LRIG3 And Soluble LRIG3 Ameliorate Tumorigenesis Of Glioblastoma Via Regulation Of MET/PI3K/Akt Pathway

PART ? Expression of LRIG3 protein in glioma tissues and its correlation with prognosis in glioma patientsObjective:To investigate the expression levels of LRIG3 protein in different WHO grade glioma tissue and to investigate the correlation of the LRIG3 expression level and the overall survival of the patients.Methods:Sixty-five paraffin-embedded glioma samples(from Tongji Hospital,Wuhan,China,20 cases of grade ?,17 cases of grade ?,28 cases of grade ?)were prepared for immunohistochemistry(IHC)staining with primary rabbit anti-LRIG3 antibody and then semi-quantitatively stratified and scored by percentage positivity of tumor cells(0–10% = 1,11–30% = 2,31–50% = 3,and 51–100% = 4)and staining intensity(none = 0,weak = 1;moderate = 2;intense = 3).The immunoreactivity score(IRS)for each case was determined by multiplying the vlues for staining intensity and the values for the percentage of immunoreactive tumor cells.The difference of LRIG3 expression levels among different grades was analyzed through one-way ANOVA.Samples with IRS of 0–3 were considered as low LRIG3 expression,while 4–12 were considered as high LRIG3 expression.Correlation analysis of LRIG3 expression levels in tumor tissues of HGG patients and overall survival time was performed using log-rank correlation test.Results:The expression levels of LRIG3 in high-grade gliomas(WHO grades ? and ?) were significantly lower than those in low-grade gliomas(WHO grade ?),and the difference was statistically significant(p = 0.026 and p = 0.00012).For HGG patients,higher LRIG3 expression levels were significantly associated with better overall survival.Conclusion:LRIG3 is down-regulated in high-grade gliomas compared with that in low-grade gliomas.LRIG3 expression levels were positively correlated with the prognosis of HGG patients.PART ? Identification of soluble LRIG3 and establishment of cell lines overexpressing LRIG3 or soluble LRIG3 proteinObjective: To investigate the existence of soluble LRIG3 protein and to establish glioblastoma cell lines overexpressing full-length LRIG3 and soluble LRIG3 extracellular domain protein for further investigation of the functions of LRIG3 and sLRIG3.Methods: The existence of soluble LRIG3 protein was identified by western blotting in the concentrated culture supernatants of glioma cell lines with inducible expression of LRIG3 and in the patients' serum and tumor cystic fluid.The expression vectors p LVX-Puro-3 × FLAG-LRIG3-full and p LVX-Puro-3 × FLAG-LRIG3-ecto were constructed according to the protocol in our laboratory.These two vectors and the empty control vector p LVX-Puro were stably transduced into GL15,U87 and patient-derived adherent Pri GBM glioblastoma cell lines using Lenti-X Lentiviral expression system according to manufacturer's instructions.These cells lines were selected with culture medium containing 1 ?g/ml puromycin for 1 week and 3 ?g/ml puromycin for another 2 weeks to get the stably transduced cell populations.Western blotting and in situ immunofluorescence staining were used to detect the expression of the target gene.Results: The existence of soluble LRIG3(sLRIG3)protein was detected in the culture supernatants of glioma cell lines overexpressing LRIG3,and in the patients' serum samples and tumor cystic fluid through western blot.As the LRIG3 antibody we used recognizes the ectodomain of the total protein and the molecular weight of sLRIG3 is about 110 k D,which is smaller than that of the full-length LRIG3 protein and in line with the predicted molecular weight of LRIG3 ectodomain,we conclude that the detected sLRIG3 is the ectodomain of the full-length LRIG3 protein.After the transduction of the expression vectors and the selection with puromycin,western blotting and in situ immunofluorescence staining confirmed the expression of the target genes.Conclusion: These results indicated the existence of sLRIG3 in the cell culture supernatants and in the patients' serum samples and tumor cystic fluid.Moreover,glioma cell lines stably expressing FLAG-tagged LRIG3 and LRIG3 ectodomain(sLRIG3)were successfully established.PART ? The effects of LRIG3 and sLRIG3 proteins on the biological characteristics of glioma cellsObjective: To investigate the effects of full-length LRIG3(LRIG3-full)and LRIG3 ectodomain(LRIG3-ecto,sLRIG3)proteins on proliferation,invasion and tumor formation of glioma cell lines in vitro and in vivo.Methods: The CCK-8 assay,cell-cycle assay,migration and invasion assay were carried out to detect the differences in the proliferation and invasion capacity.Soft-agar assay and subcutaneous tumor xenograft model in nude mice were carried out to determine the influence on tumorogenesis in vitro and in vivo.Western blotting was performed to assertain the possible mechanism and singnaling pathways for these phenomena.We further clarified the differential expression of the moleculars related the pathway through western blotting for clinical glioma tissue samples and through IHC for tumor xenografts.Results: Overexpression of LRIG3 or sLRIG3 inhibited the proliferation,cell cycle progression,soft agar colony formation,migration and invasion characteristics of glioblastoma cells(p < 0.05).Mechanistic studies confirmed that LRIG3 and sLRIG3 inhibited the activation of the MET and the downstream moleculars Akt and m TOR,but not ERK.For protein samples extracted from frozen glioma tissue,western blotting further confirmed that the phosphorylation levels of MET and Akt were inversely correlated with LRIG3 expression levels(p = 0.0144,r2 = 0.3378 and p = 0.0423,r2 = 0.2473,respectively).The subcutaneous xenografts tumors in the experimental groups overexpressing LRIG3 or sLRIG3 were smaller than that in the control group.Moreover,IHC staining of these tumors showed the phosphorylation levels of MET,Akt and proliferation marker Ki67 were all lower in the experimental groups than that in the control group.Conclusion: LRIG3 and sLRIG3 attenuated the proliferation,migration,invasion and tumorigenesis of glioma cells through the downregulation of MET/PI3K/Akt pathway both in vitro and in vivo.PART ? Serum sLRIG3 levels and the correlation with the prognosis of HGG patientsObjective: To investigate the correlation between the serum sLRIG3 expression levels and the tumor grades,and the correlation between the serum sLRIG3 levels and overall survival of high-grade glioma(HGG)patients.Methods: A total of 50 serum samples of glioma patients(6 grade I cases,11 grade ? cases,6 grade ? cases,27 grade ? cases)and 14 non-tumoral control samples were obtained.These samples subjected to enzyme-linked immunosorbent assay(ELISA)for quantitative analysis.Then the differences among different grade group and control group were analyzed using one-way ANOVA.For HGG samples,they were grouped according to their sLRIG3 levels: the high sLRIG3 group(sLRIG3-high)included samples with levels higher than the mean level of 3.9 ng/ml,whereas the rest of the samples were classified in the low sLRIG3 group(sLRIG3-low).Kaplan–Meier method was used to construct survival curves with a Log-rank test.Results: Serum sLRIG3 levels ranged from 0 to 21 ng/ml.Among non-tumoral controls and patients with grade I-? gliomas,the average level of sLRIG3 increased gradually.When compared grade ? group with the control group,the difference is statistically significant(p = 0.012),whereas the differences between the other groups were not statistically significant.Serum sLRIG3 levels showed no significant correlation with sex.There is significanct difference between HGGs and LGGs,and control group(HGG vs LGG,p = 0.048;HGG vs Control,p = 0.010).According to the threshold mean level 3.9 ng/ml,HGGs samples were grouped into sLRIG3-high group(13 cases)and sLRIG3-low group(20 cases)and survival analysis demonstrated that higher serum sLRIG3 levels were associated with better overall survival.Conclusion: There is no significant differences of serum sLRIG3 levels among different grade(grade I-?)glioma patients.Only in grade ? cases and HGG cases(grade ?+?),sLRIG3 levels were statistically different from that of the non-tumoral control group.For HGG patients,higher serum sLRIG3 levels were associated with better overall survival.