| ObjectiveNeuroblastoma (NB) is the most common extracranial solid cancer in childhood. Clinically it is characterised by rapid progression and early distant metastasis. Despite constant progress in its therapies, the prognosis remains poor. Up to now many studies have demonstrated that a significant proportion of high-risk NBs are accompanied by the abnormal changes in the MYCN and ALK genes; meanwhile, the strong synergic effects between such changes are closely associated with the rapid proliferation, metastasis, and poor prognosis of NB.With a strong target gene-silencing capability, RNAi can regulate various cellular activities by down-regulating or closing gene expressions; also, it may be involved in many functional gene deletion studies. TAE684 was the first discovered small molecule inhibitor of ALK kinase. Currently, quite a few ALK kinase inhibitors have been applied in the molecular targeted therapies for tumors and yielded good anti-tumor activities. However, to our knowledge, no study has investigated the role of the combination of RNAi and ALK kinase inhibitor targeting these two abnormally changed genes in human NB.In our current study, we selected a cell line that highly expressed MYCN and ALK in human NB cells; with MYCN and ALK as the target genes and by using the RNA i and the construction technology for stably transfected cell lines, we established a stably MYCN-transfected cell line with the lentivirus as the gene vector. The effects of MYCN knockdown on the proliferation and invasion of the cultured human NB cells in vitro were explored. Also, the effects of the combination of the MYCN knockdown and ALK inhibitor TAE684 on the apoptosis of human NB cells and on the expressions of MYCN protein, ALK protein, and some apoptosis-related proteins (Bcl-2, Bax, and Cleaved Caspase 3), as well as their underlying mechanisms, were investigated. Furthermore, by using tumor formation technology in nude mice, we established human-derived NB-bearing animal models to explore the tumorigenesis potentials. We further detected the combined application of the MYCN knockdown and TAE684 treatment in the experiments in vivo, so as to clarify the roles of MYCN and ALK in the development of human NB. By doing so, we wish to provide more experimental evidences for the use of MYCN and ALK genes as the targets of multidisciplinary treatment, particularly for NB with multiple genetic changes, so as to improve the efficiency and effectiveness of the molecular targeted therapies for tumors.Methods1. The expressions of MYCN mRNA and protein and ALK protein in three human neuroblastoma (NB) cell lines including SH-SY5Y, SK-N-SH, and SK-N-BE (2) were detected using fluorescence quantitative PCR(qPCR), so as to screen a human NB cell line with high MYCN and ALK expressions.2. Four interference plasmids targeting MYCN was designed and constructed based on human MYCN gene information. After screening and identification, they were restructured with the pLenti6.3/V5 Dest vector, and packed with the lentivirus to construct the Lenti-EGFP-MYCN-miR lentiviral vector, which then infected the human NB cells with high MYCN and ALK expressions. The silencing effects of these four Lenti-EGFP-MYCN-miR lentiviral vectors on the MYCN mRNA and protein expressions were detected using qPCR and Western blotting, so as to screen the lentiviral vector with the best interference target sequence.3. After the drug resistance was screened by blasticidin, the stably MYCN-transfected human NB cell line was obtained. The mRNA and protein expression of MYCN in the cell line were determined using qPCR and Western blotting, so as to confirm the successful establishment of the stably transfected cell line.4. In the experiment in vitro, we used CCK-8 to detect the proliferation of human NB cells after the knockdown of MYCN in the stably MYCN-transfected human NB cell line; also, Transwell chamber and crystal violet staining were applied to determine the cell invasion capability.5. In the experiment in vitro, after the combined application of MYCN knockdown and TAE684, we detected the apoptosis of human NB cells using flow cytometry, and detected the expressions of MYCN, ALK protein, as well as Bcl-2, Bax, and Cleaved Caspase-3 protein expressions using Western blotting. The potential underlying mechanisms were also analyzed.6. Human-derived NB-bearing nude mouse models were established using the human NB cell line with high expression of both MYCN and ALK. Tumor growth was detected after the combined application of MYCN knockdown and TAE684. The protein expressions of MYCN and ALK were detected by Western blotting.Results1. The MYCN mRNA and protein expression levels of three cell lines including SH-SY5Y, SK-N-SH, SK-N-BE (2) were 0.090±0.010,0.340±0.026,87.867±2.316 and 0.0064±0.0003,0.3887±0.0027,1.2447±0.0301,respectively. The ALK protein expression for these three lines were 0.0175±0.0005,0.0173±0.0006,and 0.2100±0.0200, respectively. SK-N-BE (2) was found to be with high expressions of both MYCN and ALK, and therefore was used for further experiments.2. Digestion and sequencing confirmed that four MYCN-targeting Lenti-EGFP-MYCN-miR lentiviral vectors were successfully constructed. These four lentiviral vectors were transfected into the SK-N-BE(2) cells. As shown by qPCR and Western blotting, the mRNA and protein expression levels of MYCN were 0.577±0.015, 0.333±0.016,0.090±0.010,0.197±0.015 and 0.833±0.049,0.563±0.040, 0.300±0.020,0.387±0.015, respectively, among which the Lenti-EGFP-MYCN-3-miR showed most potent inhibitory effect on MYCN and therefore was selected for the the best RNAi target sequence.3. The stably MYCN-transfected SK-N-BE (2) cell line was selected following the drug resistance test using blasticidine. As shown by qPCR and Western blotting, the mRNA and protein expressions of MYCN in the stably transfected cell line were 0.363±0.012,and 0.467±0.035, respectively, which were significantly lower than those in the normal control group and negative lentivirus stably transfected control group (P<0.05); thus, the stably MYCN-transfected SK-N-BE (2) cell line was established.4. CCK-8 was used to detect the proliferation of cells in the experiment in vitro, which showed that the stably MYCN-transfected SK-N-BE (2) cell line had OD value of 0.258±0.012,0.335±0.016,0.383±0.076,0.597±0.023,0.767±0.021, and 0.989±0.060 in the first day to the sixth day, which was significantly lower than those in the normal control group and negative lentivirus stably transfected control group in the second day to the sixth day (P<0.05); the OD value was slightly lower in the negative lentivirus stably transfected control group than in the normal control group, but the difference was not statistically significant (P>0.05). As shown by the cell invasion experiment, the OD value was 0.042±0.009, which significantly decreased than those in the normal control group and negative lentivirus stably transfected control group (P<0.05).5. In the experiment in vitro, The flow cytometry was used to detect the apoptosis levels and Western blotting was applied to detect the protein expressions by MYCN knockdown alone; use of TAE684 alone; TAE684 combined with MYCN knockdown. The flow cytometry showed that the cellular apoptosis levels were 28.867±0.764,22.593±0.075 and 51.623±0.137, respectively. The expressions of MYCN protein were 0.247±0.006,0.403±0.012 and 0.130±0.002, respectively; the expressions of ALK protein were 0.068±0.007,0.036±0.003 and 0.021±0.001, respectively; the expressions of Bcl-2 protein were 0.192±0.005,0.190±0.015 and 0.056±0.010, respectively; and the expressions of Bax protein were 0.314±0.008, 0.161±0.003 and 0.435±0.024, respectively; and the expressions of Cleaved Caspase-3 protein were 0.807±0.041,0.949±0.003 and 1.452±0.020, respectively, which were significant difference compared with the normal control group, DMSO group, and negative lentivirus stably transfected control+DMSO group (all P<0.05). The changes in the combined application of TAE684 and MYCN knockdown were more significant than those in the groups of MYCN knockdown alone and TAE684 alone (P<0.05).6. The tumor-bearing nude mouse models were successfully established using the SK-N-BE (2) cell line. Detection of the tumor size showed the tumor size was 0.1097±0.008cm3,0.0930±0.0060cm3 and 0.0024±0.0003cm3 in the MYCN knockdown group; TAE684 group and TAE684 combined with MYCN knockdown group. As shown by Western blotting, the MYCN and ALK protein expression levels were 0.345±0.020,0.650±0.033,0.217±0.023 and 0.054±0.002,0.042±0.005, 0.010±0.001 in the tumor, which were significantly lower than those in the normal control group, DMSO group, and negative lentivirus stably transfected control+ DMSO group (all P<0.05). The changes in the combined application of TAE684 and MYCN knockdown were more significant than those in the groups of MYCN knockdown alone and TAE684 alone (P<0.05).Conclusion1. Human SK-N-BE (2) cells was NB cell line wells with high expressions of both MYCN and ALK.2. The successfully constructed MYCN gene-targeting Lenti-EGFP-MYCN-3-miR lentiviral vector was highly efficient infecting SK-N-BE (2) cells and inhibiting the expression of the target gene MYCN. After the drug resistance was screened by blasticidin, the stably MYCN-transfected SK-N-BE (2) cell line could be established.3. After the MYCN knockdown in the stably MYCN-transfected SK-N-BE (2) cell line, the proliferation and invasion capabilities of the cultured SK-N-BE (2) cells in vitro dramatically decreased.4. The combined application of MYCN knockdown and TAE684 could remarkably down-regulate the protein expressions of MYCN, ALK, and Bcl-2 in the cultured SK-N-BE (2) cells in vitro and up-regulate the protein expressions of BAX and Cleaved Caspase-3, along with obviously increased apoptosis. The combined application of MYCN knockdown and TAE684 might induce the apoptosis by regulating the apoptotic molecules.5. The tumor-bearing nude mouse models could be successfully established using the SK-N-BE (2) cell line. The combined application of ALK inhibitors TAE684 and MYCN knockdown by RNAi could enhance the suppression of the target gene MYCN and ALK expression in tumor tissues and inhibit the tumor growth in human-derived NB -bearing animal models, which showed a good anti-tumor effect in vivo. |
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