COL6A6 Mediated PI3K/Akt Signaling Pathway Inhibits The Proliferation And Invasion Of AtT20 And HP75 Cells In Pituitary Adenoma

Objectives:Pituitary adenoma(PA)is a common and frequently-occurring disease in skull base neurosurgery.45-55%of PA has obvious invasiveness because of over proliferation.It often invades important tissue structures such as cavernous sinus,sphenoid sinus and internal carotid artery,which is difficult to deal with clinically.At present,there are few reliable biomarkers that can predict the occurrence and progression of pituitary adenomas.The purpose of this study is to explore the expression of COL6A6 and the possible regulatory pathways and mechanism of PA malignant biological behaviors such as proliferation,invasion of pituitary adenoma,which will help to further clarify the pathogenesis of PA and find new molecular targets for establishing possible intervention methods in the future.Methods:1)Based on GEO biological database,the microarray of pituitary adenoma GSE26966 was used for data analysis,through gene differentiation analysis and gene enrichment analysis,the potential target gene COL6A6 and its closely related gene P4HA3 were screened for further research.From June 2017 to March 2018,30 clinical specimens of pituitary adenomas and 15 effective paratumoral tissues were collected from patients with pituitary adenomas treated by Neurosurgery,the First Affiliated Hospital of Kunming Medical University.RT-qPCR and immunohistochemical techniques were used to analyze the difference of target gene expression between pituitary adenomas and paratumoral tissues.In addition,the basic clinical data of 30 cases were collected.The correlation between COL6A6 expression and clinical characteristics were analyzed.Also,the correlation between Knosp classification of 30 cases and surgical total(near total)resection rate and operation related complications was evaluated.2)At the same time,pituitary adenoma AtT-20 and HP75 cell lines were subcultured in vitro and transfected with target gene plasmid.The two cell lines were divided into three groups:NC(Negative Control,NC group,COL6A6 group(pc-DNA-COL6A6 plasmid transfection group)and silent group(si-COL6A6 plasmid transfection group)The expression of COL6A6 was analyzed by Western blotting;the proliferation ability of tumor cells was evaluated by CCK-8 adsorption;the invasion ability of tumor cells was detected by Transwell invasion test;the migration ability of tumor cells was evaluated by wound healing test;meanwhile,the detection of apoptosis by flow cytometry,the expression of Ncadherin,Ecadherin and Vimentin protein was detected by Western blot to evaluate the ability of epithelial mesenchymal transformation in each group.In addition,we also established the subcutaneous tumor model of ectopic pituitary adenoma in nude mice with AtT-20 and HP75 cell lines,and divided them into NC group and COL6A6 group(transfected with pc-DNA-COL6A6 plasmid).The tumor growth size was measured dynamically,and the mice were killed 4 weeks after tumor growth in the model,and the changes of tumor volume and weight in each group were compared.Western blot analysis of the difference of COL6A6 expression,Ncadherin,Ecadherin and Vimentin protein expression between the two groups;immunohistochemical detection of Ki67.3)In addition,the molecular mechanism of action to COL6A6 on PI3K/Akt signal pathway was further evaluated.In vitro cell culture,HP75 and AtT cell lines were transfected with blank plasmid and pcDNA-COL6A6 plasmid and divided into four groups:NC group(blank plasmid transfection group),COL6A6 group(pcDNA-COL6A6 transfection group),COL6A6+P4HA3 group and COL6A6+IGF-1 group.The protein expressions of COL6A6,P4HA3,PI3K/Akt and p-PI3K(p85)/p-Akt(ser473)were detected by Westernblotting to evaluate the relationship between PI3K/Akt signal pathway and COL6A6 and P4HA3.At the same time,CCK-8 adsorption,Transwell invasion test and wound healing test were used to further evaluate the proliferation,invasion and migration of tumor cells in each group.Finally,Westernblotting was used again to detect the expression of Ncadherin,Ecadherin and Vimentin protein in each group to evaluate the ability of epithelial mesenchymal transformation.In addition,we also established the subcutaneous tumor model of ectopic pituitary adenoma in nude mice with AtT-20 and HP75 cell lines,and divided them into NC group,COL6A6 group(transfected with pc-DNA-COL6A6 plasmid)and COL6A6+IGF-1 group.The growth size of tumor was measured dynamically,and the mice were killed 4 weeks after tumor growth in vivo,and the changes of tumor volume and weight in each group were compared.Results:1)Gene analysis based on GSE26966 microarray of pituitary adenomas showed that there were 20307 genes expressed,and 1403 genes were differentially expressed between pituitary adenomas and normal pituitary,of which 588 genes were up-regulated and 815 were down-regulated;COL6A6 was significantly different and the expression of P4HA3 was the lowest in pituitary adenomas;gene enrichment analysis showed that PI3K/Akt signal pathway was significantly activated and P4HA3 expression was up-regulated;COL6A6 is closely related to P4HA3 in protein Action Network.RT-qPCR analysis of clinical pituitary tumor samples showed that COL6A6 expression was down regulated(1.40±0.98,2.73 ± 1.06,P<0.001)and P4HA3 expression was up regulated(4.41 ± 1.34,2.02 ± 0.75,P<0.001)in PA tissue compared with that in paratumoral tissue.The expression of COL6A6 was negatively correlated with PA tumor size(P=0.035)and Knosp grade(P=0.013),but not with age(P=0.464),gender(P=0.491)and tumor subtype(P=0.984).The knosp grade(3-4)of 30 PA was significantly lower than that of grade(1-2)patients(9/19,10/11,respectly.P=0.020).And Knosp grading was not associated with surgery-related complications in this study.2)The results of cell experiment in vitro showed that the expression of COL6A6 was significantly decreased and the expression of P4HA3 was up-regulated in PA cell line PA cells.Compared with NC group,the expression of COL6A6 protein in COL6A6 group/si-COL6A6 group increased/decreased significantly(P<0.001),and the ability of cell proliferation was inhibited/enhanced(P<0.001).Compared with NC group,COL6A6 group/si-COL6A6 group down-regulated/up-regulated the expression of N-cadherin and Vimentin,up-regulated/down-regulated the expression of E-cadherin(P<0.01),promote/reduce apoptosis(P<0.05),respectively.In the nude mouse model of ectopic pituitary adenoma,compared with NC group,the expression of COL6A6 protein in pc-COL6A6 group was significantly increased(P<0.01);the growth of tumor size,volume(P<0.01)and tumor weight(P<0.001)in AtT-20 and HP75 was significantly inhibited,respectively;meanwhile,the expression of N-cadherin and vimentin in tumor tissue in pc-COL6A6 group was significantly decreased(P<0.01),respectively.And the expression of Ki-67 in pc-COL6A6 group was decreased.3)In the evaluation of COL6A6 and PI3K/Akt signal pathway,the results of cell experiment in vitro are as follows:in COL6A6 group,the expression of P4HA3 protein,p-PI3K(P85)and p-Akt(ser473)were significantly decreased,the ratio of p-PI3K/PI3K and p-Akt/Akt was significantly different from that of NC group,the expression of p-PI3K and p-Akt was reversed and up-regulated,and the ratio of p-PI3K/PI3K and p-Akt/Akt in COL6A6+P4HA3 group was significantly higher than that in COL6A6 group(P<0.05).After COL6A6+IGF-1,p-PI3K and p-Akt increased significantly,and the ratio of p-PI3K/PI3K and p-Akt/Akt was significantly different from that of COL6A6 group(P<0.01).At the same time,there was co-immunoprecipitation between COL6A6 and P4HA3.Compared with COL6A6 group,COL6A6+P4HA3 group and COL6A6+IGF-1 group significantly promoted the ability of cell proliferation,invasion and migration,but there was no statistical significance between these two groups and NC group(blank matter transfection group).Compared with COL6A6 group,the expressions of N-cadherin and Vimentin in COL6A6+P4HA3 group and COL6A6+IGF-1 group were significantly up-regulated,while the expression of E-cadherin was down-regulated(P<0.01),but there was no statistical significance between the two groups with the NC group.In the model of ectopic pituitary adenoma of nude mice:compared with COL6A6 group,the size,volume and weight of tumor in COL6A6+IGF-1 group were significantly increased(P<0.001),but there was no statistical significance compared with NC group.Conclusion:(1)The expression of COL6A6 in PA is lower than that in normal pituitary tissues or para-tumoral tissues while the expression of P4HA3 is high in PA.(2)COL6A6 was negatively correlated with Knosp grade of PA,and Knosp grade was negatively correlated with total(near total)resection rate.(3)Up-regulation of COL6A6 can negatively regulate the proliferation,invasion and migration of pituitary adenoma AtT-20 and HP75 cells in vitro and in vivo.(4)Up-regulation of COL6A6 can negatively regulate the expression of P4HA3 in PA AtT-20 and HP75 cell lines,and further negatively regulate the expression of Ser473,thus blocking the activation of PI3K/Akt Signal pathway,thus inhibiting the proliferation of AtT-20 and HP75 cells in vitro and in vivo.(5)P4HA3 can reverse the inhibitory effect of COL6A6 on pituitary adenoma AtT-20 and HP75 cells.(6)There is a close relationship between COL6A6 and P4HA3,and there is an interaction between them.(7)COL6A6 maybe plays a role of "tumor suppressor gene" in PA,which can provide a new potential molecular target for the establishment of PA prediction and intervention in the future.