The Expression Of EIF3C In Human Brain Gliomas And Its Effect On The Proliferation Of Glioma U87MG Cells In Vitro And In Vivo

Brain gliomas are the most common, uncontrolled and lethal human intracranial tumors, which account for more than 40 percent of primary neoplasms in central nervous system. Based on the neuropathological classification of the World Health Organization(WHO), gliomas are classified as astrocytomas, oligoastrocytomas, ependymoma, medulloblastoma, glioblastoma multiforme, oligodendrogliomas, papilloma of choroid plexus and so on. According to their degree of malignancy, gliomas are categorized as grade I, grade II, grade III and grade IV by the WHO. Grade I gliomas are biologically benign and can be cured after complete surgical excision; grade II gliomas are low malignant and may follow long clinical courses, but early diffuse infiltration of the surrounding brain renders them incurable by surgery due to postoperative recurrence; grade III gliomas show increased anaplasia and malignant proliferation over grade II and are more rapidly fatal; grade IV gliomas exhibit more advanced features of malignancy and tend to have much more shorter lives. The most aggressive astrocytoma is referred to as glioblastoma multiforme or GBM for short, which is regarded as grade IV. The malignant gliomas(grade III and grade IV) accounting for approximately 60 percent of all primary brain tumors in adults are among the most difficult cancers to treat, which are recalcitrant to radiotherapy or chemotherapy and have high recurrence rates after operation. Although microsurgery, radiotherapy and chemotherapy have improved greatly in recent years, the median survival time of patients with malignant gliomas remains within the range of 9 to 12 months, and very few patients are cured from these diseases. It was reported that fewer than 3 percent of glioma patients are still alive at 5 years after diagnosis, with the exception of pilocytic astrocytomas(grade I). The prognosis of glioma patients is still very poor and has not changed significantly in recent years. Moreover, elderly patients have been reported to have a 3.18-fold higher relative risk of brain tumor compared with young adults. Gliomas show vascularized and invasive features in brain, whether they are low-grade or high-grade. Because of their invasive nature in the adjacent brain, gliomas usually fail to be cured with routine methods of treatment. Nowadays, many chemotherapy drugs are found to give severe side effects and consequently affect the results of glioma treatment. Approximately 30 percent of patients may be inclined to forgo these treatments due to severe side effects of modern chemotherapy drugs. For that, many attempts have been made to increase the therapeutic effects and decrease the side effects by developing new therapeutic approaches and finding new mechanisms of drug action. Nowadays, although a number of drugs have been used to treat brain gliomas, there still remains enormous challenges for us how to improve therapeutic efficacy, how to reduce complications and how to lower the recurrent rates.Molecular genetic researchers have confirmed that genetic changes are involved in the oncogenesis and progression of malignant tumors, which include loss of heterozygosity 10(LOH10), phosphatase and tensin homolog(PTEN), tumor protein p53(TP53), epidermal growth factor receptor(EGFR), p16 and so on. The mechanisms of tumorigenesis are believed to be associated with the accumulation of a variety of genetic alterations and the changes of gene expression pattern. By understanding the altered genetic molecular pathways of glioma oncogenesis and progression, it may be possible to develop new effective targets for glioma treatment in the near future, which will enhance the survival conditions of patients with glioma and improve the medical prognoses of them.Disrupted protein translation is prevalent in human tumors. In recent years, people have found that eukaryotic translation initiation factors(e IFs) play an important role in a wide variety of human tumors. However, the role and mechanism of e IFs in human gliomas remain unclear and need to be elucidated by further study. The present study explored the expression and function of e IF3 c in human gliomas, which is the most main and core subunit of e IF3. Firstly, immunohistochemistry SP and RT-PCR were applied to detect the expression of e IF3 c in human glioma samples and normal brain samples. Then, the impact of e IF3 c knockdown on U87 MG cells was explored in vitro by using lentivirus-mediated si RNA technique to investigate the effect of e IF3 c on human glioma proliferation, apoptosis, cell cycle, colony formation and so on. Afterward, a glioma xenograft model was also established in nude mice and the impact of e IF3 c knockdown on brain glioma formation and progression was investigated in vivo. Based on the above experimental results, it was confirmed that e IF3 c may play an important role in the oncogenesis and progress of human gliomas, and all this has laid an important theoretical underpinning of the gene therapy as a treatment for human brain gliomas in clinical practice.The content of this study contains the following three parts: Part one The expression of e IF3 c in human brain gliomas and normalbrain tissuesObjective: To explore the role of e IF3 c in human gliomas, both the immunohistochemical SP technique and the reverse transcription polymerase chain reaction technique were performed to detect the expression of e IF3 c in human brain glioma samples and normal brain samples. And the correlation was analyzed between the expression of e IF3 c in gliomas and the clinical characteristics of the patients including pathological grades, tumor position, tumor size, patient’s age and patient’s sex.Methods:1 The clinical data of patients and the disposal of tumor specimensGlioma specimens were obtained from 83 patients diagnosed with glioma receiving surgical treatment at the Second Hospital of Hebei Medical University from January, 2008 to December, 2013. Another 27 normal brain tissues were employed as the control group which were taken from 27 patients who suffered from severe brain injury and received inline decompression surgery within the same period. All specimens have been formally diagnosed by two or more nerve pathologists. No patients received any treatment including radiation, chemotherapy and immunotherapy before surgery. Among 83 patients with brain gliomas, there were 44 males and 39 females, with the average age was 44.54 ± 12.53 years(ranging from 16 to 73 years). These tumor samples were divided into two parallel parts: one part was frozen immediately and stored at-80°C in liquid nitrogen for RNA extraction of the target gene e IF3 c and the other part was fixed in 10 percent formalin and embedded in paraffin for the detection of e IF3 c expression by immunohistochemistry SP. Detailed records of clinical data of all specimens were made that consisted of the patient’s age, sex, tumor size, tumor position and pathological grade, and so on. From those specimens stored in liquid nitrogen, 31 gliomas and 5 normal brain tissues were chosen to examine the expression of endogenous gene e IF3 c by RT-PCR.2 Expression of e IF3 c protein in human brain gliomas and normal brain tissues by IHC analysisThe expression of e IF3 c was examined in human brain gliomas and normal brain tissues by immunohistochemical(IHC) staining. Paraffin blocks were cut in 4 μm sections and mounted on glass slides. After being dewaxed in xylene, the sections were rehydrated in ethanol gradients and immerged in water. Antigen retrieval was performed by microwave exposure in 0.01 mmol/l sodium citrate buffer solution. Afterwards, the sections were blocked with 10 percent bovine serum albumin for 30 min and incubated overnight with rabbit anti-human polyclonal e IF3 c antibodies, followed by secondary anti-goat antibodies for 60 min. Then, the sections were stained with DAB and counterstained with hematoxylin. Digital images were captured using microscope.3 Analysis of the relationships between e IF3 c expression in gliomas and the clinical characteristicsThe relationships were analyzed between the expression of e IF3 c protein and the patient’s age, sex, tumor size, tumor position and pathological grade.4 Analysis of e IF3 c gene expression in human brain glioma samples and normal brain samples by real-time PCRTatal RNAs were extracted from 31 human glioma samples and 5 normal brain samples and reversely transcribed into c DNA. e IF3 c was detected by real-time PCR. Data were analyzed by Graph Pad Prism 4.0 software and the 2-ΔΔCt method was used to quantitate the relative gene expression.Results:1 e IF3 c protein was overexpressed in human brain gliomasThe expression of e IF3 c protein was mainly located in cytoplasm. At high magnification, the positive cells displayed brownish yellow granules or diffused yellow in cytoplasm. Of 83 glioma specimens, 69 cases had positive expression of e IF3 c protein and 14 cases negative; and the positive expression rate of e IF3 c was 83.13 percent(69/83). However, of 27 normal brain tissues, only 1 case had weak expression of e IF3 c protein and the rest negative, and the positive expression rate of e IF3 c was 3.7 percent(1/27). There was a significantly higher positive percentage of e IF3 c expression in human gliomas than that in normal brain tissues(χ2=57.02, P<0.0001). Therefore, e IF3 c protein was overexpressed in human brain gliomas.2 The correlations between the expression e IF3 c protein in gliomas and the clinical characteristicsThe positive rates of e IF3 c protein in high grade gliomas(WHO grade III and IV) and low grade gliomas(WHO grade I and grade II) were 91.07 percent(51/56), 66.67 percent(18/27) respectively, and the difference was statistically significant(χ2=6.0950, P=0.0136<0.05). However, the positive rates of e IF3 c protein in gliomas were not associated with patient’s age, gender, tumor site and size(P>0.05 for all). Taken together, these findings demonstrate that the positive rates of e IF3 c were correlated with glioma grade.3 Overexpression of e IF3 c m RNA in human glioma specimensReal-time PCR was performed to detect the m RNA levels of e IF3 c in 31 fresh glioma samples and 5 normal brain tissue specimens preserved in liquid nitrogen. The results show that m RNA expression level of e IF3 c in gliomas is significantly higher than that in normal brain tissues(P<0.01). And the m RNA expression level of e IF3 c gene are considered to be not associated with the pathological degrees of glioma(P>0.05).4 Fold change analysis of e IF3 c gene regulation in brain gliomas and normal brain tissuesOf 31 glioma samples, there are 30 samples in which the relative quantitative expression of e IF3 c gene was up-regulated at 2.6-fold change on average compared with that in the normal brain tissues. Therefore, the e IF3 c gene can be chosen for genecard experiment.Conclusions:1 e IF3 c protein is overexpressed in human brain gliomas.2 High expression of e IF3 c is correlated with malignant phenotypes in human gliomas. However, e IF3 c expression was not correlated with patient’s age, gender, tumor site and size.3 The level of e IF3 c m RNA is overexpressed in human gliomas, but there is no correlation with the glioma pathological grades.4 e IF3 c gene expression is up-regulated in human gliomas. Part two Effects of e IF3 c on the proliferation of brain glioma U87MGcells in vitroObjective: To explore the function of e IF3 c in human gliomas, a series of experiments of cell function were performed to investigate the effects of e IF3 c on the proliferation, apoptosis, cell cycle and colony formation of human U87 MG cells through the RNA interference.Methods:1 Glioma cell cultureHuman glioma cells(U87, U251, A172 and U373) were purchased from the National Platform of Experimental Cell Resources for Sci-Tech. The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10 percent fetal bovine serum, 2 m M L-glutamine, penicillin(100 U/ml) and streptomycin(100 μg/ml). All the cells were cultured at 37?C in a humidified atmosphere containing 5 percent CO2.2 Assessment of e IF3 c gene expression in human glioma cell lines by semi-quantitative RT-PCRThe total RNA was extracted from the four human glioma cell lines(U87, U251, A172 and U373). Afterwards, the expression level of e IF3 c m RNA was tested through semi-quantitative RT-PCR approach. The data confirmed that e IF3 c was expressed in human glioma cell lines.3 Construction and transfection of the e IF3c-si RNA lentivirusThe sequences of the si RNA for e IF3 c was synthesized as 5′-GACCAT CCGTAATGCCATGAA-3′, and the sequences of the si RNA for scrambled si RNA was synthesized as 5′-TTCTCCGAACGTGTCACGT-3′. These nucleotide sequences were inserted into the plasmid using si RNA expressing vector p GCSIL-GFP and lentivirus packing e IF3c-si RNA. Then U87 MG cells and 293 T cells were stably infected with associated lentivirus.4 Knockdown efficiency of e IF3 c m RNA by q PCR techniqueU87 MG cells were infected with lentiviral RNAi expression vectors based on group. The expression of GFP was observed under a fluorescent microscope three days after infection. The U87 MG cells were collected five days after infection. Then, q PCR technique was applied to detect the knockdown efficiency of e IF3 c m RNA.5 Western blot analysisFor protein isolation, the cells were harvested with RIPA buffer. Proteins were separated by SDS-PAGE, transferred onto PVDF membranes, and stained with primary antibodies and secondary antibodies, detected using the electrochemiluminescence(ECL) kit. Bands were obtained after exposure to X-ray film.6 Cell proliferation assay using Brd UDNA synthesis in proliferating cells was determined by Brd U incorporation. The U87 MG cells infected with e IF3 c or scrambled si RNA-expressing lentivirus were cultured for 24 or 48 h. The cells were then resuspended and seeded into 96-well plates and cultured for another one or four days. Cells were collected at the first day or the fourth day after the diluted bromodeoxyuridine(Brd U) reagent was added to 96-well plates. The absorbance(OD) was determined by ELISA at a wavelength 450 nm. Brd U signals and cell proliferation were analysed according to the absorbance value.7 Cell cycle analysis by flow cytometryThe U87 MG cells infected with lentivirus expressing e IF3c-si RNA or scrambled si RNA were incubated at 37℃ for 96 h. Then, the cells were resuspended, seeded in 6 cm dishes. The cells were harvested and fixed when they grew until 80 percent confluency. After being washed with PBS, the cells were treated with the staining solution containing propidium iodide(PI), RNase and PBS. Finally, the cells were analyzed for the cell cycle phase by flow cytometry.8 Colony formation assayThe U87 cells that were transfected with e IF3c-si RNA lentivirus or scrambled si RNA lentivirus for five days were harvested and seeded in 6-well plates. After two weeks of culture, the cells were fixed with 4 percent paraformaldehyde and stained by adding diluted Giemsa stain for 20 min. Finally, the cells were rinsed with distilled water and colonies with > 50 cells were counted by fluorescence microscopy.9 Detection of cell apoptosis by flow cytometryCell apoptosis was assayed by staining using Annexin V-APC and detected by flowcytometry. For analysis of apoptosis, the U87 MG cells infected with lentivirus expressing e IF3c-si RNA or scrambled si RNA were incubated for five days, harvested and washed with PBS. Then, the cells were resuspended in staining buffer containing 5μl Annexin V-APC solution at room temperature in the dark for 10-15 min. Finally, cell apoptosis was analyzed by flow cytometry.Results:1 The expression of e IF3 c in brain glioma cell linesThe expression of e IF3 c m RNA was detected in glioma cell lines(U87, U251, A172 and U373) by semi-quantitative RT-PCR. e IF3 c m RNA was expressed in all four cell lines.2 Construction of the lentiviral vector expressing e IF3 c or scramble si RNALentiviral vector expressing e IF3c-si RNA or scrambled si RNA has been successfully constructed and maintained high expression in U87 MG cells.3 Lentiviral-mediated si RNA inhibits e IF3 c efficiently in glioma cell linesThe e IF3 c m RNA level was determined using quantitative fluorescent PCR. Treatment with e IF3 c si RNA led to a ~ 95 percent reduction in the expression level of e IF3 c m RNA. Therefore, the present results demonstrated that a highly efficient knockdown of e IF3 c expression at the m RNA level was achieved using the lentivirus-mediated RNAi strategy.4 e IF3 c protein level in 293 T cells after silencingProtein levels were determined by western blot analysis. Compared with the negative control, e IF3 c protein levels in 293 T cells were markedly reduced after e IF3 c silencing by RNAi. GAPDH was used as an internal control.5 e IF3 c knockdown inhibits the proliferation of U87 MG cellsThe cell number counting results showed that proliferation inhibition was significantly observed in U87 MG cells infected with e IF3c-si RNA after 24 h of infection. Furthermore, the inhibitory effect of e IF3 c knockdown on cell proliferation had increased four days subsequent to transfection. Brd U incorporation in U87 MG cells by e IF3 c knockdown were significantly suppressed compared with controls after four days of cell plating(P<0.05). These data suggested that e IF3 c knockdown markedly suppressed the proliferation of the U87 MG cells.6 e IF3 c knockdown impairs the ability of U87 MG cells to form coloniesCloning formation assay showed that a ~34.8 percent reduction in colony number of U87 MG cells treated with e IF3c-si RNA. An average of 23 clones formed in U87 MG cells treated with scr-si RNA, and only 15 clones in cells with e IF3c-si RNA. There was a significant difference between them(P<0.05). This result suggested that e IF3 c gene was closely related to the clone forming ability of U87 MG cells.7 e IF3 c knockdown causes U87 MG cell cycle arrestThe cell cycle distribution revealed that e IF3 c knockdown induced a significant G0/G1 phase arrest and led to the inhibition of the G1-S transition in U87 MG cells, with a corresponding reduction in the percentage of S-phase cells. The proportion of cells in the G0/G1 phase increased between 71.42 percent and 79.05 percent in the e IF3 c si RNA treatment group, while the ratio in the S phase decreased between 17.09 percent and 7.83 percent. There was a significant difference between e IF3c-si RNA group and Scr-si RNA group(P<0.05). This results indicated that e IF3 c knockdown led to the inhibition of the G1-S transition.8 e IF3 c knockdown promotes apoptosis of U87 MG cellsAn Annexin V-APC assay was employed to evaluate the apoptosis of U87 MG cells. The apoptosis rate was observed at 5.83 percent of U87 MG cells treated with e IF3 c si RNA, while at 2.78 percent of U87 MG cells treated with scrambled si RNA. It suggested that e IF3 c gene was largely related to the apoptosis of U87 MG cells and its knockdown promoted their apoptosis.Conclusions:1 The expression of e IF3 c m RNA was detected in glioma cell lines including U87, U251, A172 and U373 by semi-quantitative RT-PCR.2 Through the RNAi technique, lentivirus-mediated knockdown of e IF3 c gene significantly inhibited U87 MG cells from proliferating and promote them apoptosis.3 Knockdown of e IF3 c caused G0/G1 phase arrest and suppressed the G1-S transition in U87 MG cells. This effect of e IF3 c gene on cell cycle distribution may be one of the mechanisms that impact proliferation and apoptosis of human glioma.4 The expression of e IF3 c down-regulated can significantly inhibit the proliferation of U87 MG cells, which makes it as a potential therapeutic target for human glioma. Part three Effects of e IF3 c on the proliferation in brain glioma U87 MGcells in vivoObjective: To further investigate the role of e IF3 c in human glioma in vivo, a glioma xenograft model was established in athymic nude mice.Methods:1 Experimental groupsTwenty-four athymic nude mice were randomly divided into three groups(Negative control, scrambled and e IF3c-si RNA group), which were treated respectively.2 Cell culture of human glioma U87 MGThe untreated U87 MG cells and the U87 MG cells transfected with e IF3 c or scrambled si RNA-expressing lentivirus were cultured for 48 h. These cells were then collected and resuspended with PBS buffer.3 The establishment of the glioma xenograft model in nude miceThe athymic nude mice were injected subcutaneously with these treated or untreated U87 MG cell suspension respectively. The nude mice were allocated to three groups(Negative control, scrambled and e IF3c-si RNA group) as previously.4 The measurement of the volume and weight of tumorsThe nude mice were fed and diameter of tumor mass were subsequently measured every other day from the tenth day for 18 days. Their period of observation ended after they were fed for 28 days. Then, nude mice were sacrificed. The subcutaneous tumors were resected and theses specimens were weighed respectively.Results:1 The glioma xenograft model in nude mice was constructedThe glioma U87 MG xenograft model in nude mice was constructed successfully. The tumor inhibited experiment in vivo was performed.2 Knockdown of e IF3 c delayed tumor growth in vivoIn the initial 11 days of observation, no significant difference was observed among the three groups. However, after 13 days, the tumor growth was markedly suppressed in the group treated with lentiviral e IF3 c si RNA. In the end of observing, the mean tumor volume decreased significantly in the e IF3c-si RNA group compared to the scrambled group(P<0.01) or the negative control group(P<0.01).3 Knockdown of e IF3 c reduced tumor weight in vivoIn the end of observing, all mice were killed. The tumor bodies were removed from these mice and weighted. The results showed that tumor mass weight was significantly reduced in the e IF3c-si RNA group compared with the scramble group(P<0.01) or Control group(P<0.01).Conclusions:1 e IF3 c knockdown significantly suppressed U87 MG cell proliferation and the tumor growth in vivo.2 e IF3 c gene can be used as a promising therapy target for human brain glioma treatment.