An Experimental Study Of The Effect Of Human Gene PSMA7 On The Biological Behavior Of A549 Cell Line

Introduction:Proteasome subunit alpha type-7 is a protein that in humans is encoded by the PSMA7 gene. The proteasome is a multicatalytic proteinase complex with a highly ordered ring-shaped 20S core structure. This particular subunit has been shown to interact specifically with the hepatitis B virus X protein (HBx), a protein critical to viral replication.In addition, this core alpha subunit is involved in regulating some tumor-associated proteins, such as the hypoxia-inducible factor-1 alpha (HIF-1) and c-Abl and Arg. The protein is also involved in conveying membrane protein and regulating proteasomes activity. Gene PSMA7 is located in chromosome 20 (20q13.33) which is repeatedly related to the amplification of some oncogenes. Although human tumors are usually accompanied with the amplification of chromosome 20q, but few literatures concerned with PSMA7 functions in tumor have been reported.In order to elucidate its exact functions, we designed a series of experiments on the basis of bioinformatics analysis to verify the hypothetic functions from different aspects, for example, to observe the effects of PSMA7 overexpression on cell growth, proliferation and invasiveness of A549 cells, and to investigate the growth status of transplantation tumor in BALB/c-nu nude mice. On the other hand, small interfering RNA (siRNA) technique was performed to knock down PSMA7 expression in A549 cells to investigate the effects of down regulation of PSMA7 on the invasiveness, proliferation, and tumor growth of A549 cells. Above all, our purpose was to understand the biological function of the PSMA7 protein and to elucidate its mechanism in the development of tumors.Methods:1. Expression profile and subcellular localization of PSMA71.1 Expression of PSMA7 in a variety of tumor cell lines was detected by Western blot technique.1.2 Recombinant plasmid pDsRedl-C3/PSMA7 was constructed and fluorescence microscope was used to observe its subcellular localization in 786-0 and 293T cell lines. In addition, subcellular localization of PSMA7 in Lovo, HBE135-E6E7, A549 cells was detected by immunofluorescence technique.1.3 Immunohistochemistry was utilized to detect the expression of PSMA7 in bronchogenic carcinoma.2. Experiments on overexpression of PSMA7 gene in A549 cell line2.1 Construction of recombinant plasmid pcDNA3.1(-)/PSMA7 and establishment of stable expression cell line:Aim gene PSMA7 was amplified from normal bronchial epithelial cell line HBE135-E6E7 by RT-PCR method, and connected to vector pMD18-T. After PCR and BamHⅠ/XhoⅠdouble digestion, the aim fragment was connected to eukaryotic expression vector pcDNA3.1(-) to obtain recombinant plasmids pcDNA3.1(-)/PSMA7. And then the recombinant plasmid was digested with BamHⅠ/XhoⅠand identified by sequencing and finally transfected into A549 cells by lipofectamine 2000, screened with G418 to obtain the stable expression line. RT-PCR and Westren blot were used to identify the expression of PSMA7.2.2 The MTT assay was used to determine the effect of PSMA7 up-regulation on the proliferation of A549 cell line. Flow cytometry was performed to measure the distribution of cell cycle phase induced by starvation and cell apoptosis induced by cis-diamminedichloroplatinum. Colony formation assay was used to evaluate the clonality and transwell assay to observe the cell migration and invasive ability, and flow cytometry to detect the expression of ICAM-1.2.3 In order to establish transplantation tumor model in BALB/c-nu nude mice, A549/pcDNA3.1(-) cell and A549/pcDNA3.1(-)/PSMA7 cell were injected hypodermically in the roots of the right and left anterior limb of BALB/c-nu nude mice, respectively. The growth velocity of transplantation tumor and volume of tumor body were monitored.3. Experiments on silencing of PSMA7 gene in A549 cell line3.1 Recombinant plasmids bought from Shanghai GenePharma Co., Ltd were transfected into A549 cell and screened with hygromycin. Western blot was used to found the best recombinant plasmid which could suppress efficiently the expression of PSMA7 protein in A549 cell.3.2 The MTT assay was used to determine the effect of PSMA7 down-regulation on the proliferation of A549 cell and flow cytometry to detect cell cycle and cell apoptosis. Colony formation assay was used to evaluate the clonality and transwell assay to observe the cell migration and invasive ability, and flow cytometry to detect the expression of ICAM-1.3.3 The animal experiment on silencing of PSMA7 gene was performed as above.Results:1. Subcellular localization1.1 PSMA7 protein expression was found in many kinds of tumor cell lines by Western blot, but with tissue specificity.1.2 Eukaryotic expression vector pDsRedi-C3/PSMA7 was successfully constructed and transfected into 786-0 and 293T cell by liposome method. Fusion protein pDsRed1-C3/PSMA7 was located in the cytoplasm and nucleus, but there were differences in different cell lines.1.3 PSMA7 was found to distribute mainly in the cytoplasm of Lovo, A549 and HBE135-E6E7 cells by immunofluorescence technique, but different in the nucleus.1.4 Immunohistochemistry showed that PSMA7 was expressed in the cytoplasm and nucleus of nonsmall-cell lung cancer. 2. Overexpression of PSMA7 gene in A549 cell line2.1 Eukaryotic expression vector pcDNA3.1(-)/PSMA7 was successfully constructed and transfected into A549 cell by liposome method. And then RT-PCR and Western blot were used to identify the stable expression cell line screened with G418. RT-PCR and Western Blot showed that overexpression of PSMA7 was more significant in transfected pcDNA3.1(-)/PSMA7 group than that in transfected pcDNA3.1(-) group and control group (P<0.05). The data showed that a cell line with overexpression of PSMA7 was successfully constructed.2.2 MTT method and growth curve showed that PSMA7 overexpression could significantly decrease the proliferation of A549 cell (F=3451.158, P=0.000). Flow cytometry suggested that PSMA7 overexpression could not change the cell cycle (P > 0.05), but we found overexpression of PSMA7 could promote cell apoptosis induced by cis-diamminedichloroplatinum (F=63.413, P=0.000). Plate clone formation test showed that the clonality of A549/pcDNA3.1(-)/PSMA7 group was less than that of A549/pcDNA3.1(-) group (t=2.863, P=0.046) and transwell assay showed the invasive ability of A549/pcDNA3.1(-)/PSMA7 cell decreased (t=3.236, P=0.032). Flow cytometry also showed the significant decrease of ICAM-1 secreted by A549/pcDNA3.1(-)/PSMA7 cell (t=86.325, P=0.000).2.3 Transplantation tumor model in BALB/c-nu nude mice was established successfully. The tumor size of A549/pcDNA3.1(-)/PSMA7 group was smaller than that of A549/pcDNA3.1(-) group (t=-4.636, P=0.006)3. Silencing of PSMA7 gene in A549 cell line3.1 Both recombinant plasmid pGPU6/Hygro-PSMA7-265 and pGPU6/Hygro-PSMA7-929 were confirmed to suppress efficiently the expression of PSMA7 protein in A549 cell by Western blot (P<0.05). Recombinant plasmid pGPU6/Hygro-PSMA7-265 (abbr., shPSMA7) with higher inhibition ratio (69.37%) was selected to use in the following experiments. In the meanwhile, the negative control pGPU6/Hygro-SHNC was named as shNC.3.2 MTT method showed that shPSMA7 group had a higher growth rate than shNC group (F= 1237.794, P=0.000). Flow cytometry showed that the percent of G1 and G2 phase decreased in shPSMA7 group and inversely that of S phase increased (P < 0.05). Flow cytometry also showed that PSMA7 silencing could not change the cell apoptosis induced by cis-diamminedichloroplatinum (F=0.336, P=0.570). Plate clone formation test showed that the clonality of shPSMA7 group was more than that of shNC group (t=-2.747,P=0.033) and transwell assay showed the invasive ability of shPSMA7 cell increased (t=-3.080, P=0.037). Flow cytometry also showed that ICAM-1 secretion increased significantly in shPSMA7 cell (t=-119.827, P=0.000).3.3 The tumor size of shPSMA7 group was bigger than that of shNC group (t=3.216, P=0.024)Conclusions:Our data showed that up-regulation of PSMA7 in A549 cell significantly suppressed cellular proliferation and promoted cell apoptosis induced by cis-diamminedichloroplatinum and also decreased the clonality and invasive ability and tumor growth. At the same time, our data also showed that down-regulation of PSMA7 expression in A549 cell significantly promoted cellular proliferation rate and changed the cell cycle phase, also increased the clonality and invasive ability and tumor growth. It suggests that PSMA7 might be a tumor-associated protein involved in the carcinogenesis and development of lung cancer and a potential bio-marker for anti-cancer therapies. This would pave the way for further study of PSMA7.