Expression and biological functions of PIK3R3 in growth hormone pituitary neuroendocrine tumors
Objective To investigate the expression of phosphoinositide 3 kinase regulatory subunit 3(PIK3R3)in growth hormone pituitary neuroendocrine tumors(GH-PitNETs)and its relationship with PitNET cell proliferation,migration and invasion.Methods The expression of PIK3R3 in GH-PitNETs was analyzed using the PitNETs dataset in Gene Expression Omnibus(GEO).Using cell lines derived from different PitNETs,the expression of PIK3R3 in GH3,MMQ and AtT-20 cells was detected by Western blot.Lentiviral infection was used to knockdown the PIK3R3 gene in the GH3 cells,and the cells were divided into PIK3R3 knocked down 2 group(Sh-PIK3R3-2),PIK3R3 knocked down 3 group(Sh-PIK3R3-2)and untransfected control group(Sh-NC).Cell proliferation was detected by CCK-8,EdU and colony formation assay.Apoptosis was detected by flow cytometry.Cell migration and invasion were detected by Transwell assay.The expression of epithelial cadherin(E-cadherin),neural cadherin(N-cadherin),Vimentin,matrix metalloproteinase-2(MMP2),matrix metalloproteinase-9(MMP9),AKT and phosphory-lated AKT(P-AKT)were detected by Western blot.Results GEO database analysis showed that PIK3R3 expression was upregulated in GH-PitNETs compared to normal pituitary or paraneoplastic tissues(all P＜0.05),whereas there was no statistically significant difference in prolactin-type PitNETs,adrenocorticotropic hormone-type PitNETs,and gonadotropin PitNETs(all P＞0.05).Western blot showed that PIK3R3 was highly expressed in GH3 cells compared to MMQ and AtT-20 cells(both P＜0.05).CCK-8 assay showed that the absorbance of the cells in the Sh-PIK3R3-2 and Sh-PIK3R3-3 groups were 0.19±0.02,0.46±0.03,1.25±0.06 and 0.17±0.02,0.37±0.02,1.03±0.05 at 24 h,48 h and 72 h,respectively,which were all lower than the control group's 0.24±0.02,0.64±0.04,1.67±0.09(all P＜0.05).EdU assay showed that the rates of EdU-positive cells in the Sh-PIK3R3-2 and Sh-PIK3R3-3 groups were(16.87±1.63)％and(13.45±1.33)％,respectively,which were all lower than the control group's(34.51±2.76)％(all P＜0.05).Colony formation assay showed that the number of cell colonies in the Sh-PIK3R3-2 and Sh-PIK3R3-3 groups were 84±10 and 75±8 respectively,which were lower than that in the control group(173±19)(all P＜0.05).Flow cytometry assay showed that the apoptosis rates of the Sh-PIK3R3-2 and Sh-PIK3R3-3 groups were(25.62±2.14)％and(27.40±2.51)％respectively,which were all higher than the control group's(14.12±1.48)％(all P＜0.05).Transwell migration assay showed that the numbers of migrating cells in the Sh-PIK3R3-2 and Sh-PIK3R3-3 groups were 182±15 and 166±13 respectively,both lower than the control group's(428±27)(all P＜0.05).Transwell invasion assay showed that the numbers of invading cells in the Sh-PIK3R3-2 and Sh-PIK3R3-3 groups were 31±5 and 22±3 respectively,both lower than the control group's(64±9)(all P＜0.05).Western blot assay showed that compared with the control group,both PIK3R3 knockdown groups showed decreased expression of N-cadherin,Vimentin,MMP2,MMP9,and P-AKT(all P＜0.05),increased expression of E-cadherin(all P＜0.05),and no significant change in AKT expression(all P＞0.05).Conclusion PIK3R3 is highly expressed in GH-PitNETs and promotes cell proliferation,migration,and invasion by activating AKT signaling transduction.
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