| BackgroundGastric cancer is one of the most frequent malignant tumors in the world. It is thesecond most leading cause of cancer death worldwide. and approximately one millionpatients are diagnosed every year. Although advanced in diagnostic tools andtherapeutic techniques, the5-year survival rate is is still less than30%, because of localinvasion and metastasis, which are known to be the leading biological charcateristics ofgastric cancer. Numerous studies have demonstrated that invasion and metastasis arehighly dependent upon proliferation of tumor cells and angiogenesis, andantiangiogenic therapy could benefit cancer patients. Preoperative chemotherapy hasbeen used in the treatment of locally advanced gastric cancer, because it could shrinkthe tumor and increase the possibility of completely resection. It has been indicated thatthis treatment could significantly improve overall survival in patients with resectablegastric cancer. However, only approximately20%of the patients had complete orsubtotal tumor regression. One of the major obstacles for successful treatment waschemotherapy resistance. Therefore, in order to imporve the therapeutic efficacy ofgastric cancer, searching for specific molecules that associated with invasion, metastasis,angiogenesis and chemotherapy resistance and in-depth study of its regulationmechanism is needed.ObjectiveTo investigated the expression of Lgr5in gastric cancer and effective mechanismsin invasion, metastasis, angiogenesis, chemotherapy resistance and prognosis. Inaddition, we also explored the value of Lgr5as a specific biomarker in predicting tumorpathological response and overall survival in advanced gastric cancer patients treatedwith preoperative chemotherapy.Methods1. Follow-up was taken among318patients with gastric cancer. Immunohistochemistryfor Lgr5, MMP2, EphA3, VEGF and CD34was performed in318cases of gastriccarcinoma specimens and80distal normal gastric tissues were randomly selected from the318cases of gastric cancer as normal controls. The microvessel density (MVD) wascaculated after CD34staining. Western blot was used to determine the expression ofLgr5, MMP2, EphA3, and VEGF pretein in75cases of fresh gastric carcinoma andmatched normal mucosa tissue. The correlation between Lgr5and clinicopathologicalfeatures was analyzed. The relationships between Lgr5and invasion-related molecule(MMP2) or angiogenesis-related molecules (EphA3, VEGF, CD34) were also examed.2. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) andWestern blot was used to screen gastric cancer cell line with high expression of Lgr5.RNA interference (RNAi) technique was employed to knockdown Lgr5expression.Effects of silencing Lgr5on the expression of MMP2, EphA3, and VEGF mRNA andprotern were detected by qRT-PCR and Western blot. Effects of silencing Lgr5on theproliferation, apoptosis, invasive, migration was examed by MTT, Annexin V-FITC/PIflow cytometry, transwell and wound healing scratch assay. Furthermore, the effect ofconditioned medium form supernatant of AGS that was treated with Lgr5RNAi onproliferation and capillary tube formation of human umbulical vein endothelia cell(HUVEC) was investigated by MTT and angiogenesis two-dimensional gel model invitro.3. The effect of silencing Lgr5on cell viability and apoptosis of gastric cancer cells,which was treated by two drugs oxaliplatin and5-FU, was investigated by MTT andAnnexin V-FITC/PI flow cytometry method. Immunohistochemistry for Lgr5wasperformed in68cases of gastric cancer samples from the patients treated withpreoperative chemotherapy. The relationship between Lgr5and clinicopathologicalfeatures was analyzed.Results1. According to immunohistochemistry analysis, Lgr5positive rate was significantlyhigher in gastric carcinoma than that in normal mucosa (all P=0.001). Lgr5expressionwas more frequent in advanced T-stage cancer and was found to correlated withmetastasis in the regional lymph nodes (P=0.001) and distant (P=0.013). Multivariateanalysis using Cox regression showed that Lgr5had an independent effect on survival(P=0.001). Spearman’s correlation test showed that Lgr5was positively correlated withMMP2, EphA3,VEGF, and MVD (All P=0.001). This correlation was also confired bywestern blot analysis according to Pearson’s corretation test (Lgr5vs MMP2: P=0.001,r=0.875; Lgr5vs EphA3: P=0.001, r=0.474; Lgr5vs VEGF: P=0.001, r=0.921). 2. The expression level of Lgr5was highest in gastric cancer cell line AGS. MMP2,EphA3, VEGF mRNA and protein expression was down-regulated in the Lgr5silenced group than that in control groups（P=0.001）. Silencing Lgr5could inhibitproliferation and promote apoptosis in AGS. It also inhibited migration and invasion.When HUVECs stimulated with conditioned medium from Lgr5siRNA-transfectedAGS cells, the proliferation was significantly inhibited (P=0.001). Moreover, tubeformation of HUVEC was significantly decreased (2.51±0.19mm/mm2) compared tothe treatment with regular cell culture medium (RPMI-1640)(7.34±0.30mm/mm2) ormedium from control siRNA-transfected cells (7.18±0.33mm/mm2)(All P=0.001).3. The survival rate to Lgr5silenced AGS cells, which was treated by two drugsoxaliplatin or5-FU, was significantly lower compared to the control groups (P=0.001).The results of flow cytometric assay showed that the apoptosis rate of Lgr5silencedAGS cells, which was treated by oxaliplatin or5-FU, was significantly higher comparedto the control groups (P=0.001). The positive rate of Lgr5expression in patients withpoor tumor regression was significantly higher than those in patients with regressedtumor (P=0.001). The correlation between the expression of Lgr5in gastric cancerbefore chemotherapy and after chemotherapy was significantly difference, the Lgr5expression in before chemotherapy tissues is lower than that in after chemotherapytissues. Multivariate analysis was performed using the Cox regression. Lgr5was foundto signifcantly affect the outcome of gastric cancer after preoperative chemotherapyand appeared to be an independent prognostic factor (P=0.039).Conclusions1. Lgr5was associated with the tumorigenesis and progression of gastric cancer, andcould be used as a potential specific indicator for judging the invasive, metastasis, andangiogenesis state gastric cancer and predicting the prognosis of patients.2. Silencing Lgr5could effectively inhibit the malignant biological behavior of gastriccancer, such as proliferation, invision and migration. Conditioned medium from AGSwith Lgr5silenced could significantly supress HUVEC proliferation and decrease thecapillary tube formation of HUVEC. Lgr5siRNA could inhibit β-catenin protein andMMP2, EphA3, VEGF mRNA and protein. This indicated that Lgr5might enhancetumor cell invasion, metastasis and angiogenesis by activating the classic Wnt/β-cateninsignaling and increasing the transcription of MMP2, EphA3and VEGF mRNA.3. Lgr5could be used as an predictor for pathological response of gastric cancer patients who undergo preoperative chemotherapy, and also coud be an independentprognostic factor for survival. Silencing of Lgr5could decreased the chemotherapyresistance in AGS cells.4. Lgr5may play important roles in invasive, metastasis and angiogenesis of gastriccacner, and thus may be a useful target for therapeutic intervention. Inhibition of Lgr5with siRNA technique coule be a potential gene therapeutic method for gastric cancer. |
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