The Role And Mechanism Of NOK Gene In The Proliferation And Invasion Of Human Glioma

Glioma is the most common malignant tumor in the centrol nervous system.Approximately40percent are diagnosed as glioma in intracranial tumors.Although the treatment for glioma make better, its treatment is still notsignificantly improved, and the patients still carry the worst prognosis with anaverage survival time of less than1year. Difficulties on treatment are closelyassociated with the malignant biology phenotype of glioma. In order to furtherimprove the treatment of glioma, it has important significance and clinicalapplication value to find new biomarkers for the diagnosis and treatment ofglioma.NOK gene belongs to a member of the RTK family, which was first identifiedand cloned from the tissue of human tonsil cancer in2004[Genebank registrationnumber AAT01226]. In present, The studies about NOK gene focus on itssignificance in the malignant biological behavior and targeted therapy. Themolecule of NOK is widely expressed in a variety of epithelial tissue in theregulation of cell proliferation, differentiation, anti-apoptosis, cell invasion, cell adhesion and other aspects. In addition, NOK is also significantly high expressedin a variety of non-nervous system malignant tissues, which implied that theNOK may play a key role in the process of tumor regulation. But there is noreport about the function of NOK gene in glioma.Here, we reported our detail study on expression characteristics of NOK genein the glioma, in order to reveal a new functional dimension of the gliomaexpressed NOK. To further, we also detected the fuction of NOK on theproliferation, migration, invasion and tumorigenesis of glioma cells by inducingNOK overexpression or inhibiting NOK expression via RNAi, which wouldhelp us to understand the role of NOK in the malignant progression of glioma.Our current study was performed from four aspects as follows.1. Significance of NOK expression in human gliomaThe expression of NOK mRNA and protein were investigated in eighty-threecases of human glioma tissue, ten cases of normal human brain tissue and threehuman glioma cell lines （U251, U87and BT325） by reversetranscription-polymerase chain reaction （RT-PCR） and Western blot respectively.The results showed that, NOK mRNA and protein were both higher expressed inU251and U87than in BT325cells. The expression intensity of NOK mRNA andprotein increased with the ascending of clinical pathologic grade of tumorspecimen, but no NOK mRNA and protein were expressed in9cases of humannormal brain tissues. Further, we detected the expression of NOK protein inhuman glioma tissues by immunohistochemistry. These results indicated thatNOK protein was mainly expressed in cytoplasm of tumor cells.57（68.7%）cases of tumor specimens expressed NOK protein, whereas no NOK proteinexpression was detected in9（10%） cases of normal human brain tissues. Therewas significant difference （P<0.01） between the positive expression rate of NOKprotein in glioma tissues and that in normal brain tissues. Furthermore, there werespecially significant differences （P<0.01） between the rate of NOK proteinpositive expression in benign brain glioma and those in malignant glioma. But thepositive expression rate of NOK protein was not associated whith gender, age, tumor size and location of patients.2. NOK lentiviral expression vector and RNAi vector transfecting glioma cellsin vitroAccording to the sequence of NOK cDNA, we designed the primers andobtained the full-length of NOK cDNA from the vector of pcDNA3.1-NOK byPCR, then NOK cDNA was cloned into the vector of entry clone （pENTR/3C） ofGateway system. Next, NOK cDNA was cloned into lentiviral expression vector（pLenti-6.3/V5） from the vector of pcDNA3.1-NOK, which was namedpLenti-6.3/V5-NOK. The vectors （pLenti-6.3/V5-NOK） were identified by PCR,enzyme digestion and sequencing assay.3x105HEK-293T cells at70%confluence were transfected with1μg pLenti-6.3/V5-NOK vector and750ngpsPAX2and250ng pMD2.G using Lipofectamine2000.12h after transfection, themedium was changed. Cells were cultured for an additional36h before thelentivirus was harvested. The lentivirus was transfected into BT325cells, thencell lines were selected by blasticidin, and the successfully established cell lineswere named as BT325-NOK and BT325-cherry. The expression of NOK mRNAand protein in BT325-NOK and BT325-cherry cells were investigated byRT-PCR and Western blot. The results showed that NOK mRNA and proteinexpression in BT325-NOK cells was markedly up-regulated compared to itsexpression in BT325-cherry cells.The lentivirus system for short, hairpin RNA （shRNA） expression includedthree plasmids （pLKO.1, psPAX2and pMD2.G）. pLKO.1lentiviral nontargetingshRNA clone and NOK targeting shRNA clones（pLKO.1-NOK-1:NM₀18423.x-365s1c1; pLKO.1-NOK-2: NM₀18423.x-900s1c1） werepurchased from Sigma. We also packaged virus with the same method. ThenU251cell lines was transfected and selected by puromycin. The successfullyestablished cell lines were named as U251-sh1, U251-sh2and U251-control.PCR and western blot showed NOK mRNA and protein expression in U251-sh1and U251-sh2cells were both significantly inhibited, as compared with that ofU251-control cells. Based on the above results, lentiviral expression vector of NOK wassuccessfully constructed. NOK lentiviral expression vector and RNAi vectorwere transfected into glioma cells successfully, and in glioma cell lines, NOKmRNA and protein were upregulated or inhibited stablely. So, the successfullyestablished cell lines may be excellent cell models, which could be used to studythe effects of NOK expression in the process of regulating glioma biologicfeatures.3. Effects of upregulated or inhibited NOK expression on the proliferation,migration and invasion in glioma cellsFirstly, we observed the effect of NOK expression on the cell growth,proliferation and cell cycle. The cell growth curve drawn by MTT showed thatU251-sh1and U251-sh2cells grew slower than U251-control significantly（P<0.05）, but BT325-NOK cells grew faster than BT325-cherry cells markedly（P<0.01）. The results of Soft-Agar Assay and plate colony formation showed thatthe colony number of U251-sh1and U251-sh2cells both markedly decreasedthan that of U251-control cells （P<0.01）, but the colony number of BT325-NOKcells was notably more than that of BT325-cherry cells （P<0.01）.The cell cycle analysis by flow cytometry （FCM） showed that both inU251-sh1and U251-sh2cells, the G0/G1phase cells increased significantly, butG2/M phase cells decreased markedly, as compared with those in U251-controlcells. Whereas in BT325-NOK cells, G0/G1phase cells decreased obviously andS phase cells significantly increased. Secondly, the monolayer wound healingassay and transwell invasion assay were used to detected the invasiveness of cells.The results showed that among U251-sh1and U251-sh2cells, the cells migrationand the number of the invaded cells both declined significantly with contrast ofcontrol group（P<0.01）, whereas no obvious difference in cell migration and thenumber of invaded cells between U251-sh1and U251-sh2cells （P＞0.05）. Ascompared with those of BT325-cherry cells, the cells migration and the numberof the invaded cells in BT325-NOKcells increased notably（P<0.01）.The above results suggested that the NOK gene was closely related with the growth, proliferation, migration and invasion in human gliomas.4. Impact on tumorigenesis, proliferation and invasion of human glioma cellsin nude mice by RNAi targeting NOK geneU251-control, U251-sh1å'ŒU251-sh2cells were inoculated respectively inflank subcutaneous tissue of nude mice to establish xenograft models of humanbrain glioma. Tumor volume and tumor growth were used to observed the tumorgrowth status. The tumor weight was investigated30days after inoculation. Theresults showed that in U251-sh1å'ŒU251-sh2groups, the tumorigenesis timedelayed, tumor grew slow, both tumor volume and tumor weight decreasedsignificantly （P<0.01） as compared with U251-control group. In addition, NOKprotein expression was both inhibited in tumor tissues from U251-sh1å'ŒU251-sh2groups（P<0.01） as compared with U251-control group.In summary, NOK gene may paly an important role in initiation and progressof human glioma. NOK is not only a valuable biomarker of brain gliomas, butalso a promising candidate target for gene therapy in malignant gliomas.