The Effect Of HIF-1α On The Apoptosis And Proliferation Of Brain Glioma And Related Mechanism Study

At present, there is no any breakthrough development for the treatment of glioma. The main reason is that glioma is malignant by itself, which is more easy to be invasive, metastasis, recurrence, resistant to chemotherapy and radiotherapy. Most of the malignant characteristic is relevant with the hypoxia micro-environment. More and more researches have demonstrated that hypoxia induces the genes expression which are closely related with tumor angiogenesis, metastasis, glycolysis, drug resistance. All of this conferred the brain glioma stronger malignant behavior. HIF-1 a (hypoxia inducible factor-1α) which plays an important role is an important transcription factor in tumor hypoxia microenvironment. HIF-1 transcription complex composed of HIF-1 a and HIF-1β combined with HRE(hypoxia response elements) in gene promoter, which induce the downstream gene transcription. All of this is involved with metabolism, angiogenesis, cell differentiation, proliferation, apoptosis. It is also causes the invasion and metastasis of tumor and treatment resistance. Recently, some researches has found out that HIF-1α play an important role in tumor cells proliferation and apoptosis by bidirectional regulation. On the one hand, it promotes the transcription of proapoptotic protein including BNIP3、Bax、Bak. On the other hand, such as Bcl-2, it up-regulates the anti-apoptosis factor to inhibit the tumor cell apoptosis. It is also involved with hypoxia inducible cell apoptosis by which induce the resistance of radiotherapy and chemotherapy.It is generalized to the case that hypoxia in brain glioma. Scientist have found out that the cells can quite adapt to hypoxia environment in vivo and vitro. Moderate hypoxia can promote the cells proliferation and inhibit the cells apoptosis. Recently, some researchers reported that gradient hypoxia in solid tumor including brain glioma can cause the tumor heterogeneous. The cells which is far away from the vescular region is more malignant than other cells, meanwhile, the hypoxia degree is more severe. Due to the limitation of the lowest oxygen concentration in the incubator, there is still a long way to go for exploring the degree that brain glial cells can tolerate in hypoxia environment. Further more, it is required deep-going research on molecular mechanism of cell survival in extreme hypoxia condition. Tumor cells in hypoxia microenvironment mostly lack of the energy substance. We can infer from this that tumor cells also lack of glucose in hypoxia environment. At present, there are still no systematic studies on what influence the brain glioma cells proliferation and apoptosis in hypoxia environment that lack of glucose. Furthermore, there is no enough evidence to demonstrate that how HIF-α play the role in brain glioma cells according to different concentration of hypoxia micro-environment.In this study, by using of the regulator of hypoxia with which the lowest concentration is 0.1%, we simulate the hypoxia concentration gradient environment in which the tumor cells were cultivated. Moreover, comparing with the lack of glucose in different hypoxia concentration environment, we focus on the differences between the brain glioma cell proliferation and apoptosis in different hypoxia concentration. The data provide the evidence of the involvement that the effect of the apoptosis resistance depending on different hypoxia concentration. That will support the preclinical and clinical development of therapy by different hypoxia concentration and help us to screen out the more effective therapy target. Meanwhile, to detect the variation of HIF-1α and P53 expression level in different hypoxia concentration, we explore the role of the HIF-1α and P53 in the apoptosis resistance. Demonstration of the effect of HIF-1α silencing on the apoptosis and proliferation of glioma under different hypoxia environment contributes to speculate the if the HIF-1α is the main target for the glioma treatment.PART ⅠEffects of Gradient Hypoxia and Glucose Deprivation on Apoptosis and Proliferation of Glioma CellsObjective:To explore the effect of different levels of hypoxia and glucose deprivation on the apoptosis and proliferation on glioma T98G cell line and investigate the baseline hypoxia level inducing cell apoptosis.Method:T98G cells were cultured in normal glucose medium or glucose-free medium, then exposed to different oxygen concentrations (21% 02,3% O2,0.5%O2, 0.1% O2) for corresponding time. At post-treatment for 24h,48h and 72h, the cell apoptosis were measured by flow cytometry and hoechst stain. The proliferation were detected by MTT assay at post-treatment for 24h,48h,72h,96h and 120h. The data were expressed by mean and standard deviation and analyzed using one-way analysis. All analyses were performed with SPSS 17.0. p-value of less than 0.05 was regarded as statistically significant.Results:1. The T98G cells in all glucose groups(including different hypoxia concentration and normoxia) didn’t show obvious apoptosis (P>0.05). But in all glucose-free groups, the significant cell apoptosis was observed (P<0.01) and the apoptosis rate was highest at 21%O2 glucose-free condition within 48h post-treatment(P<0.01). To 72h post-treatment, the cell apoptosis rate in 0.1% O2 hypoxia glucose-free group was higher than that of normoxia glucose-free group instead(P<0.01). The cell morphology changed with the hypoxia prolonged.2. The results of MTT assay showed that all hypoxia level could promote proliferation of T98G cells. But the effects of moderate hypoxia(3%O2) was much more significant. The cell proliferation speed was reduced slightly in the severe hypoxia groups(0.5%O2,0.1%O2) at 72h post-treatment. In all the glucose-free groups, the proliferation of T98 cells was inhibited obviously. Within 72h, the cell proliferation inhibition of mild hypoxia-glucose-free group(3% O2 and 0.5% O2) was less than that of normoxia-glucose-free group. But in the severe hypoxia (0.1%O2), the cell proliferation inhibition rate was more than that of nomoxia condition.Conclusions:1. It suggested that hypoxia may not induce the glioma cell apoptosis as independent factor, instead mild hypoxia can promote the glioma cell proliferation. But glucose deprivation could induce apoptosis of glioma cell independently. Glioma cells are more sensitive to glucose deprivation than extreme hypoxia.2. Mild hypoxia (more than 0.5%O2) have the ability to relieve the cell apoptosis and proliferation inhibition induced by glucose deprivation. But in the extreme hypoxia, the ability diminished after a short time.part ⅡExpression Levels of HIF-la and mt-P53 Regulated by Different Hypoxia and Glucose Condition and Correlation with the Glioma Cells ApoptosisObjective:1. To investigate the expression levels of HIF-1α and mt-p53 in glioma cells cultured in different hypoxic concentration and glucose condition.2. To explore the possible correlation between the HIF-1α and mt-p53 expression levels with the cell’s apoptosis and proliferation regulated by different hypoxia and glucose condition.3. To speculate the potential role of HIF-1 and mt-p53 in apoptosis resistance in hypoxia environment.Methods:1. T98G cells were cultured under different oxygen concentration (21%O2,0.5%O2 or 0.1%O2) and glucose condition (normal-glucose or glucose-free) for 24h,48h and 72h. The total RNA was extracted at corresponding time to detect the expression levels of HIF-la mRNA using real-time PCR.2. At the corresponding time, the total protein was extracted. Western Blot method was used to detect the expression of HIF-1α protein and mt-p53 protein of the different group.3. The correlation between the cell apoptosis under different condition and the expression of HIF-1α and mt-p53 at the corresponding group were analyzed.4. Statistical significance was measured by Student’s t-tests and Pearson linear regulation with SPSS 17.0.Results:1. Real time PCR results:There was no significant change of the the T98G cells HIF-1α mRNA levels under nomoxia-glucose condition within72h. The HIF-1α mRNA levels in the nomoxia-glucose-free group increased within 24h post-treatment and declined obviously at 72h post-treatment. The HIF-1α mRNA levels of the cell under (0.1% and 0.5%) hypoxic-glucose-free condition gradually declined within 72h post-treatment. Under 0.1% hypoxia-glucose condition, it was down-regulated significantly within 72h post-treatment. But under 0.5% hypoxia-glucose condition, it was down-regulated significantly within 24h and obviously up-regulated at 72h post-treatment.2 The expression of HIF-1α protein of the nomoxia-glucose group maintained low levels within 72h. The HIF-1α protein levels of momoxia-glucose-free group increased at 24h post-treatment and gradually declined from 48h. The HIF-1α protein expression levels of 0.1% and 0.5% hypoxia-glucose group significantly was up-regulated at 48h,72h post-treatment (P<0.05). To the 0.1% and 0.5% hypoxia glucose-free group, the HIF-1α protein expression levels of the T98G cells obviously was up-regulated at 24h,48h and 72h. And the HIF-1α protein levels of 0.5% hypoxia-glucose-free group was higher than that of 0.1% hypoxia-glucose-free group.3. Under nomoxia-glucose condition, the p53 protein of the T98G cells maintained stable expression within 72h. It declined obviously at 24h post-treatment and to the lowest level at 72h post-treatment under nomoxia-glucose-free condition. Under 0.1% hypoxia-glucose and 0.5% hypoxia-glucose condition, the p53 protein level increased at 24h,48h and 72h. To 0.1% hypoxia-glucose-free and 0.5% hypoxia-glucose-free group, the p53 protein level declined from 48h post-treatment. The reduction of the p53 protein level in the 0.1% hypoxia-glucose-free group was more obvious than that of the 0.5% hypoxia-glucose-free group.4. The up-regulation of HIF-1α protein expression level of the T98G cell were negative correlation with the apoptosis under hypoxia-glucose condition. The cells apoptosis were also negative correlation with the change of HIF-1α protein expression level under glucose-free condition including 0.5% hypoxia and nomoxia. The cells apoptosis rate of 0.5% hypoxia-glucose-free group was less than that of nomoxia-glucose-free group at 72h post-treatment accompanied with the up-regulation of the HIF-1α protein expression level. It implied that cell apotosis rate reduction may be correllation with up-regulation of the HIF-1α.5. Under nomoxia-glucose-free and hypoxia- glucose-free condition, the cell apoptosis rate increased with the down-regulation of mt-p53 protein level. Under hypoxia -glucose condition, the cell apoptosis rate decreased with the up-regulation of mt-p53 protein level. The cell apoptosis rate were negative correlation with the protein level of mt-p53. The change of HIF-1α protein expression level were positive correlation with that of the mt-p53.Conclusion:1. The up-regulation of HIF-1α induced by glucose deprivation was at the mRNA level. The up-regulation of HIF-1α induced by 0.1% hypoxia was at the protein level through decreasing the protein degradation, but its mRNA level inhibited. The up-regulation of HIF-1α induced by 0.5% hypoxia was at both the protein level and mRNA level. The HIF-1α mRNA level was suppressed under 0.1% hypoxia-glucose-free and 0.5% hypoxia- glucose-free condition, but up-regulated at the protein level.2. It implied that cell apotosis rate reduction may be correllation with up-regulation of the HIF-1α. The mutant p53 protein may has the synergism effect. The glioma cell apoptosis rate under 0.5% hypoxia-glucose-free condition was less than that under the nomoxia-glucose-free condition involving in the correlation with the up-regulation of HIF-1α and p53 protein expression level.3. The up-regulation of HIF-1α protein caused by glucose deprivation may be a short time stress reaction.4. Hypoxia may indexe the up-regulation of mt-p53 protein, but glucose deprivation may cause down-regulation of the mt-p53 protein.PART ⅢConstruction of a glioma T98G cell line stably expressing HIF-1α silencing mediated by lentivirus vectorObjective:To construct a T98G cell line stably expressing HIF-1α RNAi mediated by lentivirus vector and verify the knock down efficiency at the level of mRNA and protein levels.Method:1. The lentiviral HIF-1α-sh、HIF-1α-sh10 and control vectors were packed into 293T cells to generate the corresponding lentiviruses. Transfections were performed using PEI.2. T98G cells were infected with HIF-1α-sh9, sh10 or control lentiviruses and shRNAs or scramble control lentiviruses were selected and maintained in the same medium containing 2.5μg/mL of puromycin for 72h.3. The surviving lentiviruses transfected cells were identified by real-time PCR and Western blot analysis to verify the silencing efficiency of two shRNA lentiviral vector.4. Selecting the most efficient vector for the following experiment.Results:1. The sh9, sh10 and control lentiviral vector were successfully packaged into corresponding lentiviruses and efficiently infected T98G cells. After puromycin selected, there was no obviously cell death.2. The knock down efficiency of two vector were above 90% at mRNA levels and above 75% at protein levels.Conclusion:Two kinds of recombinant lentivirus shRNA vectors can efficiently infected and interfered the expression of HIF-1α in T98G.PART ⅣThe effects of silencing HIF-1α expression by lentiviral-mediated RNAi on the glioma cell apoptosis and proliferation and the mt-p53 protein expression under hypoxiaObjective:1.To investigate the effect of silencing HIF-1α expression by lentiviral-mediated RNAi on the glioma cell apoptosis and proliferation at extreme hypoxia.2.To explore the effect of silencing HIF-1α expression by lentiviral-mediated RNAi on mt-p53 protein expression at extreme hypoxia.3. To analyze the possible interaction between HIF-1α and mt-p53 and the potential role of HIF-1 and mt-p53 in apoptosis resistance of hypoxia environment.Methods:1. The constructed T98G cell expression HIF-1α silencing(HIF-1α-shRNA T98G) and control cell(ctr) were cultivated at different oxygen concentration(21%.0.5% and 0.1%). At corresponding time, measured the proliferation inhibition rate by MTT method and detect the apotosis rate by AnexinV FITC/PI stain and flow cytometry.2. The total RNA was extracted at corresponding time to detect and contrast the expression levels of HIF-1α mRNA between pre-RNAi and post RNAi using real-time PCR.3. The total protein was extracted at corresponding time to detect and contrast the expression levels of HIF-1α protein and mt-p53 between pre-RNAi and post RNAi using Western blot method.4. To Analyze the correlation between the HIF-1α silencing and mt-p53 protein expression, the T98G cells proliferation and apoptosis.Resluts:1. Compared with the ctr cell, the proliferation speed of HIF-1α-shRNA T98G decreased obviously both in hypoxia or nomoxia condition within 72h.2. Under nomoxia, there was no obvious difference of the cell apoptosis rate between HIF-1α-shRNA T98G and ctr group. Under 0.1% and 0.5% hypoxia condition, the cell apoptosis rate of HIF-1α-shRNA T98G increased obviously at 48h post-treatment and was more significant at 72h.3. The mRNA and protein level of T98G was down-regulated notably both under nomoxia and hypoxia condition. Within 72h post-treatment, the knock down efficiency was higher at the hypoxia condition than that at the nomoxia condition.4. The HIF-1α protein expression level was negative correlation with the cell apoptosis rate, proliferation inhibition rate and was positive correlation with mt-p53 protein expression both under 0.1% and 0.5% hypoxia condition.Under nomoxia, there was no correlation between HIF-1α protein expression level and the cell apoptosis rate, proliferation inhibition rate and mt-p53 protein expression.Conclusion:1. The silencing of HIF-1α could partly could inhibit the T98G cells proliferation and promote it apoptosis.2. The silencing of HIF-1α could down-regulated the mt-p53 protein expression level. There was a positive correlation between HIF-1α and mt-p53 protein expression level.3. The HIF-1α protein expression level was negative correlation with the cell apoptosis rate, proliferation inhibition rate both under 0.1% and 0.5% hypoxia condition.PART ⅤThe effects of silencing HIF-1α expression by lentiviral-mediated RNAi on the glioma cell apoptosis and proliferation and mt-p53 protein expression under glucose deprivationObjective:1. To investigate the effect of silencing HIF-1α expression by lentiviral-mediated RNAi on the glioma cell apoptosis and proliferation under extreme hypoxia and glucose deprivation. To verify the effect of HIF-1α on the resistance to apoptosis inducied by glucose deprivation.2. To explore the effect of silencing HIF-1α expression on mt-p53 protein expression at extreme hypoxia-glucose-free and nomoxia-glucose-free condition.To analyze possible correlation between the two protein.Methods:1. The HIF-1α-shRNA T98G and control cell(ctr) were cultivated at nomoxia-glucose-free and 0.5% hypoxi-glucose-freecondition. At corresponding time, Measured the proliferation inhibition rate by MTT method and detected the apotosis rate by AnexinV FITC/PI stain and flow cytometry.2. The total protein was extracted at corresponding time to detect and contrast the expression levels of HIF-1α protein and mt-p53 between pre-RNAi and post RNAi using western blot method.3. To Analyze the correlation between the HIF-1α silencing and mt-p53 protein expression, the T98G cells proliferation and apoptosis under glucose deprivation.Results:1. The proliferation of HIF-1α-shRNA T98G and ctr cell both under hypoxia or nomoxia condition was obviously inhibited by glucose deprivation. There was no obvious difference of cell proliferation between the two group under nomoxia. But, the inhibition of proliferation was more significant in the HIF-1α-shRNA T98G than that in the ctr-T98G under hypoxia and that in the ctr-T98G under monoxia.2. The apoptosis of HIF-1α-shRNA T98G and ctr cell under nomoxia glucose deprivation condition obviously increased, but there was no significant variation between two group. After silencing HIF-1α expression of T98G cell, the cell apoptosis rate was higher under hypoxia glucose deprivation than that of the ctr cell under nomoxia glucose.3. Under hypoxia glucose-deprivation condition, the apoptosis rate of the T98G cells after silencing HIF-1α was higher than that of the ctr-T98G under nomoxia glucose-deprivation condition, which implied that the anti-apoptosis effect lost with the silencing of HIF-1α on T98G cell. At the same time, the mt-p53 protein level was down-regulated, which implied that the expression of mt-p53 protein was regulated by the HIF-1α.There was no obviously change on the apoptosis rate of T98G cell and mt-p53 expression under nomoxia and hypoxia glucose condition.Conclusion:HIF-1α was involved in resistance to the glioma cells apoptosis induced by glucose-deprivation of under hypoxia. The up-regulation of HIF-1α decreased the apoptosis rate of the T98G cell under glucose-free adversity and regulated the expression of mt-p53 protein. But it was not clear that the role of mt-p53 in the hypoxia protection T98G cell under glucose-free adversity.