The Research Of Effects And Mechanism Of GREM1 On The Phenotype And Cell Function Of Glioma

Objective: Glioma is the most common and lethal intracranial malignant tumor,and the effect of traditional therapy is not as well as people expected,especially to those types with higher malignancy.GREM1 is a glycoprotein that contributes to maintain the level of tumors.Previous studies have reported that GREM1 can be secreted by glioma stem cells.The overexpression of GREM1 can promote the proliferation and tumorigenic ability of glioma stem cells,while interference the expression of GREM1 can inhibit its proliferation and self-renewing ability.GREM1 has been reported to promote epithelial mesenchymal transdifferentiation by activing the downstream TGF-β1/Smad signaling pathways.Studies have confirmed that EMT plays an important role in cancer occurrence,invasive and metastatic thus EMT has become a hotspot at present.This study intend to transfect human glioma cells with RNA interference recombinant plasmids and to discuss its effect on glioma proliferation and metastasis.The further exploration is carried out to confirm the effect of TGF-β1/Smad pathway on glioma occurrence,invasive and metastatic caused by GREM1.MethodP: 1.30 cases of glioma patients who confirmed by histopathologic findings or clinical follow-up were collected,and patients’ medical information was recorded.Immunohistochemistry was used to detect the expression of GREM1 in pathological samples and analyzed the correlation between basic information such as age,cancer grade,sex with the level of GREM1.2.(1)Interference fragments and negative fragments that targeted GREM1 were synthesized and constructed into plasmids respectively and then vectors were used to transfect U87.G418 screening was used to obtain the stabilized interfere and control cells.Real-time PCR and Western blot were used to verify the efficiency of GREM1 gene knockout.(2)Cell proliferation was examined by MTT assay,cell cycle was detected by flow cytometry,clone formation test was used to detect oncogenicity in vitro.These experiments were carried out to verify the influence of GREM1 silence on proliferation of U87.(3)Flow cytometry was used to detect the cell apoptosis through AnnexinV/PI double-stained.Hoechst staining was used to observe the changes of nucleolus of apoptosis cell.The expression of cleaved-caspase-3,cleaved-PARP,bcl-2,bax was examined by Western blot to explore the effect of GREM1 silence on the apoptosis of U87.(4)Migration rate was measured by wound healing assay(0 h,24 h,48 h).Invasive ability was detected by Transwel chamber(Matrigel).The expression of MMP2 and MMP9 was detected by Western blot and Gelatin zymography to discuss the impact of GREM1 silence on the migration and invasion of U87.3.(1)The protein expression level of E-cadherin and N-cadherin was examined by Western blot to analyze the influence of GREM1 silence on the EMT level of glioma cell U87.(2)Western blot was used to detect the levels of TGF-β1,Smad2,p-Smad2,Smad3,p-Smad3 and BMP-7.Immunofluorescence was used to test the nuclear translocation of smad2/3 so that to confirm the inhibitory effect of GREM1 silence on the activation of TGF-β1/smad pathway.(3)The expression of p-Smad2,p-Smad3,E-cadherin,N-cadherin was detected by Western blot in U87 with 10 ng/ml TGF-β1 treatment for 48 h(Group: U87,NC,GREM1 silence,GREM1 silence+TGF-β1,U87+TGF-β1,NC+TGF-β1),and the Transwel chamber(Matrigel)was used to measure the invasion of U87,and then to analyze the influence of the activation of smad pathway induced by TGF-β1 on the invasion and EMT of glioma cell U87.Result: 1.The GREM1 protein expression of 30 cancer patients examined with immunohistochemical method showed that all of the 30 samples exist positive expression.Analysis using the rank-sum test showed that the expression of GREM1 had no significant relativity with sex and age,but significant relativity was found between GREM1 and clinical stage.The GREM1 protein expression level was considerably higher in malignant gliomas than those in low grade glioma(P<0.05).2.(1)MTT assay showed that cell proliferation was obviously decreased at 72 h,96 h and 120 h in GREM1 silence group(P<0.05).Cloning efficiency in GREM1 silence group(17.8±3.0%)was significant lower than other groups(40.8±6.0% in U87 group and 37.3±5.6% in NC group,P<0.05)(2)The number of cells invading through the Matrigel of invasion chamber in GREM1 silence group(52.60±5.86)was remarkable reduced than other groups(82.00±9.80 in U87 group and 80.40±10.24 in NC group,P<0.05)in Transwell assay.The wound healing assay indicated that the cell migration in GREM1 silence group was lower at 24 h(17.08±2.78)and 48 h(40.34±5.66)than other groups(29.61±2.73,65.84±7.20 in U87 group and 31.87±3.12,68.92±5.24 in NC group,P<0.05)(3)Western blot showed that the expression of cleaved-caspase-3,cleaved-PARP and bax was obviously increased in GREM1 silence group than U87 group and NC group(P<0.05),while the expression of bcl-2,MMP2,MMP9 decreased than the other two groups(P<0.05).(4)Flow cytometry revealed that more cells stop in the period of G1 in GREM1 silence group than the others(U87 group and NC group,P<0.05).The apoptosis rate of GREM1 silence group cells was more than the others(P<0.05).Hoechst staining showed the number of apoptosis cells was higher than U87 group and NC group(P<0.05).3.(1)The expression of E-cadherin in GREM1 silence group was significantly upregulated than U87 group and NC group(P<0.05),while the level of N-cadherin in GREM1 silence group was much lower(P<0.05).(2)The expression of BMP-7 detected by Western blot in GREM1 silence group was higher than U87 group and NC group(P<0.05),while TGF-β1,p-Smad2 and p-Smad3 showed an obvious decrease than the other two groups(P<0.05).But the expression of Smad2 and Smad3 have no significance among these groups.The nuclear translocation of smad2/3 identified by immunofluorescence analysis showed that the nuclear translocation in GREM1 silence group was inhibited than U87 group and NC group(P<0.05).(3)With 10 ng/ml TGF-β1 treatment for 48 h,the expression of p-Smad2,p-Smad3,N-cadherin in GREM1 silence+TGF-β1 group showed obviously increased than GREM1 silence group(P<0.05),while the expression of E-cadherin decreased(P<0.05).There was no statistical significance compaired with U87 group and NC group.In Transwell assay,the number of cells invading through the Matrigel of invasion chamber in GREM1 silence+TGF-β1 group was higher than GREM1 silence group(P<0.05),even so the number ia smaller than U87 group and NC group.Conclusion: The GREM1 protein expression of 30 cancer patients examined with immunohistochemical method showed that GREM1 was common expressed in patients with glioma,but negatively expressed in most normal brain tissues and spinal cord tissues.It was proved by statistics that the expression of GREM1 was correlated with glioma malignant degree.The GREM1 protein expression level was considerably higher in malignant gliomas(Ⅲ、Ⅳ grade)than those in low grade glioma but with no significant relativity with sex and age.Inhibition of GREM1 in U87 cell could significantly decrease the proliferation,invasiveness and metastasis,promote cell apoptosis.The results revealed that GREM1 itself may promote cell proliferation,invasiveness and metastasis,and inhibit cell apoptosis.This study also found that the inhibition of GREM1 can suppress the EMT of glioma cells.EMT plays an important role in invasion and metastasis of malignancies.Our study confirmed that GREM1 can active TGF-β1/Smad pathway in glioma cells by using classical TGF-β1/Smad pathway activation assay.The presumption is that GREM1 can antagonize BMP pathway and active TGF-β1/Smad pathway simultaneously,so that it can promote EMT process,and thus promote the proliferation,invasiveness and metastasis of glioma cells,inhibit cell apoptosis.