The Role Of Pitx1 And Retinoic Acid Pathway On The Related Mechanism Of Congenital Clubfoot And Clinical Observation

Part I.The mechanism of Pitx1 regulating Sirt1 expressionObjective: The pathogenesis of idiopathic congenital clubfoot(CCF)is unclear.Although some patients with familial clubfoot have a paired-type homeodomain transcription factor(Pitx1)gene mutation and Pitx1 +/-mouse have a clubfoot-like malformation.The mechanism of Pitx1 for the occurrence of CCF is still unclear.Investigating the downstream pathway of Pitx1 may help to understand the mechanism by which Pitx1 induces CCF.Methods: Immunohistochemical analysis of Pitx1 expression in the tibialis anterior tendon of 3 patients with idiopathic CCF and 2 patients with acquired CCF.After obtaining SD rat Achilles tendon cells,the expression of Pitx1 was knocked down by Si RNA.After 48 hours of culture,i TRAQ(Isobaric tag for relative and absolute quantitation,i TRAQ)mass spectrometry was used to quantitatively analyze proteins,Then GO analysis and KEGG analysis were used for downstream pathway of Pitx1.The relationship between Pitx1 and the promoter region of deacetylase 1(Sirtuin-1,Sirt1)was examined by Lucifrase and Ch IP assays.At the same time,the relationship between Pitx1 and the promoter region of Sirt1 was examined by Lucifrase and Ch IP methods in human chondrocytes.Simultaneously,Sirt1 as a downstream of Pitx1 to play a role in Pitx1 regulation of cell function.Results: We used acquired CCF patients as controls for patients with congenital CCF.We found that Pitx1 expression in the tendon samples of idiopathic CCF patients was downregulated.In mass spectrometry analysis,the expression of Pitx1 in SD rat tendon cells was knocked down by Si RNA,and GO analysis and KEGG analysis revealed that Sirt1 may be downstream of Pitx1.Further cellular experiment revealed that confirmed that Pitx1 binds to the promoter region of Sirt1 and promotes Sirt1 gene transcription.In human knee chondrocytes,we found that Pitx1 binds to the promoter region of Sirt1 and promotes Sirt1 gene transcription by Lucifrase and Ch IP assays.Pitx1 regulates chondrocyte senescence through Sirt1.Conclusion: Pitx1 binds to the promoter region of Sirt1 and promotes Sirt1 gene transcription in both SD rat tendon cells and human knee joint chondrocytes.Part II.Negative feedback regulation between Pitx1 and retinoic acid pathwayObjective: Sirt1 regulates the activity of retinoic acid signaling pathway,and retinoic acid treatment induces mice with like-CCF.However,it is not clear whether Pitx1 regulates the activity of retinoic acid signaling pathway through Sirt1.To explore whether knocking down the expression of Pitx1 affects the activity of retinoic acid signaling pathways.Methods: Two kinds of Si RNA were used to knock down the expression of Pitx1 in SD rats tendon cells for 48 hours.The expression of protein on the retinoic acid pathways such as Sirt1,CRABP2 and RAR?2 was detected.The distribution of CRABP2 protein was detected by immunofluorescence.The CRABP2 protein was purified and the acetylation modification of the CRABP2 protein was detected using anti-acetylated lysine.The tendon cells of knock down the Pitx1 and control cells were treated with retinoic acid and cultured for 48 hours to detect the expression of Pitx1 and RAR?2.Pitx1 expression was measured after treatment of tendon cells with different doses of retinoic acid after 48 hours treatment.Results: After the inhibition of Pitx1 in tendon cells,the expression of Sirt1 was decreased,the acetylation of CRABP2 was increased.It was responsible for the transport function of retinoic acid(RA)and the expression of RAR?2 in the downstream pathway of RA was increased.This indicates that inhibition of Pitx1 leads to activation of the retinoic acid pathway.After inhibition of Pitx1,retinoic acid treatment of tendon cells further increased the expression of RAR?2.And in the presence of retinoic acid,the expression of Pitx1 was inhibited in tendon cells.Conclusion: After inhibited Pitx1 in SD rats tendon cells,retinoic acid pathway is activated.There is a negative feedback in retinoic acid signaling pathway and Pitx1 in SD rats tendon cells.Part III.The relationship between the degree of ossification of the scaphoid and the prognosis of the foot in CCF patientsObjective: There are pathological factors for recurrence in CCF patients.Delayed ossification of the scaphoid may be a manifestation of pathological factors.It is unclear whether this persistent pathological factor has an effect on the function of the foot in patients who have ended treatment according to the Ponseti method.The general data and the functional scores of 56 patients with CCF treated with the Ponseti method were retrospectively analyzed to show whether the scaphoid ossification delay was an independent risk factor for the prognosis of the patients.Methods: Children with congenital clubfoot were enrolled at Xinhua Hospital affiliated to School of Medicine Shanghai Jiao Tong University from December 31,2011 to July 31,2013.Cases with complete data,end treatment and meeting the inclusion criteria were included in the study,and cases with multiple joint contractures,spinal meningocele,cerebral palsy and other clubfoot deformities were excluded.56 patients met the criteria.A total of 85 feet from CCF patients were enrolled.There were47 boys and 9 girls;36 left foot and 49 right foot;27 cases of unilateral CCF and 29 cases of bilateral CCF.The ratio of the projected area of the scaphoid and the first humerus on the positive position of the foot is calculated as an index for evaluating the ossification delay of the scaphoid.The 85 feet were grouped according to the scores of the International clubfoot study group(ICFSG)after the end of treatment.Results: Univariate analysis found that gender,pre-treatment Pirani score,correction of improvement(RCI),and scaphoid ossification delay were risk factors.Regression analysis found that scaphoid ossification was an independent risk factor affecting the function of the foot.Conclusion: The projected area of the first metatarsal can be used as a reference for evaluating the ossification delay of the scaphoid.The ratio of the projected area of the scaphoid to the first metatarsal is an independent risk factor for the prognosis of the foot in CCF patients.