The Expression And Significance Of Homer, Shank Gene Association With Brain Injury And Glioma

This study includes two parts.First part: The expression and significance of Homer and Shank gene in brain injuryBrain injury, which mainly included traumatic brain injury (TBI) and ischemia brain injury, has long been a major cause of disability and death to society. The pathphyciological progression of TBI maybe includes neural toxicity of glutamate, overloading of Ca2+, inflammation and obstruction of blood-cerebrospinal fluid barrier and so on, however, the assured developmental mechanism concerning the path physiology of TBI has been unclear. Our previous study indicated that a immediately early gene(IEG), Homer, can regulates many cell-molecule and cell signal pathways, and participate in the pivotal process of the developmental mechanism of TBI in mechanically injured model of cortical neurons of rat in vitro. In this study, we examined the expression and significance of Homer after brain injury in neurons in vivo (3 kinds of brain injury models were produced: diffuse axonal injury (DAI) was produced by accelerated lateral head rotation model; diffuse brain injury (DBI) was manufactured by acceletation-deceletation model of DBI by Marmarou et al; and ischemia-reperfusion injury was made by middle cerebral artery occlusion model). The present study may provide experimental basis for the mechanism and correlated therapy of brain injury. PartⅠExpression and significance of Homer after DAI in vivoObjective: To study the variational regulation of expression and significance of Homer after DAI in vivo. Methods: 120 young-adult male Sprague-Dawley rats were randomly assigned into normal control (n=10), sham-operated control (n=10), and DAI group (n=80). The DAI group was subdivided so that different time points (30min, 1h, 3h, 6h, 12h, 24h, 48h and 72h) post-injury could be studied. Accelerated lateral head rotation was used to produce DAI in rats in this study. And the rats'brainstem, cortex and hippocampus were detected for Homer by immunohistochemistry, Western blot and quantitative real-time RT-PCR after DAI. Results:①Immunohistochemical analysis showed that there was negative-expression of Homer1a in normal control and sham-operated control group. And Homer1a was increased significantly from 30 min to 72 hours after DAI, peaked at 24h (P<0.01). The positive particles were distributed in the neuronal cytoplasm, cyto-membrane and dendrites. No change of Homer-1b/c expression was found before and after injury of neurons;②Compared to the normal control and sham-operated control samples, the levels of Homer1a mRNA and protein expressed in samples obtained from DAI rats at 30min, and were strongly up-regulated from 1h to 72h after DAI, peaked at 24 h (P < 0.01), and remained at a high level until 72 h (P<0.05);③Homer1b/c could be observed and was constitutively expressed at similar levels in neurons of all three groups. There was no significant statistical difference in the intensity of Homer1b/c protein band among normal control, sham-operated control and DAI group (P>0.05);④In all three groups, the expression of Homer1a was significantly higher in brainstem than in cortex and hippocampus (P<0.05), during the early part of the post-injury period (30min-24 h). However, the significance of this difference was lost during the latter part of the post-injury period (48 h-72 h, P>0.05). Meanwhile, there was no difference of the expression of Homer1a between cortex and hippocampus (P>0.05). The expression of Homer1b/c was nearly the same in these anatomic sites (P>0.05). Conclusions: It was verified for the first time that DAI could induce Homer1a gene expression, which may be due to the excitement of neurons as a result of glutamic acid release after injury. Expression of Homer1b/c was not modulated by the neuronal exciting activity. Dynamically expressed Homer1a and constitutively expressed Homer1b/c might further modulate the distribution and function of mGluR1a. Potential mechanism might be that Homer1a, which competes with Homer1b/c to couple to mGluR1a, regulates the distribution of receptor at synapse and the efficiency of signal transduction, prevents neurons from excess excitement. Homer1a functions as a natural dominant negative to selectively regulate association of mGluR1a with Homer1b/c after neuronal excitement increased. In addition, the results which there was a significantly difference of the expression of Homer1a in different anatomic sites was indicated that in the DAI, the injured degree of axons in brainstem were more severe than that in cortex and hippocampus, because it more approached the midline of brain.Part II Expression and significance of Homer after DBI in vivoObjective: To study the variational regulation of expression and significance of Homer after DBI in vivo. Methods: 120 young-adult male Sprague-Dawley rats were randomly assigned into normal control (n=10), sham-operated control (n=10), and DBI group (n=80). The DBI group was subdivided so that different time points (30min, 1h, 3h, 6h, 12h, 24h, 48h and 72h) post-injury could be studied. DBI in rats was manufactured by acceletation-deceletation model of DBI by Marmarou et al in this study. The content and method of examine were the same as that of in partⅠ. Results:①the results of immummunohistochemical analysis were the same as that of in partⅠ. And Homer1a expressed in glia cell after DBI, also.②Compared with normal control and sham-operated control samples, there are two peak values of mRNA and protein of Homer1a following DBI, increased in 3h (P<0.01) and in 24h (P<0.01), then decreased in 72h(P<0.05)with high value.③The results of expression of Homer1b/c were the same as that of in partⅠ.④In all three groups, the expression of Homer1a was significantly higher in cortex and hippocampus than in brainstem (P<0.05), during the early part of the post-injury period (30min-24 h). However, the significance of this difference was lost during the latter part of the post-injury period (48 h-72 h, P>0.05). Meanwhile, there was no difference of the expression of Homer1a between cortex and hippocampus (P>0.05). Moreover, the expression of Homer1b/c was similar in the different anatomic sites (P>0.05). Conclusions: The expression of Homer1a reached two peak values, the mechanism of the first peak value maybe related with Homer1a competes with Homer1b/c to couple to mGluR1a, reduced the level of mGluR1a at synapse and Ca2+ influx; And the mechanism of the second higher value might connecte with Homer1a can induces apoptosis of neurons and glia, these indicated that overexpression of Homer1a might protect injured brain. Moreover, the expression of Homer1a was higher in neurons of cortex and hippocampus than in brainstem, which is different from the previous results of partⅠ, these phenomena might connect with injured models was different, the injured degree of neurons in cortex and hippocampus was severe and the mechanism of TBI was diverse.Part III Expression and significance of Homer after ischemic- reperfusion injury in vivoObjective: To study the variational regulation of expression and significance of Homer associated with ischemic-reperfusion injury in vivo. Methods: 120 young-adult male Sprague-Dawley rats were randomly assigned into normal control (n=10), sham-operated control (n=10), and ischemic-reperfusion injury group (n=80). The ischemic-reperfusion injury group was subdivided so that different time points (30min, 1h, 3h, 6h, 12h, 24h, 48h and 72h) post-injury could be studied. The ischemia-reperfusion injury of rat was made by middle cerebral artery occlusion model. The content and method of examine were the same as that of in partⅠ. Results:①The results of immunohistochemical analysis were the same as that of in partⅡ.②C ompared normal control and sham-operated control samples, there are three peak values of mRNA and protein of Homer1a following ischemia-reperfusion injury, increased in 30min (P<0.01), in 3h (P<0.01) and in 24h (P<0.01), and decreased in 72h(P<0.05)with high value.③T he results of expression of Homer1b/c were the same as that of in partⅠ. Conclusions: First, the expression of Homer1a reached three peak values, and peaked at 30min firstly, which maybe relate with both the ischemia injury and reperfusion injury stimulate jointly Homer1a expression, and overexpression Homer1a competes with Homer1b/c to couple to mGluR1a"timely", induced the injured degree of neuron, there was no the first peak value after DBI. The mechanism of other two peak value of Homer1a expression connected with Homer1a maybe regulates the distribution of mGluRs and induces pathological apoptosis of neurons and glia. Second, the characteristic spatio-temporal of expression of Homer1a after ischemia- reperfusion injury was found, which might because of the mechanism of ischemia-reperfusion injury was different from that of DAI and DBI, and maybe added and more complex cell signal pathways were involved in the pathophysiology of ischemia-reperfusion injury.PartⅣExpression and significance of isoforms of Shank after brain injury in vivoShank, a scaffold protein in the post-synaptic density (PSD), which including 3 subtypes, Shank1, Shank2 and Shank3, has multi-functional regions, link and interact with many different classes of proteins, distribute extensively in the nervous system, is believed to play an important role in the synaptic construction and function. Some previous studies suggest that Shank may be involved in synaptic plasticity and, by extension, learning and memory through modulate the signal transduction of Homer and mGluRs. However, the space-time transformation law of isoforms of Shank protein associate with brain injury in vivo, and the relationship between Shank and brain injury are still unknown.Objective: In this study, we examined the expression and significance of Homer after brain injury in rat in vivo (3 kinds of brain injury models above-mentioned). We examine the expression of isoforms of Shank, and find the"key"Shank molecule. This study may provide experimental basis for the mechanism and correlated therapy of brain injury. Methods: The rats'cortex was detected for Shank by immunohistochemistry, Western blot and quantitative real-time RT-PCR after brain injury (3 kind's models of brain injury) which mentioned in above study. Results:①Immunohistochemical analysis showed that no change of isoforms of Shank-Shank1, Shank2, Shank3 expression were found before and after brain injury of neurons; The positive particles were distributed in the neuronal cytoplasm, cyto-membrane and dendrites.②The results of protein expression and mRNA levels of Shank which examined by Western-blot and quantitative real-time RT-PCR were consistent after DAI. Shank1 could be observed and was constitutively expressed at similar levels in neurons of all three groups (P>0.05); Shank2 could be observed in neurons of all three groups, however, compared to normal control and sham-operated control samples, the levels of Shank2 mRNA and protein expressed in samples obtained from DAI rats at 30min, and were strongly up-regulated from 1h to 72h after DAI, and peaked at 6 h and 72 h (P < 0.01); Shank3 could be observed in neurons of all three groups, strongly rose from 30min to 6h after DAI (P < 0.01), then decreased, but remained the high level until 72 h (P < 0.05).③the results of protein expression and mRNA levels of Shank which examined by Western-blot and quantitative real-time RT-PCR were consistent after DBI. Except that Shank2 reached the peak from 3 h to 6 h (P < 0.01), other results were coincident with the expression of isoforms of Shank after DAI.④The results of protein expression and mRNA levels of Shank which examined by Western-blot and quantitative real-time RT-PCR were consistent after ischemia- reperfusion injury. Shank1 could be observed in neurons of all three groups, and compared to normal control and sham-operated control samples, the levels of Shank2 mRNA and protein expressed rose at 30min after injury, and reached to the peak at 3 h and 12 h (P < 0.01), then decreased, but remained the high level until 72 h (P < 0.05); Shank2 could be observed and was constitutively expressed at similar levels in neurons of all three groups (P>0.05); Shank3 could be observed in neurons of all three groups, and the levels of Shank3 increased from 30min after ischemia-reperfusion injury (P<0.05), peaked at 3h, then decreased markedly from 12 h to 72 h (P>0.05); The variational trends of isoforms of Shank were different obviously among ischemia- reperfusion injury, DAI and DBI. Conclusions: We firstly study the expression and function of isoforms of Shank after brain injury in vivo. Firstly, isoforms of Shank all expressed in brain cortex before and after brain injury and the expression levels of Shank changed remarkable proved that Shank maybe play important role in keep and management of the excitement of neurons, and every isoforms of Shank binded with different signal molecules, participated in different signal pathways and regulated the function of neurons together; Secondly, injury induce Shank2 and Shank3 gene expression associated with DAI and DBI, which may be due to the lots of release of glutamic acid after injury, and Shank combined with dynamically expressed Homer1a to affect the efficiency of signal transduction and the excitement of neurons, meanwhile, expression of Shank1 was not modulated by the neuronal exciting activity, these indicated that dynamically expressed Shank2 and Shank3 and constitutively expressed Shank1 might further modulate the distribution and function of Homer, mGluRs and other signal molecules; Thirdly, there were various different between the expression of isoforms of Shank after ischemia-reperfusion injury and that of associated with traumatic brain injury, which suggested suggested that different kinds of brain injury has various developmental mechanism respective, and complex signal pathway play different effects in pathphyciological progression of brain injury; In conclusion, differently variational regulation expression of isoforms of Shank in different types of brain injury prompted that Shank may be become a potential target of therapy and diagnosis of brain injury. Second part: The expression, function and mechanism of Homer gene in human brain gliomaBrain glioma is the most common malignancy of central nervous system, which occupies almost half of all intracranial tumors. The postoperative median survival is less than one year for patients with malignant brain glioma. The main reason for difficulties on treatment is associated closely with the malignant biology phenotype of brain glioma, which including excessive proliferation, antiapoptosis and abundant angiogenesis. So, it has important science significance and clinical application value to identify the key genes involved in the malignant biology behavior of this malignancy for overcoming it. Combined literature and our pre-experiment'results, we found Homer play important role in antiapoptosis and angiogenesis of brain glioma, which suggested there is close relationship between Homer and human tumor. At present, the research on the relationship between Homer and the pathological mechanism of brain glioma is lack, and the role of Homer in malignant proliferation, antiapoptosis and angiogenesis of brain glioma is not completely clear. For this, the current study was performed from three aspects as follows.PartⅠExpression and significance of Homer in human brain gliomaObjective: To investigate the expression and significance of Homer in human brain glioma. Methods: Three human brain glioma cell lines (U251, U87 and SHG-44), 60 cases of human brain glioma tissue and 5 cases of normal human brain tissue were investigated by immunohistochemistry method for protein expression of Homer. Results:①Homer1a protein was highly expressed in U87 and SHG-44 cells, and low expression of Homer1a protein in U251 cells, meanwhile, there was no expression of Homer1b/c in 3 human brain glioma cell lines. It was 61.8%, 4.35±3.99 and 1.2%, 0.09±0.35 respectively that the positive expression percent and immunoreactivity score (IRS) of Homer1a in brain glioma tissues and normal brain tissues, there were especially significant difference (P<0.01 for both) respectively in the positive expression percent and IRS of Homer1a between them. There were'U' relationship between the positive expression percent and IRS of Homer1a and type pathological grades of glioma, there were specially significant difference (P<0.01 for both) between gradeⅠand gradeⅡ,Ⅲ, and between gradeⅣand gradeⅡ,Ⅲ, also, there were significant difference (P<0.05 for both) between gradeⅠand gradeⅣ, and between gradeⅡand gradeⅢ. In addition, there was specially significant difference in the positive expression percent and IRS of Homer1a between benign and malignant brain glioma. Moreover, there were no significant difference (P>0.05 for all) respectively in the positive expression percent and IRS of Homer1b/c between benign and malignant brain glioma and among different pathological grades of glioma.②The results of Western blot and quantitative real-time RT-PCR were the same as that of immunohistochemistry. Conclusions: The results suggested that, Homer1a not Homer1b/c protein be expressed in brain glioma, and which may associate closely with initiation, progress, malignant proliferation, antiapoptosis and angiogenesis of brain glioma.PartⅡConstruction and identification of human Homer1 shRNAObjective: To construct the eukaryotic expression vector pGCsi-H1 of Homer1 shRNA and transfected human brain glioblastoma U87 cells. Methods: According to Homer1 cDNA sequence, the specific RNAi fragments targeting Homer1 were designed and synthesized, which were cloned into pGCsi vector, and the eukaryotic expression vector pGCsi-H1 of Homer1 shRNA was constructed. The pGCsi-H1 vector, blank pGCsi vector and pGCsi-NC were transient transfected respectively into human brain glioblastoma U87 cells by lipofectin medium, and temporarily called them: U87-H1,U87-P and U87-NC. Results: Quantitative real-time RT-PCR results showed that mRNA expression of Homer1 was inhibited markedly, while the results of Western blot indicated that protein expression of Homer1a was also suppressed significantly in U87-H1 cells. Conclusions: The Homer1 shRNA can suppress significantly expression of mRNA and proteins of Homer1, which offers a basis for study of biological significance of glioma with lack of Homer1a.PartⅢConstruction of recombinant plasmid pEGFP-N1- Homer1-GFP in humanObjective: To clone human Homer1 cDNA and construct eukaryotic expression vector carrying human Homer1 gene. Methods: The cDNA encoding the human brain glioma Homer1 was isolated by using RT-PCR method with total RNA extracted from human brain glioma. Homer1 gene was cloned into eukaryotic expression vector pEGFP-N1 of EGFP reported gene encoding green fluorescence protein in the form of fusion protein. The expression vector of recombinant plasmid pEGFP-N1-Homer1-GFP is successfully constructed. Results: Automatic DNA sequence analysis demonstrated that the sequence of the recombinant plasmid pEGFP-N1-Homer1-GFP was in accordance with that published in GenBank. Conclusions: pEGFP-N1-Homer1-GFPis constructed successfully, which may provide a novel and important means for gene therapy of glioma.