Study On The Roles Of SND1 Abnormal Increase In Gliomagenesis And Progression

Objectives: Gliomas are the most common primary brain tumors which account for 60%~70% of all intracranialtumors, and 60% of gliomas are malignant(WHO grade III and grade IV). Gliomas are hard to be excised completely and hence easy to relapse after neurosurgical operation due to their special anatomic location and infiltrative growth pattern. Meanwhile, they are also resistant to radio- and chemotherapy. Therefore, the outcome of gliomas is usually poor, and prognosis of malignant gliomas is even worse. Malignant gliomas are characterized by excessive proliferation and invasive growth, which are also the main cause of their poor prognosis and high recurrence rate. It has been widely accepted that multiple genes, proteins and factors modulate the invasion and proliferation of malignant gliomas. SND1 is a highly conserved multifunctional protein. It not only acts as a synergistic transcriptional activator of multiple pathways, but also participates RNA metabolism, and therefore its proper expression sounds particularly important for normal cell function maintenance. Recent studies has primarily demonstrated that SND1 is overexpressed in various kinds of extra-cranial malignant tumors, such as hepatocyte carcinoma and ovarian cancer, which suggests a close link between its overexpression and tumorigenesis. However, how SND1 involve in glioma-genesis and progression is still unclear. The aim of this study is to explore the above questions in human glioma samples and human glioblastoma cell lines.Methods: 1. The expression levels of SND1、 RHOA、MMP2 andKi-67 in 147 gliomas of various malignant grades and 20 non-tumoural control brain tissues were measured by means of immunohistochemistry. The expression changes of the above proteins and the relationships among these changes were analyzed. An independent cohort of 142 patient specimens from The Cancer Genome Atlas(TCGA) database(https://tcgadata.nci.nih.gov/tcga) was used to validate the correlation between SND1 expression and RHOA or MMP2 expression in GBM. Patients’ survival was analyzed with Kaplan-Meier method and compared with log-rank test. The Cox’s proportional hazards regression model was used for univariate and multivariate survival analyses.2. To investigate the role of SND1 in GBM malignant progression, we established SND1-downexpressing(U87MG-SND1-sh and U251-SND1-sh) and SND1-overexpressing(U87MG-SND1 and U251-SND1) GBM sub-cell lines by selectively knocking down or exogenously expressing SND1 in U87 MG and U251 cells, respectively. The efficiencies of SND1 knockdown or overexpression in U87 MG and U251 cells were checked by Western blot. The proliferation abilities of the SND1-overexpression sub-cell lines, SND1-downexpression sub-cell lines and their corresponding controls were assessed by MTT assay、colony formation assay, the migratory and invasive abilities of the SND1-overexpression sub-cell lines, SND1-downexpression sub-cell lines and their corresponding controls were assessed by transwell in vitro migration and invasion assays, and further validated the effect of glioblastoma biological behavior by different expression levels of SND1.3. Ch IP experiments, electrophoretic mobility shift assay, qRT-PCR and Western blot were adopted to confirm whether SND1 promotes RHOA and MMP2 expression via gene transcription activation and further clarify the mechanism by which SND1 up-regulated the expression of RHOA and MMP2.4. We used flow cytometry assay to detect the change of cell cycle after SND1 knockdown. The expression levels of cyclin, cyclin-dependent kinase and cyclin-dependent kinase inhibitor were measured by Western blot. To determine whether the excessive proliferation of GBM cells induced by SND1 is mediated by RHOA, U87 MG and U251 cells were co-transfected with control scramble sequence and control empty plasmid pSG5(scramble/pSG5), RHOA siRNA and pSG5(RHOA-si/pSG5), scramble and SND1 expression plasmid pSG5-SND1(scramble/SND1) or RHOA siRNA and pSG5-SND1(RHOA-si/SND1), respectively. The proliferation abilities of the 4 sub-cell lines were assessed by MTT assay, The expression levels of cyclin, cyclin-dependent kinase and cyclin-dependent kinase inhibitor were measured by Western blot. and further validated whether SND1 could facilitate the proliferation of the glioblastoma by up-regulating the expression of RHOA.5. To determine whether the excessive invasiveness of GBM cells induced by SND1 is mediated by MMP-2, U87 MG and U251 cells were co-transfected with control scramble sequence and control empty plasmid pSG5(scramble/p SG5), MMP-2 siRNA and pSG5(MMP-2-si/pSG5), scramble and SND1 expression plasmid pSG5-SND1(scramble/SND1) or MMP-2 siRNA and pSG5-SND1(MMP-2-si/SND1), respectively. The migratory and invasive abilities of the 4 sub-cell lines were assessed by transwell in vitro migration and invasion assays, The expression levels of SND1、RHOA、MMP2 were measured by Western blot.6. Western blot were adopted to confirm the expression levels of MMP-2、c-Jun、and p-c-Jun in SND1-downexpression sub-cell lines and their corresponding controls. The migratory and invasive abilities of the 4 sub-cell lines(scramble/pSG5, MMP-2-si/pSG5,scramble/SND1,MMP-2-si/SND1) were assessed by transwell in vitro migration and invasion assays, The expression levels of MMP-2、c-Jun、and p-c-Jun were measured by Western blot.Results: 1. IHC results showed that RHOA and MMP2 expressions in gliomas were higher than those in the control and that their expressions were significantly increased with the elevation of glioma grades and were the highest in GBM, and all the differences were statistically significant(P<0.05~0.001). The LI% of SND1 correlated positively with those of Ki-67(r=0.979, P<0.0001), RHOA(r=0.984,P<0.0001) and MMP2(r=0.959,P<0.0001). The positive relationship between the SND1 expression and RHOA or MMP2 expression was further validated by the GBM data from TCGA database. Kaplan-Meier analyses showed that the patients with higher level of SND1, RHOA and MMP2 had shorter disease-free survival and overall survival(P<0.0001. Univariate analysis showed that RHOA and MMP2 were the potential predictors for DFS and OS of glioma patients. Cox regression analysis showed that SND1 was an independent predictor for DFS and OS of glioma patients.2. Western blot assay of the SND1 protein levels in different transfected/infected cells showed that we established SND1-downexpressing(U87MG-SND1-sh and U251-SND1-sh) and SND1-overexpressing(U87MG-SND1 and U251-SND1) GBM sub-cell lines by selectively knocking down or exogenously expressing SND1 in U87 MG and U251 cells, respectively.3. MTT assay showed that proliferations of U87MG-SND1-sh and U251-SND1-sh were significantly reduced comparing with the control groups(P<0.05~0.01). The effect of SND1 knockdown on GBM cell proliferation was further verified by colony formation assay(P<0.001). Transwell assays demonstrated that migratory and invasive abilities of U87MG-SND1-sh and U251-SND1-sh were dramaticlly weakened, while the migratory and invasive abilities of U87MG-SND1 and U251-SND1 were significantly increased comparing with those of the control groups. These data indicate that SND1 overexpression may significantly promote the proliferation and invasion of GBM, whereas downregulating SND1 may effectively inhibit the proliferation and invasion of GBM cells.4. qRT-PCR and Western blot detections demonstated that the expressions of RHOA and MMP2 are positively correlated with SND1 levels in both U87 MG and U251 cells(P<0.001). ChIP experiments results showed that SND1 protein interacted with one major motif of RHOA promoter(-371 to-352bpl) and two major motives of MMP2 promoter(-493 to-471 bp and-251 to-219bp).The electrophoretic mobility shift assay(EMSA) results indicated that SND1 induced RHOA and MMP2 gene transcription via binding the promoter region of these two genes.5. Flow cytometry assay to detect the change of cell cycle after SND1 knockdown, the result showed that SND1 knockdown inhibited the proliferation of glioma cells by arresting cell cycle at the G1 phase. qRT-PCR and western blot results showed that the mRNA and protein of RHOA were dramatically decreased in the cells co-transfected with RHOA-si/pSG5 or RHOA-si/SND1, while significantly increased in the cells co-transfected with scramble/SND1 in comparison with the control cells. MTT assay discovered that proliferation was dramaticlly increased in the cells carrying scramble/SND1, while significantly inhibited in cells carrying RHOA-si/SND1 in comparison with control cells. Western blot results showed that after SND1 knockdown, RHOA protein level was decreased, the protein level of CDKN1 B was increased and CCND1/CCNE1/CDK4 protein were decreased. SND1 overexpression could lead to CDKN1 B decrease and CCND1/CCNE1/CDK4 increase, while co-transfection of SND1 and RHOA siRNA would block the SND1 induced CDKN1 B degradation and inhibits CCND1/CCNE1/CDK4 upregulation.6. Transwell assays discovered that migratory and invasive abilities were dramatically enhanced in the cells carrying scramble/SND1, while significantly attenuated in cells carrying MMP2-si/pSG5 and MMP2-si/SND1 in comparison with control cells. These results indicate that the targeting knockdown of MMP2 expression may efficiently prevent GBM cells from migrating and invading induced by SND1 overexpression and confirm that SND1 overexpression promotes the migration and invasion of GBM cells by upregulating MMP2 expression.7. Western blot detections demonstated that there was no difference with the expressions of c-Jun in SND1-sh sub-lines and the control groups, which was totally opposite with the expressions of p-c-Jun. The cells co-transfected experiment showed that the targeting knockdown of RHOA expression may efficiently prevent GBM cells from migrating and invading induced by MMP-2 upexpression and confirm that SND1 overexpression promotes the migration and invasion of GBM cells by upregulating RHOA and MMP2 expression. All those results showed that SND1 overexpression promotes the migration and invasion of GBM cells by upregulating RHOA expression, which promotes the activation of RHOA/JNK/c-Jun/MMP2 molecular signaling pathway.Conclusions: 1. SND1, RHOA and MMP2 overexpression is a common feature of gliomas. The expression levels of the 3 proteins increase with the malignancy degrees of gliomas, and correlate perfectly with the pathological grades of the tumors. The labeling indexes of SND1, RHOA and MMP2 can objectively reflect their expression levels, and can be used as important references to access the biological behaviors of the tumors and the prognosis of the patients. SND1 was an independent predictor for DFS and OS of glioma patients.2. In the present study, we provide supportive evidences to suggest that SND1 could promote RHOA and MMP2 gene transcription. Overexpressed SND1 upregulates RHOA and MMP2 expressions through binding to the promoter regions of RHOA and MMP2 genes, and activating their transcriptions in GBM cells.3. SND1 regulate the protein level of CCND1, CCNE1, CDK4 and CDKN1 B by promoting RHOA expression. Then SND1 could promote GBM proliferation and cell cycle G1/S phase transition via CCND1, CCNE1, CDK4 and CDKN1 B.4. SND1 overexpression promotes the migration and invasion of GBM cells by upregulating MMP2 expression. SND1 overexpression promotes the migration and invasion of GBM cells by upregulating RHOA expression, which promotes the activation of RHOA/JNK/c-Jun/MMP2 molecular signaling pathway.5. The abnormal increase of SND1 expression is one of the important factors for glioma-genesis and malignant progression. SND1-downexpression can reverse the abnormal and play the effective tumor suppression role, therefore it can exert important practical value as a multifunctional tumour promoting factors in the gene therapy of malignant gliomas.6. SND1-downexpression sub-cell lines and their corresponding controls established in this study can stably express low levels of SND1, and therefore can be used as cell models to study the tumorigenic effects of SND1.