Lgr5对胃癌的恶性生物学行为和化疗耐药性的影响及机制研究
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摘要
背景
胃癌是世界上最常见的恶性肿瘤之一，每年大约有100万新发病例。胃癌在癌症相关性死亡中排在第二位。近年来，尽管胃癌的诊疗技术不断提高，由于胃癌的侵袭和转移，导致其5年生存率仍然低于30%。大量的研究认为肿瘤的侵袭和转移高度依赖于肿瘤细胞的增殖和血管生成，抗血管生成治疗可以使胃癌患者生存获益。此外，在治疗进展期胃癌过程中，术前化疗被广泛应用，因为这种方法可以缩小肿瘤，降低肿瘤的分期，提高肿瘤的切除率，从而改善患者预后。然而，由于化疗耐药性的存在，仅有20%的胃癌患者能够完全或部分缓解。因此，为了提高胃癌的治疗效果，需要寻找与胃癌的侵袭、转移、血管生成和化疗耐药性相关的特异分子并深入研究其调节机制。
目的
研究Lgr5在胃癌中的表达情况，分析其与胃癌的侵袭、转移、血管生成、化疗耐药性及预后的关系，并进一步探讨具体作用机制。此外，我们还对接受术前化疗的胃癌患者进行研究，探讨Lgr5在评价化疗效果及判断预后方面的价值。
方法
1.收集318例经根治性手术治疗的胃癌患者，进行随访。利用免疫组织化学染色检测Lgr5、MMP2、EphA3、VEGF和CD34在318例胃癌和80例正常胃黏膜组织中的表达。CD34免疫染色用来计数微血管密度（MVD）。通过Western blot方法检测了75例胃癌新鲜组织标本及相对应的癌旁正常组织中Lgr5, MMP2, EphA3和VEGF蛋白表达情况。并对Lgr5与临床病理学特征之间的关系进行了综合分析。
2.应用qRT-PCR和Western blot技术筛选Lgr5高表达的胃癌细胞株。通过RNA干扰技术敲低胃癌细胞中Lgr5的表达。通过qRT-PCR和Western blot技术分析沉默Lgr5对MMP2，EphA3和VEGFmRNA和蛋白表达的影响。通过MTT实验检测沉默Lgr5基因对胃癌细胞增殖能力的影响。采用Annexin V-FITC/PI双染流式细胞术检测沉默Lgr5对胃癌细胞凋亡的影响。采用Transwell和细胞划痕修复实验观察Lgr5siRNA对胃癌细胞侵袭和迁移能力的影响。利用MTT法和二维凝胶血管生成体外模型探讨Lgr5沉默后的胃癌细胞培养上清液对人脐静脉血管内皮细胞（human umbulical vein endothelia cell，HUVEC）增殖和管状结构形成的影响。
3.采用MTT法和Annexin V-FITC/PI双染流式细胞技术检测Lgr5siRNA对接受奥沙利铂和5-FU作用的胃癌细胞的存活率和凋亡率。收集68例接受新辅助化疗的胃癌患者石蜡标本。通过免疫组织化学染色方法检测胃癌组织中Lgr5表达情况，并分析其表达与该类患者的临床病理学特征之间的关系。
结果
1.免疫组化染色结果显示：Lgr5在胃癌组织中的阳性表达率明显高于正常粘膜组织（P=0.001）。Lgr5阳性率随着侵润深度增加（T分期）而增高（P=0.001），并且与区域淋巴结转移（P=0.001）和远处转移（P=0.001）相关。多因素生存分析显示Lgr5是影响胃癌患者预后的独立因子（P=0.001）。通过Spearman等级相关检验显示Lgr5表达与MMP2、EphA3、VEGF和MVD呈正相关（P=0.001）。对Western blot检测结果行Pearson相关检验也进一步验证这一相关性（(Lgr5vsMMP2: P=0.001, r=0.875; Lgr5vs EphA3: P=0.001, r=0.474; Lgr5vs VEGF: P=0.001,r=0.921).
2. Lgr5在胃癌细胞系AGS中表达量最高。沉默AGS细胞中Lgr5基因可以导致MMP2、EphA3和VEGF表达明显下降（P=0.001）。同时我们还发现沉默Lgr5以后，AGS细胞的增殖、迁徙和迁移能力显著降低（P=0.001），凋亡率显著增高（P=0.001）。Lgr5沉默的胃癌AGS细胞培养液可以抑制HUVEC细胞增殖能力。此外，当利用Lgr5沉默的AGS细胞上清液培养HUVEC时，管状结构形成（2.51±0.19mm/mm2）相比空白对照组(7.34±0.30mm/mm2)和阴性对照组(7.18±0.33mm/mm2)明显减少，差异具有显著性（P=0.001，P=0.001）。
3.在化疗药物奥沙利铂和5-FU作用下，Lgr5基因沉默的AGS细胞的存活率较空白对照组和阴性对照组明显下降（P=0.001），凋亡率较空白对照组和阴性对照组显著增加（P=0.001）。肿瘤消退情况差的胃癌中Lgr5阳性表达率显著高于肿瘤消退情况好的胃癌（P=0.001）。将化疗前后两组Lgr5表达情况进行配对分析，发现化疗前Lgr5表达较化疗后低（P=0.001）。多因素分析结果显示Lgr5与接受术前化疗的胃癌患者预后相关，可以作为判断预后的独立因子（P=0.039）。
结论
1. Lgr5与胃癌的发生发展有关，可以作为判断胃癌侵袭、转移、血管生成情况以及患者预后的参考指标。
2.沉默Lgr5基因可以有效地抑制胃癌细胞的增殖、侵袭和转移等恶性生物学行为。Lgr5沉默后胃癌AGS细胞培养液可以抑制HUVEC细胞增殖和管状结构形成能力。Lgr5siRNA可以有效抑制β-catenin蛋白、MMP2、EphA3、VEGF mRNA和蛋白的表达。这表明胃癌中Lgr5的表达可以通过激活经典Wnt/β-catenin信号通路，从而促进MMP2、EphA3和VEGFmRNA的转录
3. Lgr5可以作为预测化疗疗效的有效指标，同时也可以作为独立影响因子来判断接受术前化疗胃癌患者的预后。沉默Lgr5基因以后可以显著降低AGS细胞的化疗耐药性。
4. Lgr5在胃癌的侵袭、转移和血管生成过程中扮演了重要角色，因此，它可以作为有用的治疗靶点。应用RNA干扰技术沉默Lgr5可以成为一种潜在的胃癌基因治疗的手段。
Background
Gastric 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.
Objective
To 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.
Methods
1. 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.
Results
1. 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).
Conclusions
1. 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.
胃癌是世界上最常见的恶性肿瘤之一，每年大约有100万新发病例。胃癌在癌症相关性死亡中排在第二位。近年来，尽管胃癌的诊疗技术不断提高，由于胃癌的侵袭和转移，导致其5年生存率仍然低于30%。大量的研究认为肿瘤的侵袭和转移高度依赖于肿瘤细胞的增殖和血管生成，抗血管生成治疗可以使胃癌患者生存获益。此外，在治疗进展期胃癌过程中，术前化疗被广泛应用，因为这种方法可以缩小肿瘤，降低肿瘤的分期，提高肿瘤的切除率，从而改善患者预后。然而，由于化疗耐药性的存在，仅有20%的胃癌患者能够完全或部分缓解。因此，为了提高胃癌的治疗效果，需要寻找与胃癌的侵袭、转移、血管生成和化疗耐药性相关的特异分子并深入研究其调节机制。
目的
研究Lgr5在胃癌中的表达情况，分析其与胃癌的侵袭、转移、血管生成、化疗耐药性及预后的关系，并进一步探讨具体作用机制。此外，我们还对接受术前化疗的胃癌患者进行研究，探讨Lgr5在评价化疗效果及判断预后方面的价值。
方法
1.收集318例经根治性手术治疗的胃癌患者，进行随访。利用免疫组织化学染色检测Lgr5、MMP2、EphA3、VEGF和CD34在318例胃癌和80例正常胃黏膜组织中的表达。CD34免疫染色用来计数微血管密度（MVD）。通过Western blot方法检测了75例胃癌新鲜组织标本及相对应的癌旁正常组织中Lgr5, MMP2, EphA3和VEGF蛋白表达情况。并对Lgr5与临床病理学特征之间的关系进行了综合分析。
2.应用qRT-PCR和Western blot技术筛选Lgr5高表达的胃癌细胞株。通过RNA干扰技术敲低胃癌细胞中Lgr5的表达。通过qRT-PCR和Western blot技术分析沉默Lgr5对MMP2，EphA3和VEGFmRNA和蛋白表达的影响。通过MTT实验检测沉默Lgr5基因对胃癌细胞增殖能力的影响。采用Annexin V-FITC/PI双染流式细胞术检测沉默Lgr5对胃癌细胞凋亡的影响。采用Transwell和细胞划痕修复实验观察Lgr5siRNA对胃癌细胞侵袭和迁移能力的影响。利用MTT法和二维凝胶血管生成体外模型探讨Lgr5沉默后的胃癌细胞培养上清液对人脐静脉血管内皮细胞（human umbulical vein endothelia cell，HUVEC）增殖和管状结构形成的影响。
3.采用MTT法和Annexin V-FITC/PI双染流式细胞技术检测Lgr5siRNA对接受奥沙利铂和5-FU作用的胃癌细胞的存活率和凋亡率。收集68例接受新辅助化疗的胃癌患者石蜡标本。通过免疫组织化学染色方法检测胃癌组织中Lgr5表达情况，并分析其表达与该类患者的临床病理学特征之间的关系。
结果
1.免疫组化染色结果显示：Lgr5在胃癌组织中的阳性表达率明显高于正常粘膜组织（P=0.001）。Lgr5阳性率随着侵润深度增加（T分期）而增高（P=0.001），并且与区域淋巴结转移（P=0.001）和远处转移（P=0.001）相关。多因素生存分析显示Lgr5是影响胃癌患者预后的独立因子（P=0.001）。通过Spearman等级相关检验显示Lgr5表达与MMP2、EphA3、VEGF和MVD呈正相关（P=0.001）。对Western blot检测结果行Pearson相关检验也进一步验证这一相关性（(Lgr5vsMMP2: P=0.001, r=0.875; Lgr5vs EphA3: P=0.001, r=0.474; Lgr5vs VEGF: P=0.001,r=0.921).
2. Lgr5在胃癌细胞系AGS中表达量最高。沉默AGS细胞中Lgr5基因可以导致MMP2、EphA3和VEGF表达明显下降（P=0.001）。同时我们还发现沉默Lgr5以后，AGS细胞的增殖、迁徙和迁移能力显著降低（P=0.001），凋亡率显著增高（P=0.001）。Lgr5沉默的胃癌AGS细胞培养液可以抑制HUVEC细胞增殖能力。此外，当利用Lgr5沉默的AGS细胞上清液培养HUVEC时，管状结构形成（2.51±0.19mm/mm2）相比空白对照组(7.34±0.30mm/mm2)和阴性对照组(7.18±0.33mm/mm2)明显减少，差异具有显著性（P=0.001，P=0.001）。
3.在化疗药物奥沙利铂和5-FU作用下，Lgr5基因沉默的AGS细胞的存活率较空白对照组和阴性对照组明显下降（P=0.001），凋亡率较空白对照组和阴性对照组显著增加（P=0.001）。肿瘤消退情况差的胃癌中Lgr5阳性表达率显著高于肿瘤消退情况好的胃癌（P=0.001）。将化疗前后两组Lgr5表达情况进行配对分析，发现化疗前Lgr5表达较化疗后低（P=0.001）。多因素分析结果显示Lgr5与接受术前化疗的胃癌患者预后相关，可以作为判断预后的独立因子（P=0.039）。
结论
1. Lgr5与胃癌的发生发展有关，可以作为判断胃癌侵袭、转移、血管生成情况以及患者预后的参考指标。
2.沉默Lgr5基因可以有效地抑制胃癌细胞的增殖、侵袭和转移等恶性生物学行为。Lgr5沉默后胃癌AGS细胞培养液可以抑制HUVEC细胞增殖和管状结构形成能力。Lgr5siRNA可以有效抑制β-catenin蛋白、MMP2、EphA3、VEGF mRNA和蛋白的表达。这表明胃癌中Lgr5的表达可以通过激活经典Wnt/β-catenin信号通路，从而促进MMP2、EphA3和VEGFmRNA的转录
3. Lgr5可以作为预测化疗疗效的有效指标，同时也可以作为独立影响因子来判断接受术前化疗胃癌患者的预后。沉默Lgr5基因以后可以显著降低AGS细胞的化疗耐药性。
4. Lgr5在胃癌的侵袭、转移和血管生成过程中扮演了重要角色，因此，它可以作为有用的治疗靶点。应用RNA干扰技术沉默Lgr5可以成为一种潜在的胃癌基因治疗的手段。
Background
Gastric 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.
Objective
To 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.
Methods
1. 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.
Results
1. 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).
Conclusions
1. 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.
引文
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