Abstract
Object: To investigate the change of CXCR7 protein expression after CXCR7-shRNA transfered into human colon cancer cell SW620 which is lentivirus-mediated. Methods: 1. To design and synthesis three shRNA sequence and one negative control sequence of CXCR7. To synthesis and construct recombinant lentiviral vector by pSilencerTM 4. 1 system and CXCR7. To transfer the vector into HEK293T cell for packaging viral and to detect titer. 2. To transfer the three different recombinant lentiviral vector and one negative control vector into human colon cancer cell SW620. To detect expression and silence efficiency of CXCR7 mRNA by RT-PCR. The group expressing the best silence efficiency of CXCR7-shRNA is selected as expression vector for next experiment. 3. To detect the change of SW620 cell proliferation after CXCR7-shRNA transfection by MTT. 4. To detect the change of SW620 cell invasion and migration after CXCR7-shRNA transfection by cell scratching experiment. 5. To detect the SW620 protein expression after CXCR7-shRNA transfection by Western blot. Results: 1. Packaging the three different recombinant lentiviral vector and one negative control vector is successful by sequencing and titer is 3. 16 × 108TU / ml,4. 27 × 108TU / ml,3. 93 × 108TU / ml,2. 95 × 108TU / ml respectively. 2. Expression of CXCR7 mRNA in three positive group is lower than negative control group after transfection( P 0. 05). The inhibition rate of CXCR7-shRNA-1 to CXCR7 is higher than the another group. 3. The tumor cell proliferation is reduced after CXCR7-shRNA-1 transfection into SW620 cell and there was significant difference comparing to control group( P 0. 05). 4. The migration index of control group and positive group are( 49. 92 ± 6. 41) %,( 29. 13 ± 5. 38) % respectively 24 hours after cell scratching experiment and it has statistical significance( P 0. 05). The migration index of control group and positive group are( 96. 52 ± 7. 44) %,( 72. 03 ± 8. 29) % respectively 48h hours after cell scratching experiment and it has statistical significance( P 0. 05). 5. Expression of CXCR7 is reduced after transfection into SW620 cell comparing to empty virus vector group and control group and it has statistical significance( P 0. 05). Conclusion: Expression of CXCR7 mRNA and CXCR7 protein is down-regulated effectively after CXCR7-shRNA recombinant lentiviral vector transfection into SW620 cell and proliferation and migration of tumor cell is inhibited effectively. It is a good foundation on next study of colon cancer RNAi therapy with lentiviral which target CXCR7 / CXCL12 biological axis. And it is a new direction for colon cancer gene therapy.
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