Effects Of FZD2 Gene Regulation On Cell Function Of Human Tongue Squamous Cell Carcinoma Cell Line

Oral squamous cell carcinoma is the most common malignant tumor in the head and neck,also one of the 10 most common malignancies in humans.The incidence of oral squamous cell carcinoma is about 80% in the incidence of oral malignancy.The incidence of oral squamous cell carcinoma has increased and the age of incidence is younger and younger in recent years.Oral squamous cell,especially the tongue cancer has a high transfer characteristics because of its special anatomical and physiological characteristics.The five-year survival of patients with oral squamous cell carcinoma is still low,although the measures of treatment of tongue cancer are constantly improving with the progress of science and technology,especially for patients with metastasis.Therefore,explore the molecule of invasion and metastasis of tongue cancer from molecular level,making people clearer about the mechanism of invasion and metastasis of tongue cancer.Find the measure of inhibit the invasion and metastasis and explore the effective treatment of oral squamous cell carcinoma,improve patients' survival rate.Objective Research the effect of FZD2 gene expression on the proliferation and invasion of tongue cancer,and provide some theoretical basis and clinical basis for the treatment of tongue cancer.Methods Part I Design and synthesize siRNA-FZD2 aording to FZD2 gene sequence in vitro and transfect CAL-27 cells.The expression of FZD2 on mRNA was examined by Real-time PCR,the expression of FZD2 on protein was examined by Western Blot.The proliferation of CAL-27 cells was tested by CCK8 and plate cloning assays,wound healing assays and transwell were used to evaluate cell metastasis and invasion,tongue cancer xenograft model was used the changes in tumor formation.Part II Construct overexpression plasmid of FZD2.Transfect CAL-27 cellswith lipofectemine3000 after the recombinant plasmids were tested by restriction endonuclease digestion and gene Sequenced.The expression of FZD2 on mRNA was examined by Real-time PCR,the expression of FZD2 on protein was examined by Western Blot.The proliferation of CAL-27 cells was tested by CCK8 and plate cloning assays,wound healing assays and transwell were used to evaluate cell metastasis and invasion.Results Part I Results of real-time PCR revealed that the target gene expressed less.After 48 hours when CAL-27 cell was transfected with siRNA-FZD2 and NC,Western blot analysis showed that the protein expressions of siRNA group was significantly decreased compared to the negative control group.The growth of CAL-27 cells transfected with siRNA-FZD2 was significantly suppressed in CCK8 and plate cloning assays.The metastasis and invasion ability of CAL-27 cells were verified suppressed in wound healing and transwell assays.The tumor weight and tumor volume of siRNA group less than the NC group.Part II The construct of recombinant plasmid was successed.The result of real-time PCR revealed that the target gene expressed more.After 72 hours when CAL-27 cells were transfected with pCDH-FZD2 and Vector,the expression of FZD2 on protein increased compared to the Vector group.The growth of CAL-27 cells transfected with pCDH-FZD2 was raised in CCK8 and plate cloning assays.The metastasis and invasion ability of CAL-27 cells were raised in wound healing assays and transwell assays.Conclusions The proliferation,migration and invasion of CAL-27 cells were decreased by FZD2 knockdown and increased by FZD2 overexpression.These results showed that FZD2 gene could promote the proliferation and invasion of human tongue squamous carcinoma.FZD2 may play a key role in diagnosis,prognosis and gene therapy.