Objective To construct human EpCAM eukaryotic recombinant plasmid ,and to study the expression of EpCAM pro‐tein in HepG2 cells .Methods The recombinant plasmid ,named pcDNA3 .1(+ )‐EpCAMwas constructed by cloning EpCAM gene into eukaryotic vector pcDNA3 .1(+ ) .Then HepG2 cells were transfected with EpCAMrecombinant plasmid .The eukaryotic ex‐pression of EpCAM protein was verified by immunofluorescence ,Western Blotting and flow cytometry .Results The construction of EpCAM recombinant eukaryotic plasmid was identified by restriction enzyme digestion .The results of immunofluorescence , Western Blotting ,and flow cytometry consistently indicated that EpCAM protein were successfully expressed in HepG2 cells .Con‐clusion Human EpCAM eukaryotic vector is constructed and EpCAM protein could be expressed well in HepG2 cells ,which laid a foundation for further research on the function ofhepatocarcinoma cells highly expressing EpCAM .

OBJECTIVE: To explore the genetic basis for a child with mental retardation. METHODS: Whole exome sequencing was carried out for the child. Candidate variant was screened based on his clinical features and verified by Sanger sequencing. RESULTS: The child was found to harbor a c.995_1002delAGACAAAA(p.Asp332AlafsTer84) frameshift variant in the SYNGAP1 gene. Bioinformatic analysis suggested it to be pathogenic. The same variant was not detected in either parent. CONCLUSION The c.995_1002delAGACAAAA(p.Asp332AlafsTer84) frameshift variant of the SYNGAP1 gene probably underlay the mental retardation in this child. Above finding has expanded the spectrum of SYNGAP1 gene variants and provided a basis for the diagnosis and treatment for this child.
Child ; Humans ; Intellectual Disability/genetics* ; Frameshift Mutation ; High-Throughput Nucleotide Sequencing ; Computational Biology ; Heterozygote ; Mutation ; ras GTPase-Activating Proteins/genetics*

Objective To investigate the mechanism of metformin for regulating tumor-stromal cell cross-talk in breast cancer.Methods Tumor associated fibroblasts(CAFs)co-cultured with breast cancer cells were treated with metformin,and the changes in expressions of hypoxia-inducible factor-1α(HIF-1α),p-AMPK,stroma-derived factor-1(SDF-1)and interleukin-8(IL-8)in the CAFs were detected using ELISA,RT-qPCR or Western blotting;Transwell assay was used to evaluate the invasiveness of the tumor cells and its changes following treatment with exogenous SDF-1,IL-8 and TGF-β1.The effects of HIF-1α shRNA or overexpression plasmid,AMPK shRNA,and treatment with OG(a proline hydroxylase inhibitor)or 2-OXO(a proline hydroxylase activator)were examined on p-AMPK,HIF-1α,SDF-1 and IL-8 expressions and invasiveness of the CAFs.Results Metformin treatment significantly increased the expression levels of p-AMPK,SDF-1 and IL-8(P<0.05)and decreased HIF-1α expression(P<0.05)without affecting AMPK expression level(P>0.05)in the CAFs.The invasion ability of metformin-treated breast cancer cells was significantly decreased(P<0.05).Exogenous SDF-1 and IL-8,HIF-1α overexpression,and OG-induced upregulation of HIF-1α all significantly attenuated the inhibitory effects of metformin on breast cancer cell invasion(P<0.05)and HIF-1α,SDF-1 and IL-8 expressions in CAFs(P<0.05).Transfection with HIF-1α shRNA or treatment with 2-OXO significantly decreased the invasiveness of breast cancer cells(P<0.05).P-AMPK knockdown significantly suppressed the inhibitory effect of metformin on HIF-1α expression in CAFs and on invasion of breast cancer cells(P<0.05).Treatment with TGF-β1 partially decreased the inhibitory effect of metformin on HIF-1α expression in CAFs and invasiveness of the breast cancer cells(P<0.05).Conclusion Metformin suppresses HIF-1α expression in CAFs to block tumor-stromal cross talk in breast cancer.

Objective To explore the regulation of DIRAS2 gene expression by acetylated STAT3 and its involvement in the proliferation of triple-negative breast cancer(TNBC)cells.Methods The expression levels of DIRAS2 and acetylated STAT3 in TNBC tissues and cells were analyzed by database query,Western blotting,and qRT-PCR.TNBC cell lines MDA-MB-231 and SUM159 were selected,and lentivirus or plasmid was used to construct DIRAS2 overexpression and STAT3 wild or Lys685 mutation cell lines.The CCK-8 assay was used to evaluate the effect of DIRAS2 and STAT3 acetylation on the proliferation of TNBC cells.Western blotting,pyrosequencing,ChIP and IP were employed to investigate the regulatory effect and mechanism of acetylated STAT3 on DIRAS2 expression.Results The expression of DIRAS2 was decreased in TNBC tissues and cells.Pyrosequencing analysis found that the methylation level of CpG islands in the DIRAS2 promoter was increased in TNBC cells compared with normal breast epithelial cells,which promoted the growth of cancer cells.Furthermore,TNBC cells showed an increase in STAT3 acetylation,which was accompanied by a shift in the methylation status of the DIRAS2 promoter.ChIP and IP experiments showed that acetylated STAT3 could bind to the DIRAS2 promoter,and the STAT3 Lys685 mutation disrupted the interaction between STAT3 and DNMT1.Conclusion Acetylated STAT3 induces DIRAS2 promoter methylation by recruiting DNMT1,leading to loss of DIRAS2 expression and cancer cell proliferation in TNBC.

Objective To investigate the mechanism of metformin for regulating tumor-stromal cell cross-talk in breast cancer.Methods Tumor associated fibroblasts(CAFs)co-cultured with breast cancer cells were treated with metformin,and the changes in expressions of hypoxia-inducible factor-1α(HIF-1α),p-AMPK,stroma-derived factor-1(SDF-1)and interleukin-8(IL-8)in the CAFs were detected using ELISA,RT-qPCR or Western blotting;Transwell assay was used to evaluate the invasiveness of the tumor cells and its changes following treatment with exogenous SDF-1,IL-8 and TGF-β1.The effects of HIF-1α shRNA or overexpression plasmid,AMPK shRNA,and treatment with OG(a proline hydroxylase inhibitor)or 2-OXO(a proline hydroxylase activator)were examined on p-AMPK,HIF-1α,SDF-1 and IL-8 expressions and invasiveness of the CAFs.Results Metformin treatment significantly increased the expression levels of p-AMPK,SDF-1 and IL-8(P<0.05)and decreased HIF-1α expression(P<0.05)without affecting AMPK expression level(P>0.05)in the CAFs.The invasion ability of metformin-treated breast cancer cells was significantly decreased(P<0.05).Exogenous SDF-1 and IL-8,HIF-1α overexpression,and OG-induced upregulation of HIF-1α all significantly attenuated the inhibitory effects of metformin on breast cancer cell invasion(P<0.05)and HIF-1α,SDF-1 and IL-8 expressions in CAFs(P<0.05).Transfection with HIF-1α shRNA or treatment with 2-OXO significantly decreased the invasiveness of breast cancer cells(P<0.05).P-AMPK knockdown significantly suppressed the inhibitory effect of metformin on HIF-1α expression in CAFs and on invasion of breast cancer cells(P<0.05).Treatment with TGF-β1 partially decreased the inhibitory effect of metformin on HIF-1α expression in CAFs and invasiveness of the breast cancer cells(P<0.05).Conclusion Metformin suppresses HIF-1α expression in CAFs to block tumor-stromal cross talk in breast cancer.

ObjectivesTo investigate characteristics and influencing factors of short-term variation of systolic blood pressure of community hypertension patients in Shanghai. MethodsBased on the standardized blood pressure measurement data of hypertensive patients in Shanghai （2018‒2021） and the noninfectious chronic disease management system database， coefficient of variation of systolic blood pressure （CV_S） was described and the influencing factors were analyzed. ResultsAmong 112 680 community hypertension patients， males accounted for 46.87% with a median age of 69 years. CV_S was 0.038 6 ± 0.035 0. Generalized linear model analysis showed the following influencing factors of CV_S： gender， females were higher than males， B=0.032， P<0.001； age group， 60‒69 years， 70‒79 years old group， and ≥80 years groups were all higher than <50 years group， B=0.042， 0.056， and 0.074 respectively with P values of 0.020， 0.002， and <0.001 respectively； smoking， smoking cessation and smoking everyday was lower than never smoking， B=-0.032 and-0.028 respectively with P values of 0.023 and 0.007 respectively； systolic blood pressure， 140‒159 mmHg group， 160‒179 mmHg group， and ≥180 mmHg group were lower then <140 mmHg group， B=-0.039， -0.091， and -0.175 respectively with P values all <0.001； and measurement season， autumn was lower than spring， （B=-0.056， P<0.001）. The paired test showed that CV_S calculated from the three measurements was 1.04% higher than that calculated from the two measurements （P<0.001）. ConclusionThe coefficient of variation of systolic blood pressure of community hypertension patients in Shanghai has large variation， and was influenced by many factors.