Inhibitory Effect Of ATF3-SiRNA On Human Brain Glioblastoma

Gliomas represent a series of highly malignant and highly lethal tumor, despite considerable progress in treatment, their prognosis remain extremely poor. Glioblastoma is the most common and malignant of all gliomas, it accounts for 60% of all glial tumors and its overall survival rate had no significant change in the past 20 years. Despite aggressive treatment, essentially all malignant gliomas recur and ultimately lead to death. The median survival time of glioblastoma patients was only 5-7 months after the relapse.The major cause of recurrent is tumor invasion. Tumor invasion is a complicated process of malignant transformation of various types of tissues. This involves a series of molecular-molecular, cell-cell and cell-matrix interactions, and ultimately leads to tumor cell migration and invasion to surrounding healthy brain tissues. Previous studies have shown that protease, components of extracellular matrix, cell adhesion and signaling molecules all play an important role in the migration and invasion of glioma cells and all these components can be viewed as potential therapeutic targets. Recent studies suggest that certain signaling molecules that should be activated under stress can promote tumor cell migration under certain circumstances and are likely to promote glioblastoma invasion.Activating transcription factor 3(ATF3) is one of the first expressed transcription factors. It’s an early response gene and is a key regulator of the cell’sresponse to extracellular signals including hormones, growth factors, etc. Many studies have also proved that ATF3 is an adaptive response gene, and is the center of the body’s response network when faced with disturbing signal. ATF3 is highly expressed in several malignant tumors and can induce reentering into cell cycle from the quiescent state, accelerate cell proliferation, and thus is related to invasion, metastasis and prognosis of a variety of tumors. Thus, it becomes an important hot spot in the study of malignant tumor invasion.For now, no study has referred to the effect of ATF3 in malignant glioma invasion. To investigate whether ATF3 also has a role in the progress and invasion of malignant glioma and thus becomes a therapeutic target, we used ATF3-si RNA to treat glioblastoma in vitro and in vivo. Immunohistochemistry and some other molecular biology techniques are used in this study. The study is divided into three parts.Object:1. To observe the expression of ATF3, Maspin and MMP2 in human brain gliomas of different grades at both m RNA and protein level, and speculate the possible correlation among them as well as a possible meaning of ATF3 expression in gliomas.2. To construct a ATF3-si RNA eukaryotic expression vector, observe its effects on the viability, cell cycle, apoptosis and invasion of human glioblastoma U373 cell line as well as on the expression levels of Maspin and MMP2, and evaluate the inhibitory effect of ATF3-siRNA on U373 cells in vitro.3. To observe the inhibitory effect of ATF3-si RNA on the transplanted glioma and the expression levels of Maspin and MMP2 in nude mice,and evaluate the inhibitory effect of ATF3-si RNA on U373 cells in vivo.Main Content:Part one: ATF3, Maspin and MMP2 expression in human glioma.Method:1. Protein expression and subcellular distribution of ATF3, Maspin and MMP2 of normal brain tissue and tumors of different grades were detected using immunohistochemistry.2. The relative m RNA levels of ATF3, Maspin and MMP2 of normal brain tissue and tumors of different grades were analyzed using real-time quantitative PCR, and their correlations are speculated.3. The relative protein levels of ATF3, Maspin and MMP2 of normal brain tissue and tumors of different grades were analyzed using western blot, and their correlations are speculated.Results:1. With the increase of pathological grade of glioma, the protein levels of ATF3 and MMP2 increase,and Maspin level decreases.2. Messenger RNA levels of ATF3 and MMP2 are higher in glioma of higher grade, and this difference is significant between normal brain tissue and grade II-IV glioma(P < 0.05). Maspin m RNA level is lower in glioma of higher grade, and this difference is significant between normal brain tissue and glioma of all four grades(P < 0.05).3. Protein levels of ATF3 and MMP2 are higher in glioma of higher grade, and this difference is significant between normal brain tissue and grade II-IV glioma(P < 0.05). Maspin protein level is lower in glioma of higher grade, and this difference is significant between normal brain tissue and glioma of all four grades(P < 0.05).Part two: The inhibitory effect of ATF3-si RNA on glioblastoma in vitroThe first chapter: Screening and identification of ATF3-si RNA in U373 MG cell lineMethod:1. Three pairs of 63 bp complementary oligonucleotides with Bam H I and HindIII sticky ends on each ends were synthesized. Each pair was annealed and ligated into p Silencer2.1 U6 vector. Positive colonies were selected for p Silencer/si R-ATF3 expression vector.2. Expression vector were transfected into human brain glioblastoma U373 MG cell line, and the optimum transfection condition was determined by microscopic observation of GFP expression.3. Total RNA were extracted 48 h after transfection,and real-time fluorescence quantitative PCR was used to observe the inhibitory effect of p Silencer/si R-ATF3 vector on ATF3 m RNA levels.4. Cells were harvested 72 h after transfection, and western blot was used to observe the effect of p Silencer/si R-ATF3 vector on ATF3 protein levels.Results:1. Enzyme digestion and sequencing results showed that the pSilencer/ si R-ATF3 expression vector was successfully constructed.2. Transfection efficiency was over 70%.3. Real-time PCR and western blot results showed that p Silencer-siR1-ATF3 and p Silencer-si R2-ATF3 expression factor effectively repressed the expression of ATF3 in U373 MG cell.The second chapter: Cell growth and other functional assay after ATF3-si RNA for U373 cell line.Method:Five experimental groups were set up: ① control group; ② control-siRNA group; ③ ATF3-si RNA group; ④ cisplatin treatment group; ⑤ ATF3-si RNA + cisplatin treatment group1. Cell viability and susceptibility to cisplatin of each group were analyzed by Methylcyclopentadienyl manganese tricarbonyl(MTT) method.2. Cell proliferation index and changes in cell cycle after ATF3 suppression were analyzed by flow cytometry.3. Apoptosis of each group after ATF3 suppression was accessed by terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling assay(TUNEL) assay.4. In vitro invasion ability was observed by trans-well experiment in which number of cells migrated through the membrane were counted.5. Protein expression of ATF3, Maspin, and MMP2 was detected by immunocytochemistry.6. Messenger RNA expression of ATF3, Maspin, and MMP2 was determined by real-time PCR, and relative expression levels of the three m RNAs were determined after ATF3 suppression.7. Protein expression of ATF3, Maspin, and MMP2 was determined by western blot, and relative expression levels of the three proteins were determined after ATF3 suppression.Results:1. MTT assay results: in comparison with control group and control-siRNA group,cell viability of ATF3-si RNA group was significantly decreased(P < 0.05), cell viability of cisplatin group and ATF3-si RNA plus cisplatin group decreased with time(P < 0.05) and there’s no significant difference between the two(P > 0.05).2. Flow cytometry results: in comparison with control group and control-si RNA group,ATF3-si RNA group and cisplatin groups had their cell cycle arrested at G1, cell number of S phase significantly decreased, and this phenomenon is more significant in ATF3-si RNA plus cisplatin group.3. TUNEL assay results: in comparison with control group and control-si RNA group, ATF3-si RNA group and cisplatin groups had significantly higher level of apoptosis(P < 0.05), and it’s more severe in ATF3-si RNA plus cisplatin group.4. Transwell assay results: in comparison with control group and control-si RNA group, migration ability was significantly decreased in ATF3-si RNA group and cisplatin groups(P < 0.05), and ATF3-si RNA plus cisplatin group was similar with cisplatin group(P > 0.05).5. In comparison with control group and control-si RNA group,relative m RNAand protein level was decreased in cisplatin group, even lower in ATF3-si RNA group and lowest in ATF3-si RNA plus cisplatin group(P < 0.05), as for Maspin, it’s significantly higher in ATF3-si RNA group but lower in cisplatin group and lowest in ATF3-si RNA plus cisplatin group(P < 0.05).Part three: The inhibition effect of ATF3-si RNA on glioblastoma growth in nude mice.Method:1. Human glioblastoma xenograft model was established by subcutaneously injecting 2 × 106 / ml U373 MG cells in 0.2 ml PBS into nude mice. The mice were then randomly divided into 5 experimental groups: ① control group; ② control-si RNA group; ③ ATF3-si RNA group; ④ cisplatin treatment group; ⑤ ATF3-si RNA + cisplatin treatment group, 8 mice for each group. The mice were treated with corresponding reagents by peritumoral injection, once a day for 15 days. The status of nude mice and tumor growth were observed.2. Histologic characteristics of the glioma tumor before and after treatment were analyzed by Hematein Eosin(HE) staining.3. TUNEL assay was used to assess growth inhibition of the transplanted tumor after treatment.4. Protein expression of ATF3, Maspin and MMP2 of each group was analyzed by immunohistochemistry. And the expression level was compared between before and after treatment.5. Messenger RNA expression of ATF3, Maspin, and MMP2 was determined by real-time PCR, and relative expression levels of the three m RNAs were determined before and after treatment for graft glioblastoma.6. Protein expression of ATF3, Maspin, and MMP2 was determined by western blot, and relative expression levels of the three proteins were determined after treatment for graft glioblastoma.Results:1. Histological observation of nude mice tumors before and after treatment: tumor cells of the control group and control-si RNA group are bigger and vary in size, have higher proportion of nuclear chromatin and obvious atypia. while in the ATF3-si RNA group, cisplatin group and ATF3-si RNA plus cisplatin group the tumor cells are smaller, have lower proportion of nuclear chromatin, and punctate and flaky necrosis foci can be seen which increase in size among the three groups.2. TUNEL assay results: in comparison with control group and control-si RNA group, ATF3-si RNA group and cisplatin groups had significantly higher level of apoptosis(P < 0.05), and it’s more severe in ATF3-si RNA plus cisplatin group.3. In comparison with control group and control-si RNA group, relative m RNA and protein level was decreased in cisplatin group, even lower in ATF3-si RNA group and lowest in ATF3-si RNA plus cisplatin group(P < 0.05), as for Maspin, it’s significantly higher in ATF3-si RNA group but lower in cisplatin group and lowest in ATF3-si RNA plus cisplatin group(P < 0.05).Conclusion:1. ATF3 is highly expressed in human glioma of ATF3. Expression of ATF3, Maspin and MMP2 are related. High expression of ATF3 and MMP2 and low expression of Maspin are closely related to glioma progress.2. ATF3-si RNA can effectively reduce the U373 MG cell activity, inhibit cell proliferation, arrest cell cycle progression, promote apoptosis and ultimately prevent tumor invasion.3. ATF3-si RNA can effectively down-regulate ATF3 and MMP2 expression and up-regulated Maspin expression in U373 MG cells.4. ATF3-si RNA can inhibit glioblastoma in vitro and in vivo, and is an effective treatment.