The Preliminary Study Of KIF5B-RET Fusion Gene Transfect BEAS-2B Cell Line Mediated By Lentivirus

Objective:Lung cancer is still the leading cause of cancer death in the world.Chemotherapy is the main measure of advanced lung cancer, but its efficiency has reached platform. As the development of molecular biology and the improvement of test facility, molecular target therapy for NSCLC has become the highlight in the recent years. The most wildly studied signaling is EGFR mutation, serial clinical trials has showed EGFR-TKI is in effective for patients with EGFR mutation. The gene translocation or gene fusion is also play an important part in the development of lung cancer besides gene mutation. KIF5B-RET fusion gene is the latest fusion gene in lung cancer after ALK、ROS fusion gene, some basic research has showed KIF5B-RET fusion gene can promote cell proliferation and is tumorgenic. Yet either the functional study or the drug sensitivity study of KIF5B-RET fusion gene is limited in mouse cell, the character of this gene in human cell is still unknown. BEAS-2B cell, which is the ideal cell model for lung cancer in vitro, is the normal bronchial epithelial cell, it can exclude the interference of other proto-oncogene of lung cancer cell, and the report of using this cell to research KIF5B-RET fusion gene is still lacked. The main purpose of this study is to constructa KIF5B-RET fusion gene positive BEAS-2B mediated by lentivirus, and explore the apoptosis in the transfected cells, which is useful for the functional study and the drug sensitivity study in the future.Methods:According to the BamH I cleavage site near the break point of K1F5B-RET fusion gene, we amplificated the KIF5B gene section and the RET section and then verified by electrophoresis. Sections separately connected with pEASY-Blunt Zero Cloning vector were converted into competent cell DH5a, then were verified by cleavage electrophoresis and sequence after cloning and distraction of the plasmid. The pCDH-CMV-MCS lentivirus expression vector together with the KIF5B plasmid、RET plasmid were connected and then converted into competent cell DH5a, after cloning and distraction of the plasmid, we performed cleavage electrophoresis and sequence to verify. The four plasmid system including pMDLg/pRRE, pRSV-REV、pMD2.G and KIF5B-RET-pCDH was used to transfect293T cell, the virus supernatant was collected which was to transfect BEAS-2B cell. The RNA and protein of BEAS-2B cell after transfected for72hours were distracted for RT-PCR and Western blot. We detected the apoptosis in the transfected cells by flow cytometry.Results:RET gene has two isomer, variant2and variant4, our study get KIF5B section RET variant2section and variant4section through PCR, whose electrophoresis strips match up to the theoretical size. The electrophoresis strips of plasm id pEasy-KIF5B、 pEasy-RET V2and pEasy-RET V4match up to the theoretical size,and their sequencing outcome are in accordance with the theoretical sequence. The electrophoresis strips of plasmid KIF5B-RET V2-pCDH、KIF5B-RETV4-pCDH match up to the theoretical size,and their sequencing outcome are in accordance with the theoretical sequence. The fluorescence intensity of293T cell and BEAS-2B cell after48hours transfected with the recombinantlentivirus vector pCDH-CMV-MCS-EGFP are strong,which cover more than90%.The RT-PCR method shows than the fusion gene mRNA are over erpressed in the BEAS-2B cell after transfected.WB shows that KIF5B-RET fusion protein is over expressed in BEAS-2B cell transfected by KIF5B-RET v2-PCDH lentiviirus vector.After12h serum starved, the proportion of KIF5B-RET v2transfected cells are5.49%、5.98%、5.6%, and the EGFP transfected cells are14.56%、15.28%、15.17%, the difference is of significance in statistics science.Conclusion:1. We successfully construct the KIF5B-RET-PCDH lentivirus expression vector.2. we successfully construct the stable BEAS-2B cell line with KIF5B-RET fusion gene positive.3.KIF5B-RET fusion gene can inhibit the apoptosis in BEAS-2B cell.