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Objective @#To investigate the relationship between circular RNA homeodomain interacting protein kinase 3 (circHIPK3) and the activation of rat microglia (RM) cells.@*Methods @#In vitro , RM cells were cultured and randomized into normal and oxygen⁃glucose deprivation/reperfusion (OGD/R) groups , and the expression level of circHIPK3 in each group was detected by RT⁃qPCR. The circHIPK3 lentiviral vector with puromycin resistance was constructed , and the overexpression (OE) group and negative control (NC) group were set up. The optimal multiplicity of infection (MOI) for RM cells was determined based on fluorescence expression , and puromycin was used to screen RM cells stably expressing circHIPK3. The cells of OE and NC groups were treated with OGD/R , and the expression levels of ionized calcium binding adaptor molecule 1 (Iba ⁃1) and eukaryotic tumor necrosis factor receptor superfamily (CD40) were detected by Western blot. The circHIPK3 translational protein potential was analyzed by the circRNAdb database , while the potential binding microRNAs on circHIPK3 were predicted by circBank and Starbase databases.@*Results @#OGD/R down⁃regulated circHIPK3 in RM cells ( P < 0. 000 1) . The sequencing results were accurate and the lentiviral vector of circHIPK3 was constructed successfully. The optimal MOI of RM cells was 80 , puromycin at a concentration of 2 μg/ml was used to screen RM cell lines stably expressing circHIPK3. RT⁃qPCR results showed that the expression level of circHIPK3 was significantly higher in the OE group compared with the NC group (P < 0. 01) . Western blot results revealed that the expression levels of Iba and CD40 in the OE group were markedly lower than those in the NC group (P < 0. 05) . Protein translation analysis showed that circHIPK3 encoded a polypeptide of 404 amino acids with two internal ribosome entry sites (IRES) and an open reading frame (ORF) . Database analysis uncovered that circHIPK3 could target eight specific miRNAs , namely hsa⁃miR⁃3529⁃5p , hsa⁃miR⁃379⁃5p , hsa⁃miR⁃506⁃3p , hsa⁃miR⁃33 , hsa⁃miR⁃450b⁃5p , hsa⁃miR551b⁃3p , hsa⁃miR⁃193 , and hsa⁃miR⁃508 ⁃3p.@*Conclusion @#The overexpression of circHIPK3 effectively suppresses OGD/R⁃induced activation of RM cells. It has the potential to encode peptides and may act as a miRNA sponge. These findings provide a foundation for further study of circHIPK3 functions.
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Objective To investigate the influence of Nei endonuclease Ⅷ-like protein 1(NEIL1)on H2O2-induced apoptosis and autophagy in lens epithelial cells(LECs).Methods Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expression of NEIL1 messenger ribonucleic acid(mRNA)in the anterior lens capsule and LEC line SRA01/04 cells from age-related cataract(ARC)patients and macular epiretinal membrane patients who underwent the clear lens extraction.SRA01/04 cells were then divided into the control group,OE-Vector group and OE-NEIL1 group.RT-PCR and Western blot(WB)were used to detect the overexpression efficiency.Subsequently,the cells were divided into the control group,H2O2 group,OE-Vector H2O2 group and OE-NEIL1 H2O2 group;cell viability was detected by Cell Count-ing Kit-8,WB was used to evaluate the expression of autophagy-related proteins(ATG7,P62,Beclinl,LC3-Ⅰ and LC3-Ⅱ)and apoptosis-related proteins(Bax and Bcl-2),and fluorescent staining was adopted to measure cell apoptosis and mito-chondrial membrane potential.Results The expression of NEIL1 mRNA in the anterior lens capsule samples from ARC patients was significantly lower than that of macular epiretinal membrane patients,and the expression of NEIL1 mRNA in the SRA01/04 cells in the H2O2 group was also lower than that in the control group(both P＜0.05).The results of RT-PCR and WB revealed that the expressions of NEIL1 mRNA and protein in the SRA01/04 cells of the OE-NEIL1 group were signif-icantly higher than those in the OE-Vector group(both P=0.000).Compared with the control group,the viability of SRA01/04 cells in the H2O2 group decreased,the expression of the Bax protein was up-regulated,the Bcl-2 protein was down-regulated,viable cells labeled with Mito-Tracker decreased,and apoptotic cells labeled with Annexin V-FITC in-creased,with statistical significances(all P＜0.05).Compared with the OE-Vector H2O2 group,in the OE-NEIL1 H2O2 group,the viability of SRA01/04 cells significantly improved,the autophagy-related proteins(ATG7,P62,Beclin1,LC3-Ⅰand LC3-Ⅱ)and Bcl-2 protein were significantly up-regulated,the Bax protein was down-regulated,viable cells marked with Mito-Tracker increased,and apoptotic cells labeled with Annexin V-FITC decreased(all P＜0.05).Conclusion Under the oxidative stress induced by H2O2,NEIL1 can promote autophagy of LECs,maintain homeostasis of LECs,and then increase cell viability and reduce apoptosis of LECs,participating in the occurrence and development of ARC.
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Objective:To explore the diagnostic and grading value of combination of 68Ga -1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid- D-Phe1-Tyr3-Thr8-octreotide ( 68Ga-DOTA-TATE) and 18F-flurodeoxyglucose ( 18F-FDG) dual probes in multi-parameter positron emission tomography (PET)/magnetic resonance (MR) imaging in pancreatic neuroendocrine neoplasm (PNEN). Methods:From April 9th, 2020 to February 24th, 2022, in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, the clinical data and the imaging of 68Ga-DOTA-TATE PET/MR and 18F-FDG PET/MR of 59 patients with pancreatic tumors (27 male, 32 female, aged 22 to 75 years old(51.8±13.3) years old), confirmed by surgical or biopsy pathology were retrospectively analyzed. All the cases were divided into PNEN group (42 cases) and non-PNEN group (17 cases) according to pathological results. Among which 39 patients with PNET were further divided into grade 1 group (G1 group, 27 cases) and grade 2 group (G2 group, 12 cases). Non-zero parameters were selected via the least absolute shrinkage and selection operator (LASSO) regression approach, and a logistic regression model was established by combination of the selected features and the corresponding non-zero coefficients. The measurement data with non-normal distribution were compared by Mann-Whitney U test. The receiver operating characteristic (ROC) curve were used to detemine the optimal cut off value to assess the dignostic efficiency. Results:Compared with those of non-PNEN group, the parameters of PNEN group increased, which included maximum standard uptake value of 68Ga-DOTA-TATE(SUV Gmax, 46.70 (22.37, 76.35) vs. 7.12 (4.75, 8.64)), mean standard uptake value of 68Ga-DOTA-TATE(SUV Gmean, 25.50 (13.18, 43.90) vs. 3.65 (2.89, 4.69)), peak standard uptake value of 68Ga-DOTA-TATE (SUV Gpeak, 27.17 (12.39, 46.97) vs. 5.46 (4.12, 6.56)), total lesion somatostatin receptor (SSR) expression (TLSRE, 68.21 (32.52, 440.96) vs. 26.02 (14.87, 69.57)), SUV Gmax/maximum standard uptake value of 18F-FDG (SUV Fmax, 12.71 (3.80, 21.70) vs. 1.10 (0.52, 2.35)), tumor to background ratio of 68Ga-DOTA-TATE (TBR G, 13.31 (5.54, 22.38) vs. 1.57 (1.31, 2.66)), tumor to liver ratio of 68Ga-DOTA-TATE(T/L G, 6.54 (2.90, 9.63) vs. 0.74 (0.65, 0.94)), tumor to spleen ratio of 68Ga-DOTA-TATE (T/S G, 2.36 (0.97, 3.70) vs. 0.25 (0.23, 0.38)), tumor to mediastinum ratio of 68Ga-DOTA-TATE (T/M G, 104.41 (34.03, 206.52) vs. 16.00 (12.87, 21.46)), SUV Gmax/minimum apparent diffusion coeffecient (ADC min, 55.14 (22.50, 96.37) vs. 6.76 (4.39, 12.76)) and SUV Gmean/ADC min (34.57 (13.47, 55.13) vs. 3.57 (2.46, 6.81)), and the differences were statistically significant ( U=28.00, 25.00, 32.00, 198.00, 54.00, 31.00, 28.00, 19.00, 10.00, 56.00 and 44.00, all P<0.01). The area under the curve (AUC) and diagnostic accuracy of dual-probe PET/MR imaging in the diagnosis of PNEN and non-PNEN were 0.941 and 96.6%, respectively. The AUC and diagnostic accuracy of model Y 1 in the diagnosis of PNEN and non-PNEN were 0.959 and 96.6%, respectively. There was no significant difference in AUC between model Y 1 and dual-probe PET/MR imaging in PNEN diagnosis ( P>0.05), however combining model Y 1 could improve the accuracy of PNEN diagnosis (100.0%). Compared with those of PNET G1 group, the parameters of G2 Group were higher, which included the maximum diameter of tumor (2.69 cm (2.08 cm, 5.00 cm) vs. 1.50 cm (1.20 cm, 2.50 cm)), metabolic tumor volume (MTV, 7.56 mL (4.45 mL, 53.57 mL) vs. 2.16 mL (1.22 mL, 5.48 mL)), total lesion glycolysis (TLG, 22.24 (11.95, 189.85) vs. 3.81 (2.11, 18.67)), tumor to background ratio of 18F-FDG (TBR F, 2.94 (2.00, 3.96) vs. 1.48 (1.29, 3.72)), tumor to liver ratio of 18F-FDG (T/L F, 2.32 (1.35, 2.98) vs. 1.08 (0.90, 2.17)) and SSR-expressing tumor volume (SRETV, 8.00 (3.06, 40.00) vs. 1.91 (0.95, 4.88)), and the differences were statistically significant ( U=66.00、66.00、77.00、93.00、90.00、65.50, all P<0.05). The maximum diameter of tumor was the best single parameter for the differential diagnosis of PNET G2 and G1, AUC was 0.796 and the cutoff value was 1.90 cm. The model Y 2, which combined the maximum diameter of tumor and TBR G had an AUC of 0.835 for the differential diagnosis of PNET G2 and G1. There was no significant difference in AUC between the maximum diameter of tumor and model Y 2 ( P>0.05). However the combination of the maximum diameter of tumor and model Y 2 could improve the accuracy of differential diagnosis of PNET G2 and G1 (94.87%). Conclusion:The combination of multi-parameter of 68Ga-DOTA-TATE and dual-probe 18F-FDG PET/MR imaging can improve the diagnostic and grading accuracy of PNEN, which may be helpful in the selection of clinical treatment for patients.
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Objective To evaluate the hot and cold characteristics of 10 antibiotics.Methods MTT assay was used to investigate the effects of 10 antibiotics on the growth and proliferation of cultured SMMC7721 cells and MFC-7 cells in vitro .Morphological changes were observed under the inverted microscope.Results The 10 antibiotics,namely,ampicillin,cefixime,cefpodoxime proxetil,cefaclor,cefalexin,azithromycin,clarithromycin, roxithromycin,doxycycline and oxytetracycline showed cold or cool characteristics.Morphological observation showed that cells treated with these drugs presented decreased cell density and turned round.Conclusion The results of this study demonstrated that the cytological method can be used to evaluate the hot and cold characteristics of western drugs.This simple and reliable method will promote research on Chinese medicalization of western drugs.