[Analysis of chromosome aberrations in peripheral blood lymphocyte of medical radiation workers in a tertiary hospital].

Objective: To analyze the level of chromosome aberration in lymphocytes of medical radiation workers and its influencing factors. Methods: From July to September 2020, 252 medical workers in a tertiary hospital were selected as the study subjects and 107 preserviceworkers were selected as the control group. The Chromosomal aberrations of peripheral blood lymphocytes were measured using conventional cytogenetic analysis method, and the differences were analyzed. Results: The frequencies of dicentric puls centric ring, total chromosome-type aberrations, and abnormal detection rate in the radiation group were significantly higher than those in the control group (Z=2.59, 3.74, 9.99, P<0.05). There was significant difference in the frequencies of dicentric plus centric ring and total chromosome-type aberrations among different types of work (χ(2)=8.59, 8.17, 11.39, P<0.05), and the frequencies of dicentric plus centric ring were significantly higher in the interventional radiology group than those in diagnostic radiology (χ(2)=2.90, P<0.05), While the rates of acentric fragment and total chromosome-type aberrations were significantly higher in the nuclear medicine group than those in diagnostic radiology (χ(2)=2.81, 3.19, P<0.05). The difference in the abnormal detection rate of chromosome aberrations between different types of work was statistically significant (P<0.05), and the rate in the interventional radiology group was significantly higher than that in the diagnostic radiology group (χ(2)=7.66, P<0.05). There was no significant difference in chromosome aberration level and abnormal detection rate among different working ages (P>0.05). Poisson regression analysis indicated that the type of work is a risk factor for chromosomal aberration [IRR=2.31 (nuclear medicine group), 1.66 (Radiation therapy), and 1.78 (interventional group) ; P<0.05]. Conclusion: Ionizing radiation causes certain radiation damage to medical radiology workers, and the frequencies of chromosome aberration in the radiation workers of nuclear medicine and interventional radiology groups are relatively high, so radiation protection should be strengthened to ensure the health of relevant workers.

Low levels of organic compound trace elements improve the eggshell quality, antioxidant capacity, immune function, and mineral deposition of aged laying hens.

In the egg production industry, trace elements are required as additional dietary supplements to play vital roles in performance and egg quality. Compared to inorganic microelements (ITs), appropriate dose of organic trace microelements (OTs) are environmentally friendly and sufficient to satisfy the needs of hens. In order to evaluate the extent to which low-dose OTs replace whole ITs, the effects of organic copper, zinc, manganese, and iron compound on the performance, eggshell quality, antioxidant capacity, immune function, and mineral deposition of old laying hens were investigated. A total of 1 080 57-week-old Jing Hong laying hens were assigned to five groups with six replicates of 36 layers each for an 8-week experimental period. The birds were fed either a basal diet (control treatment (CT)) or the basal diet supplemented with commercial levels of inorganic trace elements (IT 100%) or the equivalent organic trace elements at 20%, 30%, and 50% of the inorganic elements (OT 20%, OT 30%, and OT 50%, respectively). Results showed that compared with those in the CT treatment, feeding hens with inorganic or organic microelement diet had significant effects on the eggshell quality, antioxidant capacity, immune function, and mineral deposition of old laying hens (P < 0.05). The eggshell strength and ratio between OT 30%, OT 50%, and IT 100% were similar at weeks 4 and 8, and the eggshell thickness of these groups was also similar at weeks 6 and 8. At week 8, the eggshell colour in OT 50% was darker than that in IT 100%. The mineral content in the eggshells of OT 50% and IT 100% significantly increased (P < 0.001), with no significant difference in effective thickness, mammillary thickness, and mammillary knob width between groups. There were no differences in the malondialdehyde content, total antioxidant capacity, and total superoxide dismutase activity in serum between OT 30%, OT 50%, and IT100%. While the catalase activities, the interleukin-1ß, interleukin-10, immunoglobulin G, and immunoglobulin M concentrations in serum were not significantly different between OT 50% and IT 100%. The mineral contents in the faeces of the organic groups were considerably reduced compared with those in IT 100% (P < 0.001). In conclusion, dietary supplementation with 30-50% organic compound microelements has the potential to replace 100% inorganic microelements in the hen industry for improving eggshell quality, mineral deposition in the eggshell, antioxidant capacity, and immune function, and reducing emissions to the environment without negative effects on laying performance.

[Mechanism of lncRNA-SRLR induced invasion and metastasis in U2OS osteosarcoma cells].

Objective: To explore the potential mechanism of sorafenib resistance associated long non-coding RNA (lncRNA-SRLR) promoted invasion and metastasis in U2OS osteosarcoma cells. Methods: We transfected U2OS cells with negative control lentivirus (LV-NC) or lncRNA-SRLR overexpressed lentivirus (LV-over/SRLR) particles. LV-NC and LV-over/SRLR stable transfected cells (U20S/NC and U20S/SRLR) were selected by primary cell culture medium containing puromycin. The mRNA expressions of lncRNA-SRLR and procollagen-lysine, procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of lncRNA-SRLR on the invasion of U2OS cells were determined by wound-healing assay and Transwell migration assay. The effect of SRLR on the interleukin-6 (IL-6) secretion of U2OS cells was evaluated by enzyme-linked immunosorbent assay (ELISA) analysis. The subcellular distribution of SRLR in U2OS cells was detected by fluorescence in situ hybridization (FISH) analysis.The expression of PLOD2 in cells was detected by immunofluorescence (IF). The expressions of PLOD2 and focal adhesion kinase (FAK)/signal transducer and activator of transcription 3 (STAT3) signal pathway related proteins in U2OS/NC and U2OS/SRLR cells were detected by western blotting. Results: qRT-PCR assay showed that mRNA expressions of lncRNA-SRLR and PLOD2 in U2OS/SRLR cells were (3 964.97±0.05) and (2.77±0.11), respectively, significantly higher than those in U2OS/NC cells (P<0.001 or P<0.01). The results of wound-healing and Transwell migration assay showed that over-expression of SRLR markedly promoted the invasion ability of U2OS cells (P<0.05). The result of ELISA analysis showed that the IL-6 secretions in U2OS/NC or U2OS/SRLR cells were (125.38±11.22) pg/ml or (119.97±13.43) pg/ml, without statistical significance (P>0.05). The subcellular distribution assay revealed that lncRNA-SRLR is predominately located in the nucleus. The result of IF showed that compared with U2OS/NC cells, the expression of PLOD2 was up-regulated in U2OS/SRLR cells. The result of western blotting showed that over-expression of SRLR significantly increased the expression levels of PLOD2, phosphorylation (p)-FAK and p-STAT3 in U2OS cells (P<0.01). Conclusion: lncRNA-SRLR promotes invasion and metastasis of osteosarcoma by activating PLOD2-FAK/STAT3 signal axis.

[The relationship between HPV integration and prognosis of cervical cancer].

Objective: To investigate the relationship between human papillomavirus (HPV) integration and prognosis of cervical cancer patients. Methods: The data of 82 patients with cervical cancer treated in the Radiotherapy Department of Peking Union Medical College Hospital from October 2004 to June 2012 were retrospectively analyzed.The patients were divided into poor prognosis group (recurrence or metastasis after surgery and adjuvant radiotherapy) and good prognosis group based on a propensity score matching strategy.The HPV integration of the two groups were detected by whole exome sequencing to determine whether the integration sites were located in the common fragile sites (CFSs). HPV integration and integration into CFSs were compared between the two groups. Results: Among the enrolled 82 patients, 37 were divided in poor survival group and 45 in good survival group. A total of 90 integration breakpoints were identified, 30 of them occurred in poor prognosis group and 60 occurred in good prognosis group. In the poor prognosis group, HPV integration occurred in 20 patients, 13 of them were inserted in CFSs of 11 patients, and the numbers in good prognosis group were 26, 17, 11, respectively. There were no significantly statistical differences in the number of HPV integration events (P=0.289), HPV integration patients (P=0.735), CFSs integration events (P=0.427), and CFSs integration patients (P=0.591) between the two groups. In poor prognosis group, more CFSs integration events occurred in patients with metastasis than those in patients with only local recurrence (9 vs 2, P=0.003). Conclusions: No significant differences are observed in HPV integration and HPV integration into CFSs between cervical cancer patients with different prognoses. HPV integration into CFSs may be associated with distant metastasis.

Contributions of Lactobacillus plantarum PC170 administration on the recovery of gut microbiota after short-term ceftriaxone exposure in mice.

This study aimed to determine the impact of Lactobacillus plantarum PC170 concurrent with antibiotic treatment and/or during the recovery phase after antibiotic treatment on the body weight, faecal bacterial composition, short-chain fatty acids (SCFAs) concentration, and splenic cytokine mRNA expression of mice. Orally administrated ceftriaxone quantitatively and significantly decreased body weight, faecal total bacteria, Akkermansia muciniphila, and Lactobacillus plantarum, and faecal SCFAs concentration. Ceftriaxone treatment also dramatically altered the faecal microbiota with an increased Chao1 index, decreased species diversities and Bacteroidetes, and more Firmicutes and Proteobacteria. After ceftriaxone intervention, these changes all gradually started to recover. However, faecal microbiota diversities were still totally different from control by significantly increased α- and ß-diversities. Bacteroidetes all flourished and became dominant during the recovery process. However, mice treated with PC170 both in parallel with and after ceftriaxone treatment encouraged more Bacteroidetes, Verrucomicrobia, and Actinobacteria, and the diversity by which to make faecal microbiota was very much closer to control. Furthermore, the expression of splenic pro-inflammatory cytokine tumour necrosis factor-α mRNA in mice supplemented with PC170 during the recovery phase was significantly lower than natural recovery. These results indicated that antibiotics, such as ceftriaxone, even with short-term intervention, could dramatically damage the structure of gut microbiota and their abilities to produce SCFAs with loss of body weight. Although such damages could be partly recovered with the cessation of antibiotics, the implication of antibiotics to gut microbiota might remain even after antibiotic treatment. The selected strain PC170 might be a potential probiotic because of its contributions in helping the host animal to remodel or stabilise its gut microbiome and enhancing the anti-inflammatory response as protection from the side effects of antibiotic therapy when it was administered in parallel with and after antibiotic treatment.

[Mechanism of PLOD2 induced osimertinib resistance in non-small cell lung cancer HCC827 cells].

Objective: To investigate the effects of osimertinib on proliferation, migration and invasion of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) overexpressing HCC827 cells and explore the potential mechanism of PLOD2 induced osimertinib resistance. Methods: We transfected HCC827 cells with LV-vector and LV-over/PLOD2. The expression of PLOD2 was detected by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. The effects of osimertinib on the proliferation of HCC827-vector and HCC827-PLOD2 cells were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The effects of osimertinib on the migration and invasion of HCC827-vector and HCC827-PLOD2 cells were determined by Transwell assays. The expressions of E-cadherin and vimentin in cells were detected by immunofluorescence (IF). The expressions of epithelial-mesenchymal transition (EMT), FAK-PI3K/AKT and MAPK signal pathway related proteins were detected by western blotting. Results: The MTT assay showed that HCC827-PLOD2 cells were hyposensitive to osimertinib. The 50% inhibitory concentration (IC(50)) and resistance index of osimertinib for HCC827-PLOD2 cells was over 1 000 nmol/L and over 100, respectively. The result of wound healing assay showed that the migration distance of HCC827-PLOD2 was about (2.13±0.21) fold changes as that of HCC827-vector cells. The result of Transwell assay showed that the numbers of HCC827-PLOD2 passing through the matrix membrane were (212.78±10.43), significantly higher than (101.32±12.52) of HCC827-vector cells (P<0.01). The result of IF showed that compared with HCC827-vector cells, the expression of E-cadherin was down-regulated while vimentin was up-regulated in HCC827-PLOD2 cells. Osimertinb downregulated E-cadherin and upregulated vimentin expression in HCC827-vector cells but had limited effect in HCC827-PLOD2 cells. The result of western blotting showed that PLOD2 significantly increased vimentin expression level while decreased E-cadherin expression level. Osimertinib inhibited the expression of p-EGFR, but did not affect the expressions of PLOD2, p-FAK, p-AKT, p-ERK, vimentin and E-cadherin in HCC827-PLOD2 cells. Conclusion: PLOD2 confers resistance to osimertinib in HCC827 cells by regulating EMT, FAK-PI3K/AKT and MAPK signal pathways.

[Upregulation of PLOD2 promotes invasion and metastasis of osteosarcoma cells].

Objective: To investigate the relationship of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression and the clinical characteristics of osteosarcoma, and explore the potential mechanism of tumour metastasis promoted by PLOD2. Methods: The expression of PLOD2 in osteosarcoma tissues and paired adjacent tissues were detected by immunohistochemistry and qRT-PCR. Correlation of PLOD2 expression in osteosarcoma with the clinical pathologic features was analyzed by Chi square test and Kaplan-Meier analysis.Fibrillar collagen formation and collagen deposition in the tumor tissues were detected by picrosirius red staining. We transfected U-2OS cells with LV-vector, LV-over/PLOD2, sh-NC and sh-PLOD2. The expression of PLOD2 was detected by qRT-PCR. The impact of POLD2 on U-2OS cell invasion was determined by wound-healing assay and Transwell migration assay. The expressions of PLOD2/FAK/JAK2-STAT3 signal pathway related proteins were detected by western blotting. Results: The high expression level of PLOD2 in osteosarcoma tissues was 72.5%, significantly higher than 0% in paired adjacent noncancerous tissues (P<0.01), the expression of PLOD2 was positively correlated with lymph node metastasis, pulmonary metastasis and poor outcome (P<0.01). The same results were also observed in qRT-PCR assay. The median survival time of patients with high expression of PLOD2 protein was 13 months, significantly shorter than 32 months of patients with low expression of PLOD2 (P<0.05). The result of picrosirius red staining showed that the percentage of collagen fiber deposition in the osteosarcoma tissue with high level of PLOD2 was (74.43+ 9.63)%, significantly higher than (9.67±1.28)% in tissue with low expression of PLOD2 (P<0.001). The result of wound-healing and Transwell migration assay showed that over-expression of PLOD2 markedly promoted the invasion, however, knockdown of PLOD2 suppressed the invasion of U-2OS cells (both P<0.01). The result of western blotting showed that over-expression of PLOD2 significantly increased the expression levels of p-FAK, p-JAK2, p-STAT3, but knockdown PLOD2 decreased the levels of p-FAK, p-JAK2, p-STAT3 in U-2OS cells. Conclusions: Up-regulation of PLOD2 in osteosarcoma is correlated with lymphatic and distant metastasis. PLOD2 promotes invasion and metastasis of osteosarcoma might through FAK/JAK2-STAT3 signal pathway.

[Mechanism of MALAT1 induced osimertinib resistance in HCC827 lung cancer cells].

Objective: To test the effect of metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) and/or osimertinib on the proliferation and apoptosis of HCC827 cells, and explore the potential mechanism of MALAT1 induced resistance to osimertinib. Methods: We transfected HCC827 cells with LV-vector or LV-over/MALAT1. Stable transfected cells (HCC827/Vector, HCC827/MALAT1) were selected by adding puromycin. HCC827/MALAT1 cells were further transfected with the shRNA-negative control (NC) or shRNA-human epidermal growth factor receptor 3 (ERBB3) plasmid. The effects of overexpression of MALAT1, knockdown of ERBB3 and/or osimertinib on the proliferation of HCC827 cells were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. Cell apoptosis induced by MALAT1 overexpression, knockdown of ERBB3 and/or osimertinib treatment were analyzed by flow cytometry analysis. The expressions of EGFR and ERBB3 signal pathway related proteins in HCC827 cells treated with overexpression of MALAT1, knockdown of ERBB3 and/or osimertinib treatment were detected by western blot. Results: The MTT assay showed that sensitivity to osimertinib of HCC827/MALAT1 cells were significantly repressed. The 50% inhibitive concentration (IC(50)) of osimertinib >4 000 nmol/L in HCC827/MALAT1 cells. However, knockdown of ERBB3 facilitated the anti-proliferation effect of osimertinib, and the IC(50) of osimertinib in shRNA-ERBB3 cells was (17.27±3.21) nmol/L. The results of flow cytometry analysis showed that the apoptotic rate of HCC827/MALAT1 cells induced by 10 nmol/L osimertinib was (8.38±0.92)%, significantly lower than (27.17±5.83)% of knockdown of ERBB3 (P<0.01). Western blotting showed that the expression of p-ERBB3, p-AKT and p-extracellular regulated protein kinases (ERK) in HCC827/MALAT1 cells was markedly up-regulated, while the expression of p-epithelial growth factor receptor (EGFR) was inhibited. The expressions of p-ERBB3, p-AKT and p-ERK were marginally affected by osimertinb. However, osimertinib downregulated the expressions of p-EGFR, p-ERBB3, p-AKT and p-ERK in ERBB3 deleted cells. Conclusions: MALAT1 confers resistance to osimertinb in HCC827 cells by activating of the ERBB3/PI3K/AKT and ERBB3/MAPK/ERK signaling pathways.

[Mechanism of miR-221 contributes to gefitinib resistance in PC-9 cells].

Objective: To investigate the effect of microRNA-221 (miR-221) overexpression on gefitinib resistance in PC-9 cells and study its underlying mechanisms. Methods: PC-9 cells were transfected with LV-NC and LV-miR-221 to establish cell stabilizing strains (PC-9/NC and PC-9/miR-221), then they were used to detect the relative expression of miR-221 and apoptotic protease activating factor-1 (APAF-1) mRNA by real-time fluorescence quantitative PCR (qRT-PCR). The effects of gefitinib (0-4 µmol/L) on the growth and proliferation of cell stabilizing strains were detected by CCK-8 Assay. After gefitinib treatment, cell apoptosis was detected by Flow Cytometry Assays. The expression of epidermal growth factor receptor (EGFR), phosphorylated epidermal growth factor receptor (p-EGFR), APAF-1 and cleaved cysteinyl aspartate specific proteinase-3 (Cleaved-caspase-3) were detected by Western blot. Dual-Luciferase Reporter Assay was used to evaluate the relationship between APAF-1 and miR-221. Results: The relative expression of miR-221 in PC-9/NC cells was significantly lower than that in PC-9/miR-221 cells[(1.00±0.082) vs (40.24±0.017)](P<0.01). The half maximal inhibitory concentration (IC(50)) in PC-9/NC cells was significantly lower than that in PC-9/miR-221 cells[(IC(50)=0.1 µmol/L) vs (IC(50)>4 µmol/L)](P<0.05). Apoptosis rate of PC-9/NC cell was significantly higher than PC-9/miR-221[(33.42±4.28)% vs (10.27±1.12)%](P<0.05); APAF-1 mRNA expression was significantly higher in PC-9/NC cells than PC-9/miR-221[(1.000+ 0.069) vs (0.701±0.072)](P<0.05), and the expression of APAF-1 protein in PC-9/NC cells was significantly higher than that of PC-9/miR-221 cells. The dual luciferase reporter gene results showed that miR-221a inhibited luciferase activity significantly stronger than transfected miRNA negative control group after co-transfection of luciferase plasmids pmir-REPORT-APAF-1-wt and miR-221a mimics (P<0.01). p-EGFR was down-regulated in both PC-9/NC and PC-9/miR-221 cells after treatment with gefitinib. APAF-1 and Cleaved-caspase-3 proteins were significantly down-regulated in PC-9/miR-221 cells compared with PC-9/NC cells, while APAF-1 and Cleaved-caspase-3 proteins were up-regulated in PC-9/NC cells treated with gefitinib compared with PC-9/miR-221 cells (P<0.05). Conclusion: miR-221 induces resistance to gefitinib in PC-9 cells by downregulating APAF-1 expression.

SETD5-AS1 stimulates neuron death in stroke via promoting PTEN expression.

OBJECTIVE: To investigate the specific role of long non-coding RNA (lncRNA) SETD5-AS1 in regulating stroke development, and its underlying mechanism. MATERIALS AND METHODS: Middle cerebral artery occlusion (MCAO) model and OGD/R (oxygen-glucose deprivation/reoxygenation) model were constructed for exploring the mechanism of ischemia-reperfusion injury induced by ischemic stroke. SETD5-AS1 expression in brain tissues of ischemic stroke mice and control mice was detected by quantitative Real-time-polymerase chain reaction (qRT-PCR). Proliferation and apoptosis of N2a cells were detected after transfection of overexpression plasmid or siRNA SETD5-AS1. The downstream gene of SETP5-AS1 was predicted by Starbase and PTEN was screened out. Both mRNA and protein expressions of PTEN in MCAO model and OGD/R model were detected. Furthermore, the binding condition of SETD5-AS1 and PTEN was verified by dual-luciferase reporter gene assay, RNA pull-down assay and RNA binding protein immunoprecipitation (RIP). The regulatory effect of SETD5-AS1 on PI3K/AKT pathway was detected by Western blot. RESULTS: SETD5-AS1 was highly expressed in the ischemia-reperfusion injury model. Overexpression of SETD5-AS1 in N2a cells resulted in increased apoptosis and decreased proliferation. PTEN expression was upregulated in MCAO model and OGD/R model. Dual-luciferase reporter gene assay indicated that SETD5-AS1 can promote PTEN transcription. The binding condition of SETD5-AS1 and PTEN was further verified by RNA pull-down assay and RIP. Overexpression of SETD5-AS1 in N2a cells inhibited PI3K/AKT pathway. CONCLUSIONS: SETD5-AS1 is highly expressed in the ischemia-reperfusion injury model. SETD5-AS1 participates in the development of ischemic stroke by activating PTEN and inhibiting PI3K/AKT pathway.

[Response and prognosis of neoadjuvant dose-dense or standard schedule chemotherapy with anthracyclines and taxanes for Luminal B breast cancer].

Objective: To evaluate the efficacy and safety of neoadjuvant dose-dense or standard schedule chemotherapy with anthracyclines and taxanes for Luminal B (HER2-)Breast Cancer. Methods: From January 2010 to December 2014, 168 Luminal B (HER2-) breast cancer patients with stageⅡA-ⅢC confirmed by pathology were randomly assigned to receive one of the following regimens: (group A) concurrent TEC× 4 every 3 weeks, ( group B ) sequential EC× 4-T × 4 every 3 weeks, (group C ) dose-dense TEC× 4 every 2 weeks with G-CSF, (group D) sequential EC× 4(dose-dense)-T × 4 with dose-dense every 2 weeks . Results: A total of 168 patients completed the neoadjuvant chemotherapy as planned. The pathologic complete response (pCR) was 16.8% in the 4 groups.The pCR were 30.9% and 26.1% in the group C and group D respectively, significantly higher than patients with group A and group B(9.5%and 7.1%) ( P<0.05). Median follow-up was 43 months (IQR 3-63). The 3-year disease free survival (DFS) rate was 64.7%, 55.5%, 87.8% and 92.1% and the 3-year overall survival(OS)rate was 79.4%, 77.7%, 95.1%, 97.3% in the 4 groups respectively. Patients in the dose-dense group had better 3-year DFS and 3-year OS than those with the regular group.The side-effects could be evaluated in 154 patients.The incidence of neutropenia was 29.2% and 21.9% in the group C and group D versus 65.7%and 51.3% in the regular group(P<0.05), the incidence of nervous toxicity was 54.2%, 18.9%, 60.0%, 26.8% in the 4 groups respectively. The incidence of nervous toxicity in the dose-dense group was lower than that in the regular regimen group(P<0.05). Conclusion: Neoadjuvant dose-dense chemotherapy with anthracyclines and taxanes for Luminal B (HER2-)Breast Cancer was effective and can improve the pCR, DFS and OS.Comparing the two dose dense regimens, sequentially with anthracyclines and taxanes, the incidence of nervous toxicity were lower.

[Mechanism of long non-coding RNA-metastasis associated lung adenocarcinoma transcript 1 induced invasion and metastasis of esophageal cancer cell EC-109].

Objective: To investigate the effect and mechanism of long non-coding RNA-metastasis associated lung adenocarcinoma transcript 1, (LncRNA-MALAT1) on invasion and metastasis of esophageal cancer cell EC-109. Methods: EC-109 cells were transfected with lentiviral vector carrying short hairpin RNA of MALAT1( shRNA-MALAT1) or a nonspecific shRNA control (shRNA-control). The expressions of MALAT1, microRNA-200a, ZEB1 and ZEB2 were detected by qRT-PCR. The effect of shRNA-MALAT1 on invasion of EC-109 cells was determined by transwell assay. The expressions of components of epithelial-msenchymal transition pathway in EC-109 cells were determined by immunofluorescence array and western blotting. The expression relationship between MALAT1 and miR-200a in EC-109 cells was detected by dual-luciferase reporter assay. Results: The result of qRT-PCR showed that the expressions levels of MALAT1, ZEB1 and ZEB2 in shRNA-MALAT1 group were 0.43±0.06, 0.64±0.04 and 0.51±0.04, respectively, significantly lower than 0.97±0.08, 1.06±0.07 and 0.98±0.05 in shRNA-control group and 1 in control group, respectively(all P<0.05). Transwell assay showed that the number of invaded cells in shRNA MALAT1 group was (96.81±10.43) per low-power field, markedly lower than that of (278.44±13.28) per low-power field in shRNA-control group (P<0.01). Immunofluorescence staining and Western blotting showed that MALAT1 downregulation significantly reduced the expressions of proteins related to EMT signal pathway in EC-109 cells.Dual luciferase reporter assay showed that compared to negative control, the activities of luciferase reporter in EC-109 cells co-transfected with pmirGLO-MALAT1-wt and miR-200a were significantly down-regulated. While co-transfected pmirGLO-MALAT1-mut with miR-200a mimics had no effect on the luciferase reporter activities of MALAT1. Conclusion: LncRNA MALAT1 functions as a competing endogenous RNA to regulate the expressions of ZEB1 and ZEB2 by sponging miR-200a and promotes invasion and migration of esophageal cancer cells through inducing epithelial-mesenchymal transition.

Inhibitory effect and mechanism of metformin on human ovarian cancer cells SKOV-3 and A2780.

OBJECTIVE: Ovarian cancer is the most common malignant tumor in female reproductive system. Metformin is an orally taken hypoglycemic agent, which is extensively applied in the clinic. Clinical trials find that there may be a certain degree of action of the metformin in inhibiting malignant tumors. This paper aims to investigate the inhibitory effect and mechanism of metformin on human ovarian cancer cells. MATERIALS AND METHODS: Through in vitro cell experiment, the influences of metformin on the proliferation, colony formation and apoptosis of ovarian carcinoma cells were studied. Ovarian cancer cells SKOV-3 and A2780 in logarithmic growth phase were selected and cell proliferation was measured by MTT method. The metformin was processed for 48 h to calculate the survival rate of cells. Also, metformin was processed for 24 h and two weeks or stained with crystal violet, after which Quantity One (Bio-Rad, Hercules, CA, USA) method was used to quantitatively analyze the cell clone formation, meanwhile, the FCM (flow cytometry) was used for the detection and analysis. RESULTS: Intervened by metformin with different concentrations for 48 h, the cell viabilities of SKOV-3 and A2780 cells were respectively reduced by 19.49 ± 2.92%, 45.41 ± 7.95%, 53.84 ± 5.53%, 64.04 ± 4.36% and 11.45 ± 3.12%, 35.42 ± 7.55%, 43.77 ± 5.77%, 53.05 ± 5.55% as compared with that in the control group with statistical significances. After processed by metformin with different concentrations for two weeks, the cells clone numbers of SKOV-3 and A2780 were significantly reduced. Treatment of metformin on SKOV-3 and A2780 cells of human ovarian cancer showed significant apoptosis. CONCLUSIONS: The metformin has the inhibitory effect on the cells of human ovarian cancer, which may be through inducing ovarian cancer cell apoptosis.

Influence of temporomandibular joint disc displacement on mandibular advancement in patients without pre-treatment condylar resorption.

The purpose of this study was to clarify the correlation between pre-treatment anterior disc displacement and mandibular stability after orthognathic and orthodontic treatment among patients with a skeletal class II malocclusion and without pre-treatment condylar resorption. Thirty-seven patients were included (7 male, 30 female). The mean length of follow-up was 6.76±3.06 years. Patients with condylar resorption before treatment were excluded. Magnetic resonance images and lateral cephalometric radiographs were taken before treatment (T0), after treatment (T1), and at follow-up (T2). Patients were classified according to the degree of disc displacement: -10-10° 'normal', 11-50° 'slight to mild', ≥51° 'moderate to severe'. Results showed the condyle moved posterosuperiorly after treatment, and then moved anteriorly to a more concentric location during the long follow-up period. Condylar movement was found not to correlate with disc displacement. The degree of disc displacement before treatment did not correlate with the post-surgical mandibular positional change in either the sagittal or vertical direction. To conclude, the mandibular bilateral sagittal split ramus osteotomy was stable in the long-term after orthognathic and orthodontic treatment. In the absence of pre-treatment condylar resorption, the degree of initial anterior disc displacement did not have a significant influence on the stability of mandibular advancement.

[Expression of long non-coding RNA MALAT1 in osteosarcoma and its effect on invasiveness and metastatic potential of osteosarcoma cells].

OBJECTIVE: To investigate the significance of long non-coding RNA MALAT1 expression in osteosarcoma, and the potential mechanism by which MALAT1 promotes tumor metastasis. METHODS: Twenty cases of osteosarcoma in the First Affiliated Hospital of Xinxiang Medical University and Ping Ding Shan First People's Hospital were collected from January 2014 to December 2015. The expression of MALAT1 in osteosarcoma tissue and paired adjacent noncancerous tissue were analyzed by qRT-PCR. Correlation of MALAT1 expression in osteosarcoma with clinical pathologic features was performed by the Mann-Whitney U test. U-2OS cells were transfected with lenti-virus carrying MALAT1-shRNA and nonspecific shRNA (LV-vector). The expression of MALAT1 was detected by qRT-PCR. The cell activity was evaluated by MTT asssy. The impact of MALAT1-shRNA on invasion in U-2OS cells were determined by transwell migration assay. The expression of Wnt/ß-catenin signal pathway related proteins were detected by Immunofluorescence stain and Western blot. RESULTS: The expression level of MALAT1 in osteosarcoma tissue was higher than that in paired adjacent noncancerous tissue and correlated significantly with nodal and pulmonary metastasis(P<0.01). MTT assay showed that knockdown of MALAT1 with lenti virus-MALAT1 shRNA inhibited the growth of U-2OS cells, along with marked decrease of invasive ability of U-2OS cells in the transwell migration assay. By immunofluorescence stain and Western blot assay, MALAT1 significantly reduced the expression of ß-catenin, MMP7, and c-MYC in U-2OS cells. CONCLUSIONS: The expression of MALAT1 is high in osteosarcoma and correlates with tumor metastasis. MALAT1 promotes invasion and metastasis of osteosarcoma cells likely thought the Wnt/ß-catenin signal pathway.

Immunoregulatory effect of neuronal-like cells in inducting differentiation of bone marrow mesenchymal stem cells.

OBJECTIVE: To evaluate the immune activity of bone marrow mesenchymal stem cells (BMSCs), and explore the biological characteristics and capabilities of BMSCs and the potential to be differentiated into neuronal cells in vitro. MATERIALS AND METHODS: The BMSCs were isolated and proliferated in vitro to generate the xenogeneic mixed lymphocyte reaction. Moreover, peripheral BMSCs (pBMSCs) were added according to different ratios, which methods were stated as follows: 1: Dulbecco's Modified Eagle Medium (DMEM) + 10% Fetal Bovine Serum (FBS) + 1 µmol/L all-trans-retinoic acid (ATRA) + 20 µg/L basic fibroblast growth factor (bFGF) + 20 µg/L epidermal growth factor (EGF); 2: DMEM + 2% dimethyl sulfoxide (DMSO) + 100 µmol/L butylated hydroxyanisole (BHA). The immunofluorescence and immunohistochemical staining were finally used to evaluate the differentiation capabilities of human BMSCs (hBMSCs) induced in neuronal cells. RESULTS: hBMSCs inhibited the lymphocyte proliferation in the mixed lymphocyte reaction (MLR) system at a proportional inhibition rate with additional numbers of stem cells. At hour 2 after culture with method 1, the plasma of hBMSCs shrank to nuclei and perinuclear bodies and was visualized under the light microscope. At hours 3-5, most of the hBMSCs formed neuron-like cells with total cell number unchanged. Afterward, the hBMSCs turned into bipolar or multipolar shaped cells and interconnected into a large network at Day 3. With immunofluorescence and immunohistochemical staining, 60-70% of the hBMSCs showed neurospecific enolase (NSE) positive and 45-50% glial fibrillary acidic protein (GFAP) positive while the Nestin-positive cells decreased to 3.4%. However, when cultured 2 hours with method 2, the most of the hBMSCs formed bipolar or multipolar shaped cells, then died after 48 hours. 40-50% NSE and 35-40% GFAP were positively expressed. Significantly, the rate of Nestin-positive cells decreased from 63% to 1.6% from hour 2 after culture to hour 48. CONCLUSIONS: hBMSCs may be effective for cell therapy and tissue engineering for the capability of differentiating into neuronal-like cells, as well as the capability of inhibiting lymphocyte proliferation in MLR system.

Invited review: mesenchymal progenitor cells in intramuscular connective tissue development.

The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF) ß signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.

Bioinformatics analysis of miRNA expression profile between primary and recurrent glioblastoma.

OBJECTIVE: Glioblastoma (GBM) is the most malignant brain tumor with rapid relapse. The goal of this study is to identify microRNAs (miRNAs) involved in recurrent GBM. MATERIALS AND METHODS: miRNA transcription profile data (GSE32466) were downloaded, including 12 primary GBM samples and 12 recurrent GBM samples. Then, limma package was utilized to identify differentially expressed miRNAs (DEMs) with the criteria of false discovery rate < 0.05 and |log2 fold change| ≥ 1. Thereafter, miTarget and TargetScan databases were used to predict the potential target genes of DEMs. Regulatory co-expression network was constructed based on co-expressed genes and potential miRNA-gene pairs, and then, pathway analysis was conducted. Furthermore, database miRWalk was used to screen out known GBM-associated miRNAs from the identified DEMs. RESULTS: A total of 71 DEMs were identified between primary and recurrent GBM samples, and 2684 potential target genes were found. Besides, regulatory co-expression network was constructed, including 12 DEMs and 81 potential target genes. These genes significantly enriched in ECM-receptor interaction, ribosome, and focal adhesion pathways, and DEMs like hsa-miR-320a, hsa-miR-139-5p, has-miR-128, hsa-miR-140-5p, and hsa-miR-146b-5p had high degree. Notably, 7 DEMs in network were known GBM-associated miRNAs recorded in database miRWalk. CONCLUSIONS: DEMs like hsa-miR-320a, hsa-miR-139-5p, has-miR-128, hsa-miR-146b-5p, hsa-let-7c, hsa-miR-128, and hsa-let-7a might participate in recurrent GBM. These results would pave ways for further study of recurrent GBM mechanism, and for the prevention and treatment of recurrent GBM. However, more experimental verifications are required to prove these predictions.

Importance of CD44 on umbilical cord mesenchymal stem cells for expansion of hematopoietic cells.

Human umbilical cord mesenchymal stem cells (hUCMSCs) have important functions on the expansion of hematopoietic stem cells (HSCs) through providing the essential microenvironment for hematopoiesis. In order to test whether CD44 on hUCMSCs could have a key function for the ability of hUCMSCs to expand human HSCs, the soluble anti—CD44 antibody was added to the co—cultures of hUCMSCs and cord blood (CB) CD34+ cells, which blocked the ability of hUCMSCs to expand CB CD34+ cells significantly. Long—term culture initiating cell (LTC—IC) assay revealed that the ability of multipotent differentiation of CB CD34+ cells co—cultured with CD44 knockdown hUCMSCs could only retain lasting at most for 5 weeks in vitro. In vivo assay, based on non—obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice, revealed that the hematopoietic reconstitution potential of CB CD34+ cells co—cultured with CD44 knockdown hUCMSCs is significantly reduced. The hematopoietic supporting ability of hUCMSCs in vivo and in vitro is reduced upon the knockdown of CD44. CD44 has important functions on the ability of hUCMSCs to expand human HSCs in the cell— extrinsic control.