Study On Mechanisms Of Glioma Invasion By Stable Knocking Down PLCγ1 And PKCα

Glioblastoma is one of the most common brain tumors,accounting for 40 to 50 percent.The treatments of surgery,radiotherapy and chemotherapy do not have a good prognosis.One year fatality of glioblastoma is about 77 percent after the definite diagnosis.It has become well accepted that solid tumors must create a vascular system for nutrient delivery and waste removal in order to grow appreciably. This process,angiogenesis,is critical to the progression of gliomas,with vascular changes accompanying the advancement of these tumors.The cascade of events in this process of blood vessel formation involves a complex interplay between tumor cells,endothelial cells,and their surrounding basement membranes in which enzymatic degradation of surrounding ground substance and subsequent endothelial cell migration,proliferation,and tube formation occurs.It is likely that a host of growth factors is responsible for mediating these key events.These growth factors may influence glioma angiogenesis by directly stimulating endothelial cell proliferation,by mediating the expression of key proteases on endothelial cells necessary for angiogenesis,or by regulating the expression of vascular endothelial growth factor(VEGF) and of each other.Epidermal growth factor receptor(EGFR) is involved in the regulation of cell growth through apoptosis,proliferation and differentiation,and also modulates the migratory behavior of cells.Joshua et al demonstrated that glioma cells responded to Epidermal growth factor(EGF) with an increase in intra- and intercellular[Ca²⁺]_i,and sustaining for 10 minutes.The forward effect can be antagonism by Phospholipase Cγ1(PLCγ1) specific inhibitor U-73122.This consequence demonstrated that the increasing of[Ca²⁺]_i in glioma cells depended on PLCγ1 transdution pathway.Oscillatory Ca²⁺ signaling has been associated with both cell migration and cell proliferation.And tumor invasion and migration have been also associated with cell migration and proliferation.Therefore, all of these concluded that PLCγ1 transdution pathway could affect tumor invasion and migration.PLCγ1 is known to be activated in response to growth factor stimulation by a mechanism that relies on tyrosine phosphorylation and plays an important role in regulating cell proliferation and differentiation.PLCγ1 was found increased in many cancer tissues and cancer cell lines,also associated with tumor progression.It was reported that PLCγ1 may function as a downstream modulator of EGF and a key molecular switch in glioblastoma motility.Nevertheless,the signaling mechanisms of PLCγ1 in cancer invasion and metastasis are remain uncertain.PLC can catalyze phosphatidylinositol 4,5-bisphosphate(PIP₂) and results in the generation of two intracellular messengers,inositol 1,4,5-trisphosphate(IP₃) and diacylglycerol(DAG),which then activated protein kinase C(PKC) and subsequent signaling cascade.PKC family consists of at least twelve serine-threonine kinases which are classified into three major groups.PKC isoenzymes have been shown to display variable expression profiles during cancer progression depending on a particular cancer type.They were previously shown to affect tumor progression and malignant phenotype.It was reported that PKCαhad high expression in glioblastoma and mediated its migration and invasion.While the signal transduction mechanisms of it are remain unknown.Nuclear factor-kappaB(NF-κB) proteins are predominantly cytoplasmic, associating with members of the inhibitory IκB family in resting cells.NF-κB will translocate to nucleus,combining withκB motif and regulate cell transcription if the cells are undergoing activator of NF-κB.Activation of NF-κB and other transcription factors involved in the innate immune/inflammatory response can upregulate the expression of many genes,the products of which promote and support the malignant phenotype.In a word,NF-κB can promote cell proliferation,regulate cell apoptosis, promote angiogenesis,tumor invasion and migration through inducing expeession of many kinds of genes.And tumors have tolerance to radiotherapy and chemotherapy due to the activation of NF-κB with anomaly construct.So far,the mechanisms of NF-κB activation are still unclear in tumor cells.Our previous research has suggested that PLCγ1 can regulate cell motion and cell migration in high metastatic colorectal cancer cells Lovo via NF-κB. Accumulating research suggested that PKC family was critically involved in NF-κB activation in response to extracellular stimuli.In lymphocyte B,BCR can regulate its proliferation and survival through a cascade of reactions involving PLCγ2-PKC-CARMA1-NF-κB.Additionally,these inflammatory responses occurred through a cascade of reactions involving TNFR-associated factors(TRAFs), PLC,phosphatidylinositol-3- kinase(PI3K),protein kinase C(PKCαand PKCζ), and reactive oxygen species(ROS),leading to NF-κB activation.All of those hints that some interaction among PLC,PKC and NF-κB may be exist.Glioblastoma U-87MG cell line was used in this study to investigate the relationship among PLCγ1,PKCαand NF-κB.Using molecular biology technology, cell biology technology and RNA interference,the biological behaviors of glioblastoma cells are investigated,which can reveal the mechanisms of glioblastoma invasion and migration and provide theory basis for the gene therapy of glioblastoma. Firstly Oligonucleotides which can transcribe short hair RNAs(shRNAs) specific for PLCγ1 and PKCαwere synthesized,the oligonucleotides were annealed and then ligated into pSUPER.retro vector via the two special ends.Then the expression construct vectors were transfected into the U-87MG cells with liposome.U-87MG cells were transfected with each vector construct,stable transfected cells were selected by complete culture medium containing the appropriate concentration of puromycin.The protein expressions of PLCγ1 or PKCαin stable cell lines were examined by Western bloting.The relationship among PLCγ1,PKCαand NF-κB in glioblastoma was revealed through Electrophoretic Mobility Shift Assay(EMSA).The main results and conclusions are as follows:1.Recombinant eukaryotic expression plasmids pSUPER.retro/PLCγ1-shRNA-construct and pSUPER.retro/PKCα-shRNA-construct were constructed successfully. The construct plasmids were indentified by restriction endonuclease analysis and DNA sequencing.Then the recombinant expression vectors were transfected into the U-87MG cells with liposome.U-87MG cells were transduced with each construct vector,stably transduced cells were selected by complete culture medium containing the appropriate amount of puromycin.Two kinds of selected puromycin-resistant colonies were respectively named as:U-87/pSUPER.retro/PLCγ1-shRNA-construct (to abbreviate U-87 ^PLCγ1-) and U-87/pSUPER.retro/PKCα-shRNA-construct (to abbreviate U-87^PKCα-).Puromycin-resistant colonies were picked and each clone was expanded to assay for knockdown of the target gene by Western blot. This laid sound basis for the following research of PLCγ1 and PKCα.2.Glioblastoma U-87MG cell line has high expression of PKCα.Western blot was used in this investigation to prove the high expression of PKCαin U-87MG cell line, and using NIH3T3 cell line as positive control.3.Activity of NF-κB is partly PLCγ1-dependent in response to EGF in glioblastoma.EMSA assay concluded that NF-κB was hardly existence in nucleus while U-87MG cells were in resting.The level of NF-κB translocating to nucleus of U-87MG starts to increase 15 minutes after EGF stimulation,peaked 60 minutes, which could be successfully reversed in U-87 ^PLCγ1- cells.EGF can mediate the nuclear translocation of NF-κB in U-87MG partly through the signal transduction of PLCγ1.4.Activity of NF-κB is partly PKCα-dependent in response to phorbol 12-myristate 13-acetate(PMA) in glioblastoma.The level of NF-κB translocating to nucleus of U-87MG starts to increase 15 minutes after PMA stimulation,peaked 60 minutes,which could be successfully reversed in U-87 ^PKCα-cells.And we proved that PKCαhas high expression in U-87MG previously,therefore we can conclude that PMA can mediate the nuclear translocation of NF-κB in U-87MG partly through the signal transduction of PKCα.Taken together,all of the results indicate that EGF can mediate the nuclear translocation of NF-κB in U-87MG through PLCγ1-PKCα.thereby regulating the transcription of invasion and metastasis related genes,thus paving the road for improving therapeutic approaches.