| Background and significanceGlioma is the most common brain tumor in the adult population and account for 46% of the intracranial tumors. According to the report of the World Health Organization (WHO), malignant gliomas ranks the second fatal disease among the cancer patients under the age of 34, and ranks the third among cancer patients between 35 and 50 years old. Based on the Nuclear atypia, nuclear division index, endothelial cell proliferation and necrosis, the newest WHO tumor grading system for central nervous system neoplasms divides glioma to four subtypes:Grade Ⅰ, the benign type, mainly the pilocytic astrocytoma,5%; Grade Ⅱ, normal astrocytoma and oligo-astrocytoma; Grade Ⅲ, anaplastic astrocytoma,15～25%; grade Ⅳ, Glioblastoma Multiforme (GBM),30%.GBM is the most lethal type of glioma, with the annual morbidity of 3.19/10,0000 in USA. GBM is usually located beneath the cortex and diffusely infiltrates several brain lobes, even the deep structures and contralateral lobes. In statistics, the most commonly suffered focus lies in the temporal lobe. Although nonmetastasizing, the fast-growing GBM can cause severe symptoms, such as hydrocephalus and intracranial hypertension, which both could result in death. Despite combined surgical resection, radiotherapy, and chemotherapy, the survival of GBM patients is very bad, which is less than 1 year for 90% GBM patients. So far, the mechanism of GBM is still a challenge for researchers in neurosurgery and cancer. There are great breakthroughs in the field of GBM biomarkers and molecular classficication recently, such as EGFR, p53, MGMT, IDH, PTEN, MicroRNAs and LncRNAs. The deepgoing studies for biomarkers greatly promote the process of revealing the GBM pathogenesis, and contribute a chance for individualized treatments and outcome prediction.Many studies have revealed that some receptor tyrosine kinases (RTKs), including EGFR, PDGFR and MET, are involved into the tumorigenesis of GBM. These RTKs could interfere to some key signaling pathways, such as PI3K/AKT/mTOR and MAPK pathways, resulting in pathogenesis of GBM. By now, a great many tests have showed that inhibitors against these aberrant pathways present a promising potential for targeting GBM cells.Cyclin-dependent kinase subunit (CKS) proteins prevail among eukaryotes, with the molecular weight of about 9～18 KDa. In human, there exist two kinds of CKS, CKS1 and CKS2. The CKS2 gene locates at chromosome 9q22 and codes a small protein of about 9KD. It is a necessary condition to drive the process of meiotic mitosis. CKS2 has been demonstrated abnormal expression among various malignancies, including gastric, colorectal, liver, esophageal, biliary and prostate cancer, indicating that CKS2 takes part in tumorigenesis. Up to now, there is no report about CKS2 expression and effects on GBM. In this study, we aim to explore the roles that CKS2 plays in GBM.The whole dissertation can be divided into three parts. In the first part, we collected 65 GBM cases and got the clinicopathological data and post-operation survival time by follow-up. Otherwise, we got 30 normal brain tissues form patients suffering from craniocerebral trauma as the control. With consent of patients or their family, we got tissue samples. We performed IHC, RT-PCR and Western blot to explore the CKS2 expression in GBM and normal brain tissues. Then, we calculated the correlation between CKS2 overexpression in IHC and clinicopathological data.We analyzed the survival data by Kaplan-Meier analysis and Multivariate Cox regression analysis. In the second part, we explored the influence of CKS2 silencing on biological behaviors of GBM cell lines. We found that CKS2-shRNA down-regulated the CKS2 expression level in GBM cell lines and inhibit the proliferation and invasion, but induce apoptosis. In the third part, we used CKS2 silencing to explore the effect of CKS2 on signaling pathway. Our study indicates that CKS2 may contribute to the tumorigenesis and progression of GBM by affecting the PI3K-AKT-mTOR and MAPK pathways.PartⅠThe abnormal overexpression and clinical significance of CKS2 in GlioblastomaObjectives:1 To explore the expression of CKS2 in normal brain and GBM.2 To explore the correlation between CKS2 expression and clinicopathological parameters.3 To explore the influence of CKS2 on GBM patients survival.Methods:1. Specimen Collection: We selected 65 GBM cases from Shandong Provincial Hospital Affiliated to Shandong University, Jinan, P.R. China, according to the criteria:(1) Undergoing surgeries from 2010 to 2013; (2) Agreeing to contribute to this study and signing consents; (3) Preoperative Kamofsky performance score (KPS) score>70; (4) None of the patients had received chemotherapy, radiotherapy or other therpies prior to surgery. The Clinical data (including grade, age, gender and extent of resection) were obtained from patients’medical records or attending physicians. Otherwise, we got 30 normal brain tissues form 30 pateints suffering from craniocerebral trauma as the control. We divided the specimens into two parts, one part fixed in 10% formalin, the other part stored in liquid nitrogen.2. Immunohistochemical method to detect the expression of CKS2:The expression CKS2 was detected by two step immunohistochemical method in the control of pathology department. Semi-quantitative scores (TIS) were recorded according to the number of positive cells and the degree of dyeing. The difference of expression between groups was analyzed by SPSS 21.0 software.3. Correlation analysis between CKS2 overexpression and clinicopathologic parameters:We detected the Ki67 expression by IHC, and then calculated the correlations between CKS2 overexpression and clinicopathologic parameters, such as Ki67 labeling index(LI), age, gender, etc.4. Detect CKS2 mRNA expression in GBM and normal brain tissues by Real-Time PCR:Total RNA was isolated from tissues using TRIzol Reagent and cDNA was synthesized by inverse transcription System. Quantitative real-time PCR was performed using SYBR Green Supermix according to manufacturer’s instructions.The data were analyzed with 2-ΔΔCt method.5. Detect CKS2 expression in GBM and normal brain tissues by Western blot: We used RIPA lysis buffer to resolve GBM and normal brain tissues, extract protein and measure sample concentration by the BCA assay. Then protein extracts were added with the same quantity to the 10% SDS-polyacrylamide gels and electrophoresed. After probed with primary antibodies and secondary antibodies, proteins were detected using the chemiluminescence detection kit. The results were analyzed using the ImageJ2× Image software.Results:1. Patient Data:65 GBM patients with the mean age of 51.1 years old, including 37 males and 28 females; other information, such as extent of resection and adjuvant therapy, saw in the dissertation(Table 1, Part I).2. CKS2 was mainly expressed in cell nuclei. It was more overexpressed in GBM than normal brain tissue. (P<0.05)3. For GBMs, CKS2 overexpression correlated significantly with Ki-67 LI (r=0.323, P<0.05), but not with other clinicopathologic parameters.4. Survival analysis:Kaplan-Meier analysis showed that the survival of patient without CKS2 overexpression were better than those with CKS2 overexpression (Log Rank Chi-square=13.518, P<0.05); Cox Proportional-Hazards Model analysis indicated that CKS2 overexpression (Risk ratio, RR=2.627, P<0.01) was significant predictors for survival for GBM patients.5. Western blot and RT-PCR results showed that the expression of CKS2 in GBM was significantly higher than that in normal brain tissue.(both P<0.05)Conclusions:1. CKS2 is mainly expressed in cell nuclei. No matter in mRNA level and protein level, the expression of CKS2 in GBM is significantly higher than that in normal brain tissue.2. The CKS2 overexpression in GBM is correlated significantly with Ki-67 expression.3. CKS2 is valuable for predicting survival of GBM patients.Part ⅡCKS2 expression in GBM cell lines and the effects on the proliferation, invasion and apoptosis by shRNA mediated CKS2 gene silencingObjective:1 To explore the expression of CKS2 in GBM cell lines U87, U251 and A172.2 To investigate the effects of CKS2 on the proliferation, invasion, and apoptosis of GBM cell line U87/U251 by shRNA mediated CKS2 gene silencing.Methods:1. Cell culture:Human glioblastoma cell lines U87, U251 and A172 were purchased from Shanghai cell bank affiliated to Chinese academy of sciences, and cultured in Dulbecco-modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37℃ and 5% CO2 atmosphere.2. Construction of CKS2-shRNA:Design and synthesize oligonucleotide sequence of CKS2 shRNA, insert it into psuper plasmid by double digestion and then get sequenced.3. Comfirming the interference efficiency of CKS2 shRNA:Electransfect the psuper-shCKS2 plasmid into U87 cell line, collect cells after 48 hours and detect the CKS2 expression by Western blot and RT-PCR.4. Evaluate the influence of CKS2 silencing on proliferation ability of U87/U251 cells:We got three groups:non-transfection group, shCON transfection group and CKS2 shRNA transfection group. Electransfect CKS2 shRNA into U87 cells, digest cells with Tyrisin and count after 24 hours, transfer cells to 96 wells plate by 1000 cells per well, then test the proliferation of GBM cells by means of CCK-8 colorimetry(0,12h,24h,48h,72h,96h).5. Evaluate the influence of CKS2 silencing on invasion ability of U87/U251 cells:We get three groups:non-transfection group, shCON transfection group and CKS2 shRNA transfection group. After electrotransfection, we assessed the apoptosis among all there groups according to the protocol of Trans well Cell Invasion Assay Kit.6. Test the influence of CKS2 silencing on survival ability of U87/U251 cells:We got three groups:non-transfection group, shCON transfection group and CKS2 shRNA transfection group. After electrotransfection, we assessed the apoptosis among all there groups according to the protocol of TUNEL test kit.Results:1. CKS2-shRNA can down-regulated the CKS2 mRNA level (p<0.05) and protein expression (p<0.05) by GBM cell line U87.2. Influence of CKS2 silencing on proliferation ability of U87/U251:The CCK-8 test indicated that CKS2-shRNA could significantly reduce the proliferation ability compared to non-transfection group and shCON transfection group (P<0.05).U87 cell lines:From time point 12h, compared to non-transfection group or shCON transfection group, the proliferation ability of CKS2-shRNA group declined significantly (both P<0.05)U251 cell lines:From time point 24h, compared to non-transfection group or shCON transfection group, the proliferation ability of CKS2-shRNA group declined significantly (both P<0.05)3. Influence of CKS2 shRNA on invasion ability of U87/U251:Compared to non-transfection group, the Transwell Cell Invasion test show that the invasion ability of CKS2 shRNA group is inhibited significantly(PU87<0.05, PU251<0.05); Compared to shCON transfection group, the Transwell Cell Invasion test show that the invasion ability of CKS2 shRNA group is inhibited significantly(PU87<0.05, PU251<0.05).4. Influence of CKS2 shRNA on apoptosis ability of U87/U251:Compared to non-transfection group, the TUNEL (terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling) experiment showed that CKS2-shRNA could significantly induce the apoptosis index(PU87<0.05, PU251<0.05); Compared to shCON transfection group, the TUNEL(terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling) experiment showed that CKS2-shRNA could significantly induce the apoptosis index(PU87<0.05, PU251<0.05).Conclusions:1. CKS2 presents expression in GBM cell lines U251, U87 and A172.2. CKS2-shRNA down-regulates the CKS2 expression level in GBM cell lines and inhibits the proliferation and invision, but induces the apoptosis.Part III Effects of CKS2 on signaling proteins in GBMObjectives:1 To investigate the influence of CKS2 on the phosphorylation of AKT and mTOR, the key signaling proteins in PI3K/AKT/mTOR pathway.2 To investigate the influence of CKS2 on the phosphorylation of ERK1/2, the key signaling protein in MAPK pathway.Methods:1. The plasmid of CKS2-shRNA was transfected into the cell lines U87/U251 by electroporation. We got three groups:non-transfection group, shCON transfection group and CKS2 shRNA transfection group. Then extract the proteins detect the phosphorylation level of AKT and mTOR between different groups.2. The plasmid of CKS2-shRNA was transfected into the cell lines U87/U251 by electroporation. We got three groups:non-transfection group, shCON transfection group and CKS2 shRNA transfection group. Then extract the proteins detect the phosphorylation level of ERK1/2 between different groups..Results:1. Compared with non-transfection group, phosphorylation of AKT(p-AKT/AKT) in CKS2 shRNA group was inhibited significantly(PU87<0.05, Pu251<0.05); compared with shCON transfection group, phosphorylation of AKT in CKS2 shRNA group was inhibited significantly(PU87<0.05, PU251<0.05); there is no statistical difference between non-transfection group and shCON transfection group(PU87>0.05, PU251>0.05).2. Compared with non-transfection group, phosphorylation of mTOR(p-mTOR/mTOR) in CKS2 shRNA group was inhibited significantly(PU87<0.05, PU251<0.05); compared with shCON transfection group, phosphorylation of mTOR in CKS2 shRNA group was inhibited significantly(PU87<0.05, PU251<0.05); there is no statistical difference between non-transfection group and shCON transfection group(PU87>0.05, PU251>0.05).3. Compared with non-transfection group, phosphorylation of ERK1/2(p-ERK/ERK) in CKS2 shRNA group was inhibited significantly(PU87<0.05, PU251<0.05); compared with shCON transfection group, phosphorylation of ERK1/2 in CKS2 shRNA group was inhibited significantly(PU87<0.05, PU251<0.05); there is no statistical difference between non-transfection group and shCON transfection group(PU87>0.05, PU251>0.05).Conclusions:1. CKS2 may contribute to the tumorigenesis and progression of GBM by interfering in the PI3K-AKT-mTOR pathway.2. CKS2 may contribute to the tumorigenesis and progression of GBM by interfering in the MAPK(ERK) pathway. |
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