| Objective:To detect the expression of lnc RNA FOXD3-AS1 and FOXD3 gene in gastric cardia adenocarcinoma(GCA)tissues and gastric cancer cell lines,and to explore the relationship between expression level and clinicopathological parameter.The effects of FOXD3-AS1 and FOXD3 genes on the proliferation,migration and invasion of gastric cancer cells were investigated in vitro experiments.Methods:1.By searching UCSC database,the position relationship between FOXD3-AS1 and FOXD3 was analyzed.2.q RT-PCR was used to detect the expression level of FOXD3-AS1 and FOXD3 in 53 cases gastric cardia adenocarcinoma tissues and normal tissue adjacent to carcinoma and four human gastric cancer cell line(SGC-7901 and BGC-823,MGC-803,the HGC-27).3.FOXD3-AS1 knockdown and overexpression plasmids were constructed,and FOXD3 overexpression plasmids were synthesized,the transfection efficiency was verified by q RT-PCR.4.The effects of FOXD3-AS1 and FOXD3 on the proliferation,migration and invasion of gastric cancer cells in vitro were detected by cell proliferation assay,clone formation assay,scratch assay and Transwell chamber invasion assay.5.The effects of FOXD3-AS1 and FOXD3 on the m RNA and protein expression levels of Epithelial mesenchymal transition(EMT)related factors(E-cadherin,N-cadherin,Vimentin,β-catenin)were detected by q RT-PCR and Western blot.6.The regulate relationship of FOXD3-AS1 and FOXD3 on RNA and protein expression levels was detected by q RT-PCR and Western blot.Results:1.UCSC database suggested that FOXD3-AS1 and FOXD3 were correlated in gene positions.2.The expression of FOXD3-AS1 gene in gastric cancer cell lines SGC-7901,BGC-823,MGC-803 and HGC-27 were higher than those in normal control group(P<0.05);the expression of FOXD3-AS1 in gastric cardia adenocarcinoma tissues was significantly higher than that in adjacent normal tissues(Z=-2.695,P=0.007),and the expression level of FOXD3-AS1 was correlated with clinical stage(Z=-2.981,P=0.003)and lymph node metastasis(Z=-3.43,P=0.001)of GCA patients.3.The expressions of FOXD3 in gastric cancer cell lines SGC-7901,BGC-823,MGC-803 and HGC-27 were lower than those in normal control group(P<0.05);the expression of FOXD3 in cardia adenocarcinoma was significantly lower than that in adjacent normal tissues(Z=-7.649,P<0.01),and the expression of FOXD3 was correlated with the clinical stage(Z=-2.853,P=0.004)and lymph node metastasis(Z=-3.187,P=0.001)of GCA patients.4.Construction of knockdown and overexpression of FOXD3-AS1 cell lines and verification of transfection efficiency: compared with the control group,the expression level of FOXD3-AS1 in SGC-7901 cells transfected with si-FOXD3-AS1 was significantly down-regulated,and the expression level of FOXD3 in SGC-7901 cells transfected with pc DNA3.1-FOXD3-AS1 plasmid was significantly up-regulated(P<0.05).5.Construction of overexpressed FOXD3 cell lines and verification of transfection efficiency: compared with the control group,the expression level of FOXD3 in SGC-7901 cells transfected with pc DNA3.1-FOXD3 plasmid was significantly up-regulated(P<0.05).6.The results of cell proliferation assay and plate clone formation assay showed that FOXD3-AS1 knockdown inhibited the proliferation ability of gastric cancer cells SGC-7901 in vitro,while FOXD3-AS1 overexpression enhanced the proliferation ability of gastric cancer cells SGC-7901 in vitro.The results of scratch healing assay showed that FOXD3-AS1 knockdown inhibited the migration ability of gastric cancer cells SGC-7901,while FOXD3-AS1 overexpression enhanced the migration ability of gastric cancer cells SGC-7901 in vitro.Transwell compartment invasion assay showed that FOXD3-AS1 knockdown inhibited the invasion ability of gastric cancer cells SGC-7901,while FOXD3-AS1 overexpression enhanced the invasion ability of gastric cancer cells SGC-7901 in vitro.FOXD3-AS1 knockdown can up-regulate the m RNA expression of E-cadherin in gastric cancer cells SGC-7901,and decrease the m RNA expression of N-cadherin,β-catenin and vimentin,while overexpression has the opposite effect.FOXD3-AS1 knockdown could up-regulate the protein expression levels of E-cadherin in SGC-7901,and decrease the protein expression levels of N-cadherin,β-catenin and vimentin,while over-expression had the opposite effect(P <0.05).7.The results of cell proliferation assay and plate clone formation assay showed that FOXD3 overexpression could inhibit the proliferation of gastric cancer cell line SGC-7901 in vitro.The results of scratch healing assay showed that FOXD3 overexpression could inhibit the migration of gastric cancer cell line SGC-7901.Transwell compartment invasion assay showed that Fox D3 overexpression inhibited the invasion ability of SGC-7901.FOXD3 overexpression can up-regulate the m RNA expression of E-cadherin in SGC-7901,and decrease the m RNA expression of N-cadherin,β-catenin and vimentin.FOXD3 overexpression up-regulated the protein expression level of E-cadherin in SGC-7901,and decreased the protein expression levels of N-cadherin,β-catenin and vimentin(P < 0.05).8.SGC-7901 cell lines with knockdown and overexpression of FOXD3-AS1 gene were constructed to detect the expression level of FOXD3 by q RT-PCR: The relative expression level of FOXD3 gene in the knockdown group was significantly higher than that in the control group;the relative expression of FOXD3 gene in FOXD3-AS1 overexpression group was significantly lower than that in control group.9.Western blot was used to detect the protein expression of FOXD3 in gastric cancer cell line SGC-7901 after knockdown and overexpression of FOXD3-AS1: the expression level of FOXD3 protein in gastric cancer cells was significantly increased after the knockdown of FOXD3-AS1;the expression level of FOXD3 protein in gastric cancer cells was significantly decreased after the overexpression of FOXD3-AS1.Conclusion:1.The expression of FOXD3-AS1 was significantly up-regulated in GCA tissues and gastric cancer cell lines,and the expression of FOXD3 was significantly down-regulated in GCA tissues and gastric cancer cell lines,both of which were closely related to the clinical stage and lymph node metastasis of patients.2.FOXD3-AS1 knockdown can inhibit the proliferation,migration and invasion ability of gastric cancer cells in vitro,and the overexpression of FOXD3-AS1 can enhance the proliferation,migration and invasion ability of gastric cancer cells in vitro.FOXD3-AS1 may affect the invasion and metastasis process of gastric cancer cells by participating in the expression of EMT pathway related factors.3.FOXD3 overexpression can inhibit the proliferation,migration and invasion ability of gastric cancer cells in vitro,and FOXD3 may affect the invasion and metastasis process of gastric cancer cells by participating in the expression of EMT pathway related factors.4.FOXD3-AS1 negatively regulates the expression of FOXD3 at the transcriptional and post-transcriptional levels. |
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