The Roles Of Lung Tumor Oncogene ENPP1 In Lung Tumorigenesis

Objectives:By comparing the differential gene expression profile between lung normal cells and cancer cells,the differential gene ENPP1 was found.To investigate the molecular mechanism and the key target signaling pathways for ENPP1-mediated tumorigenicity,and to reveal the role and biological significance of ENPP1 in development of lung cancer.Materials and methods:1.The differential expression of wild-type and Gprc5a knockout mice with mouse tracheal epithelial cells(MTEC)was analyzed by mRNA expression profiling.The significant difference of gene ENPP1 was found and verified by RT-PCR at mRNA level.And through analyzed the Oncomine database,validation of the significance up-regulated expression of ENPP1 in lung cancer patients compare with normal.2.A series of ENPP1 high expression,knockdown and functional sequence mutations were constructed,and stable ENPP1 transfectants of HCC827 and A549 were developed for investigation the effects of ENPP1 on the growth and globing ability by in vivo and in vitro experiments.3.Flow cytometry and Westernblot techniques were used to detect changes in stemcell markers in human ENPP1 knockdown cell lines(A549,HCC827)compare with normal;ENPP1 stable transfectants of HCC827 and parental cells were subcutaneously injected into nude mice,and tumor sizes were measured as for biological effects of ENPP1 expression.4.The effect of knockdown of ENPP1 on the migration activity of human lung cancer cell lines was detected by Wound healing assay.Western blot and immunofluorescence assay were used to investigate the expression of EMT-related protein and its localization induced by TGF-? in human ENPP1 knockdown cell lines(A549,HCC827)compare with normal.5.A panel of 12 pairs of lung tumor tissues and adjacent normal lung tissues were used to detect the expression of ENPP1 by Western blot;And immunohistochemistry was used to detect the protein level of ENPP1 in 36 pairs of lung tissues from normal and tumor tissues.Results:1.The results of mRNA expression profiling analysis showed that the GRPC5A knockout MTEC cells significantly increased the expression of ENPP1 compared with wild-type cells.And its consistent with the results in Oncomine,that ENPP1 is significantly higher in lung cancer than normal.2.The knockdown of ENPP1 in human lung cancer cell lines HCC827 and A549 was significantly suppressed colony numbers compared to those in control.And the same results were obtained in soft agarose assay.3.The expression of tumor stemcell markers include NANOG,ABCG2,SOX2 and CD44 were significantly down-regulated in human lung cancer cell lines HCC827 and A549 knockdown ENPP1 compared to their control group.Subcutaneous tumor formation in nude mice showed that the HCC827 knockdown ENPP1 group had a significant decrease in the tumor volume compared with the control group.4.In human lung cancer cell lines HCC827 and A549,the migratory activity was significantly suppressed in the group of knockdown ENPP1 as compared to parental cells,the EMT phenomenon induced by TGF-? and EMT related protein expression were also decreased.5.ENPP1 is significantly overexpression in human lung tumor tissues compared to normal lung tissues.Conclusions:1.ENPP1 is overexpression in human lung cancer2.Knockdown ENPP1 could significantly suppressed colonogenic formation and soft agarose assay in human lung cancer cell lines HCC827 and A549.3.ENPP1 knockdown reduces the stem cell phenotype of lung tumors and tumorigenicity in human lung cancer cell lines.4.ENPP1 knockdown reduces the EMT phenotypes induced by TGF-?.