Objective:To investigate the effects of cardiomyocyte-specific TSHR knockout on myocardial insulin resistance in a mouse model of heart failure.Methods:A cardiomyocyte-specific TSHR knockout(TSHR CKO) mouse model was generated by crossing TSHR flox/flox mice with α-MHC-Cre transgenic mice. F1 offspring(TSHR flox+ /-α-MHC-Cre+ mice) were interbred to obtain TSHR CKO mice, and littermate TSHR flox/flox mice served as controls. Fasting blood glucose levels were measured using a Roche glucometer, and fasting insulin levels were determined using a mouse insulin ELISA kit. Cardiac function and the expression of ANP, BNP, β-MHC, IRS-1, IRβ, GLUT-4, phosphorylated IRS-1, phosphorylated IRβ, and TSHR in myocardial tissues were assessed by echocardiography, RT-qPCR, Western blot, and immunohistochemistry(IHC). IHC was also used to evaluate the myocardial expression of IRS-1 and GLUT-4, while Masson′s trichrome staining was performed to assess the degree of myocardial fibrosis. Comparisons between groups were made using ANOVA. Results:The insulin resistance index indicated no systemic insulin resistance in all groups. Echocardiography revealed that compared with the FLOX group, the FLOX-ISO group exhibited significant reductions in left ventricular ejection fraction(LVEF), left ventricular fractional shortening(LVFS), left ventricular end-systolic volume(LVESV), and left ventricular end-diastolic volume(LVEDV), along with increases in heart weight-to-body weight(HW/BW), left ventricular end-systolic diameter(LVESD), and left ventricular end-diastolic diameter(LVEDD). Compared with the FLOX-ISO group, the CKO-ISO group showed significantly increased LVEF and decreased LVESV, LVEDV, LVESD, and LVEDD. Immunohistochemistry results demonstrated that myocardial TSHR knockout increased the expression of IRS-1 and GLUT-4. Additionally, RT-qPCR and Western blotting showed that ANP, BNP, and β-MHC expression levels were reduced, while IRS-1, IRβ, and GLUT-4 expression levels were elevated in TSHR CKO mice. Conclusion:Cardiomyocyte-specific TSHR knockout improves myocardial insulin resistance in mice with heart failure.

Objective:To investigate the effects of cardiomyocyte-specific TSHR knockout on myocardial insulin resistance in a mouse model of heart failure.Methods:A cardiomyocyte-specific TSHR knockout(TSHR CKO) mouse model was generated by crossing TSHR flox/flox mice with α-MHC-Cre transgenic mice. F1 offspring(TSHR flox+ /-α-MHC-Cre+ mice) were interbred to obtain TSHR CKO mice, and littermate TSHR flox/flox mice served as controls. Fasting blood glucose levels were measured using a Roche glucometer, and fasting insulin levels were determined using a mouse insulin ELISA kit. Cardiac function and the expression of ANP, BNP, β-MHC, IRS-1, IRβ, GLUT-4, phosphorylated IRS-1, phosphorylated IRβ, and TSHR in myocardial tissues were assessed by echocardiography, RT-qPCR, Western blot, and immunohistochemistry(IHC). IHC was also used to evaluate the myocardial expression of IRS-1 and GLUT-4, while Masson′s trichrome staining was performed to assess the degree of myocardial fibrosis. Comparisons between groups were made using ANOVA. Results:The insulin resistance index indicated no systemic insulin resistance in all groups. Echocardiography revealed that compared with the FLOX group, the FLOX-ISO group exhibited significant reductions in left ventricular ejection fraction(LVEF), left ventricular fractional shortening(LVFS), left ventricular end-systolic volume(LVESV), and left ventricular end-diastolic volume(LVEDV), along with increases in heart weight-to-body weight(HW/BW), left ventricular end-systolic diameter(LVESD), and left ventricular end-diastolic diameter(LVEDD). Compared with the FLOX-ISO group, the CKO-ISO group showed significantly increased LVEF and decreased LVESV, LVEDV, LVESD, and LVEDD. Immunohistochemistry results demonstrated that myocardial TSHR knockout increased the expression of IRS-1 and GLUT-4. Additionally, RT-qPCR and Western blotting showed that ANP, BNP, and β-MHC expression levels were reduced, while IRS-1, IRβ, and GLUT-4 expression levels were elevated in TSHR CKO mice. Conclusion:Cardiomyocyte-specific TSHR knockout improves myocardial insulin resistance in mice with heart failure.

Objective·To examine the expression level of protein arginine methyltransferase 6(PRMT6)in breast carcinoma tissues and to assess its impact on the proliferative and migratory behaviors of breast cancer cells.Methods·The PRMT6 transcriptome sequencing data between 33 tumor tissues and normal tissues from The Cancer Genome Atlas(TCGA)database was analyzed through the R language.The gene expression profile interactive analysis(GEPIA2)online database was used to analyze the difference of PRMT6 expression in normal breast tissues and breast cancer tissues.By using the immunohistochemistry(IHC)data of human normal breast tissues and breast cancer tissues from Human Protein Atlas(HPA)database to analyze the protein expression of PRMT6.IHC was used to detect the expression of PRMT6 in breast cancer tissues and paired para-tumor tissues from 27 clinical samples.After PRMT6 was knocked down with small interfering RNA(siRNA)in MDA-MB-231 and MCF-7 cells,the expression of PRMT6 was detected by qRT-PCR and Western blotting.The proliferation ability of breast cancer cells was measured with cell counting kit-8(CCK-8)assay and colony formation assay.The effect of PRMT6 on the migration ability of breast cancer cells was detected by wound healing assay and Transwell assay.By using the RNA-sequence data from GSE210948 of Gene Expression Omnibus(GEO)database,differentially expressed genes were analyzed in control and low expression groups of PRMT6.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was performed to reveal the signaling pathways associated with PRMT6.Cell cycle analysis was detected by flow cytometry.The expressions of cyclin D1 and EMT-related proteins(E-cadherin,N-cadherin and Vimentin)were detected by Western blotting after knocking down PRMT6.Results·Bioinformatics analysis and IHC results showed that PRMT6 was highly expressed in breast cancer tissues compared with normal tissues(P=0.000)and para-tumor tissues(P=0.001).qRT-PCR and Western blotting results verified that the siRNA significantly reduced the expression level of PRMT6 in MDA-MB-231 and MCF-7 cell lines compared with the control group(mRNA:P=0.006,P=0.004;P=0.001,P=0.043.Protein:P=0.035,P=0.001;P=0.003,P=0.002).After knocking down PRMT6,the proliferation(P=0.014,P=0.000;P=0.003,P=0.003)and migration(P=0.000,P=0.000;P=0.000,P=0.002)ability of breast cancer cells were inhibited significantly.The KEGG pathway enrichment analysis showed that the expression of PRMT6 affected the cell cycle pathway.After knocking down PRMT6,the expression of cyclin D1 decreased in protein level(P=0.021,P=0.000;P=0.034,P=0.014)and transcription level(P=0.036,P=0.001;P=0.044,P=0.000).Knock down of PRMT6 increased the number of cells in G0/G1 phase(P=0.000;P=0.003)and decreased the number of cells in G2/M phase of the cell cycle.The expression level of E-cadherin increased(P=0.002,P=0.012;P=0.043,P=0.003),while the expression levels of N-cadherin(P=0.004,P=0.041;P=0.032,P=0.034)and Vimentin(P=0.028,P=0.005;P=0.024,P=0.001)decreased in PRMT6 knockdown cells.Conclusion·PRMT6 is highly expressed in breast cancer,which can promote the proliferation and migration of breast cancer cells.

Objective To investigate the overall and subgroup efficacy,subgroup differences,predictors of efficacy and safety of roxadustat in the treatment of refractory non-severe aplastic anemia(NSAA)and low-risk myelodysplastic syndromes(LR-MDS).Methods Patients with refractory NSAA and LR-MDS who were admitted to the Department of Hematology,Peking Union Medical College Hospital from August 2020 to December 2022 were enrolled.All patients received first-line standard treatment,including recombinant human erythropoietin(rhEPO)for at least 3 months before roxadustat treatment.All patients received roxadustat 2.5 mg/kg every other day for at least 3 months,and were followed up for at least 8 months.The clinical characteristics of patients,roxadustat efficacy,predictors of efficacy,adverse effects,relapse and disease clonal evolution were analyzed.Results A total of 40 patients including 24 refractory NSAA and 16 LR-MDS were included.median age was 56(18～81)years and 40%were males.65%of the patients were transfusion dependent.Median follow-up was 21(9～34)months.22.5%,25.0%,47.5%,55.0%,57.5%,60.0%and 50.0%of the patients achieved haemato-logical improvement-erythroid(HI-E)at months 1,2,3,4,5,6,and end of the follow-up period,respectively,and no factors affecting HI-E were identified.The hemoglobin change from baseline was statistically different between the two groups at the end of the follow-up period.50%of patients were relieved from transfusion dependence.Adverse reactions were reported in 22.5%of patients.28.5%of patients relapsed after achieving HI-E,with a median time to relapse of 7(4～12)months.No clonal evolution was observed at the end of the follow-up period.Conclusions Our preliminary findings suggested that Roxadustat may be effective for patients with NSAA or LR-MDS refractory to conventional therapies and rhEPO,with mild adverse effects and low relapse rate.The degree of hemoglobin improvement may be better in the refractory NSAA patients.

Humans ; Cyclosporine/therapeutic use* ; Anemia, Aplastic/drug therapy* ; Neoplasms/drug therapy* ; Immunosuppressive Agents/therapeutic use* ; Benzoates/therapeutic use*

Non-coding RNAs (ncRNAs) are a class of functional RNAs that play critical roles in different diseases. NcRNAs include microRNAs, long ncRNAs, and circular RNAs. They are highly expressed in the brain and are involved in the regulation of physiological and pathophysiological processes of central nervous system (CNS) diseases. Mounting evidence indicates that ncRNAs play key roles in CNS diseases. Further elucidating the mechanisms of ncRNA underlying the process of regulating glial function that may lead to the identification of novel therapeutic targets for CNS diseases.
Humans ; RNA, Untranslated/genetics* ; MicroRNAs/genetics* ; RNA, Long Noncoding/genetics* ; RNA, Circular ; Central Nervous System Diseases/genetics*

Objective: To investigate the anti-tumor effect of CTL cells on colon cancer xenograft in nude mice after knocking out the immune check point CTLA-4 by CRISPR/Cas9 technology. Methods: A specific small guide RNA (sgRNA) for CTLA-4 was designed to construct sgRNA/Cas9 plasmid, which was then transfected into CTL using a lentiviral vector to obtain CTL cells with CTLA-4 deletion (CTLA-4 KO CTL). The transfection efficiency of the plasmid and the deletion efficiency of CTLA-4 were verified. BALB/c nude mice were randomly divided into two groups to prophylactically inoculate CTLA-4 KO CTL (experimental group) or CTL (control group); 3 days later, the animals of two groups were inoculated with colon cancer cell line LS174-T to observe the tumor formation rate and tumor formation time. After constructing colon cancer xenograft model in nude mice, the animals were randomly divided into two groups, respectively treated with CTLA-4 KO CTL (experimental group) and CTL (control group) cells to observe the tumor growth volume and survival time of mice. The serum levels of TNF-α and IFN-γ in nude mice were detected. Results: sgRNAwas designed and CRSIPR/Cas9 system with lentivirus as vector was successfully constructed. CTL cells were transfected with the established CRSIPR/ Cas9 system, and the highest transfection efficiency was up to (28.80±0.62)%. After transfection, the deletion efficiency of CTLA-4 was detected by Flow cytometry. The CTLA-4 expression of CTLA-4 KO CTL group was significantly lower than that of CTL group [(0.91±0.25)% vs (42.70±2.72)%, P<0.05]. In prophylactic assay, the formation rate of colon cancer xenografts in the experimental group was significantly lower than that in the control group(33.33%vs100%,P<0.05). In treatment assay, the tumor volume in the experimental group was significantly inhibited compared with the control group ([503±23.9] vs [911.2±51.4] mm3, P<0.05), and the survivaltimeoftheexperimentalgroupwassignificantlyprolonged (mediansurvivaltime:78dvs42d,P<0.05); Moreover, the secretion levels of serumTNF-α([268.93±17.04]pg/mlvs[148.26±20.07]pg/ml,P<0.05) and IFN-γ(315.38±18.67 pg/ml vs 202.92±29.32 pg/ml, P<0.05) in the experimental group were significantly higher than those in the control group. Conclusions: The lentiviral vector CRSIPR/Cas9 system is an effective gene editing method; its successful deletion of CTLA-4 in CTL cells can significantly inhibit the tumor formation rate of colon cancer xenografts in nude mice and enhance the anti-tumor effect of CTLon colon cancer xenografts.