Objective:To investigate the effect of nuclear receptor subfamily 2 group F member 2(NR2F2)on the biological behaviors of human ovarian cancer SKOV3 cells,and to clarify its molecular mechauism and provide the new idea for treatment of ovarian cancer.Methods:Gene Expression Profiling Interactive Analysis(GEPIA)Database analyse the expression level of NR2F2 gene in ovarian tissue,and analyse its correlation with clinical prognosis of ovarian cancer patients.The human ovarian cancer SKOV3 cells were divided into control group and NR2F2 over-expression(NR2F2 OE)group,which were transfected with mCherry control virus and NR2F2 OE over-expression virus,respectively,when the cell deusity reached 70％,and the stable transfection SKOV3 cell lines were screened with puromycin(puro)48h lafter.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the transfection efficiencies of the cells;RT-qPCR method was used to detect the expression levels of NR2F2 and sex-determining region Y-box 2(SOX2)mRNA in the cells in two groups;Western blotting method was used to detect the expression levels of NR2F2,ATP-binding cassette superfamily G member 2(ABCG2),and programmed cell death 1-ligand 1(PD-L1)protcins in the cells in two groups.CCK-8 assay was used to detect the proliferation activities of the cells in two groups;Wound assay was used to detect the migration rates of the cells in two groups;Transwell chamber assay was used to detect the number of transmembrane cells;Spheroidization assay was used to detect the numbers of spheroids in the cells;peripheral blood mononuclear cells(PBMCs)-mediated tumor cell killing assay was used to detect the relative densities of surviving tumor cells;CCK-8 assay was used to detect the half maximal inhibitory concentration(IC50)of paclitaxel(PTX)and carboplatin(CBP).Results:Compared with normal ovarian tissue,the expression level of NR2F2 gene in ovarian tumor tissue was decreased(P＜0.05),and decreased with the improvement of clinical pathological grading of ovarian tumor.The patients with higher expression level of NR2F2 gene had better clincal prognosis.The SKOV3 cells with NR2F2 over-expresson were successfully constructed,and the expression levels of NR2F2 mRNA and protein in the cells in NR2F2 OE group were increased compared with control group(P＜0.001).The CCK-8 assay results showed that compared with control group,the proliferation activities of the cells in NR2F2 OE group were decreased at different time points(1,2,3,and 4 d)(P＜0.05 or P＜0.01).The cell wound assay results showed that compared with control group,the migration rate of the cells in NR2F2 OE group was decreased(P＜0.001).The Transwell assay results showed that compared with control group,the number of transmembrane cells in NR2F2 OE group was decreased(P＜0.01).Compared with control group,the number of the spheroids in NR2F2 OE group was decreased(P＜0.05),and the expression levels of SOX2 mRNA(P＜0.01)and protein(P＜0.001)were increased.Compared with control group,the relative density of surviving tumor cells in NR2F2 OE group was decreased,but the difference was not significant(P＜0.05),and the expression level of PD-L1 protein was decreased(P＜0.05).Compared with control group,the proliferation activities of cells in NR2F2 OE group were decreased(P＜0.05),and the drug sensitivities of the cells to PTX and CBP were enhanced(P＜0.05);the IC50 of PTX was significantly reduced,while the IC50of CBP could not be calculated due to excessively high drug concentration;the expression level of ABCG2 protein was decreased(P＜0.05).Conclusion:The over-expression of NR2F2 may inhibit the proliferation,migration,and invasion of the human ovarian cancer SKOV3 cells,decrease the expression levels of SOX2,PD-L1 and ABCG2 proteins,suppress the stemness and immune evasion ability of the SKOV3 cells,and enhance the sensitivities of the SKOV3 cells to PTX and CBP.

The Hedgehog(HH)signaling pathway is involved in various developmental processes of vertebrates and invertebrates,including embryogenesis,stem cell renewal,tissue regeneration,the occurrence and development of tumor and chemotherapy resistance.Overactivation and abnormal expression of key components in the HH signaling pathway are associated with the development of various malignant tumors.Phosphorylation plays an important role in regulating the activity and function of proteins in normal cells,and the disorder of phosphorylation-dephosphorylation cascade is closely related to tumorigenesis.The abnormal activation of this signaling pathway is also related to protein phosphorylation,while its complex underlying mechanism needs to be further explored.This article reviewed the research progress of the HH signaling pathway and protein phosphorylation modification in malignant tumors,focusing on the regulatory effect of phosphorylation modification on the activity of HH signaling pathway and its effect on the occurrence and development of malignant tumors,in order to provide a theoretical basis for the in-depth development and clinical application of targeted drugs for phosphatase/dephosphatase.

Objective:To discuss the effect of urolithin C(UC)on the proliferation,apoptosis,and autophagy of the acute myeloid leukemia(AML)HL-60 cells,and to clarify its mechanism.Methods:The HL-60 cells were divided into different concentrations(20,40,60,80,and 100 μmol·L-1)of urolithin A(UA)groups,urolithin B(UB)groups,and UC groups.CCK-8 assay was used to detect the proliferation activity of the cells in various groups;the morphology of the cells in different concentrations of UC groups was observed under optical microscope.The HL-60 cells were divided into different concentrations(0,20,40,and 80 μmol·L-1)of UC groups and 3-methyladenine(3-MA)combined with different concentrations(0,20,40,and 80 μmol·L-1)of UC groups.CCK-8 assay was used to detect the proliferation activities of the cells in various groups.The HL-60 cells were divided into control group(0 μmol·L-1)and different concentrations(20,40,and 80 μmol·L-1)of UC groups.The live/dead cell staining method was used to detect the dead rates of the cells in various groups;flow cytometry was used to detect the apoptotic rates of the cells in various groups;the autophagy of the cells was detected by autophagy staining kit(monodansylcadaverine,MDC)method;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of Beclin 1,autophagy related gene 9(ATG9),and autophagy related gene 7(ATG7)mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of cysteinyl aspartate specific proteinase-3(Caspase-3),cleaved cysteinyl aspartate specific proteinase-3(Cleaved Caspase-3),microtubule-associated protein 1 light 3(LC-3),extracellular regulated protein kinases(ERK),phosphorylated ERK(p-ERK),AMP-activated protein kinase(AMPK),and phosphorylated AMPK(p-AMPK)in the cells in various groups.Results:The CCK-8 assay results showed that after cultured for 24,48,and 72 h,compared with 0 μmol·L-1 UA,UB,and UC groups,the proliferation activities of the cells in different concentrations of UA,UB,and UC groups were decreased(P＜0.01)with a concentration-and time-dependent manner;at 48 h,compared with UA and UB,the half-maximal inhibitory concentration(IC50)of UC was the lowest.The cell morphology observation results showed that compared with control group,the intercellular connection and the number of the cells were decreased with the increasing of UC concentration,and the cell fragment was increased.The CCK-8 assay results showed that compared with 40 and 80 μmol·L-1 UC groups,the proliferation activities of the cells in 3-MA combined with 40 and 80 μmol·L-1 UC groups were increased(P＜0.05 or P＜0.01).The live/dead cell staining results showed that compared with control group,the dead rates of the cells in 40 and 80 μmol·L-1 UC groups were increased(P＜0.01).The flow cytometry results showed that compared with control group,the apoptotic rate of the cells in 80 μmol·L-1 UC group was increased(P＜0.01).The MDC method results showed that with the increasing of UC concentration,the green fluorescence in the cells in different concentrations of UC groups was gradually intensified.The RT-qPCR results showed that compared with control group,the expression levels of Beclin 1,ATG9,and ATG7 mRNA in the cells in 80 μmol·L-1 UC group were increased(P＜0.01).The Western blotting results showed that compared with control group,the expression levels of Cleaved Caspase-3 protein in the cells in 20,40,and 80 μmol·L-1 UC groups were increased(P＜0.01),the ratio of membrane LC3/cytoplasmic LC3(LC3-Ⅱ/LC3-Ⅰ)in the cells in 80 μmol·L-1 UC group was increased(P＜0.05),and the ratios of p-AMPK/AMPK and p-ERK/ERK in the cells in 40 and 80 μmol·L-1 UC groups were increased(P＜0.01).Conclusion:UC can inhibit the proliferation of the AML HL-60 cells,induce the apoptosis and autophagy,and increase the phosphorylation levels of ERK and AMPK proteins in the cells.

Objective To study the effects and mechanism of norcantharidin(NCTD)on proliferation and apoptosis of NB4 and K562 human leukemic cells by regulating phosphoprotein phosphatase 5 catalytic(PPP5C).Methods PC3.1 and PPP5C-PC3.1 plasmids were electroporated into NB4 and K562 cells.Stable NB4 and K562 cell lines were selected with geneticin(G418).Protein and mRNA expression levels of PPP5C were measured by Western blot and RT-qPCR,respectively.Proliferation,migration,and apoptosis of NB4 and K562 cells were determined by a CCK-8 assay,transwell assay,and Live & Dead? animal cell viability/toxicity detection kit,respectively.NB4 and K562 cells were divided into 0 μg/mL NCTD group and various NCTD dose groups,and cultured in RPMI 1640 medium containing 0,8,16,or 32 μg/ml NCTD.The Live & Dead? animal cell viability/toxicity detection kit measured the numbers of dead and live cells,and cell morphology was observed under a microscope.Western blot was used to measure protein expression levels of caspase 3,Cleaved caspase 3,JNK,p-JNK,p38,p-p38,and α-Tubulin.Results Proliferation,migration,and apoptosis of NB4 and K562 cells were enhanced by overexpression of PPP5C.Compared with 0 μg/mL NCTD group,NCTD promoted apoptosis in a dose-dependent manner.PPP5C overexpression antagonized the killing effect of NCTD on leukemic cells.Mechanistic investigations showed that PPP5C reduced the protein level of p-JNK by dephosphorylating and regulating the expression of apoptosis-related protein Cleaved caspase 3.Conclusions NCTD promotes apoptosis of NB4 and K562 cells and inhibits their proliferation by inhibiting PPP5C.

Objective To investigate the effects of atorvastatin calcium on thyroid function, immune response and C-Jun N-terminal kinase/p38 mitogen-activated protein kinase (JNK/p38 MAPK) signaling pathway in rats with hypothyroidism. Methods A total of 30 healthy adult male SD rats were randomly divided into control group, hypothyroid group (PTU group) and atorvastatin calcium treatment group (ACT group), with 10 rats in each group. Rats in the PTU group and the ACT group were injected with PTU subcutaneously at the dorsum of the neck every day for 28 consecutive days; instead of PTU, rats in the control group were injected subcutaneously with 0.3 mL of saline. After 2 weeks of PTU treatment, rats in the ACT group were gavaged with 3 mL of atorvastatin calcium saline solution (containing 5 mg/kg of atorvastatin calcium), which was administered once daily; the control group was gavaged with an equal amount of saline in the same way. The body weight, food intake and water intake of rats were measured weekly. The histopathological changes of the thyroid gland were observed in histopathological sections of rats in each group. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the levels of triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH), interferon γ (IFN-γ) and interleukin-4 (IL-4) in serum; quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to detect the mRNA expression levels of IFN-γ, IL-10, Foxp3 and IL-4; western blot was performed to determine the levels of p-JNK/JNK and p-p38/p38 MAPK. Results Compared with control group, PTU-induced hypothyroidism rats showed a significant decrease in body mass and food and water consumption (P < 0.05). After 2 weeks of treatment with atorvastatin calcium, the body mass loss of PTU rats was inhibited, food and water consumption was improved, and the differences were statistically significant (P < 0.05). Atorvastatin calcium was able to significantly increase the serum T3 and T4 levels and decrease the serum TSH level in hypothyroid rats (P < 0.05). Atorvastatin calcium treatment was able to significantly alleviate histopathological changes such as follicular cell proliferation and related hypertrophy induced by PTU, and increase follicular size (P < 0.05). After treatment with atorvastatin calcium, the spleen mass of PTU rats increased significantly, and the expression of IFN-γ mRNA in hypothyroid rats decreased significantly, but the expression levels of IL-10 mRNA, Foxp3 mRNA and IL-4 mRNA increased significantly (P < 0.05). After treatment with atorvastatin calcium, the levels of p-JNK/JNK and p-p38/p38 MAPK in thyroid tissue of hypothyroid rats decreased significantly (P < 0.05). Conclusion Atorvastatin calcium treatment for hypothyroidism has the function of promoting the normalization of thyroid hormone imbalance, balancing Th1/Th2 cytokines, and inhibiting the activation of JNK/p38 MAPK signaling pathway.

OBJECTIVE:To observe effects and safety of Kangfuxin solution combined with intense pulsed light in the treat-ment of rosacea. METHODS:A total of 50 rosacea patients in our hospital during May 2014-Jun. 2016 were divided into control group(25 cases)and observation group(25 cases)according to random number table. Based on oral administration of Metronida-zole tablets,control group received intense pulsed light. Observation group was additionally given Kangfuxin solution for local wet compress after 4 to 6 layers of gauze saturated with liquid,5-10 min,qn. Both groups received treatment for 4 weeks. Clinical effi-cacies,as well as symptom score and DLQI score were compared between 2 groups before and after treatment,and the occurrence of ADR was recorded. RESULTS:The response rate of observation group was 92.0%,which was significantly higher than 64.0%of control group,with statistical significance (P<0.05). Before treatment,there was no statistical significance in erythema,pap-ules,pustules,itching,telangiectasia score and total score,DLQI score with before treatment(P>0.05). After treatment,erythe-ma,papules,pustules,itching,telangiectasia score and total score,DLQI score of 2 groups were decreased significantly,and the observation group was significantly lower than the control group,with statistical significance(P<0.05). The incidence of ADR in observation group was 16.0%,which was significantly lower than 40.0% in control group,with statistical significance(P<0.05). CONCLUSIONS:Kangfuxin solution combined with intense pulsed light show significant efficacy for rosacea,and can effectively improve erythema,papules,pustules,itching and telangiectasia,and improve the quality of life with good safety.

Objective To research the action mechanism of Chinese FormulaXuetongling on MDA-MB-231 human breast cancer cell invasion.Methods Transwell Invasion assay was applied to investigate the inhibitory effects on cancer cell invasion ofXuetongling extract in different concentrations;MMP-9 secretion activity was detected by zymography assay after the treatment of XTL extract in MDA-MB-231;Western blot was used to detect the effect of XTL extract on MMP-9 protein expression in MDA-MB-231.ResultsXuetongling extract in different concentrations significantly suppressed the cell invasion, MMP-9 secretion and MMP-9 protein expression in dose dependent manner.Conclusion The inhibitory effect ofXuetongling on MDA-MB-231 cell invasion may be due to the down-regulation of both MMP-9 secretion and MMP-9 protein expression.