| In recent years,the forkhead box family of transcription factors has received more and more attention in cancer prevention and treatment research.FOXP4 belongs to the forkhead box family.It has been shown that FOXP4 is up-regulated in a variety of tumor tissues,indicating that abnormal expression of FOXP4 may be associated with malignant tumor pathogenesis.However,its role and mechanism in gastric cardia adenocarcinoma(GCA)have not been reported.Long non-coding RNAs(lnc RNAs)can regulate the expression of genes,and recent studies have shown that the abnormal expression of lnc RNAs is closely related to a variety of tumors,while the regulatory mechanism of FOXP4-AS1 gene in cardiac adenocarcinoma and its relationship with FOXP4 gene have not been reported.In this study,we first detected the expression of FOXP4 and FOXP4-AS1 genes in gastric cancer cell lines and cardiac adenocarcinoma tissues,and further constructed knockdown and overexpression plasmids to investigate their effects on the proliferation,migration and invasion of gastric cancer cell lines and their effects on epithelial-mesenchymal transition(EMT)in gastric cancer cell lines.Main study contents and results are as follows:Part one Expression and correlation of transcription factor FOXP4 and long non-coding RNA FOXP4-AS1 in gastric cancer cell lines and cardiac adenocarcinoma tissuesObjective: To investigate the expression and correlation of long-chain non-coding RNAFOXP4-AS1 and transcription factor FOXP4 genes in gastric cancer cell lines and gastric cardia adenocarcinoma tissues,respectively,and to analyze the clinical significance of their expression.Methods: 1.The expression of FOXP4 and FOXP4-AS1 genes in 4 gastric cancer cell lines(BGC-823,SGC-7901,MGC-803 and HGC-27)was detected by real-time reverse transcription-polymerase chain reaction(qRT-PCR).2.The expression and correlation of FOXP4 and FOXP4-AS1 genes in 48 cases of cardiac adenocarcinoma and their corresponding adjacent normal tissues were detected by qRT-PCR,and statistically analyzed in combination with clinicopathological parameters.Results: 1.qRT-PCR results showed that FOXP4 gene was highly expressed in all four gastric cancer cell lines(P < 0.05),and the expression level was the highest in SGC-7901 cells.2.The m RNA expression of FOXP4 gene was up-regulated in cardiac adenocarcinoma tissues(P < 0.05),and was closely related to TNM stage and lymph node metastasis(P < 0.01).3.qRT-PCR results showed that FOXP4-AS1 gene was highly expressed in all four GC cell lines(P < 0.05),and the expression level was the highest in BGC-823 cells.4.FOXP4-AS1 gene expression in cardiac adenocarcinoma tissues was lower than that in their corresponding adjacent normal tissues(P < 0.05),and was closely related to TNM stage and lymph node metastasis(P < 0.01).5.Correlation between m RNA expression of FOXP4-AS1 and FOXP4 genes in cardiac adenocarcinoma tissues: There was no significant correlation between m RNA expression of FOXP4-AS1 and FOXP4 genes in cardiac adenocarcinoma tissues(P > 0.05).Part Two Effect of FOXP4 gene on the biological behavior of gastric cancer cellsObjective: To investigate the mechanism of knockdown and overexpression on the effect of FOXP4 gene on gastric cancer cellsMethods: 1.si-FOXP4 was designed and synthesized and transfected into gastric cancer cell line SGC-7901.Transfection efficiency of cell lines was verified using the method of qRT-PCR.2.The overexpression vector pc DNA3.1-FOXP4 was constructed and transfected into gastric cancer cells SGC-7901,and transfection efficiency of cell lines was verified using the method of qRT-PCR.3.MTS assay was used for verification the effects of knockdown and overexpression of FOXP4 gene on the proliferation ability of SGC-7901 cells in vitro.4.Scratch assay was used for verification the effects of knockdown and overexpression of FOXP4 gene on the migration ability of SGC-7901 cells in vitro.5.Transwell chamber invasion assay was used for verification the effects of knockdown and overexpression of FOXP4 gene on the invasive ability of SGC-7901 cells in vitro.6.qRT-PCR was used for verification the effects of knockdown and overexpression of FOXP4 gene on the m RNA expression levels of EMT-related factors(E-cadherin,N-cadherin,vimentin,β-catenin,Twist,ZEB1,MMP2 and MMP9).7.Westernblot was performed to examine the effect of knockdown and overexpression of FOXP4 gene on the protein expression levels of EMT-related factors(E-cadherin,N-cadherin,vimentin and β-catenin).Results: 1.Design of si RNAsi-FOXP4 and verification of transfection efficiency: si RNAsi-FOXP4 was successfully designed and si-FOXP4 was transfected into gastric cancer cell SGC-7901,and verification of transfection efficiency revealed that the relative expression of FOXP4 gene in the knockdown group was lower than that in the blank control group(P < 0.01).2.Construction of overexpression vector pc DNA3.1-FOXP4 and verification of transfection efficiency: The overexpression vector pc DNA3.1-FOXP4 was transferred into gastric cancer cells SGC-7901.Validation of transfection efficiency by qRT-PCR revealed that the relative expression of FOXP4 gene in the overexpression group was higher than that in the blank control(P < 0.01).3.Effect of knockdown and overexpression of FOXP4 gene on the proliferation ability of GC cells: MTS assay results showed that knockdown of FOXP4 gene significantly inhibited the proliferation ability of SGC-7901,while overexpression of FOXP4 gene had the opposite effect(P < 0.05 or P < 0.01).4.Effect of knockdown and overexpression of FOXP4 gene on the migration ability of GC cells: The results of scratch assay showed that knockdown of FOXP4 gene significantly inhibited the migration ability of SGC-7901,while overexpression of FOXP4 gene had the opposite effect.(P < 0.05 or P < 0.01).5.Effect of knockdown and overexpression of FOXP4 gene on the invasive ability of GC cells in vitro: The results of transwell chamber invasion assay showed that knockdown of FOXP4 gene significantly inhibited the invasive ability of SGC-7901,while overexpression of FOXP4 gene had the opposite effect.(P < 0.05 or P < 0.01).6.Effect of FOXP4 gene on the m RNA expression level of EMT-related factors: qRT-PCR results showed that in SGC-7901 cells,after knockdown of FOXP4 gene,the m RNA expression level of E-cadherin was increased(P < 0.01),and the m RNA expression levels of N-cadherin,vimentin,Twist,β-catenin and MMP9 were decreased(P < 0.05 or P < 0.01).The m RNA expression levels of E-cadherin were decreased(P < 0.01),and the m RNA expression levels of N-cadherin,vimentin,Twist,β-catenin and MMP9 were increased(P < 0.05 or P < 0.01)after overexpression of FOXP4 gene.7.Effect of FOXP4 gene on the protein expression level of EMT-related factors: Westernblot assay results showed that in SGC-7901 cells,after knockdown of FOXP4 gene,the protein expression level of E-cadherin increased,and the protein expression levels of N-cadherin,vimentin,Twist and β-catenin decreased.However,after overexpression of FOXP4 gene,the protein expression level of E-cadherin was decreased,and the protein expression levels of N-cadherin,vimentin and β-catenin were increased.Part Three Effect of Long Noncoding RNAFOXP4-AS1 Gene on the Biological Behavior of Gastric Cancer CellsObjective: To investigate the effect of knockdown and overexpression of long non-coding RNA FOXP4-AS1 on gastric cancer cells and its mechanism.Methods: 1.Sh-FOXP4-AS1 was designed and synthesized and transfected into gastric cancer cell lines SGC-7901 and BGC-823,transfection efficiency of cell lines was verified using the method of qRT-PCR.2.Overexpression pc DNA3.1-FOXP4-AS1 was constructed and transfected into gastric cancer cells SGC-7901 and BGC-823,transfection efficiency of cell lines was verified using the method of qRT-PCR.3.MTS assay was used for verification the effects of knockdown and overexpression of FOXP4-AS1 gene on the proliferation of SGC-7901 and BGC-823 cells in vitro.4.Scratch assay was used for verification the effects of knockdown and overexpression of FOXP4-AS1 gene on the migration ability of SGC-7901 and BGC-823 cells in vitro.5.Transwell chamber invasion assay was used for verification the effects of knockdown and overexpression of FOXP4-AS1 gene on the invasion of SGC-7901 and BGC-823 cells in vitro.6.The effects of knockdown and overexpression of FOXP4-AS1 gene on the m RNA expression levels of EMT-related factors(E-cadherin,N-cadherin,vimentin,β-catenin,Twist,ZEB1,MMP9,MMP2)were detected by qRT-PCR.7.Westernblot assay was used for verification the effects of knockdown and overexpression of FOXP4-AS1 gene on the protein expression levels of EMT-related factors(E-cadherin,N-cadherin,vimentin,β-catenin).Results: 1.Screening of FOXP4-AS1 sh RNA plasmid and verification of knockdown efficiency: SGC-7901 cells and BGC-823 cells knocked down FOXP4-AS1 gene were successfully constructed.The results of qRT-PCR analysis showed that the relative expression level of FOXP4-AS1 gene in the sh1-FOXP4-AS1 transfection group was lower than that in the sh-NC transfection group and the blank control group,and the difference had statistical significance(P < 0.01).2.Construction of overexpression vector pc DNA3.1-FOXP4-AS1 and verification of transfection efficiency: The overexpression vector pc DNA3.1-FOXP4-AS1 was transferred into gastric cancer cells SGC-7901 and BGC-823.Verification of transfection efficiency by qRT-PCR revealed that the relative expression level of FOXP4-AS1 gene in the overexpression group was significantly higher than that in the control group(P < 0.01).3.Effect of knockdown and overexpression of FOXP4-AS1 gene on the proliferation of GC cells: MTS assay results showed that knockdown of FOXP4-AS1 gene significantly inhibited the proliferation of cells SGC-7901 and BGC-823,while overexpression of FOXP4-AS1 gene had the opposite effect(P < 0.05 or P < 0.01).4.Effect of knockdown and overexpression of FOXP4-AS1 gene on the migration ability of GC cells: The results of scratch assay showed that knockdown of FOXP4-AS1 gene significantly inhibited the migration ability of cells SGC-7901 and BGC-823,while overexpression of FOXP4-AS1 gene had the opposite effect.(P < 0.05 or P < 0.01).5.Effect of knockdown and overexpression of FOXP4-AS1 gene on the invasive ability of GC cells: The results of transwell chamber invasion assay showed that knockdown of FOXP4-AS1 gene significantly inhibited the invasive ability of cells SGC-7901 and BGC-823,while overexpression of FOXP4-AS1 gene had the opposite effect.(P < 0.05 or P < 0.01).6.Effect of FOXP4-AS1 gene on the m RNA expression level of EMT-related factors: qRT-PCR results showed that in SGC-7901 cells and BGC-823 cells,after knockdown of FOXP4-AS1 gene,the m RNA expression level of E-cadherin was increased(P < 0.01),and the m RNA expression levels of N-cadherin,vimentin,Twist,MMP9 and β-catenin were decreased(P < 0.05 or P < 0.01).The m RNA expression levels of E-cadherin were decreased(P < 0.01)and those of N-cadherin,vimentin,Twist,MMP9 and β-catenin were increased(P < 0.05 or P < 0.01)after overexpression of FOXP4-AS1 gene.7.Effect of FOXP4-AS1 gene on the protein expression level of EMT-related factors: Western blot assay results showed that in SGC-7901 cells and BGC-823 cells,after knockdown of FOXP4-AS1 gene,the protein expression level of E-cadherin increased,and the protein expression levels of N-cadherin,vimentin and β-catenin decreased.However,after overexpression of FOXP4 gene,the protein expression level of E-cadherin was decreased,and the protein expression levels of N-cadherin,vimentin and β-catenin were increased.Conclusions: 1.Long non-coding RNA FOXP4-AS1 and transcription factor FOXP4 genes are highly expressed in gastric cancer cells and gastric cardiac adenocarcinoma tissues,and are closely related to the mechanism of gastric cardia adenocarcinoma 2.FOXP4 gene can affect the malignant biological behavior of SGC-7901 cells in vitro,and FOXP4 gene promotes the invasion and metastasis of GC cells may be achieved through the EMT pathway.3.FOXP4-AS1 gene can affect the malignant biological behavior of SGC-7901 and BGC-823 cells in vitro,and FOXP4-AS1 gene promotes the invasion and metastasis of gastric cancer cells may be by regulating the expression of EMT pathway-related factors. |
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