MiR-412-5p Targets Xpo1 To Regulate The Vascular Proliferation Of Hemorrhoids And The Mechanism Of Zhixuening Mixture Intervention

objective:The specific role of micro RNAs in the pathological process of hemorrhoid patients was elucidation from the perspective of hemorrhoid tissue epigenetic regulation mechanism.Xpo1/p53/p66shc/p16 signal transduction pathway to explore the possible mechanism of hemorrhoids;To clarify the target and regulation of zhixuening mixture in the prevention and treatment of hemorrhoids.Methods:Part I: the hemorrhoid tissue samples of 30 cases of hemorrhoid surgery patients and normal perianal tissue samples 3cm away from fistula of 30 cases of anal fistula surgery patients were collected from the department of anus and intestines of shuguang hospital affiliated to Shanghai university of traditional Chinese medicine.1.Hemorrhoid tissues were stained with hematoxylin-eosin to observe the pathological changes of hemorrhoid tissues.Meanwhile,changes of VEGFR2 protein,a marker of vascular endothelial cells,were observed by immunofluorescence staining.2.Differential expression of micro RNA in the imprinted region of dlk1-dio3 genome between the disease group and the normal group was analyzed by using RNA-seq high-throughput sequencing results,and the RNA-seq detection results were verified by micro RNA q PCR to determine the micro RNA with differential expression.3.Bioinformatics analysis was used to predict the binding sites of differentially expressed micro RNA between the disease group and the normal group,and luciferase reporting system analysis was used to verify the binding sites.4.The effects of miR-412-5p on the function of vascular endothelial cells were analyzed by cell transfection,in vitro angiogenesis analysis,Transwell chamber experiment,flow cytometry,Northern blot and Western blot.5.The molecular mechanism of miR-412-5p targeted inhibition of Xpo1 on the biological function of vascular endothelial cells was analyzed by co-ip and Ch IP technology.Part ii: hemorrhoid samples were collected from 30 hemorrhoid surgery patients in the department of proctology,shuguang hospital affiliated to Shanghai university of traditional Chinese medicine.In the 15 cases of zhixuining drug intervention group,each time 20 ml,3 times a day,1 course of treatment for 1 week,1 week after surgery.Fifteen patients in hemorrhoid group were treated by operation without medicine.Fifteen normal group specimens were obtained from normal perianal tissues3 cm away from the side of fistula in patients with anal fistula.1.Samples of each group were taken and photographed after operation,and the pathological changes of hemorrhoid nucleus were observed by hematoxylin-eosin staining.2.mRNA expressions of Xpo1,P53,p66 shc,p16 and VEGF in the normal group,hemorrhoid group and drug intervention group were detected by mRNA q PCR,and the therapeutic effect of zhixuening mixture on hemorrhoids was verified by regulating the expression of Xpo1/ P53 / p66 shc /p16 pathway.3.The patients in each group were collected venous blood before operation and stored in the serum biochemical test tube containing coagulant promoter.They were immediately sent to the laboratory of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine for serum biochemical test to observe the effect of Zhixuening Mixture on liver and kidney function.Results:Part I:compared with normal tissues,miR-412-5p was significantly lower expressed in hemorrhoid tissues of patients with hemorrhoids,while miR-412-5p in normal tissues degraded Xpo1 mRNA by targeting Xpo1 mRNA 3’UTR,there by affecting the function of vascular endothelial cells.Conversely,in vascular endothelial cells,overexpression of miR-412-5p reduces the ability of vascular endothelial cells to form blood vessels.During the biological function of vascular endothelial cells,miR-412-5p regulates the p53-p66shc-p16 signaling pathway by targeting the expression of Xpo1Part ii:Zhixuening Mixture has obvious inhibitory effect on vascular proliferation in hemorrhoid patients.zhixueling mixture can significantly reduce the expression of Xpo1 mRNA and VEGF mRNA in hemorrhoid patients’ tissues,and zhixueling mixture can significantly promote the expression of p53 mRNA,p66 shc mRNA and p16 mRNA in hemorrhoid patients’ tissues.Zhixuening Mixture had no significant effect on liver and kidney function.Conclusion:1.miR-412-5p is lowly expressed in hemorrhoid tissues.2.Xpo1 is a potential target of miR-412-5p.3.Overexpression of miR-412-5p affects the function of vascular endothelial cells.4.Overexpression of miR-412-5p activates the expression of p53-p66shc-p16 pathway.5.Zhixuening Mixture can achieve therapeutic effect by inhibiting the proliferation ofblood vessels in the tissues of patients with hemorrhoids.6.Zhixuening mixture has therapeutic effects on hemorrhoids by regulating the expression of Xpo1/p53/p66Shc/p16 pathway:7.Zhixuening Mixture is a safe and effective traditional Chinese medicine compound mixture for the treatment of hemorrhoids.