|
|
|
| Experimental Study of Specific Targeted Inhibition from RNAi Silencing CXCR7 to Human Colon Cancer Cell SW620 |
| WANG Xin, CHEN Ling, SUN Fei, LU Hang |
| The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, China |
|
|
|
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 3.16×108 TU/ml, 4.27×108 TU/ml, 3.93×108 TU/ml, 2.95×108 TU/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.
|
|
Received: 17 October 2013
Published: 25 February 2014
|
|
|
|
|
[1] 王宁, 孙婷婷, 郑荣寿, 等. 中国2009年结直肠癌发病和死亡资料分析. 中国肿瘤, 2013, 22(7): 515-520. Wang N,Sun T T ,Zheng R S ,et al.An analysis of incidence and mortality of colorectal cancer in China,2009. China Cancer,2013,22(7):515-520.
[2] Li F Y, Lai M D. Colorectal cancer, one entity or three. J Zhejiang Univ Sci B, 2009, 10(3): 219-229.
[3] Szajda S D, Jankowska A, Zwierz K. Carbohydrate markers in colon carcinoma. Dis Markers, 2008, 25(4-5): 233-242.
[4] Lu H, Sun H Z, Li H, et al. The clinicopathological significance of Bmi-1 expression in pathogenesis and progression of gastric carcinomas. Asian Pac J Cancer Prev, 2012, 13(7): 3437-3441.
[5] Frieden T R, Myers J E, Krauskopf M S, et al. A public health approach to winning the war against cancer. Oncologist, 2008, 13(12): 1306-1313.
[6] Wang Y, Li G, Stanco A, et al. CXCR4 and CXCR7 have distinct functions in regulating interneuron migration. Neuron, 2011, 69(1):61-76.
[7] Liu L, Zhao X, Zhu X, et al. Decreased expression of miR-430 promotes the development of bladder cancer via the upregulation of CXCR7. Mol Med Rep, 2013, 8(1):140-146.
[8] Schanz A, Baston-Bust D, Krussel J S, et al. CXCR7 and syndecan-4 are potential receptors for CXCL12 in human cytotrophoblasts. J Reprod Immunol, 2011, 89(1):18-25.
[9] Xue T C, Chen R X, Ren Z G, et al. Transmembrane receptor CXCR7 increases the risk of extrahepatic metastasis of relatively well-differentiated hepatocellular carcinoma through upregulation of osteopontin. Oncol Rep, 2013, 30(1):105-110.
[10] Zeng Z, Zhang C, Chen J. Lentivirus-mediated RNA interference of DC-STAMP expression inhibits the fusion and resorptive activity of human osteoclasts. J Bone Miner Metab, 2013, 31(4): 409-416.
[11] Liu L, Zhang N, Liu J, et al. Lentivirus-mediated siRNA interference targeting SGO-1 inhibits human NSCLC cell growth. Tumour Biol, 2012, 33(2): 515-521.
[12] Ying J, Xu Q, Zhang G, et al. The expression of CXCL12 and CXCR4 in gastric cancer and their correlation to lymph node metastasis. Med Oncol, 2012, 29(3): 1716-1722.
[13] Balabanian K, Lagane B, Infantino S, et al. The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J Biol Chem, 2005, 280(42): 35760-35766.
[14] Wang J, Shiozawa Y, Wang J, et al. The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer. J Biol Chem, 2008, 283(7): 4283-4294.
[15] Miao Z, Luker K E, Summers B C, et al. CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature. Proc Natl Acad Sci U S A, 2007, 104(40): 15735-15740.
[16] Raggo C, Ruhl R, McAllister S, et al. Novel cellular genes essential for transformation of endothelial cells by Kaposi's sarcoma-associated herpesvirus. Cancer Res, 2005, 65(12): 5084-5095.
[17] Elbashir S M, Lendeckel W, Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev, 2001, 15(2): 188-200.
[18] Mohan R R, Rodier J T, Sharma A. Corneal gene therapy: basic science and translational perspective. Ocul Surf, 2013, 11(3): 150-164.
[19] Miyata Y, Iwata T, Ohba K, et al. Expression of matrix metalloproteinase-7 on cancer cells and tissue endothelial cells in renal cell carcinoma: prognostic implications and clinical significance for invasion and metastasis. Clin Cancer Res, 2006, 12(23):6998-7003. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
