| Objective:According to statistical data of the World Health Organization(WHO),annual new cases of cancer increase to more than 900 million in the worldwide,the death of 500 million,and there is a rising trend year by year.Gastric cancer is one of the common malignant tumor,incidence of which ranks third,second only to lung cancer,liver cancer.China is a high-incidence area of gastric cancer,accounted for 42% of the total incidence in the world,in which there are 160 thousand deaths each year,the incidence rate of gastric cancer of middle-aged and young people is rising in recent years.Patients can not often be diagnosed with gastric cancer until the disease progresses to the middle and late term.At this time,they would not only lost the opportunity of surgical treatment but also made radiotherapy and chemotherapy more difficult,which led to be a poor prognosis and greatly low 5-year survival rate,eventually.The leading death cause of gastric cancer is extensive invasion and metastasis of cancer cell,especially distant vital organs metastasis to be suppressed difficultly Therefore,if we could fully and comprehensively understand molecular mechanisms,while finding sensitive and specific markers of gastric cancer invasion and metastasis-associated,there would be clinically important significance to suppress gastric distant organ metastasis and improve survival of patients.Tumor invasion and metastasis is a complex process,involved in multiple genes qualitative and quantitative changes.Studies have shown that tumor metastasis is related to tumor metastasis gene activation or metastasis suppressor gene inactivation.Metastasis suppressor genes are proposed on the basis of molecular cloning technology development.Such genes was highly expressed in non-metastatic tumor,showed low expression in metastatic tumors,but does not affect tumorgenesis.Recent studies have found kiss-1 gene to be a new tumor metastasis suppressor gene,associated with a variety of tumor invasion and metastasis.The binding of kisspeptins,known as kiss-1 gene-related peptide also,to GPR54 receptor can suppress metastasis of a variety of malignant tumors,such as melanoma,bladder cancer,kidney cancer;ovarian cancer,endometrial cancer,choriocarcinoma;esophageal cancer,gastric cancer et al.Lack of kiss-1 gene expression is correlated positively with highly malignancy,high metastatic potential capacity,poor treatment and prognosis among these tumor described above.Kiss-1 gene,as a tumor metastasis suppressor gene suppresses tumor metastasis and invasion by inhibited cell proliferation,increased cell adhesion and induced cell apoptosis,which is regulated by several signaling pathways and many relevant factors.Kiss-1 gene could become a new therapeutic target for anti-tumor metastasis.As of now,the reports on the kiss-1 gene expression and metastasis of gastric cancer is still a minority,its mechanism remains unclear.We analyzed the relationship between kiss-1 expression and clinical pathological data and prognosis in TCGA database and concluded that the low expression of kiss-1 gene is closely related to the metastasis of gastric cancer,and uses genetic engineering technology to construct high expression of kiss-1 gene.The vector was transfected into gastric cancer cell lines,and the effects of kiss-1 gene on the proliferation,migration and invasion of gastric cancer cells were evaluated by in vitro and in vivo experiments,and the relationship between kiss-1 gene and invasion and metastasis of gastric cancer was revealed..Methods: 1 Analysis of the relationship between kiss-1 expression and clinicopathological factors and prognosis in TCGA database 1.1 The expression of kiss-1 m RNA in 354 gastric cancer patients was downloaded from the Cancer Genome Atlas(TCGA)(https://cancergenome.nih.gov)database,and the cut-off value for distinguishing low expression and high expression of kiss-1 was set to 8.85 FPKM.Correlation analysis of clinical pathological parameters and prognosis was performed using SPSS software.1.2 Collection of gastric cancer and gastric cancer cell lines: we selected 10 pairs cases of gastric cancer and paired distal cancer normal gastric mucosa that were obtained from surgical resection specimens of the First Affiliated Hospital of of China Medical University Surgical Oncology.Once specimens left the body,its should be selected and placed to RNA enzyme free Eppendorf tubes as quickly as possible,stored in-80° C refrigerator spare.Five gastric cancer cell lines including BGC823,MGC803,SGC7901,NCI-N87,MKN45,were collected and placed to RNA enzyme free Eppendorf tubes stored in-80 ° C refrigerator spare.1.3 q RT-PCR method for detection of human gastric cancer tissues,paired distal cancer normal gastric mucosa and gastric cancer cell line kiss-1 gene m RNA,a preliminary assessment of the kiss-1 gene expression levels in gastric cancer tissues and normal gastric mucosa and screening low kiss-1 gene expression cell lines as a candidate cell lines to be transfected 2 kiss-1 gene recombinant eukaryotic expression vector construction,identification and stably transfected gastric cancer cell clones screened 2.1 By PCR technology,Kiss-1 fragment was obtained from the pre-build good plasmid.p EGFP-C1-kiss-1 recombinants was constructed by gene cloning technology and identified by double digestion and sequencing.Using calcium chloride co-precipitation method transforming E.coli Competent Cell JM109,moderate amount of p EGFP-C1 kiss-1 plasmid was extracted and stored in-80 ° C refrigerator spare after miniprep plasmid was indentificated by restriction enzyme digestion.2.2 Using Lipofectamin2000 to transfect BGC823 gastric cell,stably transfected positive clones were successfully screened by FCM and G418 dual approach,named for p EGFP-C1BGC823(BGC823/vector)and p EGFP-kiss-1BGC823(BGC823/kiss-1),respectively.Kiss-1 gene expression was detected by q RT-PCR and Western blot methods.3 Study on impact of BGC823 gastric cancer cell biological characteristics by kiss-1 gene and its mechanism involved 3.1The detection of cell biological characteristics of kiss-1 gene overexpression in BGC823: MTT assay was used to determine the 490 nm absorbance value from untransfected group,the BGC823/vector group and BGC823/kiss-1 transfected group for six consecutive days,and calculate the relative growth rate,meanwhile to draw proliferation curve.Cell cycle and apoptosis were detected by FCM.The impact of kiss-1 gene on cell migration and invasion was assessed by scratch healing assay and transwell chamber.The colony formation assay was used to observe whether the kiss-1 gene can inhibit cell colony formation 3.2 Western blot method was used for semi-quantitative ERK1/2/p-ERK,Akt/p-AKT,YAP1,MMP9,VEGF,Sp1,and ? E-catenin protein expression,by which kiss-1 gene suppressed gastric cancer cell proliferation,migration,invasion,apoptosis and angiogenesis 4 Study on mechanism and effect of kiss-1 gene in hematogenously disseminated metastasis of gastric cancer cell and the growth of gastric carcinoma xenograft tumor in nude mice 4.1 Establish subcutaneous xenograft models with BGC823,BGC823/vector and BGC823/kiss-1 cells,respectively.There were three nude mice in each group(a total of 9);Establish nude mice hematogenous disseminated models with BGC823/vector and BGC823/kiss-1cells,respectively,There were three nude mice in each group(a total of 6).4.2 After measurement of subcutaneous tumor size in every week for 5 weeks,we calculated subcutaneous tumor volume,drew the tumor growth curve and calculated inhibition rate.After tumor-bearing 5 week,Nude mice were sacrificed,followed by complete stripping subcutaneous tumor and weighing tumor heavy,and finally calculate the rate of tumor formation.The differences between each group were compared by subcutaneous tumor volume and tumor weight.4.3 Following the completion of paraffin sections of subcutaneous tumor,as well as liver and lung of hematogenous spread nude mice,we observed the morphological characteristics of the tumor tissue and EGFP expression during HE staining.4.4 Immunohistochemistry was used to detect kiss-1,YAP1,ki67,CD31,VEGF and Sp1 expression of subcutaneous tumor.Weidner counting standard detection of CD31 positive cells was used to judge the microvascular density(MVD).Counting metastatic tumor lesions in live and lung of hematogenous spread model nude mice under a fluorescence microscope(the largest cross-section of organ),the number of metastases was compared between two groups.Statistical analysis: SPSS17.0 statistical package for data analysis.Results: 1 Low expression of kiss-1 gene is associated with distant metastasis of organs.Compared with normal gastric mucosa and GES-1,kiss-1 gene in human gastric cancer and BGC823 gastric cancer cell line were down-regulated 1.1 The low expression of kiss-1 gene in gastric cancer patients was closely related to the distant metastasis of organs(P<0.05).1.2 Kiss-1 gene expression in gastric cancer tissue was significantly lower than that in the normal gastric mucosa(P<0.05).1.3 Among five gastric cancer cell lines incuding BGC823,MGC803,SGC7901,NCI-N87 and MKN45,kiss-1 gene m RNA level in BGC823 was lowest,suggesting that BGC823 is appropriate as a candidate for the transfection with kiss-1 gene.2 Kiss-1 gene recombinant eukaryotic expression vector was successfully constructed,and a stably transfected clone of BGC823 obtained 2.1 Double restriction enzyme digestion and sequencing showed that p EGFP-C1-kiss-1 eukaryotic expression vector was successfully constructed.2.2 A stably transfected clone named BGC823/kiss-1 with liposome-mediated transient transfection was obtained by FCM and G418 double screening.The results of q RT-PCR and Western bloting confirmed that kiss-1 gene overexpressed in tumor cells of the clone.3 Kiss-1 gene suppressed the proliferation,colony formation,migration and invasion of BGC823 gastric cancer cell,and induced G0/G1 phase arrest and apoptosis,but also significantly down-regulated YAP1,p ERK1/2,p Akt,MMP-9,VEGF and Sp1 protein expressions but up-regulated ? E-catenin expression in vitro 3.1 The results of MTT assays showed that the proliferative activity of tumor cells of BGC823/kiss-1 group was significantly suppressed compared with two controls of BGC823/vector group and BGC823 group(P<0.01).3.2 The results of cell cycle assay by FCM showed that BGC823/kiss-1 group appeared cell cycle arrest in G0/G1 phase,the difference is very significant in contrast to BGC823/ vector group and BGC823 group(P<0.01).3.3 Apoptosis detection by FCM dispayed higher early apoptosis rate of BGC823/ kiss-1 group than those of BGC823/vector group and BGC823 group,the both differences were statistically significant(P<0.05).3.4 In scratch heal assay,24 h,48h and 72 h wound width were measured on three groups of cells to calculate the healing rate.Compared with BGC823/vector group and BGC823 group,there was a significantly lower wound healing rate at three time points in BGC823/kiss-1 group(P<0.05).3.5 Transwell chamber invasion assay indicated that penetrating memberan cells of BGC823/kiss-1 group were significantly less than these of BGC823/vector group and BGC823 group,P<0.01,respectively.3.6 Using colony formation assay,we counted the number of colony-forming and calculated colony formation rate.The results show that clone formation rate of BGC823/kiss-1 group was significantly less than these of BGC823/vector group and BGC823 group,statistical difference was very significant(P <0.01).3.7 WB detection suggested that there was no significant change in the expression of ERK1/2 and Akt protein in the BGC823/kiss-1 group,but the phosphorylated p-ERK1/2 and p-Akt expression levels were significantly decreased.Expression of YAP1,MMP9,VEGF and Sp1 was decreased in BGC823/kiss-1 group,while expression of ? E-catenin was increased.4 Kiss-1 gene significantly suppressed the growth of gastric xenografts and lung,liver metastasis of hematogenous disseminated gastric cancer cell in nude mice,reduced YAP1,Sp1,VEGF protein expression and xenografts microvessel density in vivo 4.1 Nude mice subcutaneou tumor-bearing experimental results were indicated that tumor formation rate in three groups of nude mice is 100%.In addition,subcutaneous tumor volume and weight of BGC823/kiss-1 group were less than those of BGC823/vector group and BGC823 group(P<0.05),while there was no significant difference between BGC823 group and BGC823/vector group.4.2 Subcutaneous tumor detection by ICH was showed that kiss-1 protein was positively expressed in BGC823/kiss-1 group,and negatively expressed in BGC823 group and BGC823/vector group.Meanwhile,Sp1 and VEGF protein expression was significantly enhanced in BGC823/kiss-1 group,compared to controlgroup(P<0.05).Furthermore,MVD of control group was significantly more than that of BGC823/kiss-1 group(P <0.05).4.3 Counting lung and liver metastatic lesions in hematogenous disseminated nude mice model in vivo: metastatic lesions were found in lung and liver of all three nude mice in BGC823/vector group,just only one of three nude mice had metastatic lesions in BGC823/kiss-1 group;Lung metastatic lesions can be seen an average of 5.5 up to 7 in the BGC823/vector group,but only four cancerous emboli in micro blood vessels from the lung of a mouse were found in the BGC823/kiss-1 group;The liver has suspicious micrometastases with an average of 4.3 up to 5 in BGC823/vector group.In BGC823/kiss-1 group,only two suspicious micro cancerous emboli in a micro vein in the liver of the same mouse with lung emboli,while the other two in this group have no metastasis in neither the lung nor the liver.Conclusions: 1.The expression level of kiss-1 was correlated with the metastasis of gastric cancer by TCGA database.Compared with normal human gastric mucosal epithelial cells,the expression of the metastasis suppressor gene kiss-1 was significantly down-regulated in gastric cancer tissues and BGC823 gastric cancer cells.2.Successfully constructed a stable gastric cancer cell line BGC823/kiss-1 with overexpression of kiss-1 gene.3.Kiss-1 gene overexpression significantly inhibited the proliferation,colony formation,migration and invision of BGC823 gastric cancer cells,which may be assiociated with inducing G0/G1 arrest and apoptosis,down-regulating ERK1/2 and PI3K-Akt signal transduction pathway activity,as well as affecting the biological functions of molecules such as YAP1,? E-catenin? MMP-9,and so on.4.Kiss-1 gene overexpression significantly suppressed the growth of gastric xenografts and lung,liver metastasis of hematogenous disseminated gastric cancer cell in nude mice,mechanisms of which were involved with that kiss-1 gene down-regulated transcription factor Sp1 and inhibited VEGF transcription,thereby suppressed tumor angiogenesis,besides induction of G0/G1 arrest and apoptosis,inhibition of ERK1/2 and PI3K-Akt signal transduction pathway activity,as well as impact on the biological function of molecules such as YAP1?? E-catenin? MMP-9,and so on.Up to now,it has not been reported that Ki SS-1 may inhibit the hematogenous disseminated organ metastasis of BGC823 gastric cancer cells by down-regulating YAP1. |
PDF Full Text Request