The Identification And Functional Analysis Of GOLT1A-Kiss1 Fusion Gene In SACC

Fusion gene is a new gene formed by the fusion of some or all sequences of two genes.They share the same set of regulatory sequences(promoters,enhancers,terminators,etc.).The most common mechanism for the formation of fusion genes is chromosome rearrangement,which includes chromosomal translocation,deletion,trans-splicing,and inversion.Fusion proteins are the expression products of fusion genes.Cancer metastasis is the fact that tumor cells from the primary tumor invade into lymphatic vessels,blood vessels or other pathways and continue to grow to form the same type of tumor as the primary tumor.Generally,tumor metastasis occurs in the later stage of the disease,but it can also occur early in lung cancer,breast cancer,thyroid cancer,and prostate cancer.It has been found that fusion proteins produced by certain specific fusion genes promote tumorigenesis and cell proliferation.For the GOLT1A-KISS1 fusion gene,it is unclear whether their fusion will have a certain role or what role in tumorigenesis.Objective: Our study is to systematically study the specific gene fusion events that occur in SACC metastasis and explain the specific mechanisms involved in tumor metastasis,and to develop molecular markers that can be used for early clinical diagnosis of tumor metastasis and provide for drug targets of suppressing tumor metastasis.Methods: We confirmed Fusion genes associated with metastasis by using RNA-Seq high-throughput sequencing technology and bioinformatics,at the same time we used sanger sequencing,PCR and other techniques to re-confirm metastasis-associated fusion genes,and confirmed their specificity in highly metastatic tumor cells;then the full-length c DNA sequence of the fusion gene was cloned by RACE and other techniques,analyzed by sequence analysis software,and GOLT1A-Kiss1 fusion gene and KISS1 gene related vectors were constructed by molecular cloning technology;finally,the constructed vector was transfected into cells using transfection method,Western blotting was used to detect the expression of KISS1 protein,and dual luciferase reporter gene system was used to detect the effect of fusion genes' out of frame u AUGs on protein expression.Results: The GOLT1A-Kiss1 gene fusion event was confirmed by PCR and sanger sequencing,and confirmed to be present in tumor cells of the salivary adenoid cystic carcinoma(SACC)high metastatic cell line ACC-M;the full-length c DNA sequence of the fusion gene was obtained by RACE and other techniques,at the same time,three types of the fusion gene were discovered after cloning;p GL3-Fusion1-KISS1-GFP,p GL3-Fusion2-KISS1-GFP,p GL3-Fusion3-KISS1-GFP,p GL3-KISS1-GFP,Fusion1-Luc,Fusion2-Luc,Fusion3-Luc,KISS1-Luc,and their fusion gene u AUGs were all mutated after the carrier were successfully constructed using molecular cloning techniques;we used PCR and Western blotting to test the KISS1 gene m RNA levels and endogenous protein expression levels,which suggested the high m RNA level and low protein expression level in the metastatic cell line ACC-M.Using the dual luciferase assay showed that the luciferase activity was up-regulated when all the u AUGs of the fusion gene were mutated.Conclusion: The identification of GOLT1A-Kiss1 provided a novel mechanism of gene fusion : it suppressed the expression of the tumor suppressor gene Kiss1 by gene fusion altering the genes' 5'UTR,thereby activating cancer progression or promoting tumor metastasis.At the same time,in many tumor models including adenoid cystic carcinoma,non-small cell lung cancer,breast cancer and liver cancer,the expression of GOLT1A-Kiss1 was highly correlated with tumor metastasis,which is promising as a clinical stage diagnosis and new targets of drug development for multiple tumors.