Hedgehog Sigaling Pathway Regulates Migration And Invasion By Matrix Metalloproteinase (MMP)-7in Ovarian Cancer

Background and PurposeDue to the incidence of occult,early diagnosis is very diffcult.,most of patientaswere present with disease that has distant metastasis.Hedgehog signaling pathwaymakes great sense in organism development by regulating the embryonicdevelopment and adult body homeostasis.In recent date shoeed that Hh signalingpathway play an important role in tumorigenesis and development of many malignanttumors,Aberrant active Hh signaling pathway was found in colorectalcancer,including ovarian cancer.but its exact mechanism in the regulation of invasionand metastasis of ovarian cancer still remains unclear.using cellular biologicalmethod,animal experiment,we will illustrate the exact influence of Hh signalingpathway is involved in the molecular mechanisms of ovarian cancer invasion andmetastasis and looking for early diagnosis and therapeutic targets of ovariancancer.Our final objective is offer theoretical guidance of drug development targetingto ovarian cancer.Research Methods:(1) Three miRNA sequences targeting different fragments of MMP7mRNAwere designed and synthesized based on the Invitrogen website according to MMP7cDNA sequence obtained from NCBI. The synthesized oligos were cloned intopcDNA6.2-GW/EmGFP-miR vector to construct interference plasmids of MMP7（three recombinant construct respectively named miRMMP7-158,314,688).Interference plasmids were transfected into human ovarian cancer SKO-V3cells usingLipofectamine2000, and the transfection efficiency was detected by an invertedfluorescence microscope. Western blot was performed to detect effective interferenceplasmids.(2) The optimized transfection condition [plasmid (ug): Lipofectamine2000(ul) =1:3] was used to transfect the effective interference plasmids into human ovariancancer skov3. Detect the cell invasion and metastasis capabilities by wound healingand Transwell invasion assay.(3) Real-Time PCR and Western blot analysis were performed to detect bothmRNA and protein levels in human ovarian cancer skov3treated with GANT61which HH signaling pathway inhibitor or SHH ligand which activates SHH signalingpathway, respectively.(4) After treatment with GANT61,Immunohistochemical detection of MMP7protein protein changes in xenografts.Results:(1) Interference plasmids were successfully constructed, the fluorescence ratio ofthree plasmids (miRMMP7-158,314,688) was greater than80%in transfectedSKO-V3cells. Western blot detecting MMP7expression showed that miRMMP7-158and were the best interference plasmids(p <0.05).(2) Negative control plasmid transfection group of its invasion and metastasiswere significantly strong interference in transfected plasmid group(p <0.05);Transfected with the negative control plasmid SKO-V3cells, adding SHH ligandconditioned medium group of its invasion and migration was stronger than adding thecontrol medium group (p <0.05); miRNAMMP7-158plasmid transfected SKO-V3cells, add SHH ligand conditioned medium group of its invasion and migration thanadd control medium group no difference(3) Activation of Hh Signaling pathway in SKO-V3cells leads to higher mRNAand protein levels of MMP7in a time-dependent manner(p <0.05), while GANT61（20μM）, which is a inhibitor of SHH signaling pathway transcription factor,suppresses both mRNAand protein of MMP7in a time-dependent manner(p <0.05).(4) Immunohistochemical assays indicated that the expression of MMP7protein in xenografts that treated with GANT61was significantly lower than control group (p<0.05).Conclusion:1. SHH ligand condition medium is used to activated Hh signaling pathway，invasion and migration in ovarian cancer capability significantly enhanced，interference MMP7expression, can effectively inhibit the invasion and migration ofovarian cancer cells, interference MMP7expression and SHH ligand conditionmedium is used to activated Hh signaling pathway in SKO-V3cell,decreasedinvasion and migration of ovarian cancer, these results suggest Hh signaling pathwayregulates invasion and metastasis of ovarian cancer by expression of MMP72． Gli-specific small molecule inhibitors GANT61inhibited SKO-V3cells,down-regulation of MMP7in mRNA and protein expression levels; Hhsignaling pathway activated by SHH ligand conditions medium in ovarian cancerSKO-V3cells, enhanced of MMP7of mRNA and protein expression levels;GANT61reduce tumor volume and weight of xenograft in nude mice, protein levelsof MMP7decreased in the experimental group; MMP7may be Hh signaling pathwayof downstream target genes, Hh pathway may become a new promising target ofovarian cancer and offer theoretical guidance of trearment strategies and drugdevelopment targeting to ovarian cancer.