| Neuroblastoma(NB)is a malignant tumor commonly found in the peripheral nervous system of infants and young children.The most common origin is the embryonic solid tumor of the sympathetic nervous system.Neuroblastoma is genetically characterized by various chromosomal abnormalities.Its typical feature is metastasis,especially multi-site,multi-focal early metastasis.Early bone marrow metastasis is most common in clinic,and its proliferation and metastasis mechanisms are complex.The tyrosine kinase receptor and its ligand are thought to play a regulatory role in the development,differentiation,apoptosis,cell proliferation,angiogenesis and metastasis of neuroblastoma.At present,the main treatment methods for neuroblastoma in China are comprehensive treatments such as surgery,radiotherapy and chemotherapy,stem cell transplantation,chemotherapy and so on,which greatly improve the five-year survival of children with advanced NB.The five-year survival rate of the child is maintained at around 50%.However,the treatment of children with advanced NB has entered the bottleneck period,and NB is a rare cancer species.The understanding and treatment level of NB is quite different,which increases the complexity and refractory nature of NB.Compared with domestic,specific treatments such as immunotargeting therapy and 131I-MIBG radiotherapy have been applied in western developed countries,and have good clinical efficacy for the treatment of advanced NB.Therefore,in the future clinical treatment should pay more attention to the application of targeted therapy with greater specificity and sensitivity,and it is necessary to find new and effective potential NB molecular targets.Ribosomes are the main places for the synthesis of proteins in cells.Their main components are proteins and RNA,which play an important role in cell growth and proliferation.The most important biological behavior of ribosomes is the biosynthetic function of proteins.The ribosomal synthesis regulatory protein 1(RRS1)was first found in yeast.The human RRS1 gene is located on chromosome 8q13.1,and the chromosomal location is 66429028 – 66430733 bp.The human RRS1 protein is mainly localized in nucleolus and neurogenic endoplasmic reticulum.RRS1 is a multifunctional protein that participates in the processing of Pre-rRNA and is involved in the transport of the 60 S ribosomal subunit from the nucleolus to the cytosol.RRS1 regulates the rate of ribosome synthesis according to the state of the cell,thus maintaining intracellular homeostasis.RRS1 is an important protein in the signal transduction pathway of protein secretion and ribosome synthesis.In recent years,the research on the relationship between RRS1 gene and disease has been incrsasing.As early as 2009,Carnemolla et al reported that RRS1 is associated with the development of human Huntington's disease.The results showed that the expression of RRS1 gene in Huntington's disease was significantly increased..Recent studies have found that the abnormal expression of RRS1 gene is closely related to cancers such as cervical cancer,liver cancer,and colorectal cancer,and other cancers.Previous studies in our group have shown that RRS1 is associated with breast cancer proliferation.There are no reports about the function of RRS1 gene in neuroblastoma,so in this study we will explore the function of RRS1 gene in neuroblastoma and its regulatory mechanisms for apoptosis in neuroblastoma SH-SY5Y cells.Objective:The aim of this study is:(1)Detect the expression level of Regulator of ribosome synthesis 1(RRS1)in neuroblastoma tissues,and explore its relationship with clinicopathological features,survival prognosis and dead rate of neuroblastoma.(2)Study on the proliferation,apoptosis and invasion ability of human neuroblastoma SH-SY5Y cells after knocking out and knocking down the expression of RRS1 gene.(3)Research on the changes in apoptosis-related mechanisms of neuroblastoma SH-SY5Y cells after knocking out and knocking down the expression of RRS1 gene.Methods:(1)Immunohistochemistry(IHC)was used to detect the expression of RRS1 gene in neuroblastoma tissues,and the correlation between SSR1 expression and clinicopathological characteristics and survival prognosis of patients were statistically analyzed.(2)The neuroblastoma cell line SH-SY5Y,breast cancer cell line MCF-7,MDA-MB-231,BT549 and normal breast cell HMEC were cultured in vitro,and the mRNA expression level of RRS1 gene in each cell line was detected.(3)The lentiviral vector was used to construct the neuroblastoma SH-SY5Y cells by CRISPRCas9 and RNA interference technology,and the expression of RRS1 gene was knocked down.(4)The knockout efficiency of Cas9 was detected by Cruise digestion,and the knockdown efficiency of RRS1 gene at mRNA level was detected by qPCR and Western Blot.Cell proliferation and cell apoptosis were detected by MTT and clone formation methods;cell cycle and apoptosis levels were detected by flow cytometry;cell migration ability was detected by scratch and Transwell assay;invasion ability was detected by invasion assay.(5)The expressions of p53,p21,Bcl-2 and Bax in neuroblastoma cell lineSH-SY5Y were detected by Western blotting.The molecular mechanism of apoptosis was determined after RRS1 gene knockdown.Results:(1)Immunohistochemical results showed that RRS1 protein was positive in 16 of 45 cases of neuroblastoma,and the positive rate was 35.6%.(2)Survival prognosis.The majority of the patients with neuroblastoma were under 10 years old,and the distribution of RRS1 protein positive in the metastatic and non-metastatic populations was different(P<0.05).There was a significant correlation between RRS1 expression and death(P<0.01).).It is suggested that RRS1 positive may be an independent risk factor for neuroblastoma metastasis(P<0.05)and was also a key factor for death(P<0.01).(3)The ?Ct values of RRS1 mRNA in neuroblastoma SH-SY5Y cells,normal breast HMEC cells and breast cancer MDA-MB-231,BT-549,MDA-MB-453 and MCF-7 cells were 3.46±0.30,13.18±0.24,12.44±0.08,9.95±0.48,11.11±0.60,7.42±0.22,respectively.The expression of RRS1 gene in neuroblastoma SH-SY5Y cells was significantly higher than that of normal breast cells and breast cancer cells.(4)After the CRISPRCas9 lentivirus infected the neuroblastoma SH-SY5Y cells,the expression of RRS1 gene in SH-SY5Y cells was knocked out,and the expression of cellular protein was detected by WB assay.The expression of RRS1 protein in Control group was 1.428±0.350,while KnockOut group RRS1.The protein expression was 0.198±0.128.The results showed that the expression of RRS1 protein in Control group was significantly higher than that in KnockOut group(p<0.01).(5)The expression of RRS1 gene in the neuroblastoma SH-SY5Y cells was knocked down,after the sh-RNA lentivirus was transfected into the cells.The mRNA expression of the NC group was 1.002 ± 0.084,and the expression of the RRS1-shRNA group was 0.536 ± 0.016(P < 0.01).),and the amount of RRS1-shRNA histone expression was also significantly reduced.(6)CRISPRCas9 lentivirus infection of neuroblastoma SH-SY5Y cells,knockout of RRS1 gene in cells: MTT assay showed a significant decrease in cell proliferation ability(P<0.01);colony formation experiments showed that the number of single cell clones in NC group was 102±10,and the number of clones in the Knockout group was 8±3,the colony forming ability was significantly decreased(p<0.01).The cell cycle experiment showed that the number of cells in the G1 phase was 43.75% ±0.743 in the NC group,in the Knockout group that the number of cells in the G1 phase was 57.53% ±0.580,and the cell cycle regulation was disordered(p<0.01).The results of scratch test showed that the mobility of cells in NC group was 80.93% ±0.0775 after 48 h,and the migration rate of cells in Knockout group after 48 h was 63.00%±0.0505,the cell migration ability decreased significantly(p<0.01).Transwell results showed that the number of cells migrated to the lower chamber in the NC group was 180±3.85,and the number of cells in the Knockout group that migrated to the lower chamber was 24±4.46,the cell migration ability was significantly decreased(p<0.01).The results of the invasion experiment showed that the number of cells invading through the matrigel membrane in NC group was 157±3.36,and the number of cells invading the matrix membrane in Knockout group was 7±0.36,and the invasion and metastasis ability of cells was significantly decreased(p<0.01).(7)shRNA lentivirus infects neuroblastoma SH-SY5Y cells and knocks down RRS1 gene expression: MTT assay showed that the cell proliferation ability decreased significantly(p<0.05).The clone experiment showed that the number of single cell clones in NC group was 75±3,while the number of clones was 10±2 in RRS1-shRNA group,and the cloning ability was significantly decreased(p<0.01).The cell cycle experiment showed that the number of cells in the G1 phase was 32.15% ±0.7199 in the NC group and the number of cells in the G1 phase in the RRS1-shRNA group was 69.36% ±0.3879,the cell cycle was significantly blocked in G1 phase(p<0.01).The results of apoptosis experiment showed that the proportion of early necrotic cells in NC group was 2.21%±0.1234,and the proportion of early necrotic cells in RRS1-shRNA group was 7.24%± 0.3465,the early apoptotic rate of cells increased significantly(p<0.01).The results of scratch test showed that the mobility of cells in NC group was 16.31% ±0.0181 after 24 h,and the migration rate of cells in RRS1-shRNA group after 24 h was 9.79%±0.0346,the cell migration ability was significantly decreased(p<0.01).Transwell results showed that the number of cells in the NC group that migrated to the lower chamber was 219±4.72,and the number of cells in the RRS1-shRNA group that migrated to the lower chamber was 11±1.10,the cell migration ability was significantly decreased(p<0.01).The results of the invasion experiment showed that the number of cells invading through the matrigel membrane in NC group was 191±2.17,and the number of cells invading the matrix membrane in RRS1-shRNA group was 60±2.81,and the invasion and metastasis ability of cells was significantly decreased(p<0.01).(8)After knocking down the RRS1 gene of neuroblastoma SH-SY5Y cells,the expression levels of p53,p21 and Bax proteins were increased significantly,and the expression of oncogene Bcl-2 protein was decreased significantly,and the interaction between p53 protein and MDM2 is reduced.Conclusions:(1)The RRS1 gene is positively expressed in neuroblastoma tissues,mainly localized in the nucleus,showing pale yellow to brownish yellow particles,and partially weakly positive in cytoplasm.(2)The positive expression of RRS1 is closely related to metastasis and death in patients with neuroblastoma.(3)The RRS1 gene is highly expressed in neuroblastoma SH-SY5Y cells.After knocking out and knocking down the expression of RRS1 gene,the proliferation,migration and invasion of SY5Y cells were significantly decreased,and the level of apoptosis was significantly increased.(4)knocking down neuroblastoma SH-SY5Y cells RRS1 gene can induce the activation of p53,p21 protein,leading to cell cycle arrest and increased apoptosis.The RRS1 gene may regulate cell apotosis and cycle through p53-MDM2 signaling pathway. |
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