| ObjectiveAmong childhood liver malignancies,hepatoblastoma(HB)is the most common and usually develops within 3 years of age.Although the overall cure rate is high,the overall survival rate of children with recurrence or metastasis is not satisfactory,and the side effects of chemotherapy drugs also affect the prognosis and long-term outcome of children with HB.To further improve the treatment of hepatoblastoma,targeted drugs and immunotherapies are expected to be new therapeutic avenues.With improved understanding of the hepatoblastoma genome and transcriptomics,new therapeutic mechanisms can now be explored to improve the outcome of poorly prognosed hepatoblastoma and to reduce the toxicity of current treatments.The aim of this article is to explore the underlying mechanisms of hepatoblastoma development.MethodsMy team performed single-cell sequencing of one normal liver tissue and three tumor tissues from the same patient,and comprehensively analyzed the sequencing results to screen a candidate gene,AHCY gene,based on the bioinformatics analysis results and domestic and international literature.To address the involvement of the AHCY gene in the pathology of hepatoblastoma,immunohistochemical staining of wax blocks containing HB tumor and paraneoplastic tissues was performed to detect the difference in expression of AHCY protein between tumor and paraneoplastic tissues.Thereafter,we further characterized the implications of the AHCY gene on HB proliferation and apoptosis in vitro and in vivo.We first transiently transfected plasmids to interfere with the expression of AHCY gene and screened the best interfering fragment.Lentivirus-mediated short hairpin RNA(shRNA)was then constructed to consistently knock down the AHCY gene in human hepatoblastoma cell lines HUH6 and Hep G2 based on this interfering fragment,and the impacts of knockdown of AHCY gene on the proliferation and apoptosis of human hepatoblastoma cell lines were investigated;The effect of AHCY on the proliferation of human hepatoblastoma cell lines was investigated by CCK-8 assay and clone formation assay;the effect of AHCY on apoptosis of human hepatoblastoma cell lines was investigated by Tunel fluorescence staining assay and Annexin V combined with PI method to detect changes in cell membrane components by flow cytometry.The influence of AHCY on proliferation and apoptosis of hepatoblastoma cells in vivo was investigated by tumorigenesis assay in nude mice,and the effect of AHCY on the proliferation of hepatoblastoma cells in vivo was verified by tumor growth curve and immunohistochemical staining of nude mice tumor tissues to detect ki-67 protein expression.The effect of AHCY on the apoptosis of hepatoblastoma in vivo was verified by the detection of caspase-3 protein expression in nude mice.Next,we further investigated the mechanism of AHCY gene regulating the proliferation and apoptosis of human hepatoblastoma by transcriptome sequencing of human hepatoblastoma cell line shRNA-NC group and shRNAAHCY group cells,and applied bioinformatics to analyze the differentially expressed genes between groups of sequencing data,followed by enrichment analysis of differential genes by GO and KEGG,based on which we predicted the biological process and possible regulation of AHCY gene Based on this,We predicted the biological processes and possible pathways involved in AHCY gene regulation,and finally verified the pathway-related protein manifestation by Western blot(WB)experiments.Results1.In this study,we examined the expression of AHCY protein in 45 pairs of hepatoblastoma tumor and paraneoplastic tissue samples,and the results showed that the expression of AHCY protein in tumor tissues was significantly higher than that in paraneoplastic tissues.2.In vitro experiments,plasmid transient transfection and lentiviral vector-mediated short hairpin RNA(shRNA)were applied to stably knock down the AHCY gene in human hepatoblastoma cell lines(HUH6,Hep G2),and the down-regulation of AHCY gene expression resulted in reduced proliferation of human hepatoblastoma cells and increased apoptotic cells.3.In vivo experiments,the lentiviral vector-mediated short hairpin RNA(shRNA)was applied to human hepatoblastoma cell lines(HUH6,Hep G2)after stable knockdown of AHCY gene and injected subcutaneously into nude mice,and after downregulation of AHCY gene expression,the growth of nude mice hepatoblastoma tissue was slowed down and apoptotic cells were increased.4.The shRNA-AHCY group and shRNA-NC group cells of human hepatoblastoma cell line HUH6 were examined by transcriptome sequencing technology,and the results showed that the AHCY gene regulates cell proliferation and apoptosis mainly through PI3K/AKT pathway.5.Finally,the sequencing results were validated,and the expression of PI3K/AKT pathway star molecules was detected by Western blot(WB)assay,and the results confirmed that the AHCY gene regulates cell proliferation and apoptosis mainly through PI3K/AKT pathway.ConclusionAHCY is expected to be a potential biomarker for early diagnosis and a possible novel destination for molecularly tailored therapy of hepatoblastoma.Obiective Hisense CAS computer-assisted surgery system can compensate for the deficiencies of traditional CT and MRI two-dimensional images,clearly display the size and location of the lesion and the spatial adjacency of the surrounding vessels in three dimensions,understand the vascular alignment and anatomical variants,determine the optimal surgical boundary through virtual lesion excision,develop the corresponding surgical approach,and ensure the maximum remaining functional tissue.The purpose of this study is to investigate the application and significance of the Hisense CAS computer-assisted surgical system 3D reconstruction technology in pediatric surgical diseases.Methods Retrospective analysis The clinicopathological data of 323 children with preoperative 3D reconstruction in our hospital from March 2016 to October 2022.Among them,146 were boys and 177 were girls,aged 1 month-12 years old,with an average age of 3.6 years.In the collected cases,the children were preoperatively reconstructed in three dimensions using the Hisense CAS computer-assisted surgery system to precisely understand the relationship between the vessels and the lesion,simulate resection,calculate the volume of the remaining organs,plan the surgical plan,and observe the vascular pathways and variants of the children.In each case,at least two doctors at the attending level or above evaluate whether the child can undergo stage I surgery based on the 3D images,and further plan the surgery and perform simulated resection for children who can undergo stage I surgery.The application and value of the computer-assisted surgical system in pediatric surgery were discussed by combining the treatment plan of the child and the volume of the simulated tumor resection performed by the computer-assisted surgical system.Results1.323 cases of preoperative 3D reconstruction and surgical treatment were performed in our pediatric surgery department from March 2016 to October 2022,and the number of reconstructed cases reached 372.323 children were successfully operated according to the preoperative design.Among them,there were 87 cases of congenital common bile duct dilatation,62 cases of hepatoblastoma,46 cases of neuroblastoma,33 cases of teratoma,20 cases of retroperitoneal mass,16 cases of nephroblastoma,14 cases of other tumors,11 cases of mesenchymal tumors,10 cases of adrenal mass,9 cases of hydronephrosis,9 cases of mediastinal tumors,and 6 cases of pancreatic mass.2.Among the 323 cases collected,28 lesions were located in the chest,accounting for 8.67% of the total number of cases.14 boys and 14 girls were among the 28 cases;9 cases of mediastinal tumors,8 cases of neuroblastoma,7 cases of teratoma,4 cases of mesenchymal tumors,11 cases were operated openly and 17 cases were operated thoracoscopically.Based on the three-dimensional images,25 cases could be resected in stage I.Three cases of neuroblastoma required chemotherapy first and then the feasibility of surgery was evaluated again.3.Among the 323 cases,282 lesions were located in the abdomen,including 125 boys and 157 girls;87 cases of congenital common bile duct dilatation,62 cases of hepatoblastoma,36 cases of neuroblastoma,23 cases of teratoma,20 cases of retroperitoneal masses,16 cases of nephroblastoma,10 cases of adrenal masses,9 cases of hydronephrosis,6 cases of pancreatic masses,5 cases of renal masses,4 cases of mesenchymal tumors,and 4 cases of benign hepatic tumors.There were 4 cases of benign liver tumors.The results are shown in Table 3.132 open surgeries,139 laparoscopic surgeries,8 intra-laparoscopic conversions to open surgery,and 3 da Vinci robotic surgeries were performed.According to the assessment of 3D image results,stage I resection was feasible in 248 cases,and 34 cases needed to be reevaluated for surgical feasibility after chemotherapy.4.Among the 323 cases,13 lesions were located in the pelvis,including 7 boys and 6 girls;5 ovarian tumors,3 mesenchymal tumors,2 neuroblastomas and 3 teratomas;5 lesions were resected by open surgery and 8 by laparoscopic surgery.Based on the results of threedimensional reconstruction,all eight tumors were resected in stage I and treated according to the preoperative planning of surgery.Conclusion Hisense CAS computer-aided surgical system is an important tool for preoperative planning,intraoperative operation and postoperative recovery.Hisense CAS computerassisted surgical system is an important aid for preoperative planning,intraoperative operation and postoperative recovery,and has a certain clinical application. |
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