| Background and objectivePancreatic ductal adenocarcinoma(PDAC)is one of the most lethal solid malignancies,with 5 year survival rate of nearly 9%.Chemotherapy,radiotherapy and targeted therapy are still struggled with limited improvement of prognosis.Research of molecular mechanism of PDAC is likely to provide new treatment target of PDAC.Hematopoietic pre-B-cell leukemia transcription factor interacting protein(HPIP)is the co-repressor of pre-B-cell leukemia homeobox 1(PBX1),which is also known as a scaffold protein of cytoskeleton system.Recent years,HPIP was found acting as an oncogene in different kinds of malignant tumor.HPIP was reported to be associated with cell proliferation,cell cycle regulation,apoptosis,cell migration and invasion,chemoresistance and cell adhesion dynamics in several cancers.However,the role HPIP plays in PDAC remains unknown.The purpose of this research was to unravel the role of HPIP in the cell proliferation,cell cycle distribution,apoptosis and cell motility of PDAC cells.Methods1.HPIP expression in PDAC tissue was determined by immunohistochemistxy staining.2.Stable HPIP knockdown and overexpression cell lines were constructed in MIA PaCa-2 and BxPC-3 cell lines by lentivirus transfection and validated by western blotting.In vitro,cell proliferation was assessed using the cell coxmting kit-8 assay and colony formation assay.Cell cycle and cell apoptosis was assessed by flow cytometry analysis.Cell migration and invasion were assessed by Transwell migration and invasion assay.Expression level of cyclins,cleaved caspase 7,cleaved PARP,FAK/Src and EMT markers were assessed by western blotting.3.In vivo,nude mice xenograft model was used to investigate tumor gro,wth after knockdown of HPIP in MIA PaCa-2 cell lines.4.RNA sequencing was used to identify the change of mRNA expression profile in stable HPIP knockdown MIA PaCa-2 cell lines and control stable cell lines.KEGG analysis was used to identify signaling pathways highly related to HPIP.GO analysis was used to identify biological process,cellular component and molecular functions related to HPIP.Protein interaction network were constructed to identify core nodes of HPIP associated protein.Results1.HPIP was overexpressed in PD AC tissue compared with adjacent pancreatic tissue(P=0.031).HPIP overexpression was correlated with poor differentiation of PD AC(P=0.044).High HPIP expression is correlated with poorer prognosis in patient over 55 years old(P=0.028).2.Knockdown of HPIP inhibited cell proliferation and colony formation ability in MIA PaCa-2 and BxPC-3 cells(P<0.05).Overexpression of HPIP enhanced cell proliferation and colony formation ability in MIA PaCa-2 and BxPC-3 cells(P<0.05).Knockdown of HPIP decreased percentage of S phase cells(P=0.008;P<0.001)and increased percentage of G0/G1 phase cells(P<0.001;P=0.001)in MIA PaCa-2 and BxPC-3 cells.Overexpression of HPIP increased percentage of S phase cells(P=0.011;P<0.001)and decreased percentage of G0/G1 phase cells(P=0.034;P<0.001)in MIA PaCa-2 and BxPC-3 cells.Knockdown of HPIP increased overall apoptosis rate in MIA PaCa-2 and BxPC-3 cells(P<0.001;P=0.034).Overexpression of HPIP decreased overall apoptosis rate in MIA PaCa-2 and BxPC-3 cells(P=0.023;P=0.035).Overexpression of HPIP enhanced cell migration and invasion in MIA PaCa-2 and BxPC-3 cells(P<0.05).Knockdown of HPIP decreased cell migration and invasion in MIA PaCa-2 and BxPC-3 cells(P<0.05).Overexpression of HPIP lead to downregulation of p27 expression,upregulation of cyclin D1 expression,enhanced activity of Src.Overexpression of HPIP lead to downregulation of E-cadherin expression,upregulation of N-cadherin and Vimentin expression,enhanced activity of p-FAK.Knockdown of HPIP lead to upregulation of cleaved caspase 7 and cleaved PARP.3.In nude mice xenograft models,HPIP knockdown group showed a significant lower tumor volume(P<0.001)and tumor wet weight(P=0.002).4.RNA sequencing and KEGG analysis showed that the top 3 related signaling pathways were TGF-β,FOXO and Hippo signaling pathway.CDKN1B,MYC,CCND2,BCL2L1,TGFBR1,TGFBR2 and PPP2CA were core nodes in HPIP associated protein interaction network.GO analysis showed that the most related biological process was the cell response to laminar fluid shear stress,the most related molecular function was the regulation of transmembrane receptor kinase activity and the most related cell component was the p-catenin destruction complex.ConclusionsHPIP is overexpressed in PD AC tissue when compared with matched adjacent noncancerous tissues.High HPIP expression is correlated with low differentiation of PD AC.In PD AC patients over 55 years old,high HPIP expression is correlated with poor prognosis.HPIP enhances cell proliferation and colony formation ability.HPIP promotes G0/G1 to S cell cycle transition in PDAC cells.HPIP overexpression leads to upregulation of cyclin D1 and Src activity and downregulation of p27 expression.HPIP inhibits PDAC cell apoptosis,accompanied by overexpression of eleaved PARP and cleaved caspase 7.HPIP promotes cell migration,invasion and EMT process,accompanied by enhanced phosphorylation of FAK and enhanced activity of Src.HPIP was associated with TGF-P,FOXO and Hippo signaling pathway.CDKN1B,MYC,CCND2,BCL2L1,TGFBR1,TGFBR2 and PPP2CA were proposed as the core nodes in HPIP associated protein interaction network. |
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