| Objectives:Peritoneal metastasis of gastric cancer(GC)is the most common and intractable way of metastasis in patients with GC peritoneal metastasis after operation,and it is an important cause of postoperative death in patients with advanced GC.The molecular mechanism of GC peritoneal metastasis is not fully understood.Revealing the molecular mechanism of GC peritoneal metastasis and providing valuable molecular targets for the diagnosis and treatment of GC peritoneal metastasis is focus of the current research.Cancer-associated fibroblasts(CAFs)highly express Galectin-1(encoded by LGALS1gene)in GC microenvironment,which affects a variety of malignant biological behaviors of GC cells.The purpose of this study is to clarify the regulatory mechanism of Galectin-1 on GC peritoneal metastasis.Methods:1.Detection of clinical specimens:The specimens of GC tissues and corresponding peritoneal tissues were divided into Ⅰ-Ⅱ stage group,Ⅲ stage group and Ⅳ stage group.The expression of Galectin-1 in GC tissues were analyzed by Immunohistochemistry(IHC)and Immunoreactivity score(IRS),and the peritoneal collagen deposition was observed by HE staining and Masson staining.2.Cell experiment in vitro:(1)LGALS1 overexpression / empty lentiviral vector was used to transfect HGC-27 and SGC-7901 cells,and HMr SV5 cells were cultured in conditioned medium(CM)of GC cells with different LGALS1 expression levels.(2)Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of COL1A1,COL3A1 and FN1 mRNA.Western Blot(WB)was used to detect the expression of type I collagen(Collagen Ⅰ),type Ⅲ collagen(Collagen Ⅲ)and fibronectin 1(FN1).(3)The adhesion ability of Human peritoneal mesothelial cells(HPMCs)to GC cells were detected by cell adhesion test.3.In vivo experiments in animals:(1)The GC peritoneal metastasis models of nude mices were established by using GC cells with different LGALS1 expression levels.(2)Peritoneal metastatic nodules and peritoneal specimens were collected.(3)The metastatic nodules were counted,and the collagen thickness at the base of the nodules were measured by HE staining and Masson staining.(4)Peritoneal collagen deposition was observed by HE staining and Masson staining.(5)The expression of Collagen Ⅰ,Collagen Ⅲ and FN1 in peritoneum were measured by IHC.Results:1.Galectin-1 expression in GC is related to clinical staging and peritoneal fibrosis The expression of Galectin-1 in GC was detected by IHC.The results showed that the IRS of Galectin-1 in Ⅳ stage group was significantly higher than that in Ⅰ-Ⅱ stage group and Ⅲ stage group(P < 0.01),and the expression of Galectin-1 in Ⅲ stage group was significantly higher than that in Ⅰ-Ⅱ stage group(P < 0.01).The results of HE staining and Masson staining showed that the thickness of collagen in peritoneal tissue of Ⅳ stage group was significantly higher than that of Ⅰ-Ⅱ stage group and Ⅲ stage group(P < 0.01),and the collagen thickness of Ⅲ stage group was significantly higher than that of Ⅰ-Ⅱ stage group(P < 0.01).Pearson correlation coefficient showed that there was a significant positive correlation between the expression of Galectin-1 and the thickness of peritoneal collagen deposition in GC(r = 0.8793,P < 0.01).2.LGALS1 promotes collagen expression in peritoneal mesothelial cells The results of qRT-PCR showed that the expressions of LGALS1(P < 0.01),COL1A1(P < 0.05),COL3A1(P < 0.01)and FN1(P < 0.05)mRNA in HMRs V5 cells of OE-LGALS1-CM group were significantly higher than those in SGC-7901-CM group and NC-CM group.The above experiments were repeated with HGC-27 cells,the mRNA expression of LGALS1(P < 0.01),COL1A1(P < 0.05),COL3A1(P < 0.01)and FN1(P < 0.05)in OE-LGALS1-CM group was significantly higher than that in HGC-27-CM group and NC-CM group.The results of WB showed that the expressions of Galectin-1(P < 0.01),Collagen Ⅰ(P< 0.01),Collagen Ⅲ(P < 0.05)and FN1(P < 0.05)in HMRs V5 cells of OE-LGALS1-CM group were significantly higher than those in SGC-7901-CM group and NC-CM group.The above experiments were repeated with HGC-27 cells,The expression of Galectin-1(P < 0.05),Collagen Ⅰ(P < 0.05),Collagen Ⅲ(P < 0.05)and FN1(P < 0.05)in OE-LGALS1-CM group were higher than that in HGC-27-CM group and NC-CM group.3.LGALS1 enhances HPMCs adhesion to GC cellThe IOD of SGC-7901 cells adhering to HMRs V5 cells in OE-LGALS1-CM group was1958.20 ± 283.53,which was higher than that in WC-CM group(675.97 ± 291.57)and NC-CM group(602.80 ± 113.97)(P < 0.01).We repeated the adhesion experiment on HMRs V5 cells with HGC-27 CM.The IOD of HGC-27 cells adhering to HMRs V5 cells in OE-LGALS1-CM group was 2303.10 ± 919.11,which was higher than that in NC-CM group(659.53 ± 219.32)and WC-CM group(764.76 ± 390.20)(P < 0.05).4.LGALS1 promotes GC peritoneal metastasis through peritoneal fibrosis There was no significant difference in the number of nodules between WC group and NC group(P > 0.05),but the number of nodules in OE-LGALS1 group was significantly higher than that in control groups(P < 0.01).The basal collagen deposition thickness in WC group was 31.33 ± 12.63 μm and 29.00 ± 8.08 μm in NC group.There was no significant difference between the two groups(P > 0.05).The thickness of the OE-LGALS1 group(146.67 ± 47.84 μm)was significantly thicker than that of the control groups(P < 0.01).5.LGALS1 promotes GC peritoneal metastasis by enhancing peritoneal collagen depositionThe results of Masson staining in the peritoneum of nude mices showed that the thickness of peritoneal collagen in OE-LGALS1 group was 131.67 ± 44.50 μm,which was significantly thicker than that in WC group(32.33 ± 9.29 μm)and NC group(32.33± 10.83 μm)(P < 0.01).IHC showed that the mean IOD of Collagen Ⅰ in OE-LGALS1 group was higher than that in WC group and NC group(P < 0.01).The mean IOD of Collagen Ⅲ in OE-LGALS1 group was higher than that in WC group and NC group(P < 0.01),and the mean IOD of FN1 in OE-LGALS1 group was also higher than that in control groups(P< 0.01).Conclusions:Galectin-1/LGALS1 can promote GC peritoneal metastasis by inducing collagen deposition in peritoneal tissue,peritoneal fibrosis and increasing the adhesion of GC cells to peritoneum.Galectin-1/LGALS1 is a potential molecular marker for GC peritoneal metastasis. |
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