| Background and PurposeLung cancer is one of the most malignant tumors with the highest incidence and mortality in the world.NSCLC(Non-small cell lung cancer)accounts for 80% to 85% of lung cancer.Primary tumor metastasis is the primary cause of death in patients with lung cancer.EMT(Epithelial-mesenchymal Transition)is a prerequisite for the invasion and metastasis of epithelial tumor cells.DAL-1(Differentially expressed in Adenocarcinoma of the Lung)is a tumor suppressor gene which inhibits EMT in lung cancer.HSPA5(Heat Shock Protein 5)is a tumor oncogene which promotes EMT in lung cancer.HSPA5 protein could be directly bound to the DAL-1 protein.This study was to reveal the expression of DAL-1 and HSPA5 in NSCLC and their relationship with EMT.Methods ? Expression of DAL-1 and HSPA5 in non-small cell lung cancer cells1.The protein expressions of DAL-1 and HSPA5 in different lung cancer cell lines were detected by Western blot assays respectively.2.The co-localization of DAL-1 protein and HSPA5 protein in H460 cells was observed by immunofluorescence assay and laser confocal scanning microscope.3.Lentiviruses overexpressing DAL-1(LV-DAL-1)and negative control virus(LV-CON1)were transfected into A549 cells.This two group cells were named as A549 / LV-DAL-1(experimental group)and A549 / LV-CON1(negative control group).RTq-PCR and Western blot assays were used to detect the mRNA and protein expressions of HSPA5.4.The A549 / LV-DAL-1 cells were infected with lentiviral vectors(LV-HSPA5)and negative control virus(LV-CON2).This two group cells were named as A549/LV-DAL-1/LV-HSPA5(experimental group)and A549/LV-DAL-1/LV-CON2(negative control group).RTq-PCR and Western blot assays were used to detect the mRNA and protein expressions of DAL-1.II Effects of DAL-1 and HSPA5 on EMT,proliferation,invasion and migration of non-small cell lung cancer cells1.The experiment was divided into two groups: A549 / LV-DAL-1(experimental group)and A549 / LV-CON1(negative control group).The mRNA and protein levels of E-cadherin,?-catenin,N-cadherin and Vimentin were detected by RTq-PCR and Western blot assays.The proliferation ability was detected by CCK-8 cell proliferation assay and plate clone formation assay.The invasion and migration abilities were detected by cell scratch test,transwell invasion and migration assays.2.A549 cells were infected with lentiviruses interference with HSPA5 expression(LV-HSPA5 RNAi)and negative control virus(LV-CON3).The cells were named as A549 / LV-HSPA5 RNAi(experimental group 1)and A549 / LV-CON3(negative control group 1).A549 / LV-HSPA5 RNAi cells were infected with lentivirus(LV-HSPA5)and negative control virus(LV-CON2)respectively.This two group cells were named as A549 / LV-HSPA5 RNAi / LV-HSPA5(experimental group 2)and A549 / LV-HSPA5 RNAi / LV-CON2(negative control group 2).The mRNA and protein levels of E-cadherin,?-catenin,N-cadherin and Vimentin were detected by RTq-PCR and Western blot assays.The proliferation ability was detected by CCK-8 cell proliferation assay and plate clone formation assay.The invasion and migration abilities were detected by cell scratch test,transwell invasion and migration assays.? To investigate whether DAL-1 affects EMT,proliferation,invasion and migration of non-small cell lung cancer cells through HSPA51.The experiment was divided into two groups: A549 / LV-DAL-1 / LV-HSPA5(experimental group)and A549 / LV-DAL-1 / LV-CON2(negative control group).The mRNA and protein levels of E-cadherin,?-catenin,N-cadherin and Vimentin were detected by RTq-PCR and Western blot assays.The proliferation was detected by CCK-8 cell proliferation assay and plate clone formation assay.The invasion and migration abilities were detected by cell scratch test,transwell invasion and migration assays.2.A549 / LV-DAL-1 cells were infected with lentiviruses(LV-HSPA5 RNAi)and negative control virions(LV-CON3).This two group cells were named as A549 / LV-DAL-1 / LV-HSPA5 RNAi(experimental group)and A549 / LV-DAL-1 / LV-CON3(negative control group).The mRNA and protein levels of E-cadherin,?-catenin,N-cadherin and Vimentin were detected by RTq-PCR and Western blot assays.The proliferation ability was detected by CCK-8 cell proliferation assay and plate clone formation assay.The in vitro invasion and migration abilities were detected by cell scratch test,transwell invasion and migration assays.IV Effects of DAL-1 and HSPA5 on PI3K/Akt/Mdm2/p53 signaling pathway and EMT of non-small cell lung cancer cells1.The experiment was divided into two groups: A549 / LV-DAL-1(experimental group)and A549 / LV-CON1(negative control group).The protein expressions of PI3 K,p-PI3 K,Akt,p-Akt,Mdm2,p-Mdm2,p53,E-cadherin,?-catenin,N-cadherin and Vimentin were detected by Western blot assay.2.The experiment is divided into four groups: A549 / LV-HSPA5 RNAi / HSPA5(experimental group 1),A549 / LV-CON3(control group 1),A549 / LV-HSPA5 RNAi / HSPA5(experimental group 2)and A549 / LV-HSPA5 RNAi / LV-CON2(control group 3).The protein expressions of PI3 K,p-PI3 K,Akt,p-Akt,Mdm2,p-Mdm2,p53,E-cadherin,?-catenin,N-cadherin and Vimentin were detected by Western blot assay.V To investigate whether DAL-1 can affect the expression of PI3K/ Akt/Mdm2/p53 signaling pathway protein and EMT-related protein through HSPA5The experiment was divided into two groups: A549 / LV-DAL-1 / LV-HSPA5(experimental group)and A549 / LV-DAL-1 / LV-CON2(negative control group).The protein expressions of PI3 K,p-PI3 K,Akt,p-Akt,Mdm2,p-Mdm2,p53,E-cadherin,?-catenin,N-cadherin and Vimentin were detected by Western blot assay.Results ? Expression of DAL-1 and HSPA5 in non-small cell lung cancer1.The protein expressions of DAL-1 were higher in H520 and H460 cells,while lower in A549,SPCA-1,H1975 and H1299 cells.The protein expressions of HSPA5 were higher in all NSCLC cells.2.DAL-1 protein and HSPA5 protein were co-localized in NNSCLC cells,manaly in cytoplasm.3.DAL-1 down-regulated the mRNA and protein expressions of HSPA5(P<0.05).4.HSPA5 did not changed the mRNA expressions of DAL-1 significantly(P>0.05),while down-regulated the protein expressions of DAL-1 significantly(P<0.05).II The effect of DAL-1 and HSPA5 on EMT,proliferation,invasion and migration of non-small cell lung cancer cells1.DAL-1 increased the mRNA and protein expressions of E-cadherin and ?-catenin,while decreased the mRNA and protein expressions of N-cadherin and Vimentin(P<0.05).DAL-1 inhibited the proliferation,invasion and migration abilities of A549 cells(P<0.05).2.HSPA5 decreased the mRNA and protein expressions of E-cadherin and ?-catenin,while increased the mRNA and protein expressions of N-cadherin and Vimentin(P<0.05).HSPA5 promoted the proliferation,invasion and migration abilities of A549 cells(P<0.05).? To investigate whether DAL-1 affects EMT,proliferation,invasion and migration of non-small cell lung cancer cells through suppressing HSPA5 expression1.After overexpressing HSPA5,the mRNA and protein expressions of E-cadherin and ?-catenin in A549 / LV-DAL-1 cells were down-regulated(P<0.05),while the mRNA and protein expressions of N-cadherin and Vimentin were up-regulated(P<0.05),and the proliferation,invasion and migration abilities were significantly increased(P<0.05).2.After knockdown of HSPA5,the mRNA and protein expressions of E-cadherin,?-catenin and N-cadherin and Vimentin in A549 / LV-DAL-1 cells were up-regulated(P<0.05),and the proliferation,invasion and migration abilities were significantly decreased(P<0.05).IV Effects of DAL-1 and HSPA5 on the expression of PI3K/Akt/Mdm2/p53 and EMT protein1.DAL-1 decreased the protein expressions of PI3 K,p-PI3 K,Akt,p-Akt,Mdm2,p-Mdm2,N-cadherin and Vimentin,while increased the protein expressions of p53,E-cadherin and ?-catenin(P<0.05).2.HSPA5 increased the protein expressions of PI3 K,p-PI3 K,Akt,p-Akt,Mdm2,p-Mdm2,N-cadherin and Vimentin,while decreased the protein expressions of p53,E-cadherin and ?-catenin(P<0.05).VI To investigate whether DAL-1 can affect PI3K/ Akt/Mdm2/p53 signaling pathway through HSPA5 to affect EMTAfter overexpressing HSPA5,the protein expressions of PI3 K,p-PI3 K,Akt,p-Akt,Mdm2,p-Mdm2,N-cadherin and Vimentin in A549 / LV-DAL-1 cells were up-regulated(P<0.05),while the protein expressions of p53,E-cadherin and ?-catenin were down-regulated(P<0.05).ConclusionIn non-small cell lung cancer cells:1.DAL-1 protein and HSPA5 protein were co-localized,mainly in cytoplasm.2.DAL-1 can inhibit the expression of HSPA5 mRNA and protein.3.DAL-1 may inhibit the occurrence of EMT,proliferation,invasion and migration by inhibiting HSPA5 expression.4.DAL-1 may inhibit the occurrence of EMT by inhibiting the expression of PI3 K,AKT,Mdm2 protein and phosphorylation by inhibiting the expression of HSPA5. |
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