Objective To investigate the effect of the mediator of DNA damage check point protein 1(MDC1)on proliferation,migration,invasion,cell cycle and cell apoptosis in cholangiocarcinoma(CCA)and the potential molecular mechanism.Methods The small interfering RNA(siRNA)specifically targeting MDC1 was used to transiently knock down MDC1.Recombined plasmid containing MDC1 was transiently transfected into RBE and Huh28 cells for over-expression of MDC1.Real time quantitative PCR(qPCR)and Western blotting were adopted to verify the effectiveness of MDC1 knockdown or overexpression.The proliferation of CCA cells was measured via CCK-8 and cell colony formation assays.Transwell and Invasion assays were used to detect cell migration and invasion while flow cytometry assays were employed to detect cell cycle and apoptosis.Gene set enrichment analysis(GSEA)was conducted to investigate the pathways which were significantly associated with MDC1,and the expression of p53 downstream protein was verified by Western blotting assay.Co-immunoprecipitation(Co-IP)assays were used to verify the interactions between MDC1 and p53.Flow cytometry and Western blotting assays were performed to find out whether MDC1 promoted cell cycle and cell apoptosis through p53 pathway.Based on The Cancer Genome Altas(TCGA)database,the difference in MDC1 expression levels between CCA and normal tissues was analyzed,and the correlations between the MDC1 expression levels and the clinical prognosis of CCA patients were investigated.Results Knockdown of MDC1 in RBE and Huh28 cells significantly inhibited cells proliferation,migration and invasion,significantly decreased the proportion of cells in S phase,and significantly increased the proportion of cells in G0/G1 phase and apoptosis rate while overexpression of MDC1 could significantly promote cell proliferation,migration and invasion,significantly increase the proportion of cells in S phase,and significantly decrease the proportion of cells in G0/G1 phase and apoptosis rate.It was found that MDC1 interacted with p53 in RBE and Huh28 cells,and MDC1 significantly down-regulated the expressions of p53,p-p53(Ser-15),BAX,PUMA and p21,but significantly up-regulated the expression of Bcl-2,which in turn promoted the tumorigenesis of CCA.MDC1 was up-regulated in CCA tissues compared to the normal tissues,and the high expressions of MDC1 were significantly associated with poor clinical outcomes of CCA patients.Conclusion MDC1 promotes the development of CCA by suppressing the p53 pathway,and MDC1 may be a candidate marker for the poor prognosis in CCA.

Objective:To construct a risk prediction score for the needs of coronary care unit (CCU) care in stable condition acute ST-segment elevation myocardial infarction (STEMI) patients who receive percutaneous coronary intervention (PCI) treatment.Methods:The clinical data of 805 STEMI patients who accepted PCI in the First Hospital of Jilin University from November 2017 to October 2018 were retrospectively analyzed. Among the patients, 654 patients from November 2017 to July 2018 were served as the modeling group, the patients with needs of CCU had 125 cases, and the patients without needs of CCU had 529 cases; 151 patients from August 2018 to October 2018 were served as the validation group, the patients with needs of CCU had 28 cases, and the patients without needs of CCU had 123 cases. Binary Logistic regression analysis was used to establish the risk prediction model and determine the score standards. The critical value was determined according to the best Youden index of receiver operating characteristic (ROC) curve.Results:Among 805 patients with STEMI, 153 cases (19.01%) had the needs of CCU, and the most common reason was pump failure (heart failure and cardiogenic shock, 113 cases). In the modeling group, age (60 to 74 years old, OR = 1.513, 95% CI 0.945 to 2.424, P = 0.085; ≥75 years old, OR = 2.740, 95% CI 1.371 to 5.478, P = 0.004), total ischemic time>4 h ( OR = 1.701, 95% CI 1.022 to 2.831, P = 0.041), admission shock index ≥0.8 ( OR = 1.910, 95% CI 1.178 to 3.099, P = 0.009), multi-vessel disease ( OR = 2.090, 95% CI 1.272 to 3.432, P = 0.004), preoperative diseased vessels thrombolysis in myocardial ischemia (TIMI) blood flow grade 0 ( OR = 2.099, 95% CI 1.313 to 3.353, P = 0.002), acute anterior myocardial infarction ( OR = 3.696, 95% CI 2.347 to 5.819, P<0.001) and previous history of stroke ( OR = 3.927, 95% CI 2.057 to 7.500, P<0.001) were independent risk factors for CCU needs in STEMI patients undergoing PCI. The scoring criteria were as followings: age<60 years old was given 0 score, 60 to 74 years old 1 score, ≥75 years old 2 score; total ischemic time>4 h in 1 score, admission shock index ≥0.8 2 scores, multi-vessel disease 2 scores, preoperative diseased vessels TIMI blood flow grade 0 2 scores, acute anterior myocardial infarction 3 scores, previous history of stroke 3 scores, and the total score was 15 scores. The patients with 0 to 6 scores were low-risk, and the patients with 7 to 15 scores were high-risk. ROC curve analysis result showed that, in modeling group, the area under curve (AUC) of risk prediction score for predicting the needs of CCU in STEMI patients was 0.740 (95% CI 0.692 to 0.788, P = 0.580); in validation group, the AUC of risk prediction score for predicting the needs of CCU in STEMI patients was 0.755 (95% CI 0.658 to 0.853, P = 0.755). Conclusions:A predictive risk score based on seven risk factors such as age, total ischemic time, admission shock index, multi-vessel disease, preoperative diseased vessels TIMI blood flow grade, acute anterior myocardial infarction and previous history of stroke is constructed in order to predict the needs of CCU in STEMI patients with stable condition who receive PCI treatment. It can be used to help doctors to identify high-risk patients before the admission to CCU, thus providing simple and practical clinical tool for rational allocation of limited CCU resources.

Objective:To investigate the inhibitory effect of RMT1-10-induced tolerogenic dendritic cells (Tol-DCs) in vitro on high-risk corneal allograft rejection in mice and its mechanism. Methods:One hundred SPF male BALB/c mice and fifty SPF male C57BL/6 mice were selected.Bone marrow-derived immature dendritic cells (imDCs) obtained from C57BL/6 mice were divided into imDCs group, mature dentritic cells (mDCs) group, RMT1-10 group, and IgG isotype control group.The imDCs in the four groups were cultured with no intervention, lipopolysaccharide, RMT1-10 and lipopolysaccharide, or IgG isotype antibody and lipopolysaccharide for 7 days according to grouping.The expression levels of different phenotypes of DCs including CD11c, CD80, CD86, major histocompatibility complex (MHC)-Ⅱ, T cell immunoglobulin and mucin domain containing molecule (Tim)-4 and CD103 in the four groups were detected by flow cytometry.The concentrations of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in the DCs supernatants were determined by enzyme-linked immunosorbent assay.A mixed lymphocyte culture system was established, and the stimulation index (SI) of CD4 + T cell proliferation stimulated with DCs was detected by cell counting kit 8 method.Corneal neovascularization was induced by corneal stromal suture in BALB/c mice, and the 80 mice with neovascularization in 4 quadrants growing into the middle and peripheral cornea were used as recipients.The recipient mice were randomized into imDCs group, mDCs group, RMT1-10 group, and IgG isotype control group using the random number table method, with 20 mice in each group.An injection of corresponding DCs (1×10 6 cells/100 μl) was administered to the recipient mice via the tail vein according to grouping.At 7 days following the injection, C57BL/6 mice were used as donors and penetrating keratoplasty was performed.Within one month after the operation, signs of corneal grafts rejection, including opacity, edema and neovascularization, were observed by slit lamp biomicroscopy and scored every day.At 21 days after the operation, 5 recipients selected from each group were subcutaneously injected with naive C57BL/6 splenocytes (1×10 6 cells/100 μl) behind the ear.The delayed type hypersensitivity (DTH) was evaluated by ear swelling at 24 hours after the subcutaneous injection.The use and care of experimental animals complied with the Regulations on the Management of Experimental Animals promulgated by the State Science and Technology Commission.This study protocol was approved by an Ethics Committee of the Affiliated Hospital of Chengde Medical University (No.CYFYLL2020055). Results:Compared with mDCs group, the expressions of CD80, CD86 and MHC-Ⅱ, and the percentage of Tim-4-positive cells in CD11c-positive cells were significantly decreased in RMT1-10 group, showing statistically significant differences (all at P<0.001). The percentage of Tim-4-positive cells were significantly decreased in RMT1-10 group than imDCs group, and the percentage of CD103-positive cells in RMT1-10 group was significantly higher than imDCs group, mDCs group and IgG isotype control group (all at P<0.001). The concentrations of IL-10 and TGF-β in the cell culture supernatant of RMT1-10 group were significantly higher than those of the other three groups, with statistically significant differences (all at P<0.001). There were statistically significant differences in the SI of CD4 + T cell proliferation simulated by DCs ( Fgroup=1 833.00, P<0.001; Fratio=230.40, P<0.001; Finteraction=3.06, P=0.01). The SI of DCs/CD4 + T cells ratio at 1∶5, 1∶10, 1∶20 and 1∶40 were all significantly lower in imDCs group than mDCs group, and were all significantly lower in RMT1-10 group than imDCs group (all at P<0.05). There was a statistically significant difference in corneal graft survival curve among various groups ( χ2=77.69, P<0.001). The survival rate of RMT1-10 group was significantly higher than that of imDCs group ( χ2=9.74, P=0.002), and the survival rate of imDCs group was significantly higher than that of mDCs group ( χ2=31.02, P<0.001). The ear swelling of recipient mice of positive control group, mDCs group, IgG isotype control group, imDCs group and RMT1-10 group was (503.6±17.2), (475.7±17.6), (456.2±18.8), (225.2±39.4), (118.1±12.6), and (106.4±7.4) μm, with a statistically significant difference among them ( F=377.10, P<0.001). The mice ear swelling was more serious in positive control group than mDCs group, more serious in IgG isotype control group than imDCs group, and more serious in imDCs group than RMT1-10 group (all at P<0.05). Conclusions:RMT1-10 can inhibit the rejection of high-risk corneal transplantation in mice, the mechanism of which may be attributed to inducing imDCs to transform into Tol-DCs in vitro and up-regulating the expression of TGF-β and IL-10, which promotes antigen-specific immune tolerance after adoptive transfer, thereby indirectly prolongs the survival of corneal grafts.

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 identify key genes that may be regulated by fatty acid alteration in pancreatic cancer through tumor transcriptome screening,and to explore the expression of zinc finger protein 143(ZNF143)in pancreatic cancer and its effect on the migration and invasion of pancreatic cancer cells.Methods·The R language was utilized to integrate transcriptome data,including the GSE164760 dataset from the Gene Expression Omnibus(GEO)database,179 pancreatic cancer tissue samples and 4 adjacent non-cancerous tissue samples from The Cancer Genome Atlas(TCGA)database,as well as 167 normal pancreatic tissue samples from the Genotype-Tissue Expression(GTEx)database.We conducted screening and analysis of potential differential genes that may be induced by dysregulation of fatty acid metabolism in pancreatic cancer.After treating pancreatic cancer cells with palmitic acid(PA)and oleic acid(OA)for 24 hours,the mRNA levels of candidate genes were detected by qRT-PCR.According to the median expression level of the screened gene,pancreatic cancer patients in the TCGA database were divided into two groups with high and low expression of ZNF143.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway and Gene Ontology(GO)enrichment analyses were performed for the differential genes of the two groups.siRNA was used to knock down the expression of ZNF143 in pancreatic cancer cells,and the effects on cell migration and invasion were examined by wound healing assay and invasion assay.Western blotting was used to explore the impact of ZNF143 on epithelial mesenchymal transition(EMT)-related proteins and the Wnt/β-catenin pathway.Results·The bioinformatics database was processed to analyze key genes associated with the up-regulation of genes in lipid metabolism disorders in pancreatic cancer and liver cancer.Among them,ZNF143 was a potential gene associated with fatty acid accumulation in pancreatic cancer.In vitro experiments confirmed that the mRNA level of ZNF143 was significantly up-regulated after treating pancreatic cancer cells with palmitic acid or oleic acid.Both KEGG and GO enrichment analyses demonstrated that the differentially expressed genes associated with ZNF143 were predominantly enriched in adhesion pathways.In functional experiments,the migration and invasion abilities of pancreatic cancer cells transfected with ZNF143 siRNA were reduced,and the expression of EMT-related proteins was also decreased,potentially related to the activation of the Wnt/β-catenin pathway.Conclusion·Fatty acid accumulation up-regulates the mRNA expression of ZNF143 in pancreatic cancer cells,and ZNF143 may enhance the migration and invasion of these cells by facilitating EMT through activation of the Wnt/β-catenin pathway.

Objective:To evaluate the safety and efficacy of the treatment for oppressive spinal by microwave ablation combined with percutaneous fixation and open decompression.Methods:From January 2015 to September 2018, 20 patients with 26 spinal metastatic were treated with microwave ablation combined with percutaneous fixation and open decompression, including 13 males and 7 females with an average age of 43.85±18.67 years (range, 16-79 years). The locations of the lesions included: 9 in the thoracic, 11 in the lumbar. The tumors' type: myeloma 2 cases, leukemia 1 case, liver cancer 4 cases, osteosarcoma 2 cases, lung cancer 5 cases, kidney cancer 1 case, esophagus cancer 1case, cervical cancer 1 case, intestinal cancer 1 case, prostate cancer 1 case, adenoid cystic cancer 1case. Preoperatively all the patients suffered with the local pain and the spinal cord or nerve root compression symptoms. All 20 cases were examined with CT or MRI to determine the lesions and the sizes of metastasis, as well as to evaluate the ablation zone. The entry of the pedicle screws were performed by Wiltse method through paravertebral muscles. After that the lesions were treated with partial resection for decompression of spinal cord or nerve root, and followed with microwave ablation at the metastasis site. Thermometer was used to monitor the temperature at the central and posterior edges of the vertebral body. The surrounding important tissue were cooled by ice saline. 13 patients were performed with vertebroplasty for enhancement the intensity of the vertebral body. The visual analogue scale (VAS) score was used to evaluate the effect of pain relief after surgery. The postoperative neurological function and performance status were evaluated using Frankel grading and Eastern Cooperative Oncology Group (ECOG).Results:Each lesion was heated for 5.43±2.07 min (range, 3-10 min). The power of microwave ablation was 40-60 W. The mean blood loss during operation was 852.50±514.40 ml (range, 100-1 700 ml). The mean operating time was 4.11±0.99 h (range, 2.5-6.0 h). The temperature inside the lesion was 70-85 ℃. The temperature of the surrounding tissue was maintained at<43 ℃ by repeated frozen saline flush. All cases were followed up for 8.45±2.01 months (range, 6-14 months) without any recurrence. The VAS score of the 20 patients at 48 h, 1 month, 3 months and 6 months after operation were 1.55±1.23, 2.70±0.87, 2.40±1.14 and 3.05±1.00 points, which were all statistically lowerthan the preoperative score 5.95±1.18 ( P<0.05). The Frankel grading of 14 patients had at least one grade improvement 6 months after operation. There were 8 patients shown markedly improved ECOG score 6 months after surgery. Only one case suffered from reduced myodynamiaof lower limb and covered in one month after system treatment. Conclusion:The microwave ablation combined with percutaneous fixation and open decompression could resolve the spinal and nerve compression, relieve the pain in metastatic spinal oppression, reconstruct the stability, and improve the quality of lives, which is a safe and effective palliative surgical method.

Modulating Tankyrases (TNKS), interactions with USP25 to promote TNKS degradation, rather than inhibiting their enzymatic activities, is emerging as an alternative/specific approach to inhibit the Wnt/β-catenin pathway. Here, we identified UAT-B, a novel neoantimycin analog isolated from Streptomyces conglobatus, as a small-molecule inhibitor of TNKS-USP25 protein-protein interaction (PPI) to overcome multi-drug resistance in colorectal cancer (CRC). The disruption of TNKS-USP25 complex formation by UAT-B led to a significant decrease in TNKS levels, triggering cell apoptosis through modulation of the Wnt/β-catenin pathway. Importantly, UAT-B successfully inhibited the CRC cells growth that harbored high TNKS levels, as demonstrated in various in vitro and in vivo studies utilizing cell line-based and patient-derived xenografts, as well as APC^min/+ spontaneous CRC models. Collectively, these findings suggest that targeting the TNKS-USP25 PPI using a small-molecule inhibitor represents a compelling therapeutic strategy for CRC treatment, and UAT-B emerges as a promising candidate for further preclinical and clinical investigations.