| Backgroud and ObjectiveColorectal cancer and Gastric cancer are the most common malignancies in digestive tract malignant tumor,In United States and other developed countries,CRC is the third in the incidence of tumors,second in mortality rate and fourth in china cancer incidence.So far,the pathology and pathogenesis of colorectal cancer had not yet been fully understood,with the development of biology technology and understanding of disease,it is widely accepted that the development of colorectal cancer experiences multiple steps?factors and stages,it is the result of the mutual interaction of genetic factors and environmental factors.As we know,oncogenes and tumor suppressor genes both involve in the occurrence and development of tumors,including the activation of oncogenes,the deletion or mutation of tumor suppressor gene,in addition to this,the mismatch repair genes(mismatch,repair,MMR)mutation or inactivation also included.The inactivation of tumor suppressor genes play an important role in it.Data shows that,with the improvement of people's living standard and changes in diet,its incidence increases year by year.Gastric cancer as one of the world's high incidence malignant tumors,is the result of the combined effects of multiple factors,such as Helicobacter pylori infection,smoking and gastric ulcer and so on,but its exact pathogenesis is not yet clear.Although the exact pathogenesis of cancers has not been fully elucidated,further study of tumor pathogenesis makes people change their understanding of it from the past single physicalcarcinogens,chemical carcinogen,viruses caused cancer,gene mutation to multi-step,multi-factor comprehensive theory of cancer.At present,people have accepted that the occurrence of tumors is a gradual process,involving multiple oncogene activation and tumor-suppressor gene inactivation.People's understanding of the X chromosome is limited to gender for a long-time,but now its role in occurance and development of tumor is gradually recognized.There were more and more evidence showed that the X chromosome was associated with tumors,though the exact mechanism was not clear.Researchers from Masschusetts general hospital cancer center?Dana-Farber cancer institute and so on using a high resolution screen for DNA copy number alterations in Wilms tumor,they identified somatic deletions targeting a novel gene on the X chromosome.They named it WTX.WTX is inactivated in approximately one third of Wilms tumors.In 2007,Rivera et al published this result on?Science?,due to the special localization of WTX,it had unique characteristics and research value as a tumor suppressor gene,so once this scientific research achievement was published,it caused concern immediately?Beside this,researchers found that WTX also expressed in renal blastemal stem cells,this turned out that WTX played a key role in organ development.The occurrence and development of tumors are the result of the interaction of multiple genes and informations,the activation of oncogenes and(or)inactivation of tumor suppressor genes can lead to cell physiological regulation signal pathway inappropriate activation and inactivation,cell proliferation out of control and induced tumor formation.Some classical signaling pathways play important role in the development of tumor?Wnt/?-catenin signaling pathway is one of this.It is a kind of conduction growth stimulating signal pathways,it is activated in the process of embryonic development,mainly involved in various developmental modes,but closed in normal mature cells.Defects in the Wnt signal pathway can lead to a variety of early developmental defects,its abnormal activation is closely associated with a variety of tumorigenesis at the same time.There are dozens of high incidence cancers are relative with the disorder transduction of Wnt signal pathways,the abnormal activation of ?-catenin plays key role in tumorigenesis.The role of Wnt/?-catenin signal pathway in embryonic development and tumor progression has been elucidated.It was reported that WTX protein could negatively regulate Wnt/?-catenin signaling by forming a complex with AXIN1,?-catenin,APC,and?-TrCP2 to result in the degradation of P-catenin.Mutations of WTX is common in Wilms tumor and result in the loss expression of itself.At present,research on WTX gene are mainly concentrated on its mutation rate in tumors,except wilms tumor,its mutation is not common in other tumors.The expression and possible inactivation mechanisms of WTX in other tumors(including colorectal cancer and gastric cancer)have not been reported,yet need to be proved.In the aspect of non-tumor research,studies had shown that aberrant expression of WTX take part in osteopathia striata with cranial sclerosis?anti-oxidant response?cell differentiation and other disease.Base on the present study of WTX gene,our study will explore the role of WTX play in gastrointestinal tunor and its possible mechanism,to provide the basis underlie of the development of gastrointestinal tumor.Methods1.Effects and mechanisms of tumor suppressor gene WTX in colorectal cancer1.1 Identification of WTX in Colorectal cancer cell lines qRT-PCR was used to detect the expression of WTX in 6 CRC cell lines,including SW480,SW620,HT29,LOVO,HCT116 and LS174T.1.2 The functions of WTX in CRC1.2.1 The CRC-derived cell lines SW620N1/SW620W+ were established with stable overexpression of WTX using G418 selected and identificated.1.2.2 CCK8 method,colony formation assay,transwell in vitro invasion assay?wound-healing assay?western blotting and in vivo tumor growth by subcutaneous injection and in situ metastatic experiment were carried out to detect cell proliferation,invasion and metastatsis abilities in vitro and on vitro after upregulation of WTX.1.3 The molecular mechanisms of WTX involved in CRCWestern blot were used to detect the expression of EMT relative proteins(E-cadherin?Vimentin)and P-catenin in SW620N1 and SW620W+2.Effects and mechanisms of tumor suppressor gene WTX in gastric cancer2.1 Immunohistochemical and Western blot detection of WTX protein expression in gastric cancer and its paired normal tissues.2.2qRT PCR was used to detect the expression of WTX in three gastric cancer cell lines MGC803?MGC823?BGC823,and fourty three pairs of gastric cancer tissues.3.Statistical analysisSPSS 1 3.0 soRwarc Was used for statistical analysis.Data were presented as MeanąSEM of at least 3 independent experiments.Relative quantification value(2-??Ct)of Real-time PCR,colony formation assay,and transwell in vitro invasion assay were analyzed through Independent-Samples T test.The results of CCK8 assay and in vivo tumor growth by subcutaneous injection were analyzed by Factorial design analysis of variance and One-Way ANOVA.The results of immunohistochemistry was analyzed by the Mann-Whitney U Test,correlation analysis using Spearman rank correlation test.P values of<0.05 were considered statistically significant.Results1.Effects and mechanisms of tumor suppressor gene WTX in colorectal cancer1.1.Identification of the expression characteristics of WTX in CRC(Colorectal cancer)cell linesQuantitive real-time PCR was used to detect the expression of WTX in 6 CRC cell lines.The results of One-Way-ANOVA ananlysis showed that the expression of WTX in 6 cell lines was different from each other(F=10.146,P<0.01).SW620 as control group,the expression of WTX in SW480 and LOVO were higher than SW620(P<0.001,P<0.01),the expression of WTX were no difference among HT29?LS174T?HCT116 and SW620(P=0.462,P=0.188,P=0.214).Western blot result shows that the expression of WTX in SW620 lower than SW480?HCT116?LOVO andHT29,there is no significant difference between SW620 and L174T.1.2 Establishment of the stable overexpression of WTX cell line1.2.1.The CRC-derived cell lines SW620W+/SW620N1were established with stable overexpression of WTX selected using G418.1.2.2.Identification of the expression characteristics of WTX in cell lines with stable overexpression of WTX.qRT PCR results showed that WTX expression in SW620W+ was significantly higher than sw620-N1 group(t=13.695,<0.05).Western blot analysis result confirmed that the expression of WTX in SW620W+ cell line is higher than SW620N1 cell line,these results suggested that SW620W+ and SW620N1 were successfully established.1.3.The effects of WTX on biological behaviors of colorectal cancer cells1.3.1 CCK8 assays showed that cell proliferation ability was significantly different between groups(F=3.280,P<0.001),comparedto SW620N1 cells,the proliferation of SW620W+ cells was significantly decreased.1.3.2 Colony formation assay showed that SW620N1 and SW620W+colony formation rate were53.333%?16.222%respectively,the ability of SW620W+ cells to form colony was signifcantly decrease after transfection of WTX(t=18.442,P<0.001).1.3.3 In vitro invasion assays showed that invasion of SW620W+ cells were markedly decreased as compared with sw620N1 cells by use of BD Transwell Inserts(t=27.081?p<0.001).1.3.4 In vitro migration assays showed that migration of SW620W+cells were markedly decreased as compared with sw620-N1 cells by use of BD Transwell Inserts(t=12.181,P<0.001).1.3.5 Wound-healing assays showed that cells in SW620W+ group had significantly decreased motility as compared with cells in SW620N1 group..1.3.6 In vivo tumor growth was assessed by subcutaneous injection and in situ metastatic experiment was assessed by subcutaneous tumor tissue inoculating into caecum.The ability of subcutaneous tumor growth was significantly decreased after transfectedwith WTX in SW620 cells,the ability was significant difference in groups(F=35.819,P<0.001).Cells transfected with WTX got low metastatic capacity(liver metastasis).1.4 Preliminary study of WTX involved in signaling pathways in CRCWestern blot results showed that the expression of ?-catenin was decreased in SW620W+ group,the expressions of E-cadherin?Vimentin had no significant differences between SW620N1 and SW620W+ groups.2.The preliminary study of the effects and mechanisms of tumor suppressor gene WTX in gastric cancer2.1 Immunohistochemical detection of WTX protein expression in gastric cancer and its paired normal tissues.The expression of WTX in gastric cancer and normal gastric tissues were significantly different(p<0.01).The decreased expression of WTX in gastric cancer tissues was negatively correlation with cancer staging?lymph node metastasis and progonosis(p<0.01).2.2 Identification the expression characteristics of WTX in Gastric cancer cell lines and Gastric cancer tissues and its matched normal tissuesQuantitive-PCR was used to detect the expression of WTX in 3 Gastric caner cell lines.The results of One-Way-ANOVA ananlysis showed that MGC803 as control group,the expression of WTX in BGC823 was significantly higher than MGC803(P<0.01),there was no differenance between MGC803 and MGC823(P=0.942).But the western blot showed that the expression of WTX in MGC803 was lower than both MGC823 and BGC823.In 43 pairs of gastric cancer tissues and its matched normal tissues,the expression of WTX was lower in cancer tissues than in normal tissues(t=-4.731,p<0.01),western blot result showed that WTX showed high expression trend in normal tissue,low expression trend in its paired cancer tissues.Conclusions:1.The expression of WTX was down-regulated in colorectal cancer cell lines;2.Overexpression of WTX can inhibit the ability of proliferation,invasion,metastasis in colorectal cancer cells,it may be a candidate CRC tumor suppressor gene and therapeutic target;3.WTX as a tumor suppressor gene in CRC,its mechanism may be associated with the inhibition of beta-catenin expression;4.WTX gene is a tumor suppressor gene in gastric caner,and its mechanism needs further study. |
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