Objective:To elucidate the regulatory role of matrix metallopeptidase 14 (MMP14) in the migration of chondrogenic precursor cells, thereby providing data support for investigating the pathogenesis of microtia.Methods:An in vitro differentiation model was established using human induced pluripotent stem cells (iPSCs) sequentially induced into neural crest cells (iNCCs) and subsequently into chondrogenic precursor cells (iCPCs), combined with lentivirus-mediated knockdown of MMP14, to investigate the effects of MMP14 on the biological characteristics of iCPCs, including proliferation, differentiation, and migration. Collective cell migration was assessed using scratch wound healing and Transwell migration assays; directional migration was characterized via high-content live-cell imaging; single-cell adhesion force was measured using a micromanipulation system. Collagen degradation was evaluated through hydroxyproline digestion assays. Cell proliferation was analyzed using the CCK-8 assay, and the expression of osteogenic/chondrogenic-related genes (SOX5/6/9, COL1A1, COL2A1, RUNX2, TWIST1) were quantified by real-time quantitative PCR. Immunofluorescence staining was used to assess the expression of F-actin and CD44 proteins. Additionally, transcriptomic sequencing was performed on iCPCs before and after MMP14 knockdown. Results:iPSC→iNCC→iCPC differentiation model was established in vitro. The resulting iCPCs expressed osteo/chondrogenic marker genes, including SOX5, SOX6, SOX9, COL1A1, COL2A1, RUNX2, and TWIST1, and exhibited positive expression of mesenchymal stem cell markers CD90, CD105, and CD73. Upon further induction, functional cartilage spheroids were formed. Compared with normal auricular chondrocytes, auricular chondrocytes from microtia patients showed reduced expression of MMP14 at both mRNA and protein levels. Lentivirus-mediated shRNA knockdown of MMP14 in iCPCs resulted in a marked decrease in its mRNA and protein expression. MMP14 knockdown significantly impaired collective migration of iCPCs, as evidenced by reduced wound closure rates in scratch assays and decreased numbers of migrated cells in Transwell assays. High-content live-cell imaging revealed that MMP14-deficient iCPCs displayed more erratic migration trajectories and a lower straight-line migration ratio. Single-cell adhesion assays showed extracellular matrix (ECM)-dependent alterations: cell adhesion was enhanced on matrigel-coated surfaces but weakened under uncoated conditions. MMP14 knockdown also led to reduced proliferation, decreased collagen degradation, diminished F-actin expression, fewer peripheral adhesion sites, and downregulation of CD44 protein expression, without significantly affecting the expression of chondrogenic genes such as SOX6, SOX9, COL1A1, COL2A1, RUNX2, and TWIST1. Transcriptomic analysis further revealed that MMP14 knockdown significantly downregulated genes involved in extracellular matrix organization, cell adhesion, migration, and tissue development, with enrichment in pathways including ECM-receptor interaction, focal adhesion, and MAPK signaling. Conclusion:MMP14 plays a critical role in the directional migration of chondrogenic precursor cells by regulating ECM remodeling, adhesion signaling, and cytoskeletal proteins.

Objective:To investigate the effects and molecular mechanisms of Wilms tumor 1-associated proteins(WTAP)on the cell biological properties of esophageal squamous cell carcinoma(ESCC)cells.Methods:31 pairs of ESCC tissues and their paired paracancerous tissues that were surgically resected at the Second Clinical Medical College of Chuanbei Medical College between September 2019 and April 2021 were collected.The esophageal cancer cells KYSE30,KYSE410,KYSE150,KYSE510,TE-1,and normal human esophageal epithelial cells HET-1A were routinely cultured.Transfection reagents were used to transfect si-NC,si-WTAP#1 and si-WTAP#2 nucleic acids into KYSE150 and KYSE510 cells.The cells were divided into si-NC,si-WTAP#1 and si-WTAP#2 groups.The expressions of WTAP and MAP3K9 mRNA were detected in the cells of each group by qPCR assay.CCK-8 assay,clone formation assay,and scratch healing assay,Transwell assay were employed to detect the effects of knockdown of WTAP expression on ESCC cell proliferation,migration,invasion and apoptosis.WB assay was used to detect the expressions of WTAP,MAP3K9,EMT and MAPK pathway-related proteins in ESCC cells of each group knocked down of WTAP;immunohistochemistry to detect the expression of WTAP proteins in ESCC tissues,immunoprecipitation of methylated RNA(MeRIP)-qPCR assay to detect the level of MAP3K9 m6A in ESCC cells,actinomycin D assay to detect the stability of mRNA of MAP3K9,and database data to analyze the expression,target genes,functional enrichment,and interacting RNA of WTAP.Results:WTAP was highly expressed in ESCC tissues and cells(P<0.05 or P<0.01 or P<0.001)and correlated with the degree of differentiation(P<0.01);the expression of WTAP mRNA and its protein were successfully knocked down in KYSE150 and KYSE510 cells(P<0.01 or P<0.001);the knockdown of WTAP significantly inhibited the proliferation,migration and invasion of KYSE150 and KYSE510 cells(P<0.05 or P<0.01 or P<0.001),and promoted the apoptosis of KYSE150 and KYSE510 cells(P<0.05 or P<0.01).Knockdown of WTAP resulted in a significant decrease in the m6A level of MAP3K9(P<0.05),and its mRNA expression level and mRNA stability were both significantly reduced(P<0.05).Database data analysis showed that WTAP target genes clustered in the MAPK signaling pathway;the expression levels of MAP3K9,p-ERK,N-cadherin,and MMP9 were significantly reduced(P<0.05 or P<0.01),and the expression level of E-cadherin was significantly elevated(P<0.05 or P<0.01)in the KYSE150 and KYSE510 cells after knockdown of WTAP.Conclusions:WTAP is highly expressed in ESCC tissues and cells and correlates with their differentiation.It promotes the stability of MAP3K9 mRNA through m6A modification,activates the MAPK pathway and thus promotes the malignant biological behaviors of ESCC cells.

Background and Aims:Secondary hyperparathyroidism(SHPT)is a common and difficult-to-treat complication of chronic kidney disease(CKD),significantly impairing patients'quality of life and prognosis.For patients who respond poorly to medical therapy,surgical intervention remains an effective treatment option.This study aimed to evaluate the clinical efficacy and safety of total parathyroidectomy with forearm autotransplantation(tPTX+AT)in the treatment of CKD-related SHPT.Methods:A retrospective analysis was conducted on 40 patients with CKD complicated by SHPT who underwent tPTX+AT in Gaozhou People's Hospital between January 2020 and June 2023.Changes in intact parathyroid hormone(iPTH),serum phosphorus,calcium,alkaline phosphatase(ALP),and bone mineral density(BMD)were recorded preoperatively and at multiple postoperative time points.Postoperative symptom relief,complications,and follow-up outcomes were also analyzed.Results:A total of 158 parathyroid glands were removed during surgery.Among the patients,38 had four glands successfully excised,while two had only three glands removed.After operation,levels of iPTH,phosphorus,calcium,and ALP decreased significantly compared to preoperative values(all P<0.05),and BMD increased significantly at 3 months(P<0.05).Symptoms such as bone pain,pruritus,and restless leg syndrome improved markedly by 3 months postoperatively(all P<0.05).Hypocalcemia occurred in 34 cases(85.0%);one patient experienced transient recurrent laryngeal nerve injury and one had superior laryngeal nerve injury,both of which resolved after treatment.The two patients who had only three glands removed exhibited persistent SHPT postoperatively,with iPTH levels of 457 pg/mL and 609 pg/mL,respectively.Although their symptoms improved partially,the condition was medically controlled without the need for reoperation.Conclusion:tPTX+AT can effectively correct mineral metabolism disorders and improve BMD and clinical symptoms in SHPT patients.The procedure achieves a high rate of complete gland resection and stable autograft function.Although postoperative hypocalcemia is common,overall complications are manageable.This surgical approach is safe and effective for the treatment of refractory SHPT.

Background and Aims:Secondary hyperparathyroidism(SHPT)is a common and difficult-to-treat complication of chronic kidney disease(CKD),significantly impairing patients'quality of life and prognosis.For patients who respond poorly to medical therapy,surgical intervention remains an effective treatment option.This study aimed to evaluate the clinical efficacy and safety of total parathyroidectomy with forearm autotransplantation(tPTX+AT)in the treatment of CKD-related SHPT.Methods:A retrospective analysis was conducted on 40 patients with CKD complicated by SHPT who underwent tPTX+AT in Gaozhou People's Hospital between January 2020 and June 2023.Changes in intact parathyroid hormone(iPTH),serum phosphorus,calcium,alkaline phosphatase(ALP),and bone mineral density(BMD)were recorded preoperatively and at multiple postoperative time points.Postoperative symptom relief,complications,and follow-up outcomes were also analyzed.Results:A total of 158 parathyroid glands were removed during surgery.Among the patients,38 had four glands successfully excised,while two had only three glands removed.After operation,levels of iPTH,phosphorus,calcium,and ALP decreased significantly compared to preoperative values(all P<0.05),and BMD increased significantly at 3 months(P<0.05).Symptoms such as bone pain,pruritus,and restless leg syndrome improved markedly by 3 months postoperatively(all P<0.05).Hypocalcemia occurred in 34 cases(85.0%);one patient experienced transient recurrent laryngeal nerve injury and one had superior laryngeal nerve injury,both of which resolved after treatment.The two patients who had only three glands removed exhibited persistent SHPT postoperatively,with iPTH levels of 457 pg/mL and 609 pg/mL,respectively.Although their symptoms improved partially,the condition was medically controlled without the need for reoperation.Conclusion:tPTX+AT can effectively correct mineral metabolism disorders and improve BMD and clinical symptoms in SHPT patients.The procedure achieves a high rate of complete gland resection and stable autograft function.Although postoperative hypocalcemia is common,overall complications are manageable.This surgical approach is safe and effective for the treatment of refractory SHPT.

Objective:To elucidate the regulatory role of matrix metallopeptidase 14 (MMP14) in the migration of chondrogenic precursor cells, thereby providing data support for investigating the pathogenesis of microtia.Methods:An in vitro differentiation model was established using human induced pluripotent stem cells (iPSCs) sequentially induced into neural crest cells (iNCCs) and subsequently into chondrogenic precursor cells (iCPCs), combined with lentivirus-mediated knockdown of MMP14, to investigate the effects of MMP14 on the biological characteristics of iCPCs, including proliferation, differentiation, and migration. Collective cell migration was assessed using scratch wound healing and Transwell migration assays; directional migration was characterized via high-content live-cell imaging; single-cell adhesion force was measured using a micromanipulation system. Collagen degradation was evaluated through hydroxyproline digestion assays. Cell proliferation was analyzed using the CCK-8 assay, and the expression of osteogenic/chondrogenic-related genes (SOX5/6/9, COL1A1, COL2A1, RUNX2, TWIST1) were quantified by real-time quantitative PCR. Immunofluorescence staining was used to assess the expression of F-actin and CD44 proteins. Additionally, transcriptomic sequencing was performed on iCPCs before and after MMP14 knockdown. Results:iPSC→iNCC→iCPC differentiation model was established in vitro. The resulting iCPCs expressed osteo/chondrogenic marker genes, including SOX5, SOX6, SOX9, COL1A1, COL2A1, RUNX2, and TWIST1, and exhibited positive expression of mesenchymal stem cell markers CD90, CD105, and CD73. Upon further induction, functional cartilage spheroids were formed. Compared with normal auricular chondrocytes, auricular chondrocytes from microtia patients showed reduced expression of MMP14 at both mRNA and protein levels. Lentivirus-mediated shRNA knockdown of MMP14 in iCPCs resulted in a marked decrease in its mRNA and protein expression. MMP14 knockdown significantly impaired collective migration of iCPCs, as evidenced by reduced wound closure rates in scratch assays and decreased numbers of migrated cells in Transwell assays. High-content live-cell imaging revealed that MMP14-deficient iCPCs displayed more erratic migration trajectories and a lower straight-line migration ratio. Single-cell adhesion assays showed extracellular matrix (ECM)-dependent alterations: cell adhesion was enhanced on matrigel-coated surfaces but weakened under uncoated conditions. MMP14 knockdown also led to reduced proliferation, decreased collagen degradation, diminished F-actin expression, fewer peripheral adhesion sites, and downregulation of CD44 protein expression, without significantly affecting the expression of chondrogenic genes such as SOX6, SOX9, COL1A1, COL2A1, RUNX2, and TWIST1. Transcriptomic analysis further revealed that MMP14 knockdown significantly downregulated genes involved in extracellular matrix organization, cell adhesion, migration, and tissue development, with enrichment in pathways including ECM-receptor interaction, focal adhesion, and MAPK signaling. Conclusion:MMP14 plays a critical role in the directional migration of chondrogenic precursor cells by regulating ECM remodeling, adhesion signaling, and cytoskeletal proteins.

Objective:To investigate the effects and molecular mechanisms of Wilms tumor 1-associated proteins(WTAP)on the cell biological properties of esophageal squamous cell carcinoma(ESCC)cells.Methods:31 pairs of ESCC tissues and their paired paracancerous tissues that were surgically resected at the Second Clinical Medical College of Chuanbei Medical College between September 2019 and April 2021 were collected.The esophageal cancer cells KYSE30,KYSE410,KYSE150,KYSE510,TE-1,and normal human esophageal epithelial cells HET-1A were routinely cultured.Transfection reagents were used to transfect si-NC,si-WTAP#1 and si-WTAP#2 nucleic acids into KYSE150 and KYSE510 cells.The cells were divided into si-NC,si-WTAP#1 and si-WTAP#2 groups.The expressions of WTAP and MAP3K9 mRNA were detected in the cells of each group by qPCR assay.CCK-8 assay,clone formation assay,and scratch healing assay,Transwell assay were employed to detect the effects of knockdown of WTAP expression on ESCC cell proliferation,migration,invasion and apoptosis.WB assay was used to detect the expressions of WTAP,MAP3K9,EMT and MAPK pathway-related proteins in ESCC cells of each group knocked down of WTAP;immunohistochemistry to detect the expression of WTAP proteins in ESCC tissues,immunoprecipitation of methylated RNA(MeRIP)-qPCR assay to detect the level of MAP3K9 m6A in ESCC cells,actinomycin D assay to detect the stability of mRNA of MAP3K9,and database data to analyze the expression,target genes,functional enrichment,and interacting RNA of WTAP.Results:WTAP was highly expressed in ESCC tissues and cells(P<0.05 or P<0.01 or P<0.001)and correlated with the degree of differentiation(P<0.01);the expression of WTAP mRNA and its protein were successfully knocked down in KYSE150 and KYSE510 cells(P<0.01 or P<0.001);the knockdown of WTAP significantly inhibited the proliferation,migration and invasion of KYSE150 and KYSE510 cells(P<0.05 or P<0.01 or P<0.001),and promoted the apoptosis of KYSE150 and KYSE510 cells(P<0.05 or P<0.01).Knockdown of WTAP resulted in a significant decrease in the m6A level of MAP3K9(P<0.05),and its mRNA expression level and mRNA stability were both significantly reduced(P<0.05).Database data analysis showed that WTAP target genes clustered in the MAPK signaling pathway;the expression levels of MAP3K9,p-ERK,N-cadherin,and MMP9 were significantly reduced(P<0.05 or P<0.01),and the expression level of E-cadherin was significantly elevated(P<0.05 or P<0.01)in the KYSE150 and KYSE510 cells after knockdown of WTAP.Conclusions:WTAP is highly expressed in ESCC tissues and cells and correlates with their differentiation.It promotes the stability of MAP3K9 mRNA through m6A modification,activates the MAPK pathway and thus promotes the malignant biological behaviors of ESCC cells.

17q12 microdeletion syndrome is a rare genetic disease,commonly characterized by newly occurring mutations,which can cause abnormalities of the urinary and reproductive tract,diabetes mellitus,neurological and psychiatric disorders and mild deformities.This article reports a case of 17q12 microdeletion syndrome with CRYBB2 gene missense mutation,combined with menstrual abnormalities,multiple cysts in both kidneys,hypomagnesemia,hyperuricemia,small pancreatic morphology and low pancreatic enzyme levels.

Here, we report a previously unrecognized syndromic neurodevelopmental disorder associated with biallelic loss-of-function variants in the RBM42 gene. The patient is a 2-year-old female with severe central nervous system (CNS) abnormalities, hypotonia, hearing loss, congenital heart defects, and dysmorphic facial features. Familial whole-exome sequencing (WES) reveals that the patient has two compound heterozygous variants, c.304C>T (p.R102*) and c.1312G>A (p.A438T), in the RBM42 gene which encodes an integral component of splicing complex in the RNA-binding motif protein family. The p.A438T variant is in the RRM domain which impairs RBM42 protein stability in vivo. Additionally, p.A438T disrupts the interaction of RBM42 with hnRNP K, which is the causative gene for Au-Kline syndrome with overlapping disease characteristics seen in the index patient. The human R102* or A438T mutant protein failed to fully rescue the growth defects of RBM42 ortholog knockout ΔFgRbp1 in Fusarium while it was rescued by the wild-type (WT) human RBM42. A mouse model carrying Rbm42 compound heterozygous variants, c.280C>T (p.Q94*) and c.1306_1308delinsACA (p.A436T), demonstrated gross fetal developmental defects and most of the double mutant animals died by E13.5. RNA-seq data confirmed that Rbm42 was involved in neurological and myocardial functions with an essential role in alternative splicing (AS). Overall, we present clinical, genetic, and functional data to demonstrate that defects in RBM42 constitute the underlying etiology of a new neurodevelopmental disease which links the dysregulation of global AS to abnormal embryonic development.
Female ; Animals ; Mice ; Humans ; Child, Preschool ; Intellectual Disability/genetics* ; Heart Defects, Congenital/genetics* ; Facies ; Cleft Palate ; Muscle Hypotonia

In recent years, with the progress of minimally invasive treatment technology of hepatolithiasis in China, as well as the in-depth understanding of the pathological mechanism of hepatolithiasis and the integration of precision surgery concept and digital medical technology, minimally invasive surgery has gradually presented standardization and precision in the treatment of hepatolithiasis. Laparoscopic hepatectomy, as a representative of minimally invasive surgery, has become one of the most important means to treat hepatolithiasis. With the continuous accumulation of surgery, the improvement of operation technology and the innovation of instruments and equip-ment, laparoscopic hepatectomy has reached a new height in the treatment of hepatolithiasis. Com-bined with the clinical practice of the team, the authors review the relevant literature and explore the progress of laparoscopic hepatectomy in the treatment of hepatolithiasis, in order to provide clinicians with a more comprehensive and in-depth treatment reference, so as to improve the pro-gnosis and quality of life of patients.

In recent years, with the progress of minimally invasive treatment technology of hepatolithiasis in China, as well as the in-depth understanding of the pathological mechanism of hepatolithiasis and the integration of precision surgery concept and digital medical technology, minimally invasive surgery has gradually presented standardization and precision in the treatment of hepatolithiasis. Laparoscopic hepatectomy, as a representative of minimally invasive surgery, has become one of the most important means to treat hepatolithiasis. With the continuous accumulation of surgery, the improvement of operation technology and the innovation of instruments and equip-ment, laparoscopic hepatectomy has reached a new height in the treatment of hepatolithiasis. Com-bined with the clinical practice of the team, the authors review the relevant literature and explore the progress of laparoscopic hepatectomy in the treatment of hepatolithiasis, in order to provide clinicians with a more comprehensive and in-depth treatment reference, so as to improve the pro-gnosis and quality of life of patients.