Malignant tumor is a kind of integral disease which is closely related to human aging process. Tumor is the result of dynamic balance disorders between cell damage and human host's ability to repair during aging. Host factors play an important role in the occurrence and development of malignant tumor. We should not only treat human tumors, but also treat the patients with tumors. We should enhance the patient's ability of disease resistance and self-healing. To realize the long-term survival of cancer patients with tumor is an important direction of future cancer research. Delaying aging and preventing malignant tumor have much in common. Anti-aging can effectively delay the occurrence of malignant tumors and reduce their mortality.

OBJECTIVE： To induce trans-differentiation of mouse embryonic fibroblasts (MEF) into hepatic stem (iHepSCs)-like cells using a tetracycline-controlled Hnf1β and Foxa3 expression system，and to provide a model for investigations into molecular mechanisms involved in the reprogramming process. METHODS： TetO-Hnf1β-EGFP and TetO-Foxa3-mCherry tetracyclic-control vectors were constructed. 293FT cells were used to determine the optimal dosages of doxycycline (Dox) in the trans- differentiation system of MEF. After vectors were transfected into 293FT cells，expression of Hnf1β and Foxa3 genes under different dosages of Dox were detected by real-time fluorescence quantification PCR (qPCR). After 20 days of induction and then amplification of the treated cells，an iHepSCs-Dox cell line was obtained. Biological characteristics of the cell line were documented using the CCK-8 method，colon formation assay，alkaline phosphatase staining，in vitro induced differentiation and reverse transcription-PCR (RT-PCR)，etc. In addition，the induction effects of tetracyclic-controlled reprogramming system were evaluated. RESULTS： The qPCR results show that expressions of exogenous Hnf1β and Foxa3 genes were turned on at 24-h after treatment with 100 ng/mL of Dox and were turned off at 48-h after Dox was removed. After treatment with Dox，markers for bile duct cells (CK19)，common marker of liver and bile (CK18)，and liver stem/precursor cell markers (Dlk1，Sox9 and EpCAM) were detected. Positive alkaline phosphatase staining and cell clones were observed. Furthermore， iHepSCs-Dox cells could be induced into hepatic cells which were similar to iHepSCs. With cessation of Hnf1β and Foxa3 expressions after Dox was removed from the medium， proliferation rates of iHepSCs- Dox cells and expressions of CK18 and EpCAM decreased significantly. In addition， cell clones formation and inductive differentiation to hepatic cells failed as well. CONCLUSION： The trans-differentiation system of MEF which was controlled by tetracyclic has been successfully established. This model can be used for investigations into molecular mechanisms involved in cellular reprogramming processes. Additionally，our results indicate that continuous expressions of exogenous Hnf1β and Foxa3 were necessary for maintenance of iHepSCsDox cell stemness.

OBJECTIVE： To identify and analzye differentially-expressed and radiation-sensitive protein biomarkers in urines from rats after their total- body irradiation. METHODS： 30 rats were irradiated whole body with single doses of 0，1，5 Gy ⁶⁰Co γ rays at a dose rate of 1 Gy/min. Urine samples were collected at 1 and 3 days post irradiation. A label- free mass spectrometric technology was used to measure relative levels of differentially expressed proteins. Uniport and DAVID databases were conducted using Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The STRING online website was used to construct a protein-protein interaction network. RESULTS： A total of 1 069 proteins were identified. Compared with the negative control group， a total of 279 shared proteins were differentiallyexpressed in the 1 and 5 Gy irradiation groups after irradiation at 1 and 3 days， of which 190 were upregulated and 97 were down-regulated. Results from the GO analyses show that the differentially-expressed proteins were mainly involved in the biological processes such as response to drug， oxidation-reduction process，and aging. Most of the cellular components of these screened proteins were extracellular exosomes and extracellular space，and these proteins exerted protein homodimerization activity，protein binding and calcium ion binding and other molecular functions. KEGG enrichment analyses indicate that changes of the differentiallyexpressed proteins mainly participated in several signal pathways including antibiotic biosynthesis， glutathione metabolism， carbon metabolism， and metabolic pathways. CONCLUSION： Our proteomic study is a comprehensive analysis of the rat urine proteome following radiation exposure. Our analyses suggest that the up-regulated proteins Gusb，Reg3g，GCLM，AZGP1 and down-regulated protein Ly6d can be used as candidate biomarkers of radiation sensitivity for future research.

OBJECTIVE： To evaluate developmental toxicity of cerium nitrate with in vitro postimplantation whole embryo cultures (representing early，middle and late embryonic developments) using a micromass test. METHODS： Embryos of GD 9.5 SD rats were cultured at 37 ℃ for 48 h in immediately centrifuged serum which contained different doses of cerium nitrate (0.00， 0.50， 0.75 and 1.00 mmol/L). Growth，development and total morphological scores (TMS) of the embryos were documented according to the Brown's Method. Results of BALB/c 3T3 cytotoxicity and of the early embryonic development toxicity of cerium nitrate were evaluated using the whole embryo prediction model by ECVAM. In addition， limb bud cells of embryos were isolated from GD13 SD rats and cultured in micro-mass culture for 5 days with 0.03，0.06， 0.13， 0.25， 0.50， 1.00， 2.00 and 3.00 mmol/L cerium nitrate. The 50% inhibitions of cell viability and growth (IC₅₀) of limb bud cells were determined by neutral red staining and the 50% inhibition of cells differentiation (ID₅₀) was measured by alcian blue staining. The embryonic toxicities of cerium nitrate in the middle and advanced stages of embryos were evaluated with the prediction model of the micro-mass test (ECVAM). RESULTS： In the in vitro post-implantation whole embryo culture test，the no-observed-adverseeffect level (NOAEL) of cerium nitrate on embryo growth was 0.50 mmol/L. Cerium nitrate above 0.75 mmol/L inhibited embryonic growth， and reduced the yolk sac diameter (YSD) and crown-rump length (CRL) significantly (P < 0.05). Cerium nitrate at 0.75 mmol/L also reduced the number of somites (P < 0.05) which indicated developmental malformation of embryos. In the micro- mass test， the NOAEL of cerium nitrate on proliferation of limb buds was 0.25 mmol/L，the NOAEL on differentiation from limb bud cells to chondrocytes was 0.12 mmol/L， the IC50 and ID50 for limb bud cells were 1.23 and 0.76 mmol/L， respectively. CONCLUSION： Based on the prediction model of whole embryo cultures and the micro- mass test， our results indicate that cerium nitrate was a weak embryotoxic chemical.

OBJECTIVE： To investigate effects of miR- 223- 3p on proliferation and apoptosis of nonsmall cell lung cancer cells. METHODS： Relative expression levels of miR-223-3p were detected by realtime quantitative PCR in 24 patients with non-small cell lung cancer (NSCLC) and expression levels of epithelial transformation sequence 2 (ECT2) protein were analyzed by immunohistochemistry. Correlations between the expression levels of miR-223-3p and ECT2 were analyzed. Human NSCLC cells A549 were divided into normal control group， transfected control group and miR-223-3p over-expression group. The normal control group cells were not treated and cultured normally，and the transfected control group cells were transfected with empty plasmid vector using transfection reagents. The miR-223-3p over-expression group was transfected with miR-223-3p mimics. Proliferation rates of cells from each group were detected by the MTT and the plate colony formation assays. Apoptosis rates of cells in each group were detected by flow cytometry. Expression levels of the ECT2 protein were detected by Western blot assay. After ECT2 was knocked down by siRNA， A549 cells were divided into normal control， transfected control and ECT2 silenced groups. Then， changes of cell proliferation and apoptosis in each group were detected. RESULTS： Expressions of miR-223- 3p in cancer tissues were significantly lower than that in paracancerous tissues while expressions of ECT2 protein were higher， showing a negative correlation between them (r=- 0.666， P < 0.05). Compared with the normal control and the transfected control groups， proliferation rates of miR- 223- 3p in the over- expression group was decreased (P < 0.05)，expressions of ECT2 protein were significantly decreased，and apoptosis rates were significantly increased (P < 0.05). Effects from siRNA knockdown of ECT2 on cell proliferation and apoptosis were basically the same as that of miR- 223-3p over-expression group. CONCLUSION： Our data show that miR- 223-3p inhibited proliferation and induced apoptosis of NSCLC cells by negatively regulating the expression of ECT2.

OBJECTIVE： To explore the feasibility of using the micronucleus assay in human peripheral blood lymphocytes in vitro to estimate partial irradiation exposure dose. METHODS： Two human peripheral blood samples were collected and each was divided into 2 parts. One part was not exposed to radiation. The other part was divided further into 2 groups. Therefore，there were a total of 4 groups which were irradiated with 1 and 5 Gy ⁶⁰Co γ rays， respectively， at the dose rate of 1 Gy/min. Subsequently， irradiated blood samples were mixed with non-irradiated ones with ratios of 1∶3 or 3∶1 to simulate partial exposure. These mixed samples were analyzed using the micronucleus (MN) assay. The MN frequencies were used to estimate exposure doses using the Dolphin's model as recommended by the International Atomic Energy Agency (IAEA)，and to determine dose-effect curves as recommended by health industry standards. RESULTS： The observed MN frequencies did not conform to poisson distribution. In addition，the estimated partial dose from the 1 Gy groups deviated from the actual dose，and the estimated partial dose from the 5 Gy groups were close to the actual dose. CONCLUSION： MN can be used to estimate partial exposure doses in vitro. However，the estimates are more reliable for higher partial dose exposure.

OBJECTIVE： This study aims to explore induction of proliferation and migration in MCF- 7 breast cancer cells by phthalates (DEHP，DBP) as well as their inhibition by metformin. METHODS： MCF-7 cells were treated with DEHP (20，40，100，200 and 400 μg/mL)，DBP (0.14，0.28，0.7，1.4，2.8，14，28 μg/L) or metformin (2.5，5，10，20 mg/mL). Untreated cells were used as controls. 20 mg/mL metformin was added to DEHP (100 μg/mL) and DBP (0.7 μg/L) respectively. The effects of DEHP and DBP on induction of proliferation of MCF-7 cells，and inhibitory effects of metformin were detected by MTT assay. Effects of DEHP and DBP on migration of MCF- 7 cells and the inhibitory effects of metformin were detected by the wound healing and Transwell assay. RESULTS： Compared with the control group， MTT and cell migration assays revealed that DEHP (20-400 μg/mL) and DBP (0.14-28 μg/L) treatments significantly increased cell proliferation and migration in MCF-7. On the other hand，metformin (2.5-20 mg/mL) treatment diminished cell proliferation and migration (P < 0.05). Compared with the DEHP and DBP groups， the combined exposures to phthalate (100 μg/mL DEHP， 0.7 μg/L DBP) and metformin (20 mg/mL) diminished cell proliferation and migration of MCF-7 cells. CONCLUSION： The present study demonstrates that metformin inhibited proliferation and migration of MCF-7 breast cancer cells which were induced by DEHP and DBP.

OBJECTIVE： To investigate expression status， clinical implication， and functional role of neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) in esophageal squamous cell carcinoma (ESCC). METHODS： Protein expression of NEDD4 was examined by immunohistochemistry (IHC) in tissue microarrays containing 131 ESCC specimens. Correlations between NEDD4 expression levels and the clinicopathological parameters were analyzed using Pearson Chi- Square and Fisher exact tests. Alterations of NEDD4 mRNA level in ESCC specimens were evaluated using data from the TCGA and GTEx databases. Furthermore， NEDD4 expression was knocked down in ESCC cell lines which had high NEDD4 expressions and changes of cell proliferation activity were detected using the CCK8 assay. RESULTS： Over-expressions of NEDD4 were detected in 40% (53/131) of ESCC specimens，while it was not expressed or expressed at a low level in adjacent normal epithelial tissues. In addition，NEDD4 protein levels were significantly correlated with pathologic T stage and histological grades of ESCC (P < 0.05，respectively). TCGA and GTEX database analysis results show that NEDD4 mRNA levels were also prominently up-regulated in ESCC specimens (P < 0.01). Data of the CCK8 assay further demonstrate that knockdown of NEDD4 expression significantly suppressed the proliferation capacities of KYSE150 and KTSE30 cells in vitro (P < 0.01). CONCLUSION： NEDD4 was frequently up- regulated in ESCC， and its over- expression enhanced proliferation ability of ESCC cells. Our data suggest that abnormal expression of NEDD4 played an oncogenic role in the occurrence and development of ESCC.

OBJECTIVE： To investigate the role of the Nrf2/ARE signaling pathway on inhibitory effect of quercetin on mitochondrial oxidative damage which was induced by isoniazid (INH) in L- 02 hepatocytes. METHODS： L- 02 cells in cultures were randomly divided into several groups： negative control， INH (10 mmol/L)，quercetin alone (50 μmol/L) and combined (10 mmol/L INH+50 μmol/L quercetin). After cells were treated for 24 hours，cell viability was measured using the MTT method； levels of the mitochondrial reactive oxygen species (ROS) was detected by fluorescence probe DCFH-DA； contents of malondialdehyde (MDA)， glutathione (GSH) and the activity of superoxide dismutase (SOD) were measured using colorimetry to evaluate the oxidative damage status of mitochondria. In addition， the protein expressions of Nrf2 and HO-1 were detected using western blot analysis to assess changes in the Nrf2/ARE signaling pathway. RESULTS： Compared with the negative control group，cells from the INH group showed the followings：cell vitality was significantly decreased (P < 0.01)， mitochondrial ROS level and MDA content were significantly increased (P< 0.01)，GSH content and SOD activity were remarkable reduced (P < 0.01). Compared with the INH group，the other treated cells showed the followings： cell vitality was markedly increased (P < 0.01)， the level of mitochondrial ROS and the content of MDA were remarkable declined (P < 0.01)，the content of GSH and the activity of SOD were significantly elevated of the combined treatment group (P < 0.05 or P < 0.01). In addition， expressions of HO-1 protein in cytoplasm and Nrf2 protein in nucleus of the INH group were significantly higher than those in the negative control group (P < 0.01). Expressions of HO- 1 and Nrf2 proteins of the combined treatment group were markedly elevated compared with those of the INH group (P < 0.01). CONCLUSION： Quercetin inhibited mitochondrial oxidative damage which was induced by INH， and the mechanism might be mediated by quercetin-regulation of the Nrf2/ARE signaling pathway.

OBJECTIVE： To investigate relationships among neutrophil-lymphocyte ratio (NLR)，plateletlymphocyte ratio (PLR)， clinicopathological characteristics and prognosis of esophageal squamous cell carcinoma. METHODS： Retrospective analyses were conducted on 134 patients who had surgery for esophageal squamous cell carcinoma from January 1， 2010 to December 31， 2013. Relationships among NLR， PLR， clinicopathological characteristics and prognosis of these carcinomas were evaluated. RESULTS： The T stage of patients in the low NLR (<2.34) group was significantly earlier than those in the high NLR (≥ 2.34) group (P=0.001). The T stage of patients in the low PLR (<152.76) group was also significantly earlier than those in the high PLR (≥152.76) group (P < 0.01). The lesion length of patients in the low NLR (<2.12) group was significantly shorter than that in the high NLR (≥2.12) group (P < 0.01). Patients with low PLR (< 103.91) were also significantly shorter than those with high PLR (≥103.91) (P < 0.01). The 1，3，and 5 year survival rates and local control rates of the entire group were 88.1%，45.8%，33.9%，and 88.0%，69.0%， 67.4%，respectively. The 1，3，and 5 years of the transfer rates were 27.6%，54.9%，55.9%. The 1-，3-， and 5-year remote turnout rates of patients in the high NLR group were much higher than those in the low NLR group (P=0.012). The 1-，3-，and 5-year long-term turnout rates of patients in the high PLR group were much higher than those in the low PLR group (P=0.014). Multivariate analyses of survival show that only the N stage was an independent factor affecting the survival of patients (P=0.014). CONCLUSION： NLR and PLR are closely related to the clinicopathological characteristics and prognosis of patients. Patients with high NLR and high PLR had late T stage，longer lesion length and are more prone to distant metastasis.

OBJECTIVE： To analyze differentially expressed miRNAs between the triple negative breast cancer cell line MDA-MB-231 (MB-231) and its brain metastasis cell line MDA-MB-231Brm (MB-231Brm)， and to screen biomarkers associated with the brain metastases. METHODS： The Trizol method was used to extract miRNA from MB-231 and MB-231Brm，and PCR was used to construct RNA libraries. After filtering the data and comparing to known miRNA databases，the miRDeep2 method was used to make new miRNA predictions for unknown miRNAs. Then， a differential gene detection method was used to screen the differentially expressed miRNAs. Finally，bioinformatics analysis tools was used to perform functional analysis of the target genes which could be regulated by these miRNAs. Quantitative PCR (qPCR) analyses were used to validate the microarray data. RESULTS： There were differences in the types and expressions of miRNAs between the MB-231 and the MB-231Brm cells. Briefly， 961 kinds of miRNA were shared by both， 164 species of miRNAs only existed in MB-231， and 196 species of miRNAs only existed in MB-231Brm. Compared with MB-231，145 expressions were significantly up-regulated in MB-231Brm，and 64 expressions were significantly down-regulated，and there were no significant differences in 1 122 expressions. Results from differential expression analyses demonstrate that expressions of miR-199a-3p were significantly upregulated in MB-231Brm compared with MB-231 while miR-1246 and miR-4787-5p were down-regulated significantly. GO analyses reveal that genes which were regulated by differentially expressed miRNAs were related to cellular components，molecular functions and biological processes. Results from qPCR analyses confirmed that expressions of miR-1246 were down- regulated in MB-231Brm， compared with MB-231 (P < 0.05). CONCLUSION： There were differentially expressed miRNAs in MB-231 and MB-231Brm cells， and genes that were targeted by these miRNAs were associated with cellular components， molecular functions and biological processes during TNBC brain metastasis. Moreover， expressions of miR-1246 were down-regulated in MB-231Brm，compared with MB-231. Therefore， miR-1246 may participate in the occurrence and development of TNBC brain metastasis.

OBJECTIVE： To investigate expression levels of CXC chemokine receptor 2(CXCR-2) and its effect on matrix metalloproteinase- 9 (MMP- 9)， and vascular endothelial growth factor (VEGF) in esophageal squamous carcinoma cells. METHODS： From December 2016 to December 2019， 70 cases of esophageal squamous cell carcinoma tissues and adjacent normal tissues were collected from the Department of thoracic surgery，the First Affiliated Hospital of Hebei North University. Expression levels of CXCR-2，MMP-9，VEGF mRNAs and proteins were detected by real-time fluorescence quantitative PCR (qPCR) and immunohistochemistry. Pearson correlation analysis was used to analyze the correlation of CXCR- 2，MMP-9 and VEGF expression. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic significance of CXCR- 2 expression in esophageal squamous cell carcinoma. RESULTS： qPCR results show that the expression levels of CXCR-2 mRNA，MMP-9 mRNA，and VEGF mRNA were significantly higher in the esophageal squamous cell carcinoma tissues compared with their adjacent normal tissues(all P < 0.01). Similarly，the protein expressions of CXCR-2，MMP-9，and VEGF were significantly higher in the carcinoma tissues than in their adjacent normal tissues (all P < 0.01). Moreover，CXCR-2 expressions were significantly and positively correlated with expression levels of MMP- 9 and VEGF in the carcinoma tissues (r=0.488 and 0.491，P < 0.01). Results from the ROC curve analyses show that the expression levels of CXCR- 2 had auxiliary diagnostic value for esophageal squamous cell carcinoma. The area under the ROC curve was 0.938 [95% CI (0.908，0.972)]，and best cut-off values for gastric cancer was 0.77， the sensitivity was 91.2% and specificity was 80.9%. CONCLUSION： CXCR-2，MMP-9 and VEGF were highly expressed in the squamous cell carcinoma，and were closely related to TNM stage，lymph node metastasis and distant metastasis. Therefore，expression levels of CXCR-2 can be of great value in the diagnosis of esophageal squamous cell carcinoma.

OBJECTIVE： To investigate changes of Ki67 expression before and after neoadjuvant chemotherapy (NACT) in breast cancer as a predictor of therapeutic response and prognosis. METHODS： Retrospective analyses were conducted on 104 patients with breast cancer who received NACT at the Shantou University Medical College Cancer Hospital from December 2009 to September 2013. Expression of Ki67 was assessed using immunohistochemistry in pre-chemotherapy core-needle biopsy and post-surgical specimens. Clinicopathological characteristics， therapeutic responses， and survival were compared between the Ki67- reduced group and the Ki67-unchanged/increased group during NAC. RESULTS： Both the reduced and unchanged/increased groups accounted for 62.5% (65/104) and 37.5% (39/104)，respectively. Ki67 levels were significantly reduced in patients with higher Ki67 expression (63/91， P < 0.01) before NACT. Patients with reduced Ki67 after NAC showed significantly enhanced therapeutic response，such as clinical complete response and clinical partial response (59/65， P=0.005). In addition， the decreased value of Ki67 during NACT was significantly correlated with therapeutic response (r=0.302， P=0.002). The disease- free survival rates in the Ki67 reduced group was higher than that in the Ki67 unchanged/increase group (P=0.027). The overall survival rates in the Ki67 reduced group was higher than that in the Ki67 unchanged/increase group，but the difference was not statistically significant (P=0.171). CONCLUSION： The reduction of Ki67 expression after NACT could be a predictor for therapeutic response and prognosis in breast cancers.