Study On The Mechanism Of Potential Tumor Suppressor Gene TXNIP In Malignant Proliferation Of Meningiomas

Meningioma is one of the most common primary tumors of the CNS,accounting for 30% of all intracranial tumors,which is more common in women and the elderly Most meningiomas are benign and are classified as WHO I which can be cured by definite imaging diagnosis and active surgical treatment or assisted radiotherapy.In addition,meningiomas included some WHO II-III cases,although the incidence of high-grade meningioma is less than 10%,there are significant malignant proliferation,including skull and brain tissue invasion.Especially in the skull base and midline near the sagittal sinus or the falces is closely related to the operation more difficult to completely remove the case,there are high risks like mutilation and lethal.When the tumor is located in the midline,including the skull base,sagittal sinus,falx and other parts of the brain,it is difficult to achieve complete resection,the prognosis of these patients remains poor even with other treatments.Modern treatments for high-grade meningioma,many advocate the use of surgical resection combined with postoperative radiation therapy,in order to reduce the volume of residual tumor and reduce the risk of recurrence to improve the prognosis of patients after surgery,the patients could get a bad outcome after surgery and radiotherapy,but the overall treatment effect is still unsatisfactory.To study the mechanism of malignant proliferation of meningiomas is useful for discovering new therapeutic directions and helping clinicians develop new approaches for treatment of malignant meningioma,so the malignant proliferation of meningiomas is a high-grade meningioma treatment basic research direction.In our previous study,we found that retinoblastoma protein binding zinc finger structure gene RIZ is associated with meningioma cell proliferation.We used tissue microarray to detect the expression of RIZ1 in meningioma cells and found that it was inversely proportional to the malignancy of meningiomas.The higher the malignancy of meningiomas was the lower the expression of RIZ1.In order to verify the results,we used the malignant meningioma cells IOMM-LEE overexpression of RIZ1,we found that the growth of meningioma cells was significantly inhibited and the cells were arrested at G2/M phase.Therefore,we found that RIZ1 was negatively correlated with the malignancy of meningiomas.At the same time,it is found that c-Myc is a downstream molecule of RIZ,these results have been published in the《Oncogene》.Analysis of gene expression after overexpression of RIZ,we found that the expression of thioredoxin-binding protein(TXNIP)in meningioma cells was significantly increased.Through detailed literature study,we found that TXNIP is a newly discovered tumor suppressor gene,is related to the cell proliferation,apoptosis,differentiation and other processes,and also in tumor,allergy and other stress diseases.TXNIP in human breast cancer,lung cancer,liver cancer and other cancer cells has decreased performance,and it is closed to the tumor metastasis,and its deletion can lead to the proliferation of tumor cells.It can inhibit the role of apoptosis,and in anti-tumor,which can participate in cell cycle arrest,hypoxia,affect the development of NK cells and other pathways affect the occurrence and development of tumors,so TXNIP plays a key role in the outcome of development of cance.Based on the previous findings,we designed a pre-experiment for TXNIP.In the pre-experiment,we carried out the primary meningioma cells,including one WHO grade I,two WHO grade II and one WHO grade III,the original meningioma cells are strong growth,cell morphology showed bipolar or multipolar endothelial cell morphology,the growth of more than 10 generations,benign specimens tend to slow growth,and malignant specimen is in a state of continuous growth.We used 3-5 generations of primary meningioma cells,to clear RIZ1 expression in the primary cell differences between the same time,the use of gene chip technology to study the downstream expression of the protein gene,we found that over time,the changes of its downstream protein gene were progressively increased,including the expression of many tumor suppressor genes and cyclin in TXNIP.Based on the findings of previous studies and pre-experiments,we envisioned the expression of TXNIP in meningioma cells Whether its meningioma cells inhibited?If there is an inhibitory effect,is it associated with the degree of malignancy?What is the mechanism of its action?Therefore,we designed this experiment,to verify the experiment through our test,but also to further explore the mechanism of its role.Part I: Expression and prognosis of RIZ1 downstream protein TXNIP in meningiomasObjective:To study the expression level of TXNIP gene in all meningiomas and its correlation with prognosis.Method:Tumor specimens of meningiomas(N = 65)were collected by surgical examination and postoperative pathology.Tumor specimens of different grades were stained by immunohistochemical staining and Western blot.TXNIP and Ki-67 were detected in all levels of meningiomas.The expression of TXNIP was analyzed by statistic method,and the correlation between the expression of TXNIP and the clinical characteristics,pathologic features and prognosis of the patients was analyzed.The relationship between the expression of TXNIP and tumor recurrence was analyzed to determine whether TXNIP was independent risk of tumor recurrence factor.Result:The expression of TXNIP was mainly distributed in the cytoplasm by immunohistochemical staining,while Ki-67 was mainly distributed in the nucleus.Used of the IHC and western blot,the expression of TXNIP in the low grade meningioma was significantly higher than that in the high grade meningiomas(29.31 ± 18.70 vs.74.61 ± 7.51,P <0.0001).The results showed that the expression level of TXNIP was negatively correlated with the grade of meningioma(P <0.001).By using the statistical study of Kaplan-Meier,the difference was statistically significant(P <0.05).Similarly,the expression level of Ki67 was positively correlated with the recurrence of postoperative patients(P = 0.015).The expression of TXNIP was negatively correlated with the recurrence of postoperative patients(P = 0.01),Multivariate COX regression analysis showed that the high expression of TXNIP was an independent risk factor for the prognosis of glioma patients(P = 0.017).Conclusion:The expression of TXNIP gene decreased with the increase of meningioma level and was negatively correlated with the expression level of Ki67.The expression of TXNIP was positively correlated with the prognosis of meningioma patients.TXNIP gene was the potential of meningioma tumor suppressor gene.Part II: Detection and localization of TXNIP expression in primary cells of meningiomasObjective:To detect whether the expression of endogenous TXNIP was different in 4 cases of primary cultured meningioma(1 WHO grade I,2 WHO grade II,1 WHO grade III),and confirm the intracellular localization;Method:The levels of TXNIP in primary cells were detected by Western Blot by extracting the total protein in primary meningioma cells of 3-5 generations.Result:Western Blot test showed that the expression of TXNIP gene was significantly decreased or absent in the primary cells of the high grade meningioma(WHO II-III),and the expression of TXNIP gene was significantly lower than that of the control group(WHO I-III)).And the expression level of TXNIP was negatively correlated with the pathological grade of primary meningiomas.This result is consistent with the results of clinical specimens,the expression is mainly located in the cytoplasm.Conclusion: The detection of primary cells confirmed that TXNIP expression and meningioma pathological level was negatively correlated,the expression is mainly located in the cytoplasm.Part III: The effect of TXNIP overexpression vector on cell cycle,migration and invasion of meningiomas was studied.Objective:To study the function of TXNIP in primary meningioma cells.Method:To prepare TXNIP overexpressed lentiviral vector,transfected into the primary cells of the WHO grade III meningioma,the transfection group and the negative control group and the blank control group were compared to confirm the TXNIP gene in the original Overexpression in cells.The effects of TXNIP overexpression on the biological characteristics of primary cells were examined by a series of cell function experiments in the primary cells of TXNIP overexpression.Result:The TXNIP lentiviral vector was transfected into WHO grade III primary meningioma and the TXNIP in the primary cells was significantly overexpressed.The results of cell proliferation experiments(WST-1)showed that the proliferation ability of primary cells was significantly decreased after TXNIP overexpression.The results of cell cycle experiments showed that after TXNIP overexpression,The cell cycle of primary cells is stagnant on G0/G1 stage,and the effect of cell migration and invasion experiments showed that TXNIP overexpression of primary cells,its migration and invasion ability was significantly inhibited.Conclusion:TXNIP gene may play a role in inhibiting cell proliferation,cell cycle,migration and invasion in the development of meningiomas.