| ObjectiveContactin-1(CNTN-1)has been demonstrated to express in a variety of tumors and to facilitate the invasion and metastasis of cancer cells.Previously,we found that the expression of CNTN-1 was upregulated in gastric cancer tissue compared with adjacent normal tissues.The role and mechanism of its expression in the invasion and metastasis of gastric cancer is not yet clarified.Therefore,we mainly investigate the role and mechanism of CNTN-1 in the development,invasion and metastasis of gastric cancer.Materials and methods(1)The expressions of CNTN-1 and EMT-related markers(E-cadherin,snail,slug and N-cadherin)were assayed through immunohistochemical staining of human gastric cancer tissues and adjacent normal mucosa tissues,and its relationships with clinical pathological features as well as the prognosis of patients with gastric cancer were analyzed.The survival analysis was performed by Kaplan Meier method and Cox regression analysis.(2)The expressions of CNTN-1 and EMT-related markers were detected in gastric cancer cells KATO-III,SGC7901 and MKN45 by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.The invasion ability of gastric cancer cell line KATO-III,SGC7901 and MKN45 was assayed by Transwell chamber invasion test.(3)Three pairs of complementary shRNA oligonucleotides specifically targeting the CNTN-1 gene were designed,and the CNTN-1-shRNA vector was constructed and transfected into gastric cancer cell line MKN45.The assay was divided into four groups: the control group(con sh),the interference group 1(sh#1),the interference group 2(sh#2)and the interference group 3(sh#3).The expression of CNTN-1 protein in these four groups(con sh,sh#1,sh#2 and sh#3)was analyzed by Western blot and immunofluorescence technique.Screening of gastric cancer cell line sh#2 MKN45 with the highest interference efficiency was used for the follow-up study.(4)The expressions of CNTN-1 and EMT-related markers were detected by Western blot in gastric cancer cell line MKN45 untreated group(parental),control interference group(con sh)and interference group 2(sh#2);The application of Transwell invasion test was for the determination of the invasion ability of parental group,con sh group and sh#2 group.Analysis of the migration ability of parental group,con sh group and sh#2 group was performed by Transwell chamber migration assay and cell scratch test.Cell counting Kit-8(CCK-8)was used to detect the cell proliferation ability of parental group,con sh group and sh#2 group.(5)The proliferation ability of con sh and sh#2 groups was compared,with the equal amount of gastric cancer cells con sh and sh#2 transplanted subcutaneously in nude mice,respectively.Western blot was employed to detect the expressions of CNTN-1 and E-cadherin in subcutaneous transplantation tumors of con sh group and sh#2 group,respectively.Gastric cancer cells MKN45 con group and sh#2 group were intravenously injected into the lateral tail vein of nude mice,respectively,to establish the model of distant metastasis in nude mice.Micro-PET/CT technology,counting the lung metastatic nodules in nude mice and nude mice lung metastasis nodules,HE staining method were used to compare the differences in the invasion and metastasis ability between con sh and sh#2 groups.The expression level of CNTN-1 and E-cadherin in lung metastasis nodules of con sh group and sh#2 groups were assayed by immunohistochemistry.Results(1)Compared with adjacent normal mucosa tissues,the expressions of CNTN-1,Snail,Slug and N-cadherin in gastric cancer tissues was significantly higher(59.7% vs 30.6%,P < 0.001;52.8% vs 25.0%,P = 0.001;54.2% vs 36.1%,P = 0.029;52.8% vs 31.9%,P = 0.011).On the contrary,the expression of E-cadherin was decreased in gastric cancer compared with that in adjacent normal mucosa tissues(43.1% vs 72.2%,P < 0.001).The expressions of CNTN-1,E-cadherin,Slug and N-cadherin in gastric carcinoma were respectively related to the depth of tumor invasion(P = 0.001,P = 0.022,P = 0.005,P = 0.010),lymph node metastasis(P < 0.001,P < 0.001,P = 0.006,P = 0.038)and TNM stage(P = 0.002,P = 0.011,P = 0.002,P = 0.019).The expression of Snail in gastric cancer was related to the depth of tumor invasion(P = 0.036)and TNM staging(P = 0.005),and was not associated with lymph node metastasis(P = 0.260).The expressions of CNTN-1,E-cadherin,Snial,Slug and N-cadherin in gastric cancer were not related to age,sex,tumor diameter,Lauren classification,vascular invasion and lymphatic invasion of patients with gastric cancer(P > 0.05,respectively).Spearman rank correlation analysis showed the expression of CNTN-1 was positively correlated with those of slug(r = 0.324,P = 0.005)and N-cadherin(r = 0.244,P = 0.039)in gastric cancer tissue,negatively correlated with the expression of E-cadherin(r =-0.430,P < 0.001),and not correlated with the expression of snail(r = 0.131,P = 0.274).The correlation of the expression of CNTN-1 or EMT-related proteins with the prognosis of patients suffering from gastric cancer was also investigated,which showed that patients with gastric cancer,respectively,with CNTN-1 positive,Slug positive,or N-cadherin positive but E-cadherin negative survived significantly shorter than those with CNTN-1 negative(P < 0.001),Slug negative(P < 0.001),or N-cadherin negative(P = 0.012)but E-cadherin positive(P = 0.001),while there were no significant difference between patients with Snail positive and patients with Snail negative(P = 0.178).Logistic regression analysis revealed that CNTN-1 positive expression in gastric cancer tissues was an independent risk factor for lymph node metastasis(P < 0.001).Multivariate analysis with the Cox regression models showed that CNTN-1(P = 0.001)and Slug(P = 0.002)positive expressions were independent prognostic factors of postoperative survival in patients with gastric cancer.(2)Transwell invasion assay showed that the number of penetrating gastric cancer cells of MKN45 and SGC7901 were significantly more than that of KATO-III,with the difference being statistically significant(P < 0.05).The results of RT-PCR and Western blot showed that compared with those of gastric cancer cell of KATO-III,the mRNA and protein expressions of CNTN-1,slug and N-cadherin in gastric cancer cell lines MKN45 and SGC7901 were upregulated,and the mRNA and protein expressions of E-cadherin was downregulated.There was no significant difference in the mRNA and protein expressions of Snail among these three cell lines.(3)The results of Western blot and immunofluorescence assay demonstrated that the three recombinant expression vectors could effectively interfere the expression of CNTN-1 protein in gastric cancer cell MKN45,and the interference efficiency of the sh#2 was the best.(4)The results of Western blot showed that the protein expressions of Slug and N-cadherin were downregulated in the sh#2 group compared with those in the parental group and con sh group,while E-cadherin being upregulated and Snail being not significantly different.The results of Transwell invasion and migration assays showed that the number of penetrating cells in sh#2 group decreased significantly,compared with those in the parental group and con sh group(P < 0.001,respectively).The result of cell scratch assay revealed that the cell migration rate of sh#2 group was significantly lower than those of the parental group and con sh group(P < 0.001,respectively).The results of CCK-8 indicated that there was no significant difference in cell proliferation ability either between sh#2 group and parental(P > 0.05)or between sh#2 group and con sh groups(P > 0.05).(5)The results of subcutaneous transplanted tumors in nude mice showed that the weight and volume of subcutaneous transplanted tumors in sh#2 group were not significantly different from those of con sh group(P > 0.05,respectively).Based on the results of Western blot,the protein expression of CNTN-1 was found to decrease in sh#2 group,compared with that in the con sh group,while the protein expression of E-cadherin increasing.Micro-PET/CT showed that the lung tissues of nude mice were fuzzy,suggesting that the model of distant lung metastasis in nude mice was successfully established.The results of HE staining of lung metastatic nodules suggested that the nodules were originated from gastric cancer cells injected into the tail vein.Additionally,the immunohistochemical results of pulmonary metastasis nodules indicated that the protein expression of CNTN-1 in sh#2 group was decreased compared with that in con sh group,and the protein expression of E-cadherin was increased.The results of lung metastasis nodules in nude mice showed that the number of lung metastasis nodules in sh#2 group was significantly less than that in con sh group(P < 0.001).Conclusions(1)The elevated expression of CNTN-1 is associated with the invasion and metastasis of gastric cancer,with the increased expressions of Slug and N-cadherin,as well as the decreased expression of E-cadherin,which induce the poor prognosis of patients with gastric cancer.(2)In the gastric cancer cell lines,the elevated expression of CNTN-1 is associated with the invasion ability of gastric cancer cells,with the increased expressions of Slug and N-cadherin,as well as the decreased expression of E-cadherin.(3)The construction of three recombinant expression vectors can effectively interfere with the expression of CNTN-1 in gastric cancer cell line MKN45,and the CNTN-1-shRNA2 interference efficiency is the best,which lays the foundation for further research.(4)CNTN-1 inhibits E-cadherin expression by promoting the expression of Slug and N-cadherin,thereby promoting the migration and invasion of gastric cancer cell line MKN45.The down-regulation of CNTN-1 expression has no significant effect on the proliferation ability of MKN45 cells.(5)The studies in vivo confirm that the down-regulation of CNTN-1 expression could inhibit the invasion and metastasis of gastric cancer cell line MKN45,but have no significant effect on its proliferation ability.CNTN-1 may be an important molecular target,and knockdown of its expression may inhibit the invasion and metastasis of gastric cancer. |
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