Objective To explore the influence of IL-6 secreted by cancer-associated fibroblasts(CAFs)on pro-moting the proliferation and invasion of ovarian cancer cells and the possible mechanisms.Methods CAFs and normal ovarian fibroblasts(NFs)were isolated and cultured respectively from epithelial ovarian cancer and normal ovarian epithelial tissues.Cell markers alpha-smooth muscle actin(α-SMA),E-cadherin were detected by West-ern blot and immunofluorescence.CAFs and normal ovarian fibroblasts(NFs)were collected and cultured,and their supernatants were used to establish an indirect co-culture system with ovarian cancer SKOV3 cells,including SKOV3 cells alone(SKOV3)group,SKOV3 combined with the supernatants of NFs(NFs)group and SKOV3 combined with the supernatants of CAFs(CAFs)group.Cell immunochemistry was used to detect the expression of alcohol dehydrogenase 1B(ADH1B)in SKOV3 cells co-cultured with the supernatant of CAFs or NFs.Before and after treatment with the methylation inhibitor 5-aza-2'-deoxycytidine(5-Aza-dC),methylation-specific PCR(MSP),Reverse transcription quantitative real-time PCR(RT-qPCR),enzyme-linked immunosorbent assay(ELISA),and Western blot were used to detect the mRNA level and methylation status of ADH1B,and the phos-phorylation level of signal transducers and activators of transcription 3(p-STAT3).The cell counting kit-8(CCK-8)method and Transwell assay were used to investigate the effects of the IL-6 inhibitor LMT-286 and recombinant human interleukin-6(rhIL-6)on cell proliferation and invasion.Results The protein levels of α-SMA was highly expressed,however,CAFs and NFs cells almost lacked the E-cadherin protein.Compared with the SKOV3 and NFs groups,CAFs group exhibited significantly downregulated mRNA and protein expression of ADH1B.After treatment with 5-Aza-dC,ADH1B methylation was partially reversed,and the mRNA and protein expression of ADH1B increased in all groups.The phosphorylation level of STAT3 proteins was significantly reduced in CAFs group,while there were no significant changes in SKOV3 and NFs groups.Intervention with LMT-286 and rhIL-6 only inhibited or promoted the proliferation and invasion of cells in CAFs group,while there were no significant changes in SKOV3 and NFs groups.Conclusion CAFs can enhance the methylation of ADH1B in ovarian cancer cells via IL-6/STAT3 pathway,and may promote the proliferation and invasion.

Objective To investigate the genetic carrier rate of thalassemia and its gene mutation types as well as the distribution characteristics among couples of reproductive age in Dongfang City of Hainan Province,and to provide a basis for making prevention and control strategies against thalassemia.Methods Samples were collected from 1 000 couples undergoing premarital and pregestational screenings for thalassemia in Dongfang City of Hainan Province from September 2012 to March 2013,in which the positive ones in preliminary screening were further tested by genetic diagnoses and the genotypes were retrospectively analyzed.Results Among 1 000 couples,322 spouses were diagnosed with thalassemia gene mutation and the carrying rate was 16.10％ (322/2 000).In those carriers,246 spouses were α-thalassemia and the carrying rate was 12.30％ (246/2 000),accounting for 76.40％(246/322) of all thalassemia carriers,among them,there were 197 cases of α-deficiency genotype,accounting for 61.18％ (197/322),32 carried mutated α-gene,accounting for 9.94％ (32/322),17 carried both deleted and mutated α-gene,accounting for 5.28％ (17/322);43 spouse were β-thalassemia and the carrying rate was 2.15％(43/2 000);33 spouse were both α-and β-thalassemia and the carrying rate was 1.65％ (33/2 000).In spouses diagnosed with α-thalassemia,the major genotype was-α37/αα,accounting for 19.25％ (62/322);the second ranked was-α4.2/αα,accounting for 17.70％ (57/322),and the third ranked was--SEA/αα,accounting for 8.70％ (28/322).In spouses diagnosed with β-thalassemia,the major genotype was CD41-42/N,accounting for 9.63％ (31/322).Conclusions The population carrying rate of thalassemia in Dongfang City of Hainan Province is high,and its major type is α-thalassemia.For the purpose of decreasing the birth rate of thalassemia,major,local public health department should attach great importance to thalassemia prevention,and strengthen premarital and pregestational screening for thalassemia.

Objective To investigate whether omeprazole(OME)can enhance the sensitivity of epithelial ovarian cancer(EOC)cells to cisplatin(DDP)by inhibition of autophagy and to elucidate its possible mechanism.Methods Color in situ hy-bridization(CISH)and immunohistochemistry were applied to detect the expression of miR-214-3p and autophagy specific mark-ers p62 in EOC tissues,respectively.Pearson analysis showed the correlation between miR-214-3p and p62 expression levels in EOC.The half concentration(IC50)of DDP was determined by CCK-8 method.The mRNA expressions of miR-214-3p and multi-drug resistance gene 1(MDR1),the protein levels of p-gp and p62 were measured by using real-time quantitative PCR(qRT-PCR)and Western blot,respectively.Results In 43 cases,the expressions of miR-214-3p and p62 were 53.5％(23/43)and 60.5％(26/43)in patients with ovarian carcinoma,respectively.miR-214-3p was downregulated in platinum-relatively resistant OC tissue(P＜0.05).On the contrary,p62 was upregulated in platinum-relatively resistant OC tissue(P＜0.01).In ovarian cancer,the negative expression of miR-214-3p was closely related with p62(r=0.238,P＜0.05).After OME(150 μmol/L)pre-treatment,varying degrees of decrease was observed in cisplatin IC50 OV2008 and C13K cells,especially cisplatin resistant strain C13K(P＜0.01).After DDP treatment,qRT-PCR results revealed that the expression of miR-214-3p was decreased,the mRNA and protein expressions of MDR1 were greatly increased,and the protein levels of p62 were increased in C13K and OV2008 cells,compared to the blank control C13K and OV2008 cells(all P＜0.01).Compared with the blank control C13K and OV2008 cells,the IC50 of DDP was decreased after pretreatment with OME(150 μmol/L).The sensitivity of C13K and OV2008 cells to DDP was increased after OME(150 μmol/L)pretreatment,the relative expression of miR-214-3p was significantly increased,the expression of MDR1 protein and mRNA was decreased,and the expression of p62 protein was decreased(all P＜0.05).Conclu-sion OME pretreatment might enhance the sensitivity of ovarian cancer cells to DDP by downregulating miR-214-3p mediated autophagy.

Objective To investigate the correlation between Yes-associated protein(YAP)nuclear expression and tumor size with prognosis of patients with epithelial ovarian cancer(EOC)and to study the role of YAP in EOC.Methods 120 patients with EOC were selected as the experimental group,including 38 patients with early stage(Ⅰ+Ⅱ)EOC and 8 2 patients with advanced stage(Ⅲ+Ⅳ)EOC.3 0 normal ovarian tissues obtained from patients with uterine leiomyoma were enrolled as the control group.Immunohistochemical(IHC)assay was em-ployed to determine YAP expression and sub-location.The relationship between YAP expression and the pathologi-cal parameters of the 120 patients with EOC was analyzed,so as to the prognosis of these patients.EOC cells(C13K and OV2008)were cultured with varying initial cell volumes.Ki67 expression and cell proliferation were tested by immunofluorescence and cloning assay respectively.YAP expression at mRNA and protein levels were de-tected by q-PCR and Western blot respectively when the cell conference of EOC cells reached to low(60％)and high(90％)cell density.Results The YAP nuclear expression was significantly higher in the EOC group com-pared to the control group(P＜0.05).The average diameter of stage Ⅰ+Ⅱ EOC was larger than that of stage Ⅲ+Ⅳ EOC(P＜0.01).The high nuclear expression of YAP was positively associated with pathological grade,clinical stage and the level of Ca125＞1 000 IU/ml,while negatively correlated with tumor size(all P＜0.05).Survival analyses showed that smaller tumor size(＜10 cm)and higher YAP nuclear expression were negatively as-sociated with the 3-year overall survival rate of EOC patients(P＜0.01).C13K and OV2008 cells cultured in the low density group exhibited a high number of clone formation,high Ki67 and YAP expression(P＜0.01).The down-regulation of YAP expression could decrease the cell viability of EOC cells in the low-and high-density groups(P＜0.05).Conclusion Higher level of YAP nuclear expression and smaller tumour size are inversely associated with the clinical prognosis of patients with EOC.Inhibiting YAP nuclear expression leads to a decrease in the prolif-eration capacity of EOC cells.

Objective To investigate the effect of microRNA-214-3p(miR-214-3p)expression in cancer-associated fibroblasts(CAFs)on the cisplatin sensitivity of ovarian cancer cells and its mechanism.Methods Sixty-four ovarian cancer patients were selected as study subjects and divided into platinum-partially sensitive group and platinum-sensitive group based on progression-free survival after chemotherapy.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative expression of miR-214-3p in ovarian cancer tissues from the two groups,and the 2-year survival rates of patients with different clinical characteristics were compared.CAFs and normal ovarian fibroblasts(NFs)were primarily cultured,and qRT-PCR and immunofluorescence experiments were used to detect the expression of miR-214-3p and p62 protein in CAFs and NFs.The expression lev-els of SQSTM1 gene in different types of ovarian cells were searched through the CSIOVDB data-base.CAFs were transiently transfected with miR-214-3p mimic(mimic group)and miR-214-3p mimic NC(NC group),and untransfected CAFs were selected as control group.Cell culture super-natants from each group were collected,and an indirect co-culture model of ovarian cancer cell line SKOV3 with CAFs from each group was established.The CCK-8 method,DCFH-DA method,qRT-PCR,and immunoblotting were used to detect the proliferation rate,half-maximal inhibitory concen-tration of cisplatin(IC50),reactive oxygen species(ROS)content,and relative expression levels of miR-214-3p,cisplatin resistance gene CCND1,and autophagy protein p62 in SKOV3 cells under different culture conditions.Results The relative expression of miR-214-3p was lower in the plati-num-partially sensitive group than in the platinum-sensitive group(P＜0.01).There were statisti-cally significant differences in the 2-year survival rates among patients with different International Federation of Gynecology and Obstetrics(FIGO)stages,platinum partial sensitivity,and low miR-214-3p expression(P＜0.01).The relative expression of miR-214-3p was lower in ovarian CAFs than in NFs,while the expression level of p62 protein was higher in CAFs than in NFs(P＜0.01).Online analysis of the CSIOVDB database showed that the expression level of SQSTM1 gene in ovari-an CAFs was higher than that in ovarian cancer epithelial cells and NFs(P＜0.01).Compared with SKOV3 cells indirectly co-cultured with CAFs in the control group and CAFs in NC group,SKOV3 cells indirectly co-cultured with mimic CAFs showed reduced proliferation rate,cisplatin IC50,and ROS content,increased relative expression of miR-214-3p,and decreased relative ex-pression of CCND1 mRNA and p62 protein(P＜0.01).Conclusion The expression of miR-214-3p in CAFs is correlated with the sensitivity of ovarian cancer cells to cisplatin.Low expression of miR-214-3p in CAFs can promote the proliferation of ovarian cancer cells and ROS-mediated autophagy,thereby reducing the sensitivity of ovarian cancer cells to cisplatin.

Objective To investigate the effect of microRNA-214-3p(miR-214-3p)expression in cancer-associated fibroblasts(CAFs)on the cisplatin sensitivity of ovarian cancer cells and its mechanism.Methods Sixty-four ovarian cancer patients were selected as study subjects and divided into platinum-partially sensitive group and platinum-sensitive group based on progression-free survival after chemotherapy.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative expression of miR-214-3p in ovarian cancer tissues from the two groups,and the 2-year survival rates of patients with different clinical characteristics were compared.CAFs and normal ovarian fibroblasts(NFs)were primarily cultured,and qRT-PCR and immunofluorescence experiments were used to detect the expression of miR-214-3p and p62 protein in CAFs and NFs.The expression lev-els of SQSTM1 gene in different types of ovarian cells were searched through the CSIOVDB data-base.CAFs were transiently transfected with miR-214-3p mimic(mimic group)and miR-214-3p mimic NC(NC group),and untransfected CAFs were selected as control group.Cell culture super-natants from each group were collected,and an indirect co-culture model of ovarian cancer cell line SKOV3 with CAFs from each group was established.The CCK-8 method,DCFH-DA method,qRT-PCR,and immunoblotting were used to detect the proliferation rate,half-maximal inhibitory concen-tration of cisplatin(IC50),reactive oxygen species(ROS)content,and relative expression levels of miR-214-3p,cisplatin resistance gene CCND1,and autophagy protein p62 in SKOV3 cells under different culture conditions.Results The relative expression of miR-214-3p was lower in the plati-num-partially sensitive group than in the platinum-sensitive group(P＜0.01).There were statisti-cally significant differences in the 2-year survival rates among patients with different International Federation of Gynecology and Obstetrics(FIGO)stages,platinum partial sensitivity,and low miR-214-3p expression(P＜0.01).The relative expression of miR-214-3p was lower in ovarian CAFs than in NFs,while the expression level of p62 protein was higher in CAFs than in NFs(P＜0.01).Online analysis of the CSIOVDB database showed that the expression level of SQSTM1 gene in ovari-an CAFs was higher than that in ovarian cancer epithelial cells and NFs(P＜0.01).Compared with SKOV3 cells indirectly co-cultured with CAFs in the control group and CAFs in NC group,SKOV3 cells indirectly co-cultured with mimic CAFs showed reduced proliferation rate,cisplatin IC50,and ROS content,increased relative expression of miR-214-3p,and decreased relative ex-pression of CCND1 mRNA and p62 protein(P＜0.01).Conclusion The expression of miR-214-3p in CAFs is correlated with the sensitivity of ovarian cancer cells to cisplatin.Low expression of miR-214-3p in CAFs can promote the proliferation of ovarian cancer cells and ROS-mediated autophagy,thereby reducing the sensitivity of ovarian cancer cells to cisplatin.