| Objective Primary hepatocellular carcinoma(PHC)is one of the most common malignant tumors in China.Hepatocellular carcinoma(HCC)is the most common type of PHC,and the mortality rate ranks second among malignant tumors.Tumor vasculogenic mimicry is a blood supply model surrounded by tumor cells,which can increase the risk of adverse prognosis and metastasis.Long non-coding RNA n339260(lnc RNA n339260)has been found in previous studies to be a factor that promotes vasculogenic mimicry(VM)formation in tumors.Six micro RNAs were found to be mi RNA29b-1-5p、mi RNA30e-5p、mi RNA31-3p、mi RNA92a-1-5p、mi RNA519c-5p、mi RNA520c-5p.These six micro RNAs may interact with lnc RNA n339260.But the specific mechanism has not been elaborated.This study explored the molecular mechanism of the formation of VM in hepatocellular carcinoma(HCC)by lnc RNA n339260,and provided a new idea for the diagnosis and targeted therapy of HCC.Methods 1.Collect the frozen tissues of 104 patients with hepatocellular carcinoma diagnosed by senior doctors in Tumor Hospital of Tianjin Medical University.Detection of the expression of lnc RNA n339260 and mi RNAs in human frozen hepatocellular carcinoma tissues by real-time fluorescence quantitative polymerase chain reaction(q RT-PCR).The strongest differentially expressed mi RNAs,i.e.mi RNA30e-5p,were selected and their relationship with clinicopathological data was analyzed.Spearman correlation analysis was used to verify the relationship between lnc RNA n339260 and mi RNA 30e-5p.Meanwhile,the survival of lnc RNA n339260 and mi RNA 30e-5p was analyzed by Kaplan-Meier method.2.Paraffin tissue sections of 104 patients with hepatocellular carcinoma confirmed by senior doctors in Tumor Hospital of Tianjin Medical University were collected.The expressions of MMP2,MMP9,VE-cadherin,E-cadherin,vimentin and Snail were observed by immunohistochemistry.The presence of VM in the tissues was observed by CD31/PAS double staining.Based on the results of q RT-PCR,the differences between lnc RNA n339260,mi RNA 30e-5p and MMP2,MMP9,VE-cadherin,E-cadherin,vimentin,Snail and VM were analyzed by chi-square test.Pearson correlation analysis was used to analyze the correlation among lnc RNA n339260、mi RNA30e-5p and MMP2、MMP9、VE-cadherin、E-cadherin、vimentin 、Snail and VM.3.Choose one cell in the high invasive and low invasive hepatocellular carcinoma cell lines and transfect the plasmid with the up-regulated and down-regulated lnc RNA n339260 respectively,and test the transfection effect with q RT-PCR,then establish stable transfection cell lines.The effects of lnc RNA n339260 on wound healing,migration,invasion,proliferation and conduit formation of HCC cells were observed by scratch,migration,invasion,MTT and three-dimensional culture in vitro.The expression of EMT and VM-related molecules(VE-cadherin,E-cadherin,vimentin,Snail)and MMP2,MMP9 were detected by Western Blot.4.In the selected hepatocellular carcinoma cell lines,the up-regulated and down-regulated plasmids of mi RNA30e-5p were transfected,and the transfection effect was tested by q RT-PCR,then stable transfection cell lines were established.The effects of mi RNA30e-5p on wound healing,migration,invasion,proliferation and conduit formation of HCC cells were observed by scratch,migration,invasion,MTT and three-dimensional culture in vitro.The expression of EMT and VM-related molecules(VE-cadherin,E-cadherin,vimentin,Snail)and MMP2,MMP9 were detected by Western Blot.5.To verify the relationship between lnc RNA n339260 and mi RNA30e-5p by luciferase reporter gene experiment.The plasmids of lnc RNA n339260 and mi RNA 30e-5p were co-transfected to establish stable cell lines.The interaction between them was observed by scratch test,migration test,invasion test,in vitro three-dimensional culture experiment and Western Blot.6.Predict the downstream target gene TP53INP1 of selected mi RNA30e-5p by Target Scan.Pic Tar and mi RDB database.mi RNA30e-5p and TP53INP1 can bind directly by luciferase reporter gene experiment.The plasmids of mi RNA30e-5p and TP53INP1 were co-transfected to establish stable cell lines.The interaction between them was observed by scratch test,migration test,invasion test,in vitro three-dimensional culture experiment and Western Blot.7.The expression of TP53INP1 in paraffin sections of 104 patients with hepatocellular carcinoma confirmed by senior doctors in Tumor Hospital of Tianjin Medical University was observed by immunohistochemistry.The differences among the expression of TP53INP1 and MMP2,MMP9,VE-cadherin,E-cadherin,vimentin,Snail and VM were analyzed.Pearson correlation analysis was used to analyze the correlation between TP53INP1 and MMP2,MMP9,VE-cadherin,E-cadherin,vimentin,Snail and VM.Meanwhile,survival analysis of TP53INP1 was performed by Kaplan-Meier method and clinical and pathological data were analyzed.Chi-square test was used to analyze the differences between lnc RNA n339260、mi RNA30e-5p and TP53INP1.Pearson correlation analysis was used to analyze the correlation between lnc RNA n339260、mi RNA30e-5p and TP53INP1.8.Western Blot was used to detect the expression of TP53INP1 in the previously established stable cell lines with up-regulation and down-regulation of lnc RNA n339260 and mi RNA30e-5p.9.In the selected hepatocellular carcinoma cell lines,the up-and down-regulated plasmids of TP53INP1 were transfected,and Western Blot was used to test the transfection effect,then stable transfection cell lines were established.The effects of TP53INP1 on wound healing,migration,invasion,proliferation and conduit formation of HCC cells were observed by scratch,migration,invasion,MTT and three-dimensional culture in vitro.The expression of EMT and VM-related molecules(VE-cadherin,E-cadherin,vimentin,Snail)and MMP2,MMP9,TP53INP1 were detected by Western Blot.Results 1.The expression of lnc RNA n339260 in HCC tissues was significantly higher than that in adjacent tissues.The expression of mi RNA29b-1-5p、mi RNA30e-5p and mi RNA520c-5p were significantly lower than that in adjacent tissues.There was no significant difference in the expression of mi RNA31-3p、mi RNA92a-1-5p and mi RNA519c-5p among HCC tissues and adjacent tissues.Spearman correlation analysis showed that the expression of lnc RNA n339260 was negatively correlated with that of mi RNA 30e-5p(r=-0.299,P=0.002).At the same time, there were no correlation between lnc RNA n339260 and mi RNA29b-1-5p as well as lnc RNA n339260 and mi RNA520c-5p.The average survival time of patients with high expression of lnc RNA n339260 was shorter than that of patients with low expression of lnc RNA n339260.The average survival time of patients with high expression of mi RNA30e-5p was longer than that of patients with low expression of mi RNA30e-5p.There was no significant correlation between the two expressions and age,TNM stage,tumor size and pathological grade,but with metastasis(P < 0.05).2.In paraffin section of HCC,the expression of MMP2,MMP9,VE-cadherin,vimentin and Snail in cancer tissues was higher than that in adjacent tissues.The expression of E-cadherin in cancer tissues was lower than that in adjacent tissues.The number of cases with VM in cancer tissues was more than that in adjacent tissues.At the same time,the expression of MMP2,MMP9,VE-cadherin,E-cadherin,Vimentin,Snail and the presence of VM had no significant relationship with age,TNM stage,tumor size and pathological grade,but were related to the metastasis of tumors(P < 0.05).The survival time of patients with positive expression of MMP2,MMP9,VE-cadherin,vimentin and Snail was significantly shorter than that of patients with negative expression.The survival time of patients with positive expression of E-cadherin was significantly longer than that of patients with negative expression.The expression of lnc RNA n339260 was positively correlated with the expression of MMP2,MMP9,VE-cadherin,vimentin,Snail and the presence of VM,but negatively correlated with the expression of E-cadherin.The expression of mi RNA30e-5p was negatively correlated with the expression of MMP2,MMP9,VE-cadherin,vimentin,Snail and the presence of VM,and positively correlated with the expression of E-cadherin.3.Select HCCLM3 cells with high invasiveness and HepG2 cells with low invasiveness to establish stable cell lines with up-regulation and down-regulation of lnc RNA n339260.The experimental results showed that the wound healing,migration,invasion,proliferation and tube-forming abilities of HCCLM3 and Hep G2 cells up-regulated by lnc RNA n339260 were significantly stronger than those of the control group(P < 0.05).Meanwhile,the wound healing ability,migration ability,invasive ability,proliferation ability and tube forming ability of HCCLM3 cells and Hep G2 cells with down-regulation of lnc RNA n339260 were significantly weaker than those of the control group(P < 0.05).The expressions of MMP2,MMP9,VE-Cadherin,Vimentin and Snail in HCCLM3 cells and Hep G2 cells up-regulated by lnc RNA n339260 were significantly higher than those in the control group,while the expression of E-cadherin was opposite(P < 0.05).The expressions of MMP2,MMP9,VE-Cadherin,Vimentin and Snail in HCCLM3 cells and Hep G2 cells down-regulated by lnc RNA n339260 were significantly weaker than those in the control group,while the expression of E-cadherin was opposite(P < 0.05).4.Select HCCLM3 cells with high invasiveness and Hep G2 cells with low invasiveness to establish stable cell lines with up-regulation and down-regulation of mi RNA30e-5p.The wound healing ability,migration ability,invasive ability,proliferation ability and tube forming ability of HCCLM3 cells and Hep G2 cells up-regulated by mi RNA30e-5p were significantly weaker than those of the control group(P < 0.05).The expressions of MMP2,MMP9,VE-Cadherin,Vimentin and Snail in HCCLM3 cells and Hep G2 cells up-regulated by micro RNA30e-5p were significantly lower than those in the control group,while the expression of E-cadherin was opposite(P < 0.05).The results of down-regulation of micro RNA30e-5p in HCLM3 cells and Hep G2 cells were contrary to those of up-regulation of micro RNA30e-5p cells.5.In luciferase reporter gene experiment,mi RNA30e-5p can bind directly to lnc RNA n339260.Increased wound healing,migration,invasiveness and tubulogenic ability of HCLM3 cells and Hep G2 cells with up-regulation of lnc RNA n339260 and up-regulation of mi RNA30e-5p were weaker than those of HCLM3 cells and Hep G2 cells with up-regulation of lnc RNA n339260 and up-regulation of mi RNA30e-5p.Western Blot assay showed that the expressions of VE-cadherin,vimentin,Snail,MMP2 and MMP9 in HCCLM3 cells and Hep G2 cells up-regulated by lnc RNA n339260 and up-regulated by mi RNA30e-5p were weaker than those in HCCLM3 cells and Hep G2 cells up-regulated by lnc RNA n339260 and down-regulated by mi RNA30e-5p,while the expression of E-cadherin was opposite.Similarly,the results of down-regulation of lnc RNA n339260 and down-regulation of mi RNA 30e-5p were opposite in HCCLM 3 cells and Hep G2 cells.6.Target Scan.Pic Tar and mi RDB databases predicted that the downstream target gene of screened mi RNA30e-5p was TP53INP1.Luciferase reporter gene experiment confirmed that mi RNA30e-5p could bind to TP53INP1 directly.The wound healing ability,migration ability,invasive ability and tubulogenic ability of HCCLM3 cells and Hep G2 cells with up-regulation of mi RNA30e-5p and down-regulation of TP53INP1 were stronger than those of HCCLM3 cells and Hep G2 cells with up-regulation of micro RNA30e-5p and down-regulation of TP53INP1.In Western Blot experiment,we observed that the expression of VE-cadherin,vimentin,Snail,MMP2 and MMP9 in HCCLM3 cells and Hep G2 cells up-regulated by mi RNA30e-5p and down-regulated by TP53INP1 was stronger than that in HCCLM3 cells and Hep G2 cells up-regulated by mi RNA30e-5p and up-regulated by TP53INP1,but the expression of E-cadherin was contrary.Similarly,the results of down-regulation of mi RNA30e-5p and up-regulation of TP53INP1 in HCLM3 cells and Hep G2 cells were opposite.7.The expression of TP53INP1 in cancer tissues was lower than that in adjacent normal tissues.The expression of TP53INP1 was negatively correlated with the expression of MMP2,MMP9,VE-cadherin,vimentin,Snail and the presence of VM,and positively correlated with the expression of E-cadherin.The average survival time of patients with high expression of TP53INP1 was longer than that of patients with low expression of TP53INP1.The expression of TP53INP1 and the presence of VM were not related to age,TNM stage,tumor size and pathological grade,but related to metastasis(P < 0.05).The expression of lnc RNA n339260 was negatively correlated with that of TP53INP1,while the expression of mi RNA30e-5p was positively correlated with that of TP53INP1.8.The expression of TP53INP1 protein decreased in the cell lines up-regulated by lnc RNA n339260,and increased in the cell lines down-regulated by lnc RNA n339260.The expression of TP53INP1 protein increased in the cell lines up-regulated by mi RNA30e-5p and decreased in the cell lines down-regulated by mi RNA30e-5p.9.Select HCCLM3 cells with high invasiveness and Hep G2 cells with low invasiveness to establish stable cell lines with up-and down-regulation of TP53INP1.The results showed that the wound healing ability,migration ability,invasive ability,proliferation ability and tube forming ability of HCCLM 3 and Hep G2 cells up-regulated by TP53INP1 were significantly weaker than those of the control group(P < 0.05).The expressions of MMP2,MMP9,VE-cadherin,vimentin and Snail in HCCLM3 cells and Hep G2 cells up-regulated by TP53INP1 were significantly lower than those in the control group,while the expression of E-cadherin was increased(P < 0.05).In HCCLM3 cells and Hep G2 cells that downregulated TP53INP1,the results were contrary.Conclusions 1.There was a negative correlation between the expression of lnc RNA n339260 and the expression of mi RNA 30e-5p in HCC tissues.High expression of lnc RNA n339260 is associated with low expression of mi RNA30e-5p and poor prognosis of patients.2.lnc RNA n339260 and mi RNA30e-5p is associated with the phenotype of EMT.lnc RNA n339260 can promote the occurrence of EMT and VM,while mi RNA30e-5p can inhibit the occurrence of EMT and VM.3.In vitro experiments,lnc RNA n339260 can promote the migration,invasion,proliferation and pipeline formation of HCC cells,while mi RNA30e-5p can inhibit the migration,invasion,proliferation and pipeline formation of HCC cells.4.In vitro,as a downstream factor of lnc RNA n339260,mi RNA30e-5p plays a role.lnc RNA n339260 can reduce the expression of mi RNA30e-5p,thus promoting the occurrence of EMT and VM.5.TP53INP1 is the target gene of mi RNA30e-5p.Upregulation of TP53INP1 can partially enhance the inhibition of EMT and VM mediated by mi RNA30e-5p and the inhibition of biological function of tumor cells.6.TP53INP1 can inhibit HCC cell migration,invasion,proliferation and pipeline formation in vitro.7.Mi RNA30e-5p inhibits the Snail signaling pathway by targeting up-regulating the expression of TP53INP1,thus exerting the function of inhibiting EMT and VM. |
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