Splicing Variation And Function Of Gene U XV Exon In Oral Squamous Cell Carcinoma Cells

Oral and maxillofacial cancer accounts for about 3%-5%of systemic malignancies,especially Oral Squamous Cell Carcinoma(OSCC),which is one of the most invasive head and neck cancers with a high incidence and mortality,seriously affecting chewing,swallowing,speeching and threatening health.Although a variety of clinical strategies have been used to treat OSCC,including surgery,chemotherapy,radiotherapy,etc.The5-year survival rate of patients is still less than 50%.The rate can increase to 85%if diagnosed and treated early.The survival rate has not improved significantly in the past50 years,mainly due to the failure to find ideal early warning markers and therapeutic targets.Currently,OSCC related oncogenes have been reported,such as Smad7,CLDN,Kank1,etc.But these genes have low specificity and sensitivity.Therefore,in order to stop the progression of cancer,reduce the mortality and prolong patients’life,it is urgent to explore accurate and effective molecular markers of OSCC which are helpful in early screening and diagnosis.Alternative splicing is an important mechanism to regulate gene expression and produce proteome diversity.It changes the splicing way of RNA precursors,which changes the sequence of introns or exons contained in the mature RNA produced,and then leads to splicing variation.In recent years,many reports have proved that alternative splicing of genes is closely related to the oncogenesis,development and prognosis of a variety of tumors,and is considered not only an important marker of the oncogenesis and development of tumors,but also an effective target for treatment.Alternative splicing has always been a focus of research at home and abroad.This study aims to explore the function and mechanism of alternative splicing in the oncogenesis and development of OSCC,so as to provide new clues for seeking ideal early warning markers and therapeutic targets of OSCC.Objectives:To detect the biological functions of Gene X and Gene U spliceosomes in OSCC cells,study the function of alternative splicing especially 15 exon of Gene U splicing mutation in the development of OSCC,then explore the molecular mechanism of the development of OSCC,look for molecular markers and provide new clues to the effective therapeutic targets.Methods:1.Tongue squamous cell carcinoma cells CAL27 and SCC25 were used,conventional cell culture methods were used,including cell resuscitation,passage and cryopreservation.2.Extract proteins and RNAs from cells.Western Blot and qPCR were used to detect the expression of Gene X in OSCC cells.Verifying cells constructed in the earlier stage of this study,CAL27-Gene X,CAL27-ctrl,SCC25 sh-Gene X and SCC25 sh-ctrl.3.qPCR,agarose gel electrophoresis,gel extraction,purification and sequencing were carried out to detect the expression of 15 exon of Gene U in different OSCC cells and to explore the regulation mechanism of variable splicing of Gene U.4.qPCR was used to verify cells with the variable spliceosome of the 15 exon of Gene U constructed earlier,CAL27 U-DE15 and CAL27 U-E15.5.The cell cloning experiment was carried out to determine the cloning and formation ability of CAL27-ctrl and CAL27-Gene X cells,as well as CAL27 U-DE15 and CAL27U-E15 cells.Cells were seeded into 6-well plates,1000 cells per well,terminated the culture 13 days later.The number of clones was observed and counted after fixation and staining.6.The wound and healing assay was used to measure the migration ability of cells.CAL27-ctrl and CAL27-Gene X cells were seeded into 6-well plate,8×10~5 cells per well.Waited until the cells adhered to the wall,drew a vertical line every 1cm,and three parallel lines in total.Selected nine fields of vision under the microscope at 0h and 24h respectively,observed and photographed,and the cell migration rate was statistically analyzed.7.The tumorigenesis ability of cells was detected by subcutaneous tumorigenesis experiment in nude mice.CAL27 U-DE15 and CAL27 U-E15 cells were respectively injected subcutaneously into nude mice,each of which was 3.5×10~6 cells.Tumors were collected 20days later,measured,photographed and statistically analyzed,so as to study the effects of the expression of 15 exon of Gene U in tumorigenesis.Results:1.qPCR and Western Blot results showed that Gene X was highly and low expressed in the constructed CAL27 and SCC25 cells,thus cells were constructed successfully.2.The gel electrophoresis and sequencing results of qPCR products indicated that the expression of 15 exon of Gene U was diverse in SCC25 cells,there was a regulatory relationship between the splicing of Gene X and 15 exon of Gene U.3.The 15 exon of Gene U was stably expressed in different OSCC spliceosomes constructed.The cell lines of CAL27 U-E15 containing 15 exon and CAL27 U-DE15without 15 exon were constructed successfully.4.The cell cloning experiment showed that the clone formation number of highly expressed Gene X cells was 4.7 times higher than that of control cells(p<0.0001),and the difference was statistically significant.The highly expressed Gene X significantly promoted the clone formation ability of OSCC cells.The clone formation number in CAL27 U-E15 was 6 times higher than that without 15 exon,and the difference was statistically significant(p<0.0001).It was suggested that the expression of 15 exon of Gene U significantly promoted the clone formation ability of OSCC cells.5.The migration rate of CAL27-Gene X cells was 2.3 times higher than that of CAL27-ctrl cells showed by the wound and healing assay,p<0.0001,and the difference was statistically significant.The high expression of Gene X significantly promoted the migration ability of OSCC cells.6.Subcutaneous tumorigenesis experiments in nude mice showed that the volume of tumors formed by CAL27 U-E15 cells was 2.5 times higher than that in CAL27 U-DE15cells,the difference was significant(p=0.0003).The weight of tumors formed by CAL27U-E15 cells was 1.8 times higher than that of in CAL27 U-DE15,the difference was significant(p=0.0017).It was suggested that the expression of 15 exon of Gene U significantly promoted the subcutaneous tumorigenesis ability of OSCC cells.Conclusions:Gene X and 15 exon of Gene U play an important role in the oncogenesis and development of OSCC,which are expected to be effective molecular markers and treatment targets of OSCC.Moreover,Gene X regulates the alternative splicing of 15exon of Gene U,which is one of the important mechanisms of Gene X participates in the oncogenesis and development of OSCC.