Objective:To explore the mechanism by which miRNA-4469 (miR-4469) regulates the proliferation and invasion of renal cancer cells in vitro.Methods:The survival differences of patients with different expression levels of miR-4469 were analyzed based on the OncomiR database. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) method was used to detect the expression of miR-4469 in renal cancer cell lines ACHN, OS-RC-2, SK-RC-20, 769-P, A498 and normal renal tubular epithelial cell line HK-2, and the renal cancer cells with the lowest expression level of miR-4469 were divided into miR-4469 group and control group, and were transfected with miR-4469 mimic and negative control sequence, respectively. The CCK-8 assay was used to detect the cell proliferation ability (expressed as absorbance value) in the two groups, and Transwell assay was used to analyze the number of invasive cells in the two groups. TargetScan Release 8.0 software was used to predict the binding site between miR-4469 and protein disulfide isomerase A4 (PDIA4) mRNA, and dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-4469 and PDIA4 mRNA. qRT-PCR method was used to detect the expression of PDIA4 mRNA in cells of each group, and Western blotting method was used to detect the expression levels of PDIA4 protein and PI3K-AKT-m-TOR pathway proteins in cells of each group.Results:Analysis of relevant data from the OncomiR database showed that compared with patients with low miR-4469 expression, the overall survival of renal cancer patients with high miR-4469 expression was better ( P < 0.001). The relative expression of miR-4469 in each renal cancer cell line was lower than that in HK-2 cells (all P < 0.05), and the expression of miR-4469 in 769-P cells was the lowest, which were selected to perform the subsequent experiments. The proliferation ability of 769-P cells in the miR-4469 group was lower than that in the control group ( P < 0.01). The number of 769-P cell invasions in the miR-4469 group were less than that in the control group [(19±3) cells vs. (64±7) cells, t = 5.44, P = 0.002]. Compared with the co-transfection of wild-type PDIA4 and miR-4469 negative sequence group, the relative luciferase activity of cells in the co-transfection of wild-type PDIA4 and miR-4469 mimic sequence group was lower (0.42±0.07 vs. 1.01±0.08, t = 5.74, P = 0.001); there was no statistical difference in cell luciferase activity between the co-transfected mutant PDIA4 and miR-4469 negative sequence group and the co-transfected mutant PDIA4 and miR-4469 mimic sequence group (0.99±0.11 vs. 1.02±0.11, t = 0.19, P = 0.001). The relative expression levels of PDIA4 mRNA in 769-P cells in the miR-4469 group were lower than that in the control group (0.98±0.23 vs. 7.19±2.23, t = 2.77, P = 0.032). Compared with the control group, the expression of PDIA4 protein and PI3K-AKT-m-TOR pathway-related p-PI3K, p-AKT, p-mTOR, and p-SGK1 proteins in 769-P cells in the miR-4469 group were all lower (all P < 0.05). Conclusions:miR-4469 may be related to the survival of renal cancer patients, and its expression is down-regulated in various renal cancer cell lines. miR-4469 may inhibit the proliferation and invasion of renal cancer 769-P cells by regulating the PI3K-AKT-m-TOR pathway through PDIA4.

During orthodontic treatment,irregular and varying sized nodular bony protrusions may sometimes appear on the labial side of the patient's anterior alveolar bone,which is closely related to the differential bone remodeling on the inner and outer surfaces of the alveolar bone.Labial protuberances not only affect the aesthetic results of orthodontic treatment,but also pose potential risks to periodontal health.Currently,it is believed that the influencing factors of the formation of the labial protuberances may be related to the patient's gender and age,tooth movement speed,and extent of anterior teeth retraction.Labial protuberances typically resolve spontaneously,however,if persistent,alveoloplasty may be necessary for treatment.This review provides a summary on the occurrence,influencing factors of formation,potential biological mechanisms,and corresponding treatment methods of labial protuberances during orthodontic treatment.

Objective:To explore the genetic causes of abnormal isovaleryl carnitine (C5) metabolism in newborns.Methods:Retrospective study.The screening and clinical follow-up data of 34 neonates with elevated C5 levels shown by the tandem mass spectrometry test in Children′s Hospital, Zhejiang University School of Medicine from January 2018 to December 2021 were collected.Afterwards, their ethylenediaminetetraacetic acid (EDTA) anticoagulant venous blood was collected to extract genomic DNA.A total of 79 genes related to genetic metabolic diseases, such as ACADSB, IVD and ACADM, were captured by liquid-phase capture technology.High-throughput sequencing and bioinformatics analysis were used to acquire gene variation information and the genes were categorized by American College of Medical Genetics and Genomics classification standard.According to the results of genetic analysis, the newborns with C5 elevation were divided into 3 groups: non-mutation group(11 cases), ACADSB mutation group(16 cases) and IVD mutation group(7 cases). Wilcoxon rank sum test was performed to analyze the difference between these groups. Results:Among 34 neonates, 6 ACADSB variants were detected in 16 cases, and 2 of them [c.461G>A (p.G154E), c.746delC(p.P249Lfs*15)] were novel variants.Eleven IVD variants were detected in 7 cases, and 7 of them [c.118A>G(p.N40D), c.296-10C>G, c.302A>G(p.Y101C), c.537G>A(p.M179I), c.667C>T(p.R223W), c.983A>G(p.K328R), c.1147+ 5G>A] were never reported before.There was no significant difference in the C5 concentration in initial screening among the three groups ( P>0.05). Conclusions:Mutations in ACADSB and IVD genes are the main causes of augmented C5 levels in neonatal screening.For newly discovered genetic variants, functional prediction by multiple bioinformatics analysis software is recommended.And it is also important to carry out clinical follow-up and evaluation.

Objective:The relative expression of lncRNA NPIPA9 in prostate cancer tissues was analyzed, and the relative expression of miR-210-3p and its effect on the growth and migration of prostate cancer cells were detected by overexpressing lncRNA NPIPA9.Methods:The relative expression of lncRNA NPIPA9 in prostate cancer tissues was analyzed by Oncomine database. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the relative expression of lncRNA NPIPA9 in prostate cancer cell lines DU-145, PC-3, C4-2B, 22Rv1, LNCaP and normal prostate epithelial cell RWPE-1. Prostate cancer PC-3 cells were cultured in vitro and divided into control group (transfected with control vector 100 nmol/L) and NPIPA9 group (transfected with lncRNA NPIPA9 vector 100 nmol/L). The proliferation activity of PC-3 cells was detected by CCK-8 method. The migration ability of PC-3 cells was detected by Transwell method. Potential target of lncRNA NPIPA9 were predicted using bioinformatics techniques. The dual-luciferase reporter gene assay determined the target binding relationship between lncRNA NPIPA9 and miR-210-3p. The effect of lncRNA NPIPA9 on the relative expression of miR-210-3p in prostate cancer cells was detected by RT-qPCR. The effect of lncRNA NPIPA9 on the expression of nuclear factor kappa-B (NF-κB) pathway proteins in prostate cancer cells was detected by Western blotting. Measurement data were expressed as mean±standard deviation ( ± s), and t-test was used for comparison between two groups, one-way analysis of variance was used for comparison between multiple groups. Results:The expression of lncRNA NPIPA9 in prostate cancer tissue was lower than that in adjacent tissue, the difference was statistically significant ( P<0.01). The relative expression of lncRNA NPIPA9 in prostate cancer cell lines was lower than that in RWPE-1 cells, the difference was statistically significant ( P<0.01), and the relative expression of lncRNA NPIPA9 in prostate cancer PC-3 cells was the lowest, the difference was statistically significant ( P<0.01). Compared with the control group, lncRNA NPIPA9 had an inhibitory effect on the viability of prostate cancer PC-3 cells, the difference was statistically significant ( P<0.05). The migration numbers of PC-3 cells in the control group and NPIPA9 group were 101.70±8.63 and 45.97±8.83, respectively, and lncRNA NPIPA9 had an inhibitory effect on PC-3 cell migration, the difference was statistically significant ( P<0.01). lncRNA NPIPA9 can directly target miR-210-3p, the difference was statistically significant ( P<0.01). The relative expression of miR-210-3p in PC-3 cells in control group and NPIPA9 group were 5.32 ± 0.79 and 1.11 ± 0.56, respectively, and lncRNA NPIPA9 could directly down-regulate the expression of miR-210-3p in PC-3 cells, the difference was statistically significant ( P<0.01). Compared with the control group, lncRNA NPIPA9 can reduce the expression of NF-κB pathway proteins c-Myc, MMP-9, VEGF, p65, p50 in PC-3 cells. Conclusion:The expression of lncRNA NPIPA9 is down-regulated in prostate cancer tissues, and it reduces the proliferation and migration ability of prostate cancer PC-3 cells by targeting and negatively regulating miR-210-3p.

OBJECTIVES: To investigate the genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine (C4). METHODS: One hundred and twenty neonates with increased C4 levels detected by tandem mass spectrometry in the neonatal screening at Children's Hospital, Zhejiang University School of Medicine from January 2018 to June 2023 were included. The initial screening data and recalled data of C4 and C4/C3 were collected and converted into multiples of C4 reference range. Next generation sequencing was performed and the exons with adjacent 50 bp regions of ACAD8 and ACADS genes were captured by liquid phase capture technique. Variant information was obtained by bioinformatic analysis and the pathogenicity were classified according to the American College of Medical Genetics and Genomics criteria. The Wilcoxon rank sum test was used to analyze the differences in C4 levels among neonates with different variation types. RESULTS: In total, 32 variants in ACAD8 gene were detected, of which 7 variants were reported for the first time; while 41 variants of ACADS gene were detected, of which 17 variants have not been previously reported. There were 39 cases with ACAD8 biallelic variations and 3 cases with ACAD8 monoallelic variations; 34 cases with ACADS biallelic variations and 36 cases with ACADS monoallelic variations. Furthermore, 5 cases were detected with both ACAD8 and ACADS gene variations. Inter group comparison showed that the multiples of C4 reference range in initial screening and re-examination of the ACAD8 biallelic variations and ACADS biallelic variations groups were significantly higher than those of the ACADS monoallelic variations group (all P<0.01), while the multiples in the ACAD8 biallelic variations group were significantly higher than those in the ACADS biallelic variations group (all P<0.01). The multiples of C4 reference range in the initial screening greater than 1.5 times were observed in all neonates carrying ACAD8 or ACADS biallelic variations, while only 25% (9/36) in neonates carrying ACADS monoallelic variations. CONCLUSIONS ACAD8 and/or ACADS gene variants are the main genetic causes for elevated C4 in newborns in Zhejiang region with high genotypic heterogeneity. The C4 levels of neonates with biallelic variations are significantly higher than those of neonates with monoallelic variations. The cut-off value for C4 level could be modestly elevated, which could reduce the false positive rate in tandem mass spectrometry neonatal screening.
Child ; Humans ; Infant, Newborn ; Acyl-CoA Dehydrogenase/genetics* ; Genotype ; Phenotype ; Carnitine/metabolism* ; Mutation

Objective:To analyze the oral phenotype and gene variation of children with hypophosphatasia (HPP), and explore the genotype-phenotype correlations.Methods:Eight children diagnosed with HPP from January 2008 to January 2023 in The Children′s Hospital, Zhejiang University School of Medicine were recruited in this study. The pathogenic genes of 5 of them were sequentially analyzed and all of their oral manifestations, laboratory tests and genetic variation types were retrospectively analyzed.Results:A total of 8 children were recruited in the study, 3 males and 5 females, aged from 20 to 104 months, whose main complaints were premature deciduous tooth loss. Among them, 3 children were diagnosed with odonto HPP, and the other 5 children were diagnosed with childhood HPP, including 2 children was odonto HPP at the first diagnosis and modified as childhood HPP at the age of 5. The age range of first deciduous tooth loss is 9 to 18 months, and the age range of diagnosis was 20 to 104 months. The patients of odonto HPP only showed premature loss of deciduous anterior tooth, while the patients with childhood HPP also showed premature loss of multiple deciduous molars. Panoramic radiographic film revealed enlarged pulp chambers and radicular canals in some primary and permanent teeth. The enamel hypoplasia, hypoplastic short roots, and alveolar resorption of deciduous molar were observed in some cases. The serum alkaline phosphatase (ALP) (30-107 U/L) levels of all the patients were lower than that in the normal children of same age and gender, and the ALP value of the 1-3 years old girls with childhood HPP (30-33 U/L) was lower than that of the three children with odonto HPP (61-107 U/L), but there was no significant difference in statistical analysis. There were 8 variation sites of ALP liver/bone/kidney (ALPL) gene detected in 5 children and their families, all of which were missense variation, including the new variants in the mutations of c.1334C>G (p.Ser445Cys) and c.1259G>T (p.Gly420Val) that were not reported in the literature. One case was autosomal dominant inheritance and other 4 cases were complex heterozygous variation with autosomal recessive inheritance.Conclusions:Pediatric stomatologists are often the first doctors to detect childhood and odonto HPP. Diagnosis of mild HPP is often delayed. The severity of HPP is related to serum ALP level and ALPL gene mutation sites.

Objective:To explore the expression level of miR-769-3p in bladder cancer tissues, and observe the effect of silencing miR-769-3p on the migration ability and cell cycle of J82 cells by down-regulating the expression level of miR-769-3p in bladder cancer J82 cells.Methods:The OncomiR database was used to analyze the expression differences of miR-769-3p in bladder cancer tissues and adjacent tissues. J82 cells were transfected with Lipofectamine 2000 transfection reagent and divided into si-miR-769-3p group (transfected with miR-769-3p small molecule interference fragments) and control group (transfected with meaningless sequences). quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression level of miR-769-3p after transfection. The cell scratch test and flow cytometry were used to compare the migration ability and cell cycle differences between the two groups of J82 cells. The bioinformatics software MicroRNAdb was used to predict the target gene of miR-769-3p. The dual-luciferase reporter gene assay was used to verify the complementary binding of miR-769-3p to the target gene. qRT-PCR and Western blotting were used to detect the expression levels of miR-769-3p target gene. Measurement data were expressed as mean ± standard deviation ( ± s), and t-test was used for comparison between two groups. Results:The expression of miR-769-3p was significantly increased in bladder cancer tissues compared with adjacent tissues, the difference was statistically significant ( P<0.01). The relative expression of miR-769-3p in the si-miR-769-3p group (1.02 ± 0.16) was significantly lower than that of the control group (4.50 ± 0.60), the difference was statistically significant ( P<0.01). The cell migration rate of the si-miR-769-3p group [(26.67±3.98)%] was significantly lower than that of the control group [(61.86±4.70)%], the difference was statistically significant ( P<0.01). The proportion of cells in the G 0-G 1 phase in the si-miR-769-3p group [(57.66±5.74)%] was significantly higher than that in the control group [(31.26±3.24)%], the difference was statistically significant ( P<0.01). Dual-luciferase reporter gene assay confirmed that endothelin 3 ( EDN3) was the target gene of miR-769-3p. The relative expression of EDN3 mRNA in J82 cells in control group and si-miR-769-3p group was 1.99 ± 0.66 and 6.98 ± 0.76, compared with the control group, the EDN3 mRNA relative expression level of the si-miR-769-3p group was significantly higher than that of the control group, the difference was statistically significant ( P<0.01). Conclusion:Low expression of miR-769-3p can inhibit the migration of bladder cancer J82 cells and block the J82 cell cycle by promoting the expression of EDN3 gene.

Objective:To introduce the method and clinical effect of inferior pedicle flap used in reduction mammoplasty.Methods:From January 2010 to December 2019, 19 patients with moderate to severe macromastia who underwent reduction mammoplasty with inferior pedicle flap were enrolled in this study. The method of inferior pedicle flap was based on Robbins vertical inferior pedicle flap. New position of nipple areola and inferior pedicle flap were designed in terms of the breast anatomy.Results:A total of 19 patients (38 breasts) were included in this study. The average tissue reduction of one side breast was 570 g (385 g to 1 525 g). After operation, all patients were satisfied with natural appearance and nipple erection effect. There were no operation-related complications including nipple-areola necrosis. After 6 months to 8 years following-up, No inverted T hypertrophic scar occurred. Hyperpigmentation occurred on two patients with dark-skinned. The position of bilateral nipples and areola was higher in 1 patient after operation. The other 18 patients were satisfied with the position and shape of nipples and areola.Conclusions:It is a good choice for patients with moderate to severe macromastia to receive reduction mammoplasty with inferior pedicle flap. Through the method, large amount of breast tissue reduction and stable blood supply of nipple and areola complex could be obtained.

Objective: To explore procedures and clinical effect of the technique, combined reposition of rectus abdominis with abdminioplasty, for treating postpartum women with diastasis rectus abdominis and lax abdominal wall. Methods: Postpartum women with diastasis rectus abdominis and lax abdominal wall who underwent reposition of rectus abdominis and abdminioplasty were enrolled in this study. This combined technique was based on abdominoplasty, including reposition of rectus abdominis, umbilical transposition and tightening abdominal wall. Results: A total of 12 patients were included in this study. Umbilical transposition was performed in 9 patients. Skin was resected by 8 to 14 centimetre in width.There were no infection, skin necrosis, and delayed wound healing.Natural abdominal contour has been found in all patients and the abdominal perimeter as well as the waistline have shrunk.Further more, this combined technique has been shown to improve quality of life by reducing pain, restoring function, and increasing satisfaction with appearance.After 3 months to 2 years following up, all patients were satisfied with operative outcomes. Conclusions This combined technique has been shown to be a good choice for postpartum women with diastasis rectus abdominis and lax abdominal wall.

Objective: To evaluate the expression of histone H4 acetylation(Ac-H4) during the cleft palates formation induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin(TCDD) in C57BL/6J mice. Methods: Forty-eight pregnant C57BL/6J mice were completely randomly divided into two groups: ① TCDD group, mice were treated with 20ug/kg of TCDD on gestation day (GD) 10.5 by gastric perfusion; ② control group, mice were treated with an equivalent of corn oil. The head samples were collected and sliced in coronal plane on GD13.5, GD14.5 and GD15.5 respectively. Histone H4 acetylation in the palates were evaluated by immunohistochemical staining and Western Blot in the two groups. Results: Histone H4 acetylation was mainly expressed in the palatal epithelial cells and slightly expressed in mesenchymal cells. The expression level of histone H4 acetylation was 0.6002±0.2530, 0.9180±0.0941 and 0.8966±0.0908 respectively in control group on GD13.5, GD14.5 and GD15.5; while 1.0229±0.2779, 1.6095±0.2651 and 1.2758±0.1251 in TCDD group. There were statistically significant differences between the control group and TCDD group (P<0.05). Conclusions The histone H4 acetylation was involved in the cleft palate formation induced by TCDD in C57BL/6J mice.