specifically inhibit Epidermal Growth Factor Receptor (EGFR). However, low molecular tyrosine kinase inhibitors such as Gefitinib have little effect on HNSCC. As for monoclonal antibodies such as cetuximab, although they have proved to be effective as an adjuvant for radiotherapy and chemotherapy, the low response rate, high resistance rate and frequent side effect such as dermatitis and digestive symptom restrict their further use.With the improvement of molecular cloning technique, it potentially become a new anti-tumor way that specifically downregulate the expression of EGFR via RNA interference (RNAi) with the adenovirus as a vector. Now there have developed a new technique of RNAi that is artificial microRNA (amiR), or second generation shRNA. Compared to siRNA and the first generation shRNA, artificial mciroRNA adds a natural framework to shRNA construction. This new RNAi is not only of high efficiency, but can be controlled by type two promoter which make it possible that specifically downregulate the expression of EGFR under the control of tissue specific promoter.On the basis of the above background, we plan to design an EGFR targeted artificial microRNA that is controlled under laryngeal carcinoma specific promoter with the adenovirus as a vector and then evaluate the anti-tumor effect.Objective:To construct a recombinant adenovirus vector with EGFR targeted artificial microRNA under the control of SLPI promoter, and then evaluate the safety and tumor-inhibiting effect both in vitro and in vivo.Methods:1、The shuttle plasmids of adenovirus pDC312-SLPI-EGFRamiR-pA and Ad-SLPI-GFP-pA were constructed and then separately cotransfected into HEK293with the backbone plasmid of pBGHlox (delta) E1,3Cre for adenovirus packaging. Recombinant adenovirus of Ad-SLPI-EGFRamiR and Ad-SLPI-GFP were identified by PCR and western blot. Recombinant adenovirus were amplified and purified by CsCl density gradient centrifugation. The titers for recombinant adenovirus were assessed by TCID50.2、The inhibition of the growth for Hep-2and HuVEC by recombinant adenoviruses in vitro was evaluated by the microscope, MTT and flow cytometry analysis.3、The antitumor activity and side effects in vivo by recombinant adenoviruses were observed in human laryngeal carcinoma xenograft in Nude Mice.Results:1Construction for the recombinant adenoviruses for Ad-SLPI-EGFRamiR, Ad-SLPI-GFPAn obvious cytopathic effect was observed in HEK293after about13days from cotransfection. Recombinant adenoviruses were then harvested and identified by PCR. The expected fragments of142bp、284bp、426bp could be found in the amplified products of the virus supernatants from Ad-SLPI-EGFRamiR and1482bp from Ad-SLPI-GFP. The recombinant adenoviruses were successfully constructed. After amplification, purification and concentration, the titers for Ad-SLPI-EGFRamiR and Ad-SLPI-GFP reached1×1010pfu/ml,6.3×109pfu/ml respectively, which was sufficient for future studies both in vivo and in vitro.Western blot showed that the expression of170kd EGFR was significantly reduced after72hours by the infection of Ad-SLPI-EGFRamiR in Hep-2. Meanwhile, strong signals of green fluorescence could be seen in a large number of Hep-2cells after72hours by the infection of Ad-SLPI-GFP.. However, it showed absent expression in HuVEC. These results indicated SLPI promoter could effectively downregulate the expression of EGFR in laryngeal carcinoma cells. The expression of green fluorescent protein was specifically restricted in Hep-2cells and absent in normal human umbilical vein endothelial cells of HuVEC. And72hours is the appropriate time for infection.2In vitro Study on cytotoxicity of Ad-SLPI-EGFRamiR to human laryngeal carcinoma cells Hep-22.1MTT AssayThe proliferation of Hep-2cells was well inhibited by Ad-SLPI-EGFRamiR after infecting72hours at the MOI level of50(inhibition rate:22.5%). while no significant inhibition was observed in HuVEC cells(inhibition rate:-4.2%).2.2Morphological changes by infecting with Ad-SLPI-EGFRamiRAfter72hours, the infected Hep-2cells showed an obvious change in morphology: cells became round and shrunk, some even floated in cluster. However, no significant morphological change was observed in the infected HuVEC cells.2.3Flow cytometry for quantitative analysis of apoptosisThe apoptosis rate in the infected Hep-2cells by Ad-SLPI-EGFRamiR for72hours at the MOI level of35and50was32.8%and31.8%respectively, while10.2%and10.0%for Ad-SLPI-GFP. For HuVEC, after72hours infection by Ad-SLPI-EGFRamiR and Ad-SLPI-GFP at the MOI level of35, the apoptosis rate was11.2%,9.2%respectively, while at the MOI level of50, the survival rate was15.5%,4.8%respectively.3The antitumor activity by Ad-SLPI-EGFRamiR in human laryngeal carcinoma xenograft in Nude Mice3.1The antitumor activity by Ad-SLPI-EGFRamiR in human laryngeal carcinoma in vivoThe average tumor volume in the Ad-SLPI-EGFRamiR group at the13th day was smaller than Ad-SLPI-GFP group and Gefitinib group, without significant statistical difference (P>0.05). The average growth rate of tumor volume of Ad-SLPI-EGFRamiR group was lower than Ad-SLPI-GFP group and Gefitinib group, also without significant statistical difference (P>0.05). By the end of the whole observation period (20days after first injection), the average tumor weight was lower than Ad-SLPI-GFP group and Gefitinib group, without significant statistical difference (P>0.05).3.2Observation on the side effect in vivo by recombinant adenovirusDuring the whole period of study, nude mice in Ad-SLPI-EGFRamiR group and Ad-SLPI-GFP group survived in good condition. There was no significant difference in the activities and diets among them. But some mice in Gefitinib suffered from diarrhea and anorexia. The average growth rate of weight for Ad-SLPI-EGFRamiR group was higher than Gefitinib with statistically difference (P<0.05), but was not statistically different from Ad-SLPI-GFP group (P>0.05).Conclusion: 1、Recombinant adenovirus of Ad-SLPI-EGFRamiR and Ad-SLPI-GFP could infect Hep-2cells specifically and effectively, inducing the expression of target genes. The expression of EGFR could be successfully downregulated by Ad-SLPI-EGFRamiR in Hep-2cells.2、Recombinant adenovirus of Ad-SLPI-EGFRamiR could well inhibit the growth of laryngeal carcinoma cells but not normal cells in vitro.3、In xenograft nude mice, recombinant adenovirus of Ad-SLPI-EGFRamiR had the tendency to inhibit tumor growth.4、In xenograft nude mice, Ad-SLPI-EGFRamiR was much safer than Gefitinib. |
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