Study On The Expression And Differentiation Mechanism Of TIAM1 In Neuroblastoma

Neuroblastoma(NB)is a neuroendocrine tumor,which rank the first place in infants malignant tumor.It is the most common extracranial solid tumor in childhood.With the continuous development of molecular diagnostic technology,precision treatment has gradually entered NB and targeted therapy is a hot research direction in NB treatment in recent years.In the application of progressive diagnosis and treatment technology,the cure rate of the patients in the low risk group was over 90%,and the middle risk group was between 70-90%.Because of the high heterogeneity of NB,the cure rate of the high-risk group was only about 40%.Therefore,the in-depth study may well elucidate specific mechanisms and explore new therapeutic targets to provide a new direction for the future diagnosis and treatment of NB.Studies have shown that many genes are involved in the development of NB,and TIAM1 is one of them.TIAM1 is a member of the GEFs family and is an important regulator for the activity of guanosine activity conversion.Abnormal protein content was found in many malignant tumors,such as gastric cancer,hepatocellular carcinoma,breast cancer,lung adenocarcinoma,ovarian cancer and so on,but the specific mechanism is not yet very clear.The main purpose of this study is to detect the protein expression of TIAM1 gene in NB tumor cells and tissues,combine specific clinical data to analyze the relationship between TIAM1 and clinical pathological features as well as prognosis of NBs.Then through a series of experiments to further explore the mechanism of TIAM1 on the occurrence and development of NB.Methods:1.Next generation sequencing:target capture gene loci which are closely related to the occurrence and prognosis of NBThe NB panel,which includes 53 gene total exons and 3 large gene CNV regions,is first designed.Then we applied this panel to test DNA from the fresh tumor tissue and matched peripheral white blood cells of 33 NB patients in depth sequencing.2.Study on the correlation between clinicopathological features and prognosis in patients with NB and TIAM1 expresstion.There were 61 cases of NB patients who met the standard of entrying the group(specific standards see materials and methods).The TIAM1 expression of the patients in the group was detected by immunohistochemical technique.Then SPSS22.0software was used to analyze the relationship between the expression level of TIAM1and the various clinicopathological features of these patients.Finally,we analyzed whether the expression level of TIAM1 affects the survival of NB patients.3.Vitro experiment:the impact of TIAM1 on the biological behavior of NB cellsDescending and overexpression TIAM1 plasmid were constructed.Lentivirus infection was used to establish TIAM1 stable down regulated,over expressed NB cell lines and corresponding control cell lines.The effects of TIAM1 on proliferation,invasion and migration of NB cell lines were preliminarily verified by cell functional experiment such as MTT,tablet cloning and scratch test.4.Retinoic acid induced differentiationThe induced differentiation drug retinoic acid was added into the NB cell line SH-SY-5Y and SK-N-Be(2),and the morphological changes of the cells were observed under the microscope every day.The proteins of the induced cells and the control group were collected at third,fifth,seventh and ninth days,respectively.Western blot technique was used to detect the protein content of TIAM1 in the cells.5.Verify the cell signaling pathway that TIAM1 participates in the regulation of NB differentiationThe protein of pMXs3-TIAM1 cell line and control group was extracted,Western blot was used to detect the protein content of Nestin、NSE、Syn、S100、TrkA and RhoA in the cells.Results:1.The quality control analysis of the two generation sequencing data of 33 NB samples showed that the sequencing depth was very high.Most of the samples had a target capture rate of about 70%.Sequencing results showed that 17 cases of somatic cell mutation involved 14 genes,and TIAM1 gene deletion was the new variation.2.The expression level of TIAM1 was closely related with the degree of differentiation(χ~2=6.205,P=0.013).But,there was no significant relationship between TIAM1 expression and age(χ~2=0.646,P=0.421),primary site(χ~2=0.221,P=0.638),tumor size(χ~2=1.417,P=0.234),clinical stage by INSS(χ~2=0.033,P=0.856),risk grouping(χ~2=0.267,P=0.605),newly diagnosed metastasis(χ~2=0.974,P=0.324),and anemia(χ~2=0.013,P=0.920).The expression intensity of TIAM1 also had no correlations with 3-year OS(χ~2=0.158,P=0.691)and PFS(χ~2=0.131,P=0.717).3.Compared with the control cell lines,the proliferation of pMXs-TIAM1 cell line was increased,but the invasion and migration was decreased.Howver,the degree of change did not reach statistical difference.4.Compared with the control cell lines,the expression level of TIAM1 increased significantly after retinoic acid induced differentiation.5.The expression level of NSE,Syn and S100 increased significantly with pMXs3-TIAM1 cell line,while the neural stem cell marker Nestin decreased significantly.The protein contents of RhoA and TrkA in TrkA/TIAM1/Rac1 and P75NGFR/RhoA signaling pathways were significantly decreased.Conclusions:1.The expression level of TIAM1 in GN cell is significantly higher than that of the GNB and NB,suggesting that TIAM1 promotes the differentiation of NB tissue.2.Vitro experiments showed that TIAM1 had no significant effect on the proliferation,invasion and migration of NB cells.3.TIAM1 can regulate the differentiation process of NB tissues through the TrkA/TIAM1/Rac1 signaling pathway.