Objective To establish human choriocarcinoma JeG-3 cell line resistant to floxuridine (FUDR)and describe the characteristics of this FUDR-resistant subline.The thymidylate synthase (TS) expression level in FUDR-resistant subline was also discussed.Methods The FUDR-resistant sub-line JeG-3/FUDRA was established by intermitted exposure to grads increased FUDR.Reversed microscope was used to observe the morphological changes in FUDR-resistant sub-line.Population doubling time was calculated and compared based on the growth curve of these two cell lines,cell cycles and chromosomal ploidy were assayed with flow cytometry methods.The chemo-luminescence assay was used to detect the hormone secretion by two kinds of cell lines.The resistant index (RI) was measured by cell counting kit-8 (CCK-8)assay.Quantitative RT-PCR was used to detect the mRNA expression level of TS and we also detected the TS mRNA expression level in different doses exposed subline.Results The RI of JeG-3/FUDRA was 31.62.Compared with the JeG-3 cell,the FUDR-resistant cell line had gross changes in morphological,cell growth,cell cycles and chromosomal numbers.The ability of human chorionic gonadotrop(hCG) and progesterone secretion was lower in JeG-3/FUDRA subline.The trend of TS mRNA expression was:while exposed to low concentration of FUDR,the TS mRNA expression level was downregulated,then followed the increasing dose of the drug,the expression level of TS mRNA ascended gradually.When the terminal concentration was reached,the expression level of TS mRNA in JeG-3/FUDRA subline was higher than that of JeG-3 cell line (P＜0.05).Conclusions We established the FUDR-resistant subline of JeG-3 successfully.The TS mRNA expression level is stage-related to the different concentration and different phase in FUDR exposure.Our data suggested that TS mRNA expression level may not be used as a biomarker to predict the chemosensitivity in FUDR-based chemotherapy.

Objective To examine the complement component 1 Q subcomponent-binding protein (C1QBP) gene expression in human resistance choriocarcinoma cell lines and its parental cell line JeG-3,and to investigate whether silence C 1QBP by small interference RNA could reverse the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs.Methods Expression of C1QBP mRNA and protein in cells were detected by real-time fluorogenic quantitative PCR and western blot,respectively.The difference of C 1QBP expression was compared between human resistance choriocarcinoma cell lines and its parental cell line JeG-3.Sub-cellular location was proved by confocal immunofluorescence microscopy.A lentiviral vector containing short hairpin RNA (shRNA) targeting C 1QBP was constructed and cotransfected with the packaging plasmid mixture into 293T cells by lipofectamine 2000.The human resistance choriocarcinoma cell lines were infected with the packaged lentivirus.Real-time fluorogenic quantitative PCR and western blot were used to validate whether the C 1QBP gene expression was silenced.The cell counting kit 8(CCK8)was used to determine the drug sensitivity.Results (1)The C1QBP mRNA expression levels among four human resistance choriocarcinoma cell lines[JeG-3/floxuridiuum (FUDR),JeG-3/methotrexate (MTX),JeG-3/etoposide (VP),JeG-3/dactinomycin (KSM)] were 2.520±0.680,1.770±0.230,1.940±0.090 and 1.740±0.350 folds compared to that in JeG-3 cells.The C1QBP protein was higher expression level in human resistance choriocarcinoma cell lines than that in JeG-3.The immunofluorescence methods and confocal analysis showed that C1QBP localized predominantly in the mitochondrial matrix.(2)The C1QBP mRNA expression in JeG-3/FUDR cells after infected with lentiviral vector were decreased by 93.1％ (P＜0.01).The protein expression of C 1QBP in JeG-3/FUDR cells after infected with lentiviral vector were almost completely suppressed.The resistance indexes of four human resistance choriocarcinoma cell lines(JeG-3/FUDR,JeG-3/MTX,JeG-3/VP,JeG-3/KSM) were respectively 86.3％,93.9％,92.8％ and 89.9％,which were decreased remarkably by knockdown the C 1QBP expression (P＜0.05).Conclusions C1QBP is overexpressed in human resistance choriocarcinoma cell lines compared with parental cell line JeG-3.Inhibition of C 1QBP by lentivirus-mediated small interference RNA could effectively reverses the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs.

Objective:To explore the expression of programmed death ligand-1 (PD-L1) protein in grade Ⅲ non-special type invasive breast cancer and its related factors, so as to provide a basis for immunotherapy.Methods:A total of 63 surgically resected specimens of grade Ⅲ non-special type invasive breast cancer diagnosed by pathology in Cancer Hospital & Shenzhen Hospital of Chinese Academy of Medical Sciences from February 2017 to February 2021 were collected. The HE-stained sections were reviewed, and the proportion of immune cells (IC) in all invasive active tumor cells in the tumor sections was calculated. The expression of PD-L1 (SP142) protein in all specimens was detected by immunohistochemistry. The relationship between the positive expression of PD-L1 protein and the clinicopathological parameters was analyzed, and the Pearson correlation test was used to analyze the relationship between the expression of PD-L1 (SP142) and the degree of IC infiltration.Results:Among 63 patients, 19 patients (30.2%) were triple-negative type, 34 patients (53.9%) were luminal type, and 10 patients (15.9%) were human epidermal growth factor receptor 2 (HER2) overexpression type. The positive rate of PD-L1 (SP142) in grade Ⅲ non-special type invasive breast cancer was 77.8% (49/63). The positive rate of PD-L1 (SP142) in triple-negative type was 94.7% (18/19), the positive rate in non-triple-negative type was 70.5% (31/44), and the difference between the two groups was statistically significant ( P = 0.047). The positive rate of PD-L1 (SP142) in estrogen receptor (ER)-negative patients or progesterone receptor (PR)-negative patients was both 90.3% (28/31), which was higher than that of ER-positive patients or PR-positive patients (65.6%, 21/32), and the difference was statistically significant ( P = 0.018); the positive rate of PD-L1 (SP142) was not related to the patient's age, tumor site, tumor maximum diameter and number, vascular tumor thrombus, nerve invasion, lymph node metastasis, HER2 status and Ki-67 positive index (all P > 0.05). The expression of PD-L1 was positively correlated with the degree of IC infiltration ( r = 0.716, P<0.001). Conclusion:In grade Ⅲ non-special type invasive breast cancer, the patients with triple-negative type has a high positive rate of PD-L1, and the patient with negative ER or PR has a high positive rate of PD-L1; the tumor IC infiltration is positively correlated with the expression of PD-L1.