The Effect Of Gli1-OPN Axis On Biobehavior Of Pancreatic Tumor And Ectomesenchymal Stem Cells

Osteopontin(OPN)was primarily found in bone tissue and regarded as an osteogenesis marker.It was reported that OPN plays a role in promoting differentiation of bone marrow mesenchymal stem cells(BM-MSCs)into osteoblasts.Recently,OPN has also been found highly expressed in a variety of tumors including pancreatic cancer,and is involved in mediating various bio-behaviors such as tumor growth,metastasis and drug resistance.However,the accurate role of OPN in pancreatic cancer remains controversial.Similar to OPN,the transcription factor Gli1 also plays a role in both promoting osteogenic differentiation of stem cells and progression of pancreatic cancer.The classical activation pathway of Gli1 is triggered by the binding of Hh ligand to the membrane receptors Patched(Ptch),which leads to abrogation of the inhibitory effect of the latter on another membrane receptor Smoothened(Smo).Thus,through a series of intracellular signal transduction,Gli1 expression is upregulated and it translocates into nucleus to induce transcription of target genes.However,it has been recently reported that activation of Gli1 can be independent of Hh ligand.Notably,it has been found that OPN is a target gene of Gli1,which promotes the progression of breast cancer,melanoma and liver fibrosis through up-regulation of OPN expression.Nevertheless,there is no direct evidence that Gli1-OPN axis plays such a regulatory role in pancreatic cancer.Furthermore,MSCs derived from ectoderm(EMSCs)are potential seed cells for bone regeneration,wheras the mechanism of Gli1 signaling on the osteogenic differentiation of EMSCs remains unclear.In this study,pancreatic cancer cell line Bx PC-3 and nasal mucosa-derived EMSCs were selected as models to study the regulatory effect of Gli1-OPN axis on the bio-behavior of pancreatic cancer and differentiation of MSCs,respectively,and to explore the mechanism involved.It might provide laboratory basis for clinical treatment of pancreatic cancer and bone defects.The research consists of two parts:Part 1.Gli1 mediates bio-behavior of pancreatic cancer via up-regulation of OPN expressionⅠ.OPN regulates proliferation,apoptosis,migration and stemness of pancreatic cancer cellsObjective:To elucidate the effects and the mechanism of OPN on proliferation,apoptosis,migration,invasion and stemness maintenance of human pancreatic cancer cells.Methods:1.Immunohistochemistry was performed to detect the expression of OPN in human PDAC tissues.QRT-PCR,Western blot and ELISA was done to detect the levels of OPN expression and secretion by human PDAC cell lines.2.Anti-OPN neutralizing antibody was used to block the OPN protein in the culture medium of Bx PC-3 cells,and cell proliferation,apoptosis,migration and apoptosis was evaluated using CCK-8,TUNEL and transwell assay,respectively.3.The sh-OPN-Bx PC-3 cell line was constructed by transfecting the lentivirus vector of OPN sh RNA.CCK-8,flow cytometry and TUNEL,and scratch wound healing as well as transwell assay were performed to observe cell proliferation,apoptosis,migration and invasion,respectively.The expressions of CSC bio-markers was investigated using q RT-PCR and immunofluorescence.Simultaneously,rh OPN was added to the culture medium to see if the above changes caused by knockdown of OPN would be reversed partially.4.Sh-OPN-Bx PC-3 cells were inoculated into nude mice to observe the effect of OPN on xenograft growth.Results:1.OPN is highly expressed in most pancreatic cancer tissues.The PDAC cell lines all express OPN m RNA and protein,among which Bx PC-3 cells express OPN most abundantly.2.The OPN neutralizing antibody blocked cell proliferation and migration,and enhanced apoptosis of Bx PC-3 cells.3.After knockdown of OPN,sh-OPN-BXPC-3 cells showed slower proliferation,increased apoptosis,decreased migration and invasion,and lower expression of CSC markers(CD44,SOX2 and NANOG).Exogenous rh OPN can partially reverse the above changes of the bio-behavior except for invasion.4.sh-OPN-Bx PC-3 innoculation showed a lower success rate,slower growth and faster atrophy.HE staining showed most tumor cells appeared dead and immunohistochemistry revealed lower OPN positive expression.Conclusion:OPN is highly expressed in human PDAC tissues and cell lines,and it can promote the proliferation and migration,but inhibit apoptosis of Bx PC-3 cells in an autocrine/paracrine way,whereas the effect on invasion is no significant.OPN also facilitates the maintenance of the CSC marker-positive subgroup.Ⅱ.Hh independent Gli1 exerts a tumor promoting effect on Bx PC-3 cells by induction of OPN expressionObjective:To analyze and verify the mechanism with which Gli1 regulates OPN expression in human pancreatic cancer cells,and to confirm the tumor promoting effect of Gli1 is at least partially through OPN.Methods:1.The correlation between Sonic hedgehog(Shh)and OPN as well as Gli1 and OPN m RNA expression in human pancreatic cancer was analyzed using bioinformatics methods.The coexpression of Shh or Gli1 with OPN in PDAC specimens was observed using immunohistochemistry.The expression of Shh and Gli1 in Bx PC-3 cells were detected by q RTPCR and Western blot.2.The Gli1 expression in the OPN knocked down Bx PC-3 cells was investigated.Bx PC-3 cells were treated with Smo inhibitor cyclopamine and Gli inhibitor GANT61 respectively to verify the changes of OPN expression.The Gli1-sh RNA lentiviral vector was transfected into Bx PC-3 cells to construct sh-Gli1-Bx PC-3 cells.Then expression of OPN was analyzed by q RT-PCR and Western blot.The dual luciferase and Ch IP assay were used to evaluate the effect of Gli1 on OPN gene transcrition.3.Rh OPN was used to treat sh-Gli1-Bx PC-3 cells to observe whether it could reverse the changes of cell proliferation,apoptosis,migration,invasion and CSC marker expression induced by Gli1 knockdown.4.Sh-Gli1-Bx PC-3 cells were inoculated into nude mice to see the effect of Gli1 on xenograft growth and OPN expressionResults:1.Bioinformatics analysis showed positive correlation between Gli1 and OPN expression in human pancreatic cancer.Immunohistochemistry revealed Gli1 and OPN were co-expressed in 53% of pancreatic cancer tissues.QRT-PCR and Western blot indicated that Bx PC-3 cells express both Shh and Gli1,but protein expression of 19 k D active Shh is weak.2.sh-OPN only down-regulated Gli1 expression very slightly,whereas sh-Gli1 down-regulated OPN level more significantly in Bx PC-3 cells.OPN expression was also down-regulated by GANT61 but not cyclopamine.Both dual luciferase reporter and Ch IP showed the binding of Gli1 to the promoter of OPN gene,and sh-Gli1 decreased the enrichment of OPN promoters.3.sh-Gli1-Bx PC-3 cells showed lower proliferation,migration,invasion and higher apoptosis rate,as well as decreased expression of CD44,SOX2 and NANOG.Supplementation of rh OPN can partially reverse the above biological changes except cell invasion.4.sh-Gli1-Bx PC-3 xenografts were all atrophied and disappeared 20 d after inoculation.Conclusion:Gli1 activated non-canonically promotes growth,survival,migration and stemness maintenance of pancreatic cancer cells at least in part via direct up-regulation of OPN expression.Part 2.Shh-Gli1 signaling promotes osteogenic differentiation of nasal EMSCsObjective: To investigate the role of Shh-Gli1 signaling in promoting osteogenic differentiation of EMSCs so as to evaluate the feasibility of EMSCs combined with Shh activator in treatment of bone defect diseases.Methods:1.Nasal mucosa was removed from SD rats for primary EMSCs culture.Immunofluorescence was performed to detect MSCs markers CD90,CD105,vimentin and neuroectodermal markers nestin.Shh and Gli1 expression were detected by q RT-PCR and Western blot on the 7th day of osteogenic induction.2.EMSCs was treated with exsogenous Shh protein.After 14 days of induction,alizarin red staining and alkaline phosphatase(ALP)activity were performed to evaluate the effect of osteogenesis.QRT-PCR and Western blot was done to detect the expression of osteogenesis related proteins OPN,OCN,COL Ⅰ,and of Gli1 as well.3.EMSCs were infected with adenovirus to construct Shh overexpressing EMSCs(ad-Shh-EMSCs)and the control ad-GFP-EMSCs.After 14 days of osteogenic induction,mineralized area,ALP activity,expression of osteogenesis related genes as well as Gli1 were detected as mentioned above.Results:1.Primarily cultured EMSCs were fibroblast-like and expressed CD90,CD105,vimentin and nestin.After osteogenic induction for 7 days,the m RNA and protein levels of Shh and Gli1 increased.2.After 14 days of osteogenic induction,Gli1 expression was higher,and enhanced osteogenesis reaction was seen: more mineralized area and higher ALP activity,higher expression of osteogenesis related genes OPN,OCN and COL Ⅰ.Such reaction was further enhanced by treatment of exogenous Shh,but reduced by cyclopamine.3.After transfection,the level of Shh was up-regulated both in the cells and in the supernatant of ad-shh-EMSCs.Notably,ad-Shh-EMSCs showed further enhanced osteogenic reaction after 14 days of induction,whereas cyclopamine reduced it to a certain extent.Conclusion: The canonical Shh-Gli1 signaling plays a promoting role in osteogenic differentiation of nasal EMSCs,and EMSCs combined with Hh signal enhancement has potential application value in clinical treatment of bone defect diseases.