The Study Of Biological Behaviors And Mechanisms Of KLF2 In Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma(PDAC)is a common and lethal malignant tumor worldwide,with complex genomic environment and rare effective treatment.It is very necessary to explore novel bio-moleculars and their underlying molecular mechanisms to develop novel strategies to treat PDAC.KLF2(Kruppel-like factor 2)belongs to the Kruppel-like factor family who has been considered as the tumor suppressors for their inhibitory effects on several malignant tumor cells'proliferation.However,the relationship between KLF2 and pancreatic cancer is unclear.Based on the former studies,we speculate that KLF2 might participate in the abnormal pathways combining with other factors to inhibit pancreatic cancer grow.To explore possible biological functions of KLF2 in PDAC,we test expression of KLF2 in pancreatic ductal cancer patients'tumor and paired normal tissues,then observe the functions of KLF2 in PDAC pancreatic cells growth,migration,senescence,and cancer organoid growth.To clarify the molecular mechanism,we explore the role of KLF2 on beta-catenin/TCF signaling and senescence with other moleculars in the two pathways.This study provides a novel explanation for the suppressive roles of KLF2 in pancreatic cancer.Part I The study of biological behaviors of KLF2 in pancreatic cancer cellsObjectiveTo explore the biological behaviors of KLF2 to pancreatic cell growth,migration,senescence,and stem cell growth.MethodsKLF2 was investigated in 52 matched patients'PDAC tissues and histologically normal tissues using the immunohistochemistry staining,real-time PCR analysis and western blot analysis to detect the protein and m RNA levels of KLF2 to clarify the relationship of KLF2 and PDAC.The KLF2 expression vector and empty pc DNA3.1 were transfected into BXPC3 and Suit2 cells using Lipofectamine 2000 reagent following the manufacturer's instructions making those cells over-expression of KLF2.On the contrary,RNAi lenti-virus particles(si RNA con and si KLF2)were incubated with the lenti-virus particles then selected with the medium containing puromycin for low-expression of KLF2.Boyden chamber assay was used to investigate the effects of KLF2 over-expression or low-expression on the migration ability of BXPC3 and Suit2 cells,respectively.Then crystal violet assay were used to investigate the effects of KLF2 over-expression or low-expression on the proliferation ability of the two kinds of cells,respectively.Furthermore,KLF2 control plasmids(p LVX)or KLF2-expressing plasmids(Flag-KLF2)were transfected into HPAC and SW1990 cells with Lipofectamine 2000 reagent following the manufacturer's instructions for over-expression of KLF2.By contrary,RNAi lenti-virus particles(sh RNA con and sh KLF2)were incubated with the lenti-virus particles then selected with the medium containing puromycin.The following stable over-expression or low-expression of KLF2 was confirmed with Western Blot.Senescence-associated SA-?-galactosidase staining was used to detect the ratio of senescence of KLF2 level changes in HPAC and SW1990 cells.In addition,we knocked down the expression of KLF2 in BXPC3-Luci cells,labeled with the luciferase gene(BXPC3-Luci),then injected into the nude mice heart to further observe the growth and distant metastasis of BXPC3cells monitored by an in vivo image system.Similarly,SW1990 cells forced to over-expression of KLF2 were injected subcutaneously to nude mice to further observe the local growth and metastasis monitored and calculated by tumor size and weight after being sentenced to death.Furthermore,we built pancreatic cancer organoids using PDX-Cre Kras^G12D(KC)mice pancreatic tissues,then transfected those organoids to over-expression KLF2 pairing with control vector.We assessed KLF2 level on organoids growth through photographing the size and number.ResultsKLF2 m RNA level was significantly decreased in cancerous tissues,and positive staining of KLF2 was less than normal pancreatic tissues.In addition,the protein level of KLF2 in PDAC tissues was also down-regulated.In vitro,knocking down the expression of KLF2 promoted the colony formation as well as the migrate ability of BXPC3 and Suit2cells.Similarly,cell migration ability showed great increasing of BXPC3 and Suit2 cells low-expression KLF2 compared with that of control cells.Proliferation ability was up-regulated of the survival of the BXPC3 and Suit2 cells following the down-expression of KLF2.HPAC and SW1990 cells inhibited to low-expression of KLF2 were dramatically having less SA-?-gal staining cells.Pancreatic cancer cells BXPC3 with low-expression KLF2 had more metastasis tumor in vivo.On the contrary,cell migration ability was deceased of BXPC3 and Suit2 cells with over-expression KLF2 compared with that of control cells.Proliferation ability was also down regulated of the BXPC3 and Suit2 cells when forced over-expression of KLF2.HPAC and SW1990 cells forced to express of KLF2 were dramatically having more percentage of SA-?-gal staining cells.Pancreatic cancer cells SW1990 over-expression KLF2 had smaller size and lower weights tumor tissue after being injected tumor cells subcutaneously in vivo.Smaller organoids were observed when expression of KLF2 was forced in vitro.ConclusionKLF2 is decreased in pancreatic cancer compared with matched normal tissues.In vitro,over-expression of KLF2 could inhibit the growth,invasive,and migration abilities of pancreatic cancer cells in vitro and vivo,while inducing senescence.Low-expression of KLF2 could induce the growth,invasive,and migration abilities of pancreatic cancer cells in vitro and vivo,while inducing senescence in vivo.Knockdown of KLF2 gene could inhibit the stem cell growth.Part II The study of mechanisms of KLF2 in inhibiting pancreatic cancer cellsObjectiveTo explore the mechanisms of KLF2 inhihibiting the pancreatic cells.MethodsWe performed a screening using reporter gene assay with Topflash reporter induced by lithium(Li Cl)to investigate the underlying molecular mechanisms.Western Blot and q PCR were utilized to examine the expression level of p21 protein and m RNA in control HPAC and SW1990 cells with KLF2 over-expression and low-expression cells.The binding protein of KLF2 was identified using mass spectrometry after immunoprecipitation.The GST-KLF2 fusion protein was purified and incubated with the cell lysates of SW1990cells.Then GST pull-down assay was demonstrated to explore the interaction between GST-KLF2 and endogenously expressed FOXO4,and exogenously expressed FOXO4.We next excluded the probability that KLF2 regulated the expression of FOXO4 and vice versa in former study.The cooperative effects of KLF2 and FOXO4 on the expression of p21were examined using Western blot.Ch IP assay was performed to map the domain on KLF2for FOXO4 binding on the activation domain(AD)and the ID(inhibitory domain)and ZF(zinc finger)domain,respectively.FOXO4 and p21 were knocked down in the KLF2-stable HPAC cells to confirm the biological functional relevance between FOXO4and KLF2.Then SA-?-galactosidase staining was performed to positive cells percentage and colony formations were counted and photographed by a microscope to count numbers on soft agar.ResultsOver-expression of KLF2 inhibited the activation of Topflash reporter induced by lithium(Li Cl),indicating the negative regulation of beta-catenin/TCF transcription activity by KLF2.Over-expression of KLF2 in BXPC3 and Suit2 cells down-regulated the expression of beta-catenin/TCF target genes,such as c-Jun,c-Myc,cyclin D1,and Snail.HPAC and SW1990 cells forced KLF2 over-expression were found higher senescence regulators p21 both of the protein and m RNA levels.Furthermore,HPAC and SW1990cells knocked down KLF2 expressed less SA-?-gal staining and senescent inhibited the expression of p21.The GST pull-down assay demonstrated the interaction between GST-KLF2 and endogenously expressed FOXO4 in SW1990 cells.In addition,exogenously expressed FOXO4 and KLF2 in SW1990 cells can form a complex.Immunoprecipitation using the cell lysates from SW1990 cells revealed the interaction between endogenous KLF2 and FOXO4.Most importantly,FOXO4 and KLF2 cooperated to directly induce the protein p21 by binding with the p21 promoter and consistently cooperated to induce cell senescence.Ch IP assay mapped showed that the AD of KLF2mediated the interaction between KLF2 and FOXO4.Consistently,the ID and ZF domains could not cooperate with FOXO4 to induce p21 expression.Moreover,co-expression of KLF2 ID and ZF domains with FOXO4 seemed to abrogate the effect of FOXO4 alone on p21 induction.Down-regulation of FOXO4 or p21 effectively abolished cell senescence in HPAC cells with KLF2 over-expression.Knockdown of FOXO4 or p21 could effectively reverse the inhibition of anchorage-independent growth on soft agar.These results indicated that KLF2 induced senescence through FOXO4 and p21.ConclusionKLF2 interacts with beta-catenin directly or negatively regulates the beta-catenin/TCF downstream signaling genes expressing to suppress Wnt/beta-catenin pathway.KLF2 can also inhibit the tumorigenesis of pancreatic cancer through inducing expression of p21directly or combinding with FOXO4 inducing p21 expression,then lead to the senescence of PDAC cells.The GST-KLF2 has interaction with endogenously expressed FOXO4,and exogenously expresses FOXO4 and KLF2 forming a complex.FOXO4 and KLF2cooperated to directly induce the protein p21 by binding with the p21 promoter and consistently cooperated to induce cell senescence.The AD of KLF2 mediated the active interaction of KLF2 and FOXO4,while the co-expression of ID and ZF domains with FOXO4 seemed to abrogate the effect of FOXO4 alone on p21 induction.Consistent with these observations,knocking down of FOXO4 or p21 can effectively reverse the inhibition of anchorage-independent growth on soft agar,which indicates that KLF2 combined with FOXO4 inducing senescence through p21.Taken together,this study suggests the suppress functions of KLF2 in PDAC cells through inhibiting beta-catenin/TCF signaling and inducing cell senescence.