CD164 Regulates Proliferation And Apoptosis By Targeting PTEN In Human Glioma

Glioma is derived from glial cell deterioration, and is one of the most common type of primary intraeranial tumors. According to the WHO classification system, it account for the vast majority of adult malignant brain tumors that are graded, which has implicated with the prognosis and management of patients. Moreover, as for glioma is a highly malignant tumor and shows invasion and metastasis during its develepment, a majority of patients succumb to the disease within 2 years of diagnosis. The current standard therapy, including maximal safe resection followed by radiotherapy or in combination with chemotherapeutics, such as temozolomide and/or radiotherapy. Recent years, great progress have been made in surgery, radiotherapy and chemotherapy, and many studies have verified that those treatments could prolonged the survival time of some patients with high grade glioma, whereas the overall prognosis of patients with high grade glioma have no improved. Moreover, the malignant degree of glioma increases after recurrence. Together, human brain glioma was still one of the difficult dealt diseases in the neurosurgical tumor. Nowadays, along with the advance in the study of the cellular immunology and molecular etiology of gliomas, more and more novel therapeutic strategies, such as immunological therapy and genetic treatment, have emerged.CD 164 is a glycoprotein and a member of the sialomucin family, which is a mucin that contains sialic acid. CD164 was first found as a carrier of a peanut agglutinin-binding site, which is a tumor-associated carbohydrate marker expressed in human gastric carcinoma cells and bone marrow stromal reticular cells. Studies have shown that CD 164 can modulate the proliferation, adhesion and migration in hematopoietic stem and progenitor cells. It has been reported that CD 164 regulates hematopoiesis by facilitating the adhesion of human CD34+cells to bone marrow stroma. In addition to regulating normal growth and differentiation, one relatively newer but exciting dimension is the effect of CD 164 on malignant cell proliferation as well as invasion. CD 164 has been implicated in the maintenance as well as progression of several human malignant tumors, including medulloblastoma, ovarian and colon cancers. An earlier study showed that CD 164 may participate in the mediation of prostate cancer bone metastasis. Recently, CD164 has been recognized as a biomarker for the detection of acute lymphoblastic leukemia and allergy. These studies indicated that CD 164 may act as a key molecule in the modulation of tumor progression. However, the relationship between CD 164 and human glioma is yet to be learned. In a Preliminary study we used U87 human glioma cells, after culture using stem cells with normal U87 cell lines used for whole genome sequencing analysis. At the same time, gene sequencing analysis was done on 3 patients with glioblastoma tumor tissue and surrounding normal brain tissue. We compared and analyzed the genetic difference between the 2 groups and verified by using qPCR technology. We found that glioma is relatively normal brain tissue and the cancer stem cells are relatively ordinary U87 cells with a high expression of CD164. In this paper, we explored the expression profiles of CD 164 in glioma cells and the correlation between CD164 and tumorigenesis processes of glioma, including proliferation and apoptosis.1. Detect the expression of CD 164 in 50 cases of human glioma tissues and thecorrelation with clinical pathogenic charactersObjective:To explore the expression of CD 164 in 50 cases of human glioma specimens as well as several glioma cell lines.Methods:The primary glioma tissues and the adjacent brain tissues from patients undergoing decompression with traumatic brain injury were obtained from fifty glioma patients (24 men and 26 women) at the Department of Neurosurgery of the First Afficated hospital of Wenzhou Medical University in China between 2013 and 2015 were included in this study. The age of the patients ranged from 46 to 73 with a mean of 63 ± 5 years. All the 50 glioma patients gave written consent to participate in the study. No patients had received chemotherapy or radiotherapy prior to surgery. Other exclusion criteria included acute or chronic infection, dysphagia, congestive heart failure, COPD, gastrectomy and cirrhosis. According to the classification standard of central nervous system tumors of WHO in 2007. All cases were histologically confirmed as glioma by trained pathologists. There were 6 cases I grade specimen,8 cases Ⅱ and 36 cases Ⅲ, Ⅳ grade respectively were chosen in my cohort. RT-PCR was used to detect the level of CD 164 in all the human glioma tissues. Furthermore, the relative expression levels of CD 164 in glioma cell lines U251, SHG-44 and U87, along with NHA were examined by RT-PCR and western blot respectively.Results:The protein level of CD 164 was detected by found to be up-regulated in 50 cases of glioma specimens in comparison with non-tumor brain tissues tissues. The glioma tissues exhibited higher expression of CD164 mRNA (t=-18.128, P< 0.001) compared with non-tumor brain tissues tissues. Western blot analysis showed that the protein level of CD 164 was barely detectable in NHA. It also demonstrated that such level in U251, SHG-44 and U87 was significantly higher than in NHA. Furthermore, the changes in the CD 164 mRNA level revealed by RT-PCR analysis in normal and malignant cells were similar to the results of western blot. As the expression of CD 164 in U87 cells was markedly over-expressed in contrast to U251 as well as SHG-44, U87 cells was chosen for the subsequent experiments.Conclusions:A Significantly increased expression of CD 164 was observed in glioma patients and three glioma cell lines.2. Construction of the virus expression carrier targeting CD164 gene and the effects of shCD164 on the cell proliferation and apoptosis of glioma cells in vitro.Objective:Construct CD164 shRNA to disturb the lentiviral vector, and establish stable infection of silencing of CD 164 to U87 cells, and explore the effects of CD 164 shRNA on the cell proliferation and apoptosis of U87 cells.Methods:Constructed the tumor-specific lentivirus mediated shRNA targeting CD 164 gene and acquired CD 164 knockdown U87 cells. Firstly, the mRNA and protein levels of CD 164 in U87 cells were analyzed by RT-PCR along with western blot analysis. Secondly, the effects of CD 164 shRNA on the proliferation of U87 cells were evaluated by CCK-8 assay and annexin V assay. Subsequently, the activation of apoptosis was investigated by measuring cleavage caspase 3, Bax and Bcl2 in U87-shCD164 cells using western blot.Results:1. The lentiviral vector of CD 164 shRNA was constructed successfully by restriction endonuclease and DNA sequencing analyzing. The transduction of CD 164 shRNA resulted in a considerable decrease in the levels of CD 164 mRNA and protein compared with that of negative control group cells.2. Functional studies demonstrated that the proliferation of U87 cells was significantly inhibited by (45.8±7.5)% after 72 h of transfection with CD164 shRNA in comparison with the negative controls (P<0.01). Additionally, the apoptosis detection results demonstrated clearly that the loss of CD164 increased the percentages of both early and late apoptotic/necrotic cells in U87 cells.3. The knock-down of CD164 expression caused the proteolytic cleavage of caspase 3, as revealed by the appearance of the cleaved protein species. Moreover, our data demonstrated that the silencing of CD164 resulted in a decrease in Bcl2 and a significant increased level of Bax.Conclusions:The CD164 shRNA expression vectors were successfully constructed. The knockdown of CD164 inhibited the proliferation and promoted the apoptosis of human glioma cell lines U87 in vitro by mediated the apoptosis associated proteins, such as caspse 3, Bax and Bcl2.3. Inhibitory effect of CD164 silencing lentivirus vector on the growth and apoptosis of subcutaneous human glioma in nude mice.Objective:Xenograft tumor model was performed to compare the tumorigenesis of U87 cells with or without CD164 shRNA transfection, and to observe the effects of CD164 shRNA on the growth and apoptosis of glioma transplantation tumor in nude mice to establish the subcutaneous xenograft tumor model.Methods:1.30 female nude mice were randomized into three groups:U87 group, U87-shNC group and U87-shCD164 group. The stable knockdown of CD164 cell line, negative control cell line and glioma cell line U87 were injected subcutaneously into the flank of nude mice respectively. The volume of tumors from three groups was measured respectively. After 56 days of observation, the mice were sacrificed whilst the tumors were surgically excised neatly and weighed.2. The positive expression of Ki-67 in xenograft tumors was examined by immunohistochemistry. RT-RCR was used to evaluate the expression of CD164 mRNA in tumors.3. Cell apoptosis in tumor tissues was assessed by TUNEL assay and calculated the apoptotic index (AI) from three groups.Results:1. The growth rate of xenograft tumor was significantly reduced in U87-shCD164 group. At the termination of experiment, the final tumor weights showed a significant decrease in the U87-shCD164 group compared with BC or NC groups (> 50% reduction, n= 10, P< 0.001). In addition, the tumor weight in BC group was not as statistically significant as the NC group.2. The positive immunohistochemical staining for Ki-67 was presented in all of the xenograft tumors. However, the percentage of Ki-67 positive stained cells was markedly lower in U87-shCD164 xenograft tumors tissues in comparison with blank or negative controls. Furthermore, RT-PCR results demonstrated that the level of CD164 mRNA was markedly decreased in the U87-shCD164 group in comparison with BC or NC groups.3. Moreover, TUNEL staining of these tissue sections indicated significant differences in the percentage of TUNEL-positive cells in the tumors derived from the U87-shCD164 group compared with the controls.Conclusions:1. Lentivirus mediated CD164 shRNA effectively decrease the level of CD164 in xenografts tumors, and inhibits the growth of subcutaneous xenografts tumor of glioma in nude mice in vivo.2. The underlying mechanism of treatment of Lentivirus mediated CD 164 shRNA in glioma xenograft tumor may by the means of inducing the cell apoptosis.3. Lentivirus mediated gene therapy is a relatively safe and effective treatment. In this way, silencing CD 164 expression could become a novel therapeutic strategy for glioma prevention and treatment in the future.4. To explored the mechanisms involved in the anti-proliferative and apoptosis induction efficacy of lentivirus mediated CD164 shRNA on U87 cells.Objective:To investigate whether the regulation of PTEN/PI3K/AKT survival pathway is involved in the CD 164 shRNA induced anti-proliferation and apoptosis activity in U87 cells.Methods:1. The expression levels of PTEN, p-AKT and p53 were investigated using western blot.2. RT-RCR was used to evaluated the expression of PTEN mRNA in U87 cells after infection of shCD 164.3. Western blot was employed to examined the levels of PTEN and p53 in xenograft tumors.Results:1. The western blot result indicated that the down-regulation of CD 164 increased the protein level of PTEN, while the p-AKT and p53 levels were decreased and increased respectively owing to the effects of CD 164 shRNA transduction.2. Moreover, further investigation was conducted on whether CD 164 shRNA increases PTEN at transcription level in U87 cells. The expression of PTEN mRNA was increased by 6.2 fold after being transfected with CD 164 shRNA.3. Western blot analysis revealed a significant increase in the expression of PTEN in the tumors from the U87-shCD164 mice compared with those from the control groups. Additionally, there was also a dramatic increase in the p53 expression in U87-shCD164 xenograft tumors tissues compared with the controls.Conclusions:These data showed that the tumorigenic efficacy of CD 164 in the progression of glioma may be associated with the signaling pathway of PTEN/PI3K/AKT. Lentivirus mediated CD 164 shRNA effectively decrease the level of CD 164 in xenografts tumors, and inhibits the growth of subcutaneous xenografts tumor of glioma in nude mice in vivo.1. The underlying mechanism of treatment of Lentivirus mediated CD 164 shRNA in glioma xenograft tumor may by the means of inducing the cell apoptosis.2. Lentivirus mediated gene therapy is a relatively safe and effective treatment. In this way, silencing CD 164 expression could become a novel therapeutic strategy for glioma prevention and treatment in the future.In conclusion, the expression level of CD 164 was related to pathological grade and the prognosis of glioma, and the increased expression of CD 164 was regarded as a indicator of highly malignant of glioma. In this study, our results showed for the first time that the expression of CD 164 is significantly correlated with the proliferation and apoptosis of human glioma cells through up-regulation of PTEN at transcription as well as translation levels. The over-expression of PTEN, in turn, enhanced p53 protein expression and inhibited the PI3K/AKT pathway, thereby inhibiting cell proliferation in U87 cells both in vitro and in vivo. This work provided further evidence for the role of the CD164 in the functional regulation of the glioma. In this way, silencing CD 164 expression could become a novel therapeutic strategy for glioma prevention and treatment in the future.