The Regulation Of NDRG1to Drug Resistance And The Expression Of Drug Resistant Proteins In Neuroblastoma

Introduction:Neuroblastoma is one of the most common solid tumors in infants, and the available treatment approaches for this tumor include surgery, chemotherapy, and radiotherapy. Biotherapy has been applied in basic research and clinical trials for multiple tumors, and this procedure shows great potential for clinical application. However, because of factors such as safety, this approach has not been widely applied in clinical practice [1]. Resistance to chemotherapeutic drugs and tumor recurrence are the major causes of poor prognosis of many tumors, including neuroblastoma. An MYCN amplification rate of>10copies per haploid genome, which is associated with accelerated progression of the tumor, is considered as an indicator of poor prognosis in cases of neuroblastoma[2,3].The N-myc downstream regulated gene1(NDRG1) is a newly identified gene that is expressed at high levels in N-myc (MYCN) knockout mouse embryos and shows inhibition of expression after treatment with MYCN. Due to its involvement in differentiation and metastasis of tumor cells, NDRG1is associated with the metastasis and invasion ability of tumor cells. Low expression levels of NDRG1may increase the metastasis and invasion ability of tumor cells. Similarly, drug resistance in tumors is also closely related to the invasion and metastasis of tumors. On a related note, NDRGl is a tumor marker under anoxic conditions, and anoxic tumors may develop drug resistance to chemotherapy. Therefore, we hypothesized the existence of a relationship between NDRG1and drug resistance in tumors. Objective:To investigate the effect of the elevated intracellular NDRGl expression on the resistance of human neuroblastomas to chemotherapy, the expression levels of drug-resistant proteins, and the possible regulation mechanisms, and thereby explore novel approaches for biotherapy of human neuroblastomaMethods:A lentiviral human NDRG1gene expression vector was constructed, and transfection of the target gene was tested. The lentivirus vector containing the target gene NDRG1was used to transfect human neuroblastoma KP-N-Ns cell lines and establish a cell line with stable NDRG1expression (the NDRG1group). Cells transfected with the blank vector were assigned to the negative control group, and normally cultured neuroblastoma cells served as blank controls. PCR assay and western blot analysis were performed to determine the genome-and protein-level changes in NDRG1expression in the transfected cells, and thereby understand the transfection efficacy. MTT and TUNEL assays were used to detect the cells' resistance to vincristine, cyclophosphamide, cisplatin, teniposide, and epirubicin hydrochloride. In addition, the expression levels of MDR-1, LRP, and MRP1proteins were determined using western blot analysis.Results:PCR amplification of the constructed lentiviral human NDRG1gene expression vector after digestion indicated successful transfection of NDRG1, while sequencing revealed that the sequence of the constructed gene was the same as that of the human NDRG1gene; the obtained viral titer was4.3x105IFU/ml. Expression levels of both NDRG1mRNA and NDRG1protein in the established stable cell lines increased, suggesting successful establishment of the stable cell lines. MTT assay revealed that in culture with vincristine, cyclophosphamide, cisplatin, teniposide, and epirubicin hydrochloride, the cell growth in the NDRG1group was significantly greater than those in the negative control and blank control groups, while there was no significant difference between the growth in the negative control and blank control groups. TUNEL assay showed that after exposure to vincristine, cisplatin, teniposide, and epirubicin hydrochloride, the apoptosis rate in the NDRG1group was significantly lesser than those in the negative control and blank control groups, while there was no significant difference between the apoptosis rates in the negative control and blank control groups, suggesting increased resistance to vincristine, cyclophosphamide, cisplatin, teniposide, and epirubicin hydrochloride in NDRG1-transfected cells. The expression levels of MDR-1, LRP, and MRP1proteins was significantly higher in the NDRG1group than in the negative control and blank control groups, demonstrating elevated expression levels of MDR-1, LRP, and MRP1in human neuroblastoma KP-N-Ns cell lines transfected with the human NDRG1gene.Discussion:Lentivirus possesses high infectivity and can effectively infect many types of cells, including neuron cells and tumor cells, thereby making it an ideal gene therapy tool. In the present study, the human NDRG1gene sequence was cloned and amplified to construct the lentiviral expression vector pLenti6.3-NDRG1-IRES-EGFP, after which the vector and lentiviral package plasmid were co-transfected into293T cells for lentiviral packaging of the target gene to yield viral particles, which were subsequently used for infecting human neuroblastoma cells.Under normal growth conditions, NDRG1mediates a reduction in apoptotic tumor cells. A reduction in the rate of apoptosis of tumor cells in the presence of chemotherapeutic drugs indicates tolerance to chemotherapeutic drugs. In the present study, the NDRG1-transfected human neuroblastoma cells were resistant to5chemotherapeutic drugs, including vincristine, cyclophosphamide, cisplatin, teniposide, and epirubicin hydrochloride, and the expression levels of MDR-1, LRP and MRP1proteins increased in these resistant cells. These findings suggest that in human neuroblastoma cells, NDRG1may regulate the expression of MDR-1, LRP, and MRP1proteins via some pathways and thereby increase the drug resistance of the tumor cells. Conclusions:In human neuroblastomas, NDRG1positively regulates the expression of MDR-1, LRP, and MRP1proteins via some pathways, which leads to the development of resistance to chemotherapeutic drugs such as vincristine, cyclophosphamide, cisplatin, teniposide, and epirubicin hydrochloride. This study will help develop a novel approach for exploring the resistance of chemotherapeutic drugs in neuroblastoma and will provide a novel biotherapy approach for neuroblastoma.