| BackgroundTumor metastasis is an important biological feature of malignant tumors. Metastasis is a multi-stage process during which multifactorial and multiple genes to coordinate. Angiogenesis is the critical event for the process of tumor metastasis, and inhibition of angiogenesis could significantly inhibit tumor growth. Anti-angiogenic therapy is a new strategy that different from the conventional anti-tumor therapy, and it has become a hot topic in cancer research.Cancer stem cells play an improtant role in tumor metastasis. Lgr5 is a newly discovered independence molecular markers of the small intestine and colon stem cell. And as the Wnt signaling pathway and the Hedgehog signaling pathway target genes, Lgr5 in colorectal cancer, ovarian cancer, liver cancer and basal cell carcinomas were increased expression. So we can speculate that Lgr5 may play an important role in tumor development. But unfortunately, now the study on Lgr5 in cancer development process and its mechanism also rare. Based on the previous cancer stem cell study finding that Lgr5 were associated with microvessel density (MDV), we try to observe the effect of Lgr5 on tumor angiogenesis and to prelimenary investigate the possible mechanism. ObjectiveTo determine the effect of Lgr5 on tumor angiogenesis and investigate the possible mechanism.The result may provide the new therapy-target for inhibiting colorectal cancer angiogenesis and the basis of theoryMethod1. Four shRNA targeting Lgr5 mRNA and a negative control shRNA were designed and synthesized, named Lgr5 shRNA 2230, Lgr5 shRNA 2031, Lgr5 shRNA 874, Lgr5 shRNA 450 respectively. Lgr5 shRNA expression plasmids were constructed using gene engineering technique. Then the recombinant plasmids were tranfected into colon cancer cell line SW620 cells with lipofactimine 2000 transfection reagent. Expression of Lgr5 mRNA and protein were detected by RT-PCR and western blot respectively. The plasmid with the most high interference efficiency was selceted (Lgr5 shRNA874) for cell biological experiments and in vivo experiment.2. The Lgr5 shRNA 874 plasmid and NC plasmid were transfected with the SW620 cells.48 hours after transfection, the conditioned medium were collected and filtered. HMVEC endothelial cells were cultured in 6-well cluster culture plate, and the confluent cells were scratched with sterile tip to to obtain a denuded area of 1 mm wide. PBS rinse each well and the cultured with the conditioned medium respectively. Cell migration was observed under a 100×inverted microscope at Oh,12h and 24h time point.3. Transwell chamber purchased from Corning company. The membrane material under the bottom of the chamber is polyearbonate, and the chambers used for invasion assay have been laid for matrigel at the time of purchase, the chamber aperture is 8.0μm. Grow well cells were trypsinize into single cell suspension, washed with serum free medium two times, cell number were counted, and then serum free medium was used to adjust cell density into 1×106/ml. Add 100μl cell suspension into the chamber, and then add 500μl conditioned medium into the lower chamber, cultured in 37℃for 24 hours. Remove the chamber, aspirate culture medium, gently wipe cells and matrigel on the room side of the chamber with a cotton swab. Fixed in paraformaldehyde for 30 minutes, air diry and then violet crystal stainning for 5 min, gently rinse with distilled water several times, and then air dry. Counting the number of penetrate cell under an inverted microscope (100Ⅹ), randomly select five field counted.4.50μl matrigel were put into the well of 96-well culture plate and cover the bottom of the cluster well, then 1×104 HMVEC cells were planted in each well, and cultured with the conditioned medium respectively under conditon of 37℃,5% CO2. And tube formation situation was observed with inverted microscope at 8h time point.5. Five-day old fertilized eggs were sterilezed with bromogeramine, and then put into the clean bench with the air chamber end (blunt end) upside, a 1.5×1.5 cm window was opened. The shell membrane was removed, exposure the CAM (chorioallantoic membrane), As the carrier,2×2×2 mm3 gelatin sponge, which absorbed with 20μl conditioned medium, was put on the CAM. Only serum free medium in the carrier was the control group. The window was sealed and eggs were incubated again under condition of 38.5℃to 39℃with the relative humidity at 65% to 70%. Three days later, the CAM was observed under stereomicroscope and the neovascularization was quantified.6. The Lgr5 shRNA874 plasmid and NC plasmid were transiently transfeced into SW620 cells, and the Lgr5 and VEGF protein expression were detected by qRT-PCR and western blot respectively.Results1. Lgr5 shRNA expression plasmids were successfully construceted and identified by restriction enzyme and sequence analysis. Expression of Lgr5 mRNA and protein were down regulated by Lgr5 shRNA874 plasmid most efficiently.2. The relative ability of HMVEC endothelial cell migration was determined by wound healing assay. The wound in mock control group and NC control group were become great narrow in 24 hours. And in the Lgr5 shRNA874 group, cell migrated slower. The finding suggested that Lgr5 may have an effect on cell migration.3. Transwell invasion assay show that compared with NC group, the number of the cell penetrated through the membrane in the Lgr5 shRNA 874 group was significantly reduced (t=19.744, P<0.001)4. Tube formation assay shows that in NC group, the HMVEC endothelial cells were significantly cross link to form a grid structure. But in Lgr5 shRNA874 group, rare grid structures were observed (t=7.276, P=0.002).5. Chick embryo chorioallantoic membrane assay (CAM) shows that compared with SFM blank control group and NC group, the number of new blood vessels of chick embryo chorioallantoic membrane in the Lgr5 shRNA 874 group were significantly readuced (F=63,564, P=0.000), while there is no significant difference of number of new blood vessel in NC group and SFM group.6. After transient transfected with Lgr5 shRNA874 plasmid and NC plasmid into SW620 cells, the Lgr5 and VEGF protein in the Lgr5 shRNA 874 group both expressed in low level compared with NC group.ConclusionThe results confirmed that high expression level of Lgr5 could promote tumor angigenesis in colorectal cancer and result in tumor metastasis. Based on the cytology, using RNAi technology surpress the expression of Lgr5 could inhibit tumor angiogenesis in colorectal cancer. The results also show that Lgr5 control tumor angiogenesis through regulate VEGF serection of tumor cells. Therefore, Lgr5 can be used as a therapeutic target in colorectal cancer, through inhibit tumor angiogenesis to prevent tumor metastasis. |
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