Search

Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P <0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells ( P <0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P =0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P =0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P =0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P =0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P <0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P <0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells ( P <0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells ( P <0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated ( P <0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells ( P <0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment ( P >0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group ( P <0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P <0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P =0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P =0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P =0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P <0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P <0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P <0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased ( P <0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly ( P >0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.

Objective: To investigate the expression of fragile-site associated tumor suppressor (FATS) in non-small cell lung cancer and its relationship with clinicopathological features and prognosis. Methods: A total of 140 non-small cell lung cancer (NSCLC) cases and 30 adjacent normal tissues were used to detect the expression level of FATS protein, and to analyze the relationship of FATS protein expression and clinicopathological features and prognosis of NSCLC. Results: Western blot showed that the expression of FATS in adjacent normal tissues was significantly higher than that in non-small cell lung cancer tissues. The results of immunohistochemistry showed that the high expression rate of FATS in 140 cases of NSCLC was 40.0%, and the high expression rate of FATS in 30 cases of adjacent tissues was 73.3%. The difference was statistically significant ( P =0.01). Further analysis showed that the TNM stage ( P =0.044) and lymph node metastasis ( P =0.022) were significant difference between FATS high expression group and low expression group. The 6-year overall survival (OS) rates of NSCLC patients with FATS high-expression and low-expression were 57.1% and 23.8%, respectively, and the 6-year disease-free survival (DFS) rates were 53.6% and 21.4%, respectively, with statistically significant differences ( P =0.001). In Cox multivariate analysis, we found gender ( HR =1.658, P =0.028; HR =1.684, P =0.023), TNM staging ( HR =2.327, P =0.019; HR =2.332, P =0.013) and FATS expression ( HR =0.532, P =0.010; HR =0.538, P =0.009) were independent prognostic factors for both OS and DFS of NSCLC patients. Conclusions: The expression of FATS protein is associated with the development and is an independent prognostic factor of NSCLC patients. The detection of FATS protein is expected to be a new biomarker for evaluating the prognosis of NSCLC patients.

The numbers of estrogen receptor (ER) in human peripheral leucocytes in 22 women with climacteric syndrome were measured by radioligand method. The results were compared with those of 12 normal child-bearing-age women. It wat found that the contents of leucocytic ER in climacteric syndrome patients were significantly lower than normal child-bearing-age women. The authors used a Chinese prescription--Liuwei Dihuang Pills (LDP) to treat the patients for 2 months. The numbers of leucocytic ER were significantly increased after treatment. The data indicate that decrease of ER levels in cell may involve in the pathogenesis of climacteric syndrome. LDP not only increases plasma estradiol levels, but also increases the leucocytic ER levels. This may be the basis of the therapeutic effect on the disease.

The number of estrogen receptors (ER) in human peripheral leucocytes in 12 women with menopausal type II diabetes was measured with radio-ligand binding method. The results were compared with those of 12 menopausal women without diabetes and 12 normal women of childbearing age. It was found that the number of ER in the patients was significantly decreased. Our data indicate that decrease of ER level in leukocytes may be related to the pathogenesis of type II diabetes in menopausal period.

The number of estrogen receptor (ER) in human peripheral leucocytes in 13 men with gynecomastia were measured by radioligand binding method. The results were compared with those of 13 sex-and age-matched healthy subjects. It was found that the number of ER in leucocytes was significantly increased in gynecomastia (Rs of leucocytes were 1054 +/- 254 sites/cell). It suggested that increase of ER levels play an important role in the pathogenesis of gynecomastia.