Study On The Expression And Function Of ZNF750 In Oral Squamous Cell Carcinoma

BackgroundOral squamous cell carcinoma（OSCC）refers to the red lips of the mouth and the hard soft palate orthe 1/3 junction of tongue.It is the most common oral malignant tumor,which accounts for 80-90%of oral malignant tumors.Although the incidence of oral cancer has been highly variable all over the world,oral squamous cell carcinoma has ranked sixth of cancer,mainly depending on the country（and even some countries in the specific region）and gender.The main causes and inducing factors of oral squamous cell carcinoma are mainly smoking and drinking habits,and ultraviolet radiation（especially lip cancer）,but such factors as human papillomavirus（HPV）Candida infection,undernutrition and genetic susceptibility.In general,the 5year survival rate of oral squamous cell carcinoma was 50%.Early,curable lesions are rarely symptomatic;therefore,the prevention of fatal diseases needs to be found early through screening.Surgery plays a greater role in the treatment of most oral cancers,but the treatment is usually performed by surgery or radiotherapy.The deletion and mutation of zinc finger protein 750（ZNF750）is related to the biological characteristics of squamous epithelium.Zinc finger protein contains zinc finger structure,and the amino acid residue group of peptide chain is specifically combined with Zn²⁺to stabilize the polypeptide spatial configuration.The ZNF750gene（GenBank Accession No.NM₀24702）is encoded by a zinc finger protein.Bioinformatics analysis shows that the protein ZNF750 contains a ZnF-C₂H₂ type domain.Recent studies have shown that the abnormal expression of C2H2 zinc finger protein may cause different degrees of tumor occurrence.The knockout or interference of zinc finger protein related genes will have a certain effect on the proliferation and growth ability of oral squamous cell carcinoma cells.At present,the clinical cure rate of oral squamous cell carcinoma is still not high,so finding a gene with high sensitivity and specificity is very important for oral squamous cell carcinoma,and it is a new breakthrough for the treatment of oral squamous cell carcinomaIn this study,we first carried out on the ZNF750 bioinformatics analysis and prediction,and according to its design and synthesis of shRNA base sequence,using lentiviral vectors will be the effective integration of human tongue squamous cell carcinoma cell line CAL-27,the expression of the target sequence to achieve the effect of persistent,effective and interference from the finger the expression of ZNF-750 ZNF750 gene was observed by zinc,silent human tongue squamous cell carcinoma CAL-27 cell survival,proliferation and migration ability.The beta-actin for reference,the interference efficiency of transcription and interference of virus ZNF750 gene in cells by real time detection of PCR interference.Western blotting was used to detect the changes in the level of the expression of the corresponding zinc finger protein.The migratory movement and repair ability of the ZNF750 gene were observed by the scratch test.The effect of overexpression of ZNF750 on the invasiveness of cancer cells in vitro was tested by Transwell test.Routine MTT assay was used to detect the proliferation of cells under the condition that the ZNF750 gene was silenced.Meterial and Methods1.the carrier,strain and cell used in the experimentThis study used lentiviral vectors include packaging plasmid pCMV-△8.2,and pCMV-VSV-G;cloning bacteria were Escherichia coli DH5 alpha;lentivirus packaging in the experiment with HEK293T cells by other labs offered by the interference of human tongue squamous cell carcinoma cell line CAL-27 cells derived from Tianjin Saier biological company purchase.2.Bioinformatics analysis of ZNF750 and the design of shRNAApplication of NCBI query ZNF750 nucleotide and protein sequences,the prediction of physicochemical properties using the ExPASy server,the ProtScale program analysis of hydrophobicity,TMHMM Server v.2.0 predicted signal peptide,transmembrane region,protein structure prediction and SMART prediction server domain using SWISS-MODEL online.According to the results of NCBI query,the shRNA target of ZNF750 was designed and sent to the Shanghai Sangon biological company for synthesis,and then lentiviral vector was constructed to carry out the experiment.3.RNA interference and interference effect analysis for ZNF750The infected CAL-27 was infected by the packaged lentivirus,and the infection efficiency of lentivirus was estimated by fluorescence microscopy.Using qPCR and Western blotting after interference experiment on the transcriptional level of ZNF750and the expression level of squamous cell carcinoma;wound healing assay.The migration and restoration ability;influence Transwell expression by ZNF750 after experimental observation on cell invasion in vitro;value-added ability of cell MTT assay after interference.4.statistical treatmentSPSS17.0 statistics software and Graphpad Prism software are used to integrate,process and analyze the experimental data.Using the data form of mean+standard deviation,we used the statistical processing methods reasonably,including 22comparison,chi square test and ANOVA,comparing the experimental results between the experimental group and the control group,and the normal group,the test level isα=0.05.ResultsBioinformatics analysis of ZNF750 zinc finger protein and the effect of silencing on oral squamous cell carcinoma cellsThe results of ZNF750 gene analysis by bioinformatics analysis are as follows:ZNF750 is full-length 3218bp,and the size of encoded protein is about 79kDa,which contains a known ZnF-C2H2 functional domain,which contains no signal peptide,no transmembrane domain,and protein has no obvious hydrophobicity.The lentiviral vector was successfully constructed using cloning and expression technology.The restriction enzyme and colony PCR showed that the band was in the correct position and was sent to Shanghai Shenggong for sequencing.The sequencing results showed that the lentiviral vector was correctly constructed.After the successful lentivirus packaged successfully infected squamous cell carcinoma CAL-27,qPCR and Western blotting experiments were performed.The experimental results showed that the level of ZNF750 gene transcription and protein expression in CAL-27 cells was significantly more than that in the normal control group after interference by lentivirus.Decline.The scratch test results showed that the scratches were significantly repaired and the width was significantly reduced in the interference group compared to the control group,but scratches were also partially repaired in the blank group and the empty plasmid control group.When the photographs were taken again after 48 hours of culture,the scores of scratches in the blank group and the PM7.1 empty plasmid control group were nearly the same,while the scratches in the PM7.1-ZNF750sh experiment group were not completely repaired,but they were not completely repaired.It is the highest degree of repair in the three groups.It shows that the silence of ZNF750 will lead to enhanced migration and repair of squamous cell carcinoma cells.Transwell experiments showed that when the ZNF750 gene is silenced,the invasiveness of the cancer cells can be increased.The MTT assay suggested that down-regulating the expression of ZNF750 protein was beneficial to promote the proliferation and proliferation of squamous cell carcinoma cells.Conclusion1.This study designed shRNA based on the ZNF750 gene sequence and successfully constructed a lentivirus vector to interfere with the ZNF750 gene in CAL-27 cells.2.ZNF750 zinc finger protein can be used as a new breakthrough in the diagnosis and treatment of oral squamous cell carcinoma,which lays the foundation for the further study of ZNF750 gene regulation mechanism.