Expression And Roles Of PBLD In Clear Cell Renal Cell Carcinoma

PBLD(Phenazine Biosynthesis Like Protein Domain Containing)was screened and isolated from human liver cDNA library by Iriyama et al.in 2001 using yeast two-hybrid technique with STRAP(serine/threonine receptor kinase-associated protein)as a bait,then a full-length target clone was obtained.In recent years,many literatures have reported that it is widely distributed in various tissues including the brain,heart,lung,liver,pancreas and kidney et al.At the same time,more and more reports of PBLD have different expressions in various tumor tissues,and the occurrence and development of tumors have a certain correlation with each other.However,studies related to PBLD have not been reported in clear cell renal cell carcinoma.Therefore,this study first used the database to explore the expression of PBLD in renal cell carcinoma,and then used renal cell tissue specimens to verify the relationship between its expression and clinical features,prognosis,etc.,and then explored PBLD by molecular biology experiments.The biological effects on the proliferation and migration of renal cancer cell lines were finally verified in nude mice.Objective:To investigate the expression of PBLD gene in renal clear cell carcinoma and its biological function on the growth and migration of various cell lines of clear cell renal cell carcinoma,and to explore the potential mechanism of PBLD in clear cell renal cell carcinoma.The diagnosis and treatment of clear cell carcinoma provides a possible theoretical basis and experimental basis.Methods:1.Using TCGA public database to explore and analyze the expression of PBLD in clear cell renal cell carcinoma and its adjacent normal renal tissues,and the relationship between the expression of PBLD and the survival of patients with clear cell renal cell carcinoma.2.Immunohistochemical staining was used to detect the expression of PBLD in 25 pairs of clear cell renal cell carcinoma tissue specimens and adjacent tissues.3.Using RT-PCR technique to analyze the expression of PBLD mRNA in 50 pairs of clear cell renal cell carcinoma tissues and adjacent normal renal tissues,and using the x2 test to express PBLD mRNA and clinicopathological features of patients with clear cell renal cell carcinoma.The relationship between the two is further analyzed.4.Western Blot was used to detect the expression of PBLD in human normal kidney cell 293T and human clear cell renal cell carcinoma cells ACHN,A498,Caki-1,786-0.PBLD overexpression vector and empty vector were introduced into human clear cell renal cell carcinoma 786-0 and ACHN by lentiviral infection to construct PBLD overexpression and empty control stable cell line,combined with qRT-PCR,Western Blot and other experimental techniques to verify the expression of PBLD.5.The effects of overexpressed PBLD on the proliferation of 786-0 and ACHN cells were detected by WST-1 proliferation assay and plate colony formation assay;overexpression was detected by Transwell chamber in vitro migration model and Wound-healing assay.The effect of PBLD on the migration ability of both 786-0 and ACHN cells.6.The nude mice were subcutaneously inoculated with the stably constructed PBLD overexpression vector and the empty control cell lines ACHN-PBLD and ACHN?NC,and the overexpressed PBLD was observed in nude mice to express human clear cell renal cell carcinoma.The effect of cell xenograft growth.Results:1.TCGA data analysis showed that PBLD was significantly lower in clear cell renal cell carcinoma than in normal renal tissues adj acent to cancer,and patients with low expression of PBLD had lower overall survival rate.2.Immunohistochemical staining showed that the expression of PBLD in clear cell renal cell carcinoma tissues was significantly lower than that in normal renal tissues in 25 pairs of renal clear cell carcinoma tissues and adjacent normal renal tissues).3.RT-PCR results showed that in 50 pairs of clear cell renal cell carcinoma tissues and adjacent normal renal tissues,the expression of PBLD mRNA in renal clear cell carcinoma tissues was significantly lower than that in adjacent normal tissues.The lower the expression level of PBLD mRNA,the higher the clinicopathological grade of the corresponding clear cell renal cell carcinoma.4.The results of Western Blot showed that PBLD was lower than human normal kidney cell 293T in human clear cell renal cell carcinoma cell lines ACHN,A498,Caki-1 and 786-0.ACHN and 786-0 cell lines were infected with PBLD overexpression vector and empty lentivirus,and screened with puromycin to construct stable PBLD overexpression vector and empty control cells:786-O-PBLD,786-O-NC;ACHN-PBLD,ACHN-NC.The results of RT-PCR showed that the expression level of PBLD mRNA in 786-O-PBLD was significantly higher than that in 786-O-NC.The expression level of PBLD mRNA in ACHN-PBLD was significantly higher than that in ACHN-NC.Western Blot showed that the expression level of PBLD in 786-O-PBLD was significantly higher than that in 786-O-NC.The expression level of PBLD in ACHN-PBLD was significantly higher than that in ACHN-NC.5.In vitro experiments to observe the growth and migration ability of clear cell renal cell carcinoma stable cell lines.The proliferation experiment of WST-1 method showed that the proliferation rate of 786-O-NC group was significantly higher than that of 786-O-PBLD group after 48 h incubation at 37?.Similarly,the proliferation rate of ACHN-NC cells was significantly greater than that of WST-1.The ACHN-PBLD group suggested that overexpression of PBLD could significantly inhibit the cell proliferation of 786-0 and ACHN.The results of plate cloning experiments further showed that the number of cell clones in 786-O-NC group was significantly higher than that in 786-O-PBLD group.The number of clones in ACHN-NC group was significantly higher than that in ACHN-PBLD group,suggesting PBLD.Overexpression can significantly inhibit the ability of 786-0 and ACHN to clone.The Transwell chamber migration experiment showed that the number of cell migration in the 786-O-NC group was significantly higher than that in the 786-0-PBLD group after 24 hours of cell addition to the Transwell chamber.Similarly,the number of cell migration in the ACHN-NC group was significantly higher than that in the ACHN-NC group.ACHN-PBLD group.The results of Wound-healing assay showed that the cell migration distance of the 786-O-PBLD group was signi ficantly shorter than that of the 786-O-NC group after 12 hours of undifferentiated scratching in the 786-0 cell line.The ACHN-PBLD group migration distance was significantly shorter in the ACHN cell line than in the ACHN-NC group after 24 hours of undifferentiated scratches.Transwell migration assay and Wound-healing assay results also showed that overexpression of PBLD in clear cell renal cell carcinoma cells significantly inhibited cell migration.6.After 12 days of cell inoculation,the subcutaneous part of the ACHN-NC cells can reach the solid mass.However,the corresponding subcutaneous sites of two nude mice inoculated with ACHN-PBLD cells failed to reach the solid mass,and a small solid mass could be reached in about 16 days.After the nude mice were sacrificed and dissected,the tumor tissues were measured.The tumor volume of the ACHN-NC group was significantly larger than that of the ACHN-PBLD group.Experimental studies on tumor formation in nude mice have shown that overexpression of PBLD can reduce the tumorigenic ability of ACHN cells under the skin of nude mice,and inhibit the growth of tumors in vivo.Conclusions:1.PBLD protein and mRNA are lowly expressed in clear cell renal cell carcinoma,and the low expression of PBLD mRNA is associated with the histological grade of clear cell renal cell carcinoma.2.Low expression of PBLD was significantly associated with a decrease in overall survival in patients with clear cell renal cell carcinoma.3.PBLD can inhibit the growth and migration of clear cell renal cell carcinoma cells,and can inhibit the tumorigenic ability of clear cell renal cell carcinoma cells in nude mice and the growth of transplanted tumors.