Regulatory Effect And Molecular Mechanism Of T-box Transcription Factor Brachyury In Apoptosis Of Chordoma Cells

Objective The study on the relationship between the specific high expression of theBrachyury gene in chordoma cells with the apoptosis of chordoma cell. Using RNAinterference, we constructed the recombinants mediated by lentivirus vectorpLenti-Bra-shRNA to express si-Brachyury. After transfected these recombinants tochordoma cell line CHZP, we studied their resistance to Paclitaxel. We expected toexplore the possible molecular mechanism of it. This research involved three parts:(1)Construction of Brachyury gene interference v ector and study on its effect on theapoptosis.(2) Construction of si-Brachyury recombinant lentivirus and the Stablytransfected cell lines of CHZP.(3) Drug resistance of CHZP-Bra-shRNA cells.Methods Four interfering sites of Brachyury gene were designed respectively, thenchemosynthesised siRNA fragments and transfected to human Non-small cell lungcancer cell H460. Brachyury gene expression in H460cell were detected by PCR aftertransfection. Using Paclitaxel on the Brachyury RNAi H460cell, we studied the drugresistance of it. Packed the lentivirus vector using the efficient interfering sites, transfectedinto293T cell for amplification and the transfection efficiency was high. Results provedthat the plasmids could transfect293T with high efficiency and express siRNA stably.Using the pLenti-Bra-shRNA and the pLenti-Bra-NC to Infection the chordoma celllines CHZP, treating by blasticidin to screen, we got the stable transfection cell lineCHZP-Bra-shRNA and CHZP-NC. The suppression of Brachyury gene in mRNA level andprotein level were determined by real-time PCR and Western Blot respectively.Different concentration gradient paclitaxel solution treatment was used on thechordoma cells line CHZP, screening the best concentration of paclitaxel forthe apoptosis of chordoma. CHZP-Bra-shRNA and CHZP-NC cell were treated withpaclitaxel for24hours, then Dyed with the Annexinâ…¤-FITC/PI. Apoptotic cells weredetermined by FACS. We examined the changes in the expression of genes related withapoptosis CHZP-Bra-shRNA, and CHZP-NC after the decline of the Brachyury geneexpression.Results Designed siRNA fragment of Brachyury gene, after transfection into H460cell Brachyury gene was silenced specifically and efficiently. The best fragment isBra-siR-4(1093T). We observed that the Brachyury gene interference apoptosis wassignificantly increased in the H460cell which was Brachyury gene interferenceafter the application of drug. We packed one lentivirus vector using the efficient interferingsite, transfected into293T cell for amplification. CHZP was transfected bypLenti-Bra-shRNA and pLenti-Bra-NC (blank vector), the transfection efficiencies ofCHZP-Bra-shRNA and CHZP-NC were determined by real-time PCR and Western blotafter transfection. The suppression efficiency of CHZP-Bra-shRNA was obvious.CHZP-Bra-shRNA and CHZP-NC cell were treated with paclitaxel for24hours, thenDyed with the Annexinâ…¤-FITC/PI. The FACS results showed that, the sensitivity of CHZP-Bra-shRNA to paclitaxel was boosted. The rate of apoptosis was78.38%inCHZP-Bra-shRNA, compared to4.1%in CHZP-NC. For apoptosis-related genes ofbax, bcl-2and bcl-xL, PCR analysis showed that the bcl-2gene expression of theinterference group was significantly down.Conclusions The first time we used the lentivirus mediated RNAi technology in apoptosisresearch of chordoma, and First time we proved the relationship between genes Brachyuryand chordoma cells apoptosis in cytology and molecular biology level. For basic researchand clinical treatment of tumors, this study provides a new way of thinking, the resultssuggest that Brachyury genes play a crucial role in the occurrence of chordoma.