Rna Interference Of Caveolin-1via Lentiviral Vector Inhibits Growth Of Hypopharyngeal Squamous Cell Carcinoma Fadu Cells In Vitro And In Vivo

IntroductionHypopharyngeal squamous cell carcinoma is one of the most common malignant cancer in the Department of Otolaryngology Head and Neck Surgery, with the characters of hidden location, high infiltration, easy submucosal spread of the primary lesion and multi-center growth. Because of the lack of obvious symptoms, specific signs and reliable diagnostic measures in early stage, the majority of patients accept the clinical diagnosis and treatments are already advanced, often involving the larynx, cervical esophagus and tongue and other organs. As hypopharynx is lymphatic-rich area, it is easy to occur cervical lymph nodes metastasis, even outside spread of the capsule and invading of surrounding tissues and organs, such as the carotid artery and other major blood vessels. Therefore, hypopharyngeal squamous cell carcinoma is one of the worst prognosis malignant tumors. As the special nature of hypopharyngeal carcinoma, surgical treatment may result in language, breathing and swallowing organ dysfunction, which seriously impact on the patients' life quality. While there are inadequate treatments, complications and pain during therapy for patients about radiotherapy and chemotherapy, therefore, new diagnostic methods based on the molecular biology and simple and effective treatments with less toxic side effects has become an urgent issue to be resolved. Biofilm is a major based structure of cell activity, which is closely related with cell malignant transformation and tumor evolution. In1950s, Yamada discovered and named caveolae under the electron microscope, which is the cell plasma membrane vesicles. In1972, Singer and Nicolson proposed "double-liquid lipid mosaic model". In1990s, researchers found that the double lipid membrane structure was heterogeneous, and caveolin was proved to be related to micro-structural changes of some cholesterol and sphingolipid-rich areas. These areas are known as lipid rafts, which are flat-shaped when there is no structural proteins. Lipid rafts turn to caveolae which is flask-shaped membrane structure after combinding with caveolin.Caveolin-1(CAV1) is an essential structural component of caveolae located at7q31.1, the molecular weight of which is about22kd. Currently, the relationship of CAV1and growth of tumors has become a research hotspot. CAV1has been observed as a candidate tumor suppressor in breast cancer, lung cancer, cervical cancer, ovarian cancer and sarcomas. Expression of CAV1was not found in follicular thyroid carcinoma. Over-expression of CAV1is associated with development of prostate, pancreatic cancers and esophageal squamous cell carcinoma, these evidences indicate that CAV1can act as a cancer-promoting gene. Therefore, the role of CAV1may vary depending on the tissue involved.In the present study, we successfully constructed the caveolin-1-RNAi-lentivirus (CAV1-RNAi-lentivirus) and received transfected FaDu cells. And the effects of CAV1on the growth of FaDu cells in vitro were examined. Then the effects of CAV1in vivo were investigated by injecting CAV1-RNAi-lentivirus construct into tumors created with FaDu cells in the HSCCs mouse model and proceeding xenografts experiments.ObjectiveTo construct the CAV1-RNAi-lentivirus and investigate the effects of CAV1on the growth of hypopharyngeal squamous cell carcinoma (HSCC) FaDu cells in vitro and in vivo. Methods1. A pair of complementary small hairpin RNA (shRNA) oligonucleotides targeting the Caveolin-1gene were designed, synthesized, annealed and inserted into linearized pGCSIL-GFP-lentivirus vector. The recombinant plasmid was identified by double restriction digestion with Age Ⅰ and EcoR Ⅰ and DNA sequencing. The recombinant plasmid and a lentivirus packaging mix were co-transfected into293T cells to obtain packaged lentivirus particles. Viral titer was then determined.2. The CAV1-RNAi-lentivirus construct was transfected into FaDu cells. And FaDu cells with stable CAV1expression were selected using G418. RT-PCR and western blotting analysis were used to test the expression of CAV1. Transferase-medisated dUTP nick-end labeling (TUNEL) assay was used to analyze cell apoptosis.3. The rCAV1-RNAi-lentivirus construct was injected in tumors created with FaDu cells in the HSCC mouse model, with the empty-vector lentivirus as a control, to investigate tumor inhibition effect. After cultured for32days, the mice were sacrificed and the tumor specimens were collected. Tumor growth curves and the inhibition rate were analysed. RT-PCR and immunehistochemistry were used to test CAV1expression in xenografts.Results1. RT-PCR and western blot analysis demonstrated successful construction of the CAV1-RNAi-lentivirus construct producing small hairpin RNA. The titer was1×108.2. After30days of transfection, we obtained FaDu cells with stable CAV1expression using G418selection. The decreased expression of CAV1was proved by results of RT-PCR and western blotting analysis(P<0.05). Downregulation of CAV1increased cell apoptosis in TUNUL(P<0.05).3. The average tumor weight and volume in mice treated with CAV1-RNAi-lentivirus was lower than that with control treatment (P<0.05). RT-PCR revealed weak positive expression of CAV1in CAV1-construct-treated xenografts (P<0.05). Immunohistochemistry revealed CAV1expression lower in CAV1-construct-treated mice than in controls, but the difference was not significant (P>0.05).ConclusionThe growth of HSCCs FaDu cells could be inhibited by recombinant CAV1-RNAi-lentivirus in vitro and in vivo. CAV1was considered to be a cancer-promoting gene in the growth of HSCCs FaDu cells.