| Esophagus cancer(EC)is highly prevalent in Asia,particularly in China,which has become one of the leading causes of cancer-related death worldwide.Recently,advances have been made in the diagnosis and treatment of EC,which have improved the survival for patients with early EC.However,EC is usually diagnosed at an advanced stage,and its prognosis remains dismal.More importantly,EC is highly resistant to radiotherapy/ chemotherapy;as a result,it is mainly treated by esophagectomy.On this account,it is required that experts and scholars all over the world should make great efforts to understand the pathogenesis and molecular mechanism of EC more deeply,and to find the natural medicine with definite curative effect while little side effect.This study had explored the relationship of miR-203 a expression with the genesis and development of esophageal squamous cell carcinoma(ESCC)at genetic level.Moreover,nine limonoid compounds were also investigated,and their inhibition activity on the proliferation of ESCC cells was detected.In addition,four kinds of compounds with obvious inhibitory activities on the proliferation of ESCC cells were screened out,the mechanism of action by which the Xylogranatin C compound suppressed the proliferation of ESCC was preliminarily investigated,and the influence of Xylogranatin C on the miR-203 a gene expression level in ESCC cells was explored,which was important for developing the highly effective and low-toxic antitumor drugs and for the basic research of new anticancer precursor compounds.Part One Expression and significance of miR-203 a in Human Esophageal Squamous Cell CancerObjective: EC can be classified into five types based on the histological classification,including squamous cell carcinoma,adenocarcinoma,and undifferentiated carcinoma and so on.Among which,esophageal squamous cell carcinoma(ESCC)is the most common.At present,the pathogenesis of EC remains largely unclear.This study aimed to examine the expression of miR-203 a genes in ESCC tissues and normal tissues,explore the relationship of miR-203 a gene with the occurrence and development of ESCC,and identify the molecular mechanism of ESCC,which could provide a new theoretical basis for the prognosis and other aspects of ESCC.Methods:The expression of miR-203 a genes in 119 ESCC tissues and their corresponding normal tissues was detected by quantitative real-time RT-PCR(qRT-PCR).2.The relationship of the expression of miR-203 a with the prognosis of ESCC was examined using Kaplan Meier analysis.3.The statistical software SPSS19.0 was employed for statistical analysis.Results:1.miR-203 a could be detected in ESCC tissues and their normal tissues,but the expression levels of miR-203 a in ESCC tumor tissues were evidently down-regulated compared with those in the corresponding normal tissues.Additionally,miR-203 a expression was associated with TNM stage,pathological differentiation,lymph node metastasis,distant metastasis or recurrence,and the UGIC family history2.ESCC patients with low miR-203 a expression had markedly poorer 5-year survival rates.Meanwhile,TNM stage and miR-203 a expression were independently associated with the survival of ESCC patients.Conclusion:1.The miR-203 a expression levels in ESCC tumor tissues areevidently down-regulated compared with those in corresponding normal tissues.miRNAs may function as the oncogenes or tumor suppressors in ESCC.The miR-203 a expression is associated with TNM stage,degree of differentiation,lymph node metastasis,distant metastasis or recurrence,and the UGIC family history.2.TNM stage and miR-203 a expression are independently associated with the survival of ESCC patients.Part two Inhibitory Effects of Nine Limonoid Compounds on the Proliferation of Esophageal Squamous CellsObjective: Limonoid compounds are a class of highly oxidized tetracyclic triterpenes,which have attracted wide attention from experts and scholars since 1970 s and 1980 s.A preliminary study has been carried out,and the results show that they not only possess insecticidal,antimicrobial,antiviral and other biological activities,but also have the most noticeable anti-tumor effect.In this study,9 kinds of lemon-like compounds,which were isolated and purified from xylocarpus granatum plants,were selected to inhibit the proliferation activity of EC cells,so as to screen out the compounds that could inhibit the proliferation of EC cells.Methods: The inhibitory effects of limonoid compounds on the proliferation of EC cells(Eca109 cells and EC9706 cells)were detected through MTT Colorimetric assay,so as to screen the monomer compounds with significant inhibition on tumor cell proliferation.Besides,the half inhibition concentrations(IC50 values)of the compounds were also calculated.Results: EC cells(Eca109 and EC9706)were co-cultured with cisplatin(100 μmol/l),which had showed strong proliferative inhibition activity.Besides,compounds,including spicatin(3),7-deacetyl-7-oxogedunin(4),xylogranatin C(5)and xylocarponoid A(7)also displayed strong inhibitory activities on the proliferation of Eca109 cells,with the cell survival rates of 9.19%,5.01%,4.04% and 7.15%,respectively.In addition,the survival rates of EC9706 tumor cells treated with these four kinds of limonoid compounds were 21.93%,19.78%,8.25% and 12.33%,respectively.However,the inhibition rate of limonoid compounds on EC cells did not reach 50% in other experiments.Moreover,the IC50 values of four compounds(compound 3,4,5 and 7)on Eca109 and EC9706 cells were calculated using the logarithmic regression equation of tumor cell proliferation survival rate.Compound 2 is steroids,compounds 1 and 3 are original lemon bitter,compound 4 is D ring cracking lemon bitter,compound 5,6,7,8,9 for B,D ring cracking lemon bitter.Compounds 5 and 6,although the names are the same,are derived from different literature and have different structures.Compounds 3,4,5,7 because the A ring contains alpha,beta-unsaturated ketone structure fragments,showing that esophageal cancer Eca109,EC9706 cells have a strong proliferative inhibition activityConclusions: Lemon Spicatin(3),7-deacetyl-7-oxogedunin(4),Xylogranatin C(5)and Xylocarponoid A(7)show remarkable inhibitory effects on the proliferation of EC cells.Typically,the unique skeleton structure of limonoid compounds,and the α,β-unsaturated ketone are the essential structures to resist the proliferation of EC cells.Part three Mechanism of Xylogranatin C inhibition on proliferation of human esophageal carcinoma cellsObjective: A large number of experiments in vitro found that many ingredients in Chinese herbal medicine have obvious antitumor activity,and the effective components was accomplished by inducing tumor cell apoptosis.However,their influence on genes has not been reported yet.Experiment in part one discovered that,low miR-203 a gene expression in ESCC tissues predicted poor prognosis for patients.Experiment in part two had screened four compounds from Xylocarpus granatum that could remarkably inhibit the proliferation of human esophageal cancer cells.Experiment in this part would be based on Xylogranatin C as a representative of the mechanism of the action of these compounds to preliminarily study whether such compounds exerted their activities by inducing tumor cell apoptosis and up-regulating miR-203 a gene expression.Methods:1.The apoptosis of Eca109 cells was detected by flow cytometry after Xylogranatin C treatment for 12 or 24 h at the final concentrations of 10 and 20 μmol/L.2.The expression of apoptosis proteins p53,Bax,caspase-3 and GRP 78 was detected through Western Blotting.3.qRT-PCR was employed to detect the miR-203 a expression after treating Eca109 cells with xylogranatin C.Results:1.Effect of Xylogranatin C on the apoptosis of Eca109 cells: Eca109 cells were treated with 10 and 20 μmol/L Xylogranatin C for 12 h and 24 h,respectively;and the cell apoptosis rates were(8.63±0.91)% and(25.76 ± 1.44)%,as well as(11.13 ± 1.80)% and(46.17 ± 1.56)%,respectively,compared with the blank control group(P< 0.05).2.Effect of Xylogranatin C on up-regulating p53,Bax,caspase-3 and GRP78: After Xylogranatin C treatment at the final concentration of 10 or 20 μmol/L for 12 or 24 h,the expression of p53,Bax,caspase-3 and GRP78 was remarkably increased compared with the control group(P<0.05).3.qRT-PCR method was used to detect the miR-203 a expression after treating Eca109 cells with Xylogranatin C.The results indicated that,miR-203 a expression levels were up-regulated in Eca109 cellls after 10 μmol/L and 20μmol/L Xylogranatin C treatment for 12 h and 24 h,respectively,and the difference was statistically significant compared with the control group(P<0.01).Besides,with the increase in concentration and extension in time,the expression was remarkably increased,and the difference between 12 h and 24 h groups was also statistically significant.Conclusions: Findings in this part indicate that Xylogranatin C can induce the apoptosis of Eca109 cells and exert the antitumor activity.Besides,the expression of p53,Bax and Caspase-3 is up-regulated in Eca109 cells treated with Xylogranatin C,revealing that Xylogranatin C may induce Eca109 cell apoptosis through the joint effect of multiple pathways such as the death receptor and mitochondria-dependent signaling pathways.Additionally,Xylogranatin C can suppress tumor cell proliferation through up-regulating the miR-203 a expression in esophageal cancer Eca109 cells.Summing up:1.Low expression participation of mirR-203 a in the occurrence and progression of esophageal squamous cell carcinoma,and is closely related to the prognosis of patients;2.XylogranatinC has significant apoptosis-inducing activity in esophageal carcinoma cells.Xylogranatinc to achieve the proliferation of anticancer cells by raising the mir-203 a expression of Eca109 cells in esophageal cancer. |
PDF Full Text Request