Lgr5Regulates Metastasis Of Colorectal Carcinoma Through VEGF-mediated Angiogenesis

BackgroundColorectal carcinoma has become one of malignant tumors with the fastest growing incidence in our country. However, most of colorectal carcinoma-associated deaths result from metastatic carcinoma.Recently, to block tumor metastasis and reduce the mortality of metastatic carcinoma, more and more researches focus on the tumor metastasis and explore its underlying mechanisms. Tumor angiogenesis is an important process in tumor growth and metastatic progression. VEGF is one of the most important and best characterized angiogenic factors, even in the process of tumor angiogenesis. To date, it has been revealed that it’s effective to inhibit tumor angiogenesis tumorigenesis through blocking expression of VEGF, but when it comes to metastasis of colorectal carcinoma, the situation is not so satisfactory. Therefore it’s necessary to further study potential mechanisms of VEGF in tumor development and metastasis.Recently, it has been proved that Igr5is an vital stem cell marker of the small intestine, colon, hair follicle and so on, what’s more, it also overexpresses in some solid tumor including hepatocellular carcioma, colorectal carcinoma, ovarian cancer, basal cell carcinoma and gastric carcinoma. Lgr5is a downstream target for the Wnt signaling pathway and functions as a negative regulator of Wnt signaling pathway,which regulates cell growth, mobility and differentiation. Ever-increasing researches reveal that Lgr5is a candidate marker for colorectal cancer stem cells （CSC）.In addtion, Lgr5has been applied to screen colorectal cancer stem cells along with other confirmed stem cell markers. However, the underlying mechanisms for the role of Igr5in colorectal carcinoma still remain elusive.Our previous research revealed that knocking down expression of Lgr5inhibited angiogenesis in colorectal carcinoma.ObjectiveWith the purpose of avoiding results by chance in loss of Lgr5and futhering to explore the role of Lgr5in development and metastasis in colorectal carcinoma, and the effect of Lgr5on VEGF and related mechanism, we designed and conducted this experiment. What’s the clinical meaning is to supply theoretical basis to the target treatment of Lgr5in colorectal carcinoma.Method1. Construction of the Lgr5expression plasmid and stable transfectionAccording to the information of a full-length human Lgr5gene sequence （NM₀03667）, the primer was designed, and amplified into the PCR production with the length of2745bps.The PCR product was cloned into the pcDNA3.1（+） vector to create the pcDNA3.1（+）-Lgr5plasmid. The orientation of the insert and the sequence of cDNA were verified by sequencing. Cultured SW480cells were transfected with the recombinant plasmids and the empty vector using the lipofectamine2000reagent according to the manufacturer’s instructions. Transfectants were selected in medium containing1000ug-mL G418.2-3weeks later, monoclonal cells were appeared and cultivate them with600ug/mL G418. After selection and isolation of stably transfected clones, the clones were analyzed for Lgr5expression using western blotting and qRT-PCR..2. Western blottingTotal proteins in cells were extracted and then quantified by BCA protein quantitative kit.20μg total proteins were loaded onto a10%SDS-polyacrylamide gel for electrophoresis, transferred onto PVDF membranes, blocked in5%non-fat milk for1h at room temperature, incubated overnight at4℃in a primary antibody （Lgr51:500, VEGF1:200, GAPDH1:1000,β-actin1:1000） and incubated secondary antibodies for1h at room temperature. Immunoreactivity was detected ECL chemiluminescence.3. Real-time fluorescent quantitative PCR （qRT-PCR）Total RNA in cells and tissues was extracted with Trizol reagent, and2ug total RNA was subjected to the first-strand cDNA synthesis with M-MLV enzyme according to the manufacturer’s instructions. Real-time fluorescent quantitative PCR was performed with SYBR Premix Ex Taq kit （Takara）4. Immunohistochemistry （IHC）58cases of colorectal carcinoma tissues and8cases of metastatic carcinoma tissues were collrected from Nanfang hospital. Formalin-fixed paraffin-embedded tissue sections were dewaxed in xylene, rehydrated with alcohol and repaired with0.01M sodium citrate buffer （pH6.0） at the high temperature for15minutes in the microwave oven. S-P Ultra-Sensitivekit was used.The slides were subsequently incubated with the primary antibody at4℃overnight （Lgr51:50>CD311:50）,dyed with DAB substrate chromogen, and followed by haematoxylin counterstaining.5.HEFormalin-fixed paraffin-embedded tissue sections were dewaxed in xylene, rehydrated with alcohol. Hematoxylin stained the cell nuclei for5minutes and eosin stained the cell plasma for seconds to several minutes.The stained tissues were observed and photographed by optical microscope.6.Transwell migration and invasion assaysTransfected cells were resuspended in serum-free RPMI-1640medium at a density of1x106/ml.100μl cell suspension and500μl RPMI-1640containing10%FBS were respectively added to each upper and the matched lower chamber.After24h, cells in the upper chamber were fixed by4%paraformaldehyde and stained by crystal violet. Non-invading cells were removed using a cotton swab. Five random fields for each insert were counted. For invasion assay, the difference from migration assay was that there was matrigel in the insert chamber.7.Tube formation assayConditional culture medium was collected after48h transient transfection and through filtering with0.22μm pore size.50μl matrigel was added to each well in96-well plate and concreted at37℃for1hours.Then1×104HMVEC cells were planted into each well and cultured in conditional culture medium. Tube formation situation was observed by optical microscope at Oh、2h、4h、8h and the numbers of complete tube was counted.8.Elisa assayAn Elisa kit （Cusabio, China） was used to detect the VEGF concentration in the recombinant cell supernatant following the manufacturer’s instructions.9.AOM/DSS modelTwenty female balb/c mice （5-6weeks old） were randomly devided into two groups. Inject intraperitoneally AOM with concentration of1.25mg/ml and amount of1.25ml/ml for the experimental group or sterile isotonic saline for the control group. The control group didn’t undergo special treatment. The experimental group was treated as follows. Mice were fed with normal water at the first week,water with 2.5%DSS at the second week, normal water at the third and forth weeks. And the process from week2to week4was seen as one cycle,which was repeated twice from week5to week10. All mice were sacrificed at week17and colon tissues were fixed by formalin, embedded by paraffin and followed by haematoxylin-eosin staining. In addtion, total RNA in colon tissues was extracted and mRNA of Lgr5and VEGF was quantified by qRT-PCR.10. Tumorigenicity and liver metastasis assays in nude miceTumorigenicity assay Fourteen female balb/c nude mice （5-6weeks old） were randomly devided into two groups and maintained under specific pathogen-free conditions in the Experiment Animal Center of Nanfang Hospital.2×106cells, SW620Lgr5shRNA stable cells and SW620NC stable cells, were subcutaneously injected into the back of nude mice. Body weigh of each mouse and length and width of subcutaneous tumors were obtained per three days. Four weeks later, nude mice were sacrificed and tumor tissues were fixed by formalin, embedded by paraffin and followed by haematoxylin-eosin staining.Liver metastasis assays of colorectal cancer Fourteen female balb/c nude mice （5-6weeks old） were randomly devided into two groups and maintained under specific pathogen-free conditions in the Experiment Animal Center of Nanfang Hospital.Nude mice were anesthetized with1%pentobarbital sodium.1×106cells, SW620Lgr5shRNA stable cells and SW620NC stable cells, were injected into envelope of spleen. Body weigh were obtained per three days. Three weeks later, nude mice were sacrificed and spleen, lung and liver tissues were fixed by formalin, embedded by paraffin and followed by haematoxylin-eosin staining.Result1. According to preliminary results in immunohistochemical assay, expression of Lgr5increases microvessel density in primary colorectal carcinomas and metastatic cancersLgr5and CD31were detected by immunohistochemistry in primary colorectal carcinomas and lymph node metastatic cancers and liver metastatic cancers.The results revealed that Lgr5was more expressed in node metastatic cancers and liver metastatic cancers compared with primary colorectal carcinomas, the same as CD31and microvessel density.2. Stable cells overexpressing Lgr5were successfully establishedRecombinant plasmids pcDNA3.1（+）-Lgr5and the empty vector were transfected into SW480cells using the lipofectamine2000reagent according to the manufacturer’s instructions. Transfectants were selected in medium containing G418. Protein and mRNA of Lgr5were respectively detected by western blotting and qRT-PCR. The results revealed that compared with SW480vector cell, protein of Lgr5in SW480Lgr5cell was significantly increased, the same as mRNA of Lgr5（t=17.499, p<0.05）.3. Lgr5regulates migration and invasion in recombined colorectal carcinoma cellsIn migration and invasion, compared with SW480vector cell, cells of migration and invasion in SW480Lgr5cell was markedly increased （migration:t=4.291, p<0.05; invasion:t=3.127, p<0.05）. And compared with SW620NC cell, cells of migration and invasion in SW620Lgr5shRNAcell was markedly reduced （migration:t=6.557, p<0.01; t=2.297, p<0.05）.All of these results revealed up-regulation of Lgr5prompted ability of migration and invasion in recombined colorectal carcinoma cells, while knocking down of Lgr5reduced this ability.4. Lgr5mediated tube formation in endothelial cellsTube formation ability in endothelial cells was detected by the numbers of complete tube. Compared with SW480vector cell, the number of complete tube in SW480Lgr5cell was markedly increased （t=8.617, p<0.05） while the number of complete tube in SW620Lgr5shRNA cell was markedly reduced compared to SW620NCcell （t=5.670,p<0.05）.5. Lgr5affected the expression of VEGF in colorectal carcinoma cellsIn Western blotting, compared with SW480vector cell, protein of VEGF in SW480Lgr5cell was significantly increased, while protein of VEGF in SW620Lgr5shRNA cell was markedly reduced compared to SW620NC cell. In Elisa, the similar changes were obtained （overexpression group:t=12.58, p<0.01; knocking down group:t=8.104, p<0.01）).6. mRNA of Lgr5and VEGF in tumor tissues of AOM/DSS model was signifcantly increasedThe results from qRT-PCR revealed that compared with the control group, mRNA of Lgr5and VEGF of colon tissues in exprimental group with tumors was signifcantly increased （Lgr5:t=14.30, p<0.01; VEGF:t=9.213, p<0.01）7. Down-regulation of Lgr5reduced tumorigenicity and liver metastasis of colorectal carcinoma in nude miceIn tumorigenicity assay, the volume of subcutaneous tumors in SW620Lgr5shRNA group was significantly smaller than SW620NC group,which started to emerge at week3. In liver metastasis assay, compared with SW620NC group, the number of nodes in liver in SW620Lgr5shRNA group was obviously less （t=4.386, p<0.05）ConclusionFrom the above results, we could make the following conclusion:1.In vitro, Lgr5increased the ability of migration and invasion in colorectal carcinoma cells.2.1n vivo, Lgr5increased tumorigenicity and liver metastasis of colorectal carcinoma in nude mice.3.Lgr5promted angiogenesis of endothelial cells through increasing the expression of VEGF in colorecal carcinoma.Taken together, Lgr5promted angiogenesis through upregulating the expression of VEGF and futhered to increase the tumorigenicity and metastasis in colorecal carcinoma. Therefore, Lgr5will be the newly promising target of treatment in colorecal carcinoma.