Molecular Mechanism Study And In Utero Stem Cells Transplantation In Defective SMC Of ARMs Rat Embryos

Objective:Anorectal malformations（ARMs）are the most common pediatric gastrointestinal malformations.According to the World Health Organization,the incidence of ARMs is 1/5000～1/1500,thus ranking first among digestive tract deformations.Thus far,the etiology of ARMs has not been established,in part because the pathologic types and changes are numerous and complicated.With the continuous improvement in surgical methods,the overall and surgical mortalities have decreased significantly.Domestic and foreign scholars have reported that some children still have different degrees of defecation dysfunction after surgery,which negatively impacts their quality of life and mental health.Indeed,the impact will result in an enormous medical burden and economic pressure on the children’s families and society.A poor prognosis,such as constipation and incontinence,is related to the type of deformity and associated deformities,such as abnormal development of the pelvic floor muscles,abnormal development of the innervation of the pelvic floor muscles,and dysplasia of the sacral tail vertebrae.Among many factors,the pelvic floor striated muscle complex（SMC）is a critical factor for controlling bowel movements.The abnormal muscle and nerve development in SMC is the basis for the prognosis of children with ARMs.Preliminary studies have confirmed that there are varying degrees of SMC dysplasia in children with ARMs and in experimental animal models.SMC dysplasia is characterized by abnormal development of muscle fibers,abnormal development of muscle spindles,and abnormal development of motor endplates.The developmental process of the embryonic SMC is complicated,and is the result of a combination of environmental and genetic factors.The molecules,pathways,and interactions among the pathways involved are still unclear.In the SMC tissues of children with ARMs,there are not only pathologic features,such as muscle fiber disarrangement,but also abnormal developmental manifestations of decreased muscle spindle and motor endplate density.Thus far,there have been few studies on the neurodevelopmental abnormalities of the SMC in ARMs,and the developmental mechanism has not been established.Therefore,this study used high-throughput sequencing technology to screen the abnormally expressed genes in the embryonic SMC of children with ARMs at the transcriptome level and explored the possible mechanism affecting muscle fibers and neurites triggering abnormal reproduction.The wingless-type（Wnt[MMTV integration site family member]）signaling pathway is closely related to the development of skeletal muscle and participates in the regulation of skeletal muscle growth,development,regeneration,and disease occurrence.Studies have shown that the Wnt signaling pathway is related to neuromuscular junction（NMJ）development,and an abnormal NMJ is the cause of myasthenia gravis and other neuromuscular disorders.In the development of different tissues,the Wnt signaling pathway interacts with other signaling pathways,including bone morphogenetic proteins（BMPs）,Sonic hedgehog（Shh）,and Notch signaling pathways.The expression of critical molecules in the classic and non-canonical Wnt signaling pathway is unclear.The expression of downstream effector molecules is also unclear.It is still unknown how the Wnt signaling pathway regulates the development of the SMC tissue in ARMs.At the same time,during the developments of different tissues,interactions between Wnt and other signaling pathways are different.In the development of the rectal tissue of ARMs,all the signaling pathways mentioned above make differences;however,in the development of the SMC,it is unclear whether they make differences.Non-coding RNA（ncRNA）plays an essential role in embryonic development.NcRNA regulates heart,nervous system,and muscle development.During the development of the pelvic floor muscles of ARMs,research on ncRNA is still unknown.Given the essential role of ncRNA,relevant research is urgently needed.The repair of muscles and nerves is difficult,especially the repair of muscle-nerve joints,which requires a more extended period and more complicated biological process.Muscle-derived stem cells（MDSCs）of muscle origin and pluripotent stem cells,unlike muscle satellite cells,limit endoderm differentiation into cells from different germ layers,such as nerve cells,muscle cells,islet cells,and osteoblasts.Due to its proliferation ability and multi-directional differentiation potential,MDSCs are considered to be a promising gene carrier and are widely used in clinical medicine and tissue engineering.The myoblast cell line are multipotent stem cells that can differentiate into myotubes in vitro.Unlike multipotent stem cells,such as MDSCs,the myoblast cell lines can only differentiate into muscle fibers;however,the proliferative ability is enormous and the direction of differentiation is determined so the myoblast cell lines are also considered a suitable type of stem cell for repairing muscle tissue.In this study,we used ethylene thiourea（ETU）to induce a Wistar anorectal rat model.We used transcriptome sequencing（RNA sequencing[RNA-Seq]）to detect differentially expressed m RNA、lncRNA,and circRNA between control and ARM groups.Furthermore,we used enrichment analysis and co-expression networks to screen relative molecules and signaling pathways in fetal SMC abnormalities with ARMs.We explored the Wnt signaling pathway and its interactive molecules in the development of SMC.We used reporter gene experiments to verify the mutual binding of lncRNA-miRNA-m RNA.MDSCs and myoblast cell lines were cultured in vitro,and transplanted into the SMC area of fetal rat ARMs and the colonization,survival,and differentiation were noted.We explored the potential of muscle-derived stem cell transplantation in the treatment of abnormalities of the pelvic floor complex of striated muscle.Research methods:1.Animal model preparation and specimen collection.Wistar rats weighing 230～280 g at approximately 12 weeks were provided by Liaoning ChangSheng Biotechnology Company（Liaoning,China）.We caged male and female mice at a 5-to-1 ratio,obtained a vaginal smear the following morning,observed the smear under a microscope to detect sperm,and labeled the smear pregnane 0 days（embryonic day 0[E0]）.At E10,we gavage-fed 1%ETU（125 mg/kg）into the ARM group and the same amount of normal saline was given to the control group.At E19,pregnant rats were anesthetized with inhaled isoflurane and we collected fetal tissue specimens after cesarean.The specimens were prepared for immunohistochemistry with4%paraformaldehyde fixation,gradient alcohol dehydration,and embedding in paraffin.The specimens for Western blot and reverse transcriptase-polymerase quantification（RT-qPCR）,collected under a dissecting microscope,infiltrated with RNAlater protection liquid,and stored in a refrigerator.2.Transcriptome sequencing screening embryonic and fetal ARM normal SMC tissues differentially-expressed genes.We collected SMC tissues in the control and ARM groups at E19 to conduct a high-throughput sequencing experiment.Beijing CapitalBio Technology Co.,Ltd.provided technological support.We detected differentially-expressed mRNA and ncRNA in the control and ARM groups.We used GO and Pathway analysis to classify data.We speculated protein interactions,selected genes and ncRNA related to muscle and nerve development and used RT-qPCR technology to verify the differential expression.3.RT-qPCR techniques.After extraction of total RNA,a UV spectrophotometer was used and mass concentration of each sample was adjusted to the same concentration level,genomic DNA was removed after reverse transcription the cDNA,and the steps of the system were formulated according to the method recommended by the kit for mRNA,lncRNA,and circRNA Perform RT-qPCR detection.4.Western blot techniques.Total protein was extracted and determined using the BCA method.The protein concentration of each sample was adjusted to the same level.We prepared the gel system according to the kit instructions.After electrophoresis and transfer,the primary antibody was diluted according to the recommended ratio,Incubate at 4℃overnight incubated for 2 h,luminescent liquid was added dropwise,and pictures were obtained by imaging.5.Immunohistochemical techniques.Paraffin sections were gradually dewaxed with benzene and immersed in water.After antigen repair,endogenous peroxidase was blocked.The primary antibody was diluted according to the recommended ratio in the instructions and incubated overnight at 4℃.We incubated the secondary antibody at room temperature for 30 min,followed by DAB staining,hematoxylin counterstaining,alcohol gradient dehydration,and neutral gum sealing.6.Double luciferase reporter gene experiment:We used TargetScan and other websites to predict the binding site.Then,we constructed plasmids according to the sequences provided by the website,which were then sequenced and extracted after synthesis.We added the corresponding plasmids,mi RNA,and transfection reagents into293T cells.After 48 h of co-transfection,we detected Luciferase activity.We used the ratio of firefly luciferase activity-to-renilla luciferase activity for data analysis.7.The intrauterine graft of MDSCs and cell lines was used to repair the striated pelvic floor.We extracted and isolated MDSCs from the hind legs of newborn male rats using a two-step digestion and differential adhesion method.We cultivated MDSCs in vitro and identified the MDSCs by immunofluorescence staining.After transfection with adenovirus carrying a green fluorescent protein gene（Ad-eGFP）in MDSCs,we made a cell suspension by trypsin digestion,then transplanted the MDSCs into the SMC area of fetal rat ARMs.We observed the survival and differentiation of MDSCs in the SMC area of the ARMs after transplantation.At the same time,the myoblast cell line was cultured and the cell line was identified by immunofluorescence staining.After transfection with Ad-eGFP,the cell line was digested with trypsin to prepare a cell suspension,which was subsequently transplanted into the SMC area of fetal rat ARMs.Survival and differentiation of myoblasts were observed in the SMC area of fetal rat ARMs after transplantation.8.Intrauterine stem cell transplantation surgery and fetal rat ARMs microscopy surgery after birth.For intrauterine stem cell transplantation,ETU-exposed pregnant rats were anesthetized with isoflurane at E19,one side of the uterine horn was severed and covered the uterus with wet gauze soaked in warm saline.A short-tailed or tailless fetal rat was identified using a cold light source through the wall of the uterus,a suture was placed in the uterine wall,a small incision was made in the center of the suture,the uterine wall and amniotic sac were opened,and the pelvic floor was exposed.A micro-injection glass needle was used to inject the prepared MDSCs or myoblast suspension into the SMC area on both sides of the anal fossa of the ARMs.After the microinjection was completed,the fetal rat was gently replaced into the uterus.Surgical nylon thread（4-0）was used to suture the abdominal wall muscle layer and skin and the abdomen was closed layer-by-layer.The position of the surgical rat was recorded for easy identification during fetal extraction.After the operation,the pregnant rats were reheated by lamp baking.Moreover,after the rats awakened,the rats were returned to the cages for continued rearing.At E22,the fetal rats were delivered by cesarean section,dried with cotton swabs to clean the mucus from the nose,and lightly pressed on the heart until the newborn rats breathed spontaneously and their skin color began to turn red.After birth,the deformed newborn was anesthetized by hypothermia.After the anesthesia,the newborn was placed on its right side with the head low and the feet elevated.The limbs were fixed with tape,and the skin and abdominal muscles were cut into the abdominal cavity.We looked for the rectum or colon,ligated the distal rectum,and freed the colon as much as possible to ensure that the fistula was tension-free.It was vital to protect the intestinal canal and organs during the operation and avoid instruments scratching the vasculature.We placed intermittent sutures in the muscular layers and skin,avoided pulling or puncturing the muscular layers,and wiped the bloodstain with normal saline after surgery.The newborn was reheated by heating on the thermostatic plate,then buried in the old bedding,and returned to the mother rat.We expanded the fistula twice per day（once in the morning and once in the evening）under an operating microscope.The microscopic instrument removed the secretion of the fistula until yellow stool was visible.We were careful not to scratch the skin and colon by rinsing with normal saline,wiping the back,and returning to the mother rat.9.SPSS 24.0 statistical software was used for statistical analysis.We determined whether the data followed a normal distribution,then used an independent sample t-test for the differences between the control and ARM group.A Pearson test was used for relativity analysis.All of the experimental data are expressed as the mean±standard deviation and a P<0.05 was considered statistically significant.Results:1.For high-throughput sequencing screening,the screening conditions were│log₂FC│≥1 and a p-value<0.05;a log₂FC≥1 indicated that the differential gene was up-regulated and log₂FC<=-1 indicated that the differential gene was down-regulated.Compared with the control group,the ARM group expressed 886up-regulated m RNA and 522 down-regulated mRNA,218 up-regulated lncRNA and 254down-regulated lncRNA,and 191 up-regulated circRNA and 130 down-regulated circRNA.Based on preliminary data classification and enrichment analysis and RT-qPCR,we chose neural and skeletal muscle development-related mRNA and ncRNA,confirmed the sequencing accuracy in mRNA was 86.67%,accuracy of lncRNA 60.00%,and circRNA 77.78%.2.Using RT-qPCR,Western blot,and immunohistochemical staining techniques were used to detect WNT7A,β-catenin,and BMP2 of mRNA and protein expression levels and using Western blot and immunohistochemical staining to detect phospho-β-catenin,C-Myc,and cyclin D1 protein levels.We showed that WNT7A,β-Catenin,and BMP2 expression was significantly increased in fetal rat ARM SMC,which was consistent with the results of transcriptome sequencing;fetal ARM SMC in phospho-β-Catenin,C-Myc,and Cyclin D1 protein level was also increased significantly.3.Using target gene prediction software（miRanda）to predict target miRNA of differentially-expressed lncRNA and mRNA based on the high-throughput sequencing results.According to the ceRNA mechanism,there may be 3 molecule pairs with targeted regulation,as follows:TCONS＿00051629—Syt7;TCONS＿00051629—Retn;and TCONS＿00028283—Dkk3.In the SMC tissues with ARMs,we used RT-qPCR,Western blot,and immunohistochemical staining techniques to verify the expression level,and confirmed that the three molecular pairs had the same differential expression trend.We used TargetScan and other software to predict the miRNA that can bound lncRNA and the targeted mRNA.We constructed the plasmid and used a dual-luciferase reporter gene experiment to confirm that TCONS＿00051629—rno-miR-326-3p—Retn,TCONS＿00051629—rno-miR-330-5p—Retn,TCONS＿00028283—rno-miR-193a-5p—Dkk3,rno-miR-328a-3p—Dkk3,and rno-miR-3584-5p—Dkk3 had binding sites and played a regulatory role.4.The extracted muscle-derived stem cells were cultured in vitro 24 h,and PAX7protein immunofluorescence staining was positive;at 48 h Desmin protein immunofluorescence staining was positive.MDSCs transfected by Ad-eGFP and appearing green fluorescence were injected orthotopically into the fetal ARM SMC region.After transplantation,MDSCs survived in the fetal ARM and expressed specific mature muscle proteins（MYH）.The myoblast lineage was cultured in vitro and confirmed by fluorescent immunostaining experiments,PAX7,and Desmin protein-positive properties.The myoblast cell line was transfected by Ad-eGFP and appeared green fluorescence were transplanted into the fetal ARM SMC region,survived in vivo,and expressed specific marker proteins MYH.Conclusions:1.The SMC organization in ARMs had noticeable abnormal changes at the transcriptome level,including 1408 mRNA,472 lncRNA,and 321 circRNA abnormally up-or down-regulated,and mainly enriched in the pathways related to neuromuscular development and disease.2.The expression levels of Wnt7a,β-catenin,and Bmp2 mRNA and protein were significantly increased in the fetal rat ARM SMC;the levels of phospho-β-Catenin,c-Myc,and cyclin D1 protein expression was also increased significantly.3.In TCONS＿00051629—Syt7,TCONS＿00051629—Retn,and TCONS＿00028283—Dkk3,lncRNA,mRNA and protein were present in the co-expression of the trend in the ARM of SMC.TCONS＿00051629 by competitive binding with the rno-miR-326-3p and rno-miR-330-5p regulated Retn expression.TCONS＿00028283 by competitive binding with the rno-mi R-193a-5p regulated Dkk3 expression,while rno-miR-328a-3p—Dkk3 and rno-mi R-3584-5p—Dkk3 had a regulatory relationship.4.Muscle-derived stem cells and myoblast cell lines transplanted to the fetal ARM pelvic SMC rear region survived and differentiated into mature muscle cells.