| Background and objectiveColorectal cancer(CRC)is one of the most common malignant tumors,its incidence is increasing year by year,and the efficacy of treatment is still not optimistic.The molecular biology of the development of colorectal cancers is complex,and its process involved multi-step,multi-mechanism,and multiple genes,in which the inactivation of tumor suppressor genes is one of the important factors that lead to its occurrence.Tumor suppressor genes,P53,APC,DCC and MMR were known closely related to colorectal cancer.Screening and identification of new suppressor genes will help to reveal the pathogenesis of colorectal cancer,prognosis and guide clinical treatment,also provide a new entry point for tumor therapy which is an important strategy for cancer treatment.Wilms tumor gene on the X chromosome(WTX),also known as FAM123B,was the first tumor suppressor gene located in the X chromosome discovered in Wilms tumor.Human WTX gene is located on Xq11.1,full-length 7.5kb and the CDS is 3571bp,encoding a 124KD protein.As a member of the WNT/?-catenin pathway,WTX gene plays an important role in normal physiological function and tumor development.It was showed that the role of WTX included three aspects.Firstly,WTX promoted ubiquitination and degradation of ?-catenin and downregulated WNT/?-catenin signaling pathway related to the classic embryonic development and tumorigenecity as a tumor suppressor gene.Secondly,WTX played an important role in the WNT signaling transduction in the plasma membrane;Thirdly,WTX controlled cell-cell adhesion through interaction with APC in the plasma membrane,and regulated the transcription factor activity of WT1 in the nucleus.Moreover,WTX gene mutation related to sclerosing skeletal dysplasia.Our group widely detected the expression of WTX protein and RNA in the systemic multi-organ tumors and matched normal tissues.It showed that WTX overexpressed in colorectal mucosa in addition to higher expression in normal kidney tissue,and its expression in colorectal cancer was significantly reduced or no expression.Our results revealed that WTX is a tumor suppressor gene in colorectal cancer as well and the WTX gene inactivation may play an important role in the progress of colorectal cancer.On the other hand,the preliminary findings of our group suggested that WTX gene deletion/mutation and gene methylation of the promoter region were not the main cause of the WTX inactivation.As one of the major discoveries of life science research in the 21st century,microRNAs(miRNA),has become a hot area of cancer research.MiRNA is a class of non-coding small molecule RNA with a length about 19-25 nucleotides in eukaryotes involved in the regulation of gene transcription.MiRNA is complementary pairing with the target mRNA,complete or incomplete,promote the degradation of target mRNA or inhibit protein translation,and it plays an important role in cell proliferation,differentiation,apoptosis,gene regulation and diseases.The large amount of research data showed that at least 1/3 of the human genes related to miRNA regulation which has important role in regulation of the function of tumor suppressor genes.The objective of our study is to confirm if the inactivation of WTX gene is regulated by miRNA in colorectal cancer,and what impact in the process of colorectal tumorigenecity.Another part of our research,proteomics(two-dimensional electrophoresis combined mass spectrometry analysis)were used to identify differentially expressed proteins between overexpressed WTX and negative controls,detect proteins interactions with WTX and screen and confirm of WTX-related downstream signaling pathways,and find the impact on miRNA regulated WTX downstream signaling pathways.In this study,2 miRNAs(miR-106a and miR-20a)related to WTX gene were screened from colorectal cancer and matched normal mucosa by microRNA array,and revealed their role in the proliferation and invasion of colorectal cancer.Differentially expressed proteins between overexpressed WTX and negative controls were screened by two-dimensional electrophoresis and mass spectrometry analysis,WTX-related downstream signaling pathways were proven,as well as explore the impact on downstream signaling pathways.The main contents are as follows:1.Screening and verification miRNAs directly regulated WTX Gene in colorectal cancer;2.Clarification the impact of miR-106a and miR-20a mediated the inactivation of WTX gene on the biological characteristics in colorectal cancer cells in vitro and in vivo;3.Proving proteins interactions with WTX,screening and verification of WTX related downstream signaling pathways and exploring WTX inactivation impact on downstream signaling pathways regulated by miR-20a and miR-106a.The purpose of this study is to prove the role and significance of not known miRNAs regulating WTX expression in the progression of colorectal cancer,and ultimately reveal the mechanism of WTX inactivation in colorectal cancer and provide a new basis on the role of WTX.Moreover,proteomics were applied to study the functional of WTX for the whole level,and provide new clues and a new target for gene therapy in the colorectal cancer and even for the mechanisms of tumorigenecity.Methods1.Screen and verify miRNAs regulated WTX inactivation in colorectal cancer tissues(1)Screen miRNAs regulated WTX inactivation in colorectal cancer by microRNA array,and confirm miRNAs expression in colorectal cancer tissues by real-time quantitative PCR.(2)Detect the expression of WTX gene in colorectal cancer and matched normal mucosa tissues by real-time quantitative PCR and spearman relative analysis were proformed.(3)Detect of the expression of miRNAs and WTX in colorectal cancer cell lines by real-time quantitative PCR.2.Detected interaction between WTX and miRNAs by Dual luciferase reporter system.Amplified gene fragment containing the WTX 3'UTR seed region and subcloned into the pGL3-control vector to construct pGL3-control-WTX-3'UTR recombinant vector,and bulid the recombinant vector of mutation,pGL3-control-WTX-3'UTR-Mut.Detected the relationship between WTX and miRNAs by dual luciferase reporter assay.3.Dectected the impact of miRNAs mediated WTX gene inactivation on the biological characteristics of colorectal cancer cells in vitro and in vivo(1)MiRNAs inhibitor were transiently transfected into the WTX low expression of colorectal cancer cell lines and detected WTX gene expression by real-time quantitative PCR and western blot detection.(2)Detected the effect of miRNAs inhibitor in colorectal cancer cell by CCK8 array,in vitro invasion assay and apoptosis detection,;(3)Constructed stably overexpressed cell lines and detected the expression of WTX gene by real-time quantitative PCR and western blo.(4)The effects of overexpressed miRNAs on the proliferation and invasion ability in colorectal cancer cell by CCK8 array and colony formation and in vitro invasion assay.(5)In a whole-body visualizing animal model,the effect of overexpressed miRNAs were observed in nude mice.4.The impact of WTX inactivation regulated by miRNAs on downstream signaling pathways in colorectal cancer.(1)WTX interacted proteins were screened by two-dimensional electrophoresis and protein mass spectrometry analysis.(2)Factors of the WTX downstream signaling pathway were detected in miRNAs inhibitor transfected colorectal cancer cell lines by Western blot;(3)Factors of the WTX downstream signaling pathway were detected in miRNAs overexpressed colorectal cancer cell lines by Western blot.5.Statistical analysisSPSS 13.0 statistical software was used for data analysis.Data were presented asx±SD in at least 3 independent experiments.Relative quantification value(2-??Ct)of real-time QPCR was analyzed by paired sample t test.Colony formation assay,transwell in vitro invasion assay and comparisons of relative luciferase activities were analyzed by One-way ANOVA,with the SNK,LSD,or Dunnett's T3 tests for multiple comparisons.The relationship between WTX and miRNAs expression was explored by Spearman's correlation.The results of CCK8 assay and tumor volume in nude mice were analyzed by factorial design analysis of variance.P values of<0.05 were considered statistically significant.Results1.Screen and verify miRNAs regulated WTX inactivation in colorectal cancer tissues(1)The results of miRNA microarray in 5 of colorectal cancer and paired normal mucosa suggested that 93 miRNAs differentially expressed,of which 29 were up-regulated in colorectal cancer tissues,64 down-regulated.Seven miRNAs,miR-141,miR-494,miR-492,miR-612,miR-93,miR-20a,miR-106a,were significantly increased in colorectal cancer tissues,and were selected as targets for validation.The expression of miRNAs was detected in 47 pairs of colorectal cancer tissues and normal mucosa by real-time quantitative.2"??Ct were analyzed by paired samples t-test,the expression of miR-141 and miR-494 in the 16 pairs of colorectal cancer and normal mucosa were no differences(t = 0.792,P = 0.435;t = 0.959,P = 0.353);The expression of miR-492 in 8 pairs of colorectal cancer tissues was no difference(t = 0.876,P = 0.410).4 pairs of the expression of miR-612 were down-regulated and 1 pairs up-regulated and other tissues couldn't detect expression.The expression of miR-106a and miR-20a in 47 pairs of colorectal cancer was significantly higher than the normal mucosa,the difference was statistically significant(t = 3.548,P = 0.001;t = 3.473,P = 0.001).The expression of miR-93 was no difference(t = 1.550,P = 0.128).(2)The expression of WTX gene in the 54 pairs of colorectal cancer was significantly lower than that in normal mucosa,the difference was statistically significant(t =-3.161,P = 0.003).The expression of miR-106a and miR-20a was not obey to normality tested by Shapio-Wilk(P<0.001,P<0.001).The expression of WTX gene and miR-20a and miR-106a in 47 colorectal cancer tissues and normal mucosa analyzed by Spearman correlation analysis,and showed a negative correlation between WTX and miR-20a and miR-106a,and the difference was statistically significance(r =-0.606,P<0.001;r =-0.605,P<0.001).(3)The expression of miR-106a and miR-20a in 6 cell lines of colorectal cancer showed SW480 cell line as a reference,miR-106a expression homogeneity of variance(F = 2.178,P = 0.125),miR-20a expression heterogeneity of variance(F =4.254,P = 0.019).The expression of miR-106a/20a was statistically significant(F =1611.484,P<0.001;F= 777.950,P<0.001)by One-way ANOVA.Multiple comparisons showed the highest expression in HT29 cells;miR-106a expression in Lovo,HCT116 and LS174T no difference(P = 0.864,P = 0.575);the expression of miR-106a in HCT116 and LS174t was no difference(P = 0.467).The expression of miR-20a in SW620 and HCT116 cells was no difference(P = 0.062),SW620 and LS174T was no difference(P = 0.059),Lovo and HCT116 and LS174T was no difference(P = 0.944,P = 1.000),HCT116 and LS174T no difference(P = 0.623).The expression of WTX in six colorectal cancer cell lines detected by western blot showed SW480,HCT116,Lovo,HT29,SW620,and LS174t in descending.Combining above results,we selected HCT116 cell line as overexpressed miR,while the SW620 and LS174t as inhibited miR expression cell lines.2.Detected interaction between WTX and miRNAs by Dual luciferase reporter system.PGL3-control-WTX-3'UTR vector and pGL3-control-WTX-3'UTR-Mut vector were successfully constructed and confirmed by restriction enzyme digestion and sequencing.The ration of firefly luciferase value(F)and renilla luciferase value(R),F/R,was detected and regarded as the relative luciferase activity.The results showed that pGL3-control-WTX-3'UTR vector,miR-20a and miR-106a mimics,and NC co-transfected into HEK293A and HCT116 cell lines,the relative luciferase activity value of homogeneity of variance(F = 0.009,P= 0.991;F = 4.927,P = 0.054)and the differences in three groups were statistically significant(F = 72.240,P<0.001;F=13.484,P = 0.006).Compared with NC group,miR-20a and miR-106a mimics HEK293 and HCT116 cells luciferase activity were lower,the differences were statistically significant(P<0.001,P<0.001;P = 0.003,P = 0.005),luciferase activity was reduced by 63.3%and 58.2%respectively.pGL3-control-MUT-3 'UTR,miR-20a and miR-106a mimics,NC co-transfected into HEK293A and HC.T1.16 cell line,the relative luciferase activity values was homogeneity of variance(F = 1.218,P=0.36;F = 0.045,P = 0.956),and showed no significant difference(F = 0.038,P =0.963;F = 0.588,P = 0.584).The above results suggested that miR-20a and miR-106a had direct interaction with WTX 3'UTR.3.Dectected the impact of miRNAs mediated WTX gene inactivation on the biological characteristics of colorectal cancer cells in vitro and in vivo(1)The effect of MiRNAs inhibitor in vitro?The expression of miR-106a and miR-20a significantly suppressed after transiently transfected inhibitors in LS174t and SW620 cell lines and the difference has significant statistical significance(F = 3470.989,P<0.001;F = 1664.453,P<0.001);WTX protein expression was significantly increased transfected with miR-106a and miR-20a inhibitor in LS174t and SW620 cells by Western blot.?CCK8 assay results showed the proliferation ability of LS174t and SW620 cells reduced after transfected with miR-106a and miR-20a inhibitor.The growth time level difference was statistically significant(F = 366.119,P<0.001;F = 275.854,P<0.001);the difference of proliferation ability between 3 groups was statistically significant(F = 35.31,P<0.001;F = 137.132,P<0.001);Interaction effect between time and cell group difference was statistically significant(F = 1.0904,P<0.001;F=35.684,P= 0.000).The CCK8 data of 3 groups in the same day analyzed by one-way ANOVA,the difference was statistically significant from the fourth day(P<0.001).?In vitro invasion assay results showed homogeneity of variance in LS174t and SW620 cell lines(F = 6.168,P = 0.014;F = 3.570,P = 0.610).The penetrated cells in miR-106a and miR-20a inhibitor group were less than the NC group,the difference of cell migration was statistically significant(F = 122.740,P<0.001;F =641.853,P<0.001)by One-way ANOVA.The experimental results showed that transfection of miR-106a and miR-20a inhibitor supressed the migration ability in LS174t and SW620 cell lines.?Muse TM Cell Analyzer was used to detect the percentage of apoptotic cells in LS174t and SW620 cells transfected with miR-106a and miR-20a inhibitor.It was suggested that the percentage of apoptotic cells declined in LS174t and SW620 cells transfected with miR-106a inhibitor by one-way ANOVA;the percentage of apoptotic cells increased transfected with miR-20a inhibitor,the differences were statistically significant(F = 2637.734,P<0.001;F = 87.097,P<0.001).(2)The effect of MiRNAs overexpressed in vitro? The expression of miR-106a and miR-20a was up-regulated in HCT116 cell line by overexpression of miR-106a and miR-20a,the difference was statistically significant by the independent samples t test(t = 100.035,P<0.001;t = 23.262,P<0.001).The expression of WTX gene in HCT116 cells was down-reugulated,and the difference was statistically significant(t =-42.647,P<0.001;t =-20.302,P<0.001).Western blot analysis showed that the WTX protein expression was significantly increased compared with the NC group in overexpressed miR-106a and miR-20a HCT116 cells.? CCK8 assay results showed compared to NC group,the proliferation capacity of miR-106a and miR-20a overexpressed group increased in HCT116 cells,the difference of growth time level of three groups was statistically significant(F =149.400,P<0.001);And proliferative capacity differences between groups statistically significant(F= 58.208,P<0.001);interaction effect between time and cell group difference was statistically significant(F =7.851,P<0.001).The measured data of 3 groups in the same day by one-way ANOVA except the first day(P = 0.182,P = 0.230),the difference between groups in difference day was statistically significant(P<0.05);?Colony formation assay results showed that the test was homogeneity of variance(F = 4.634,P= 0.061);The differences of 3 groups were statistically significant(F = 56.573,P<0.001),and compared to NC group,the rate of colony formation increased in miR-20a/106a over-expression groups(P<0.001;P = 0.019)by one-way ANOVA.?The in vitro invasion assay results showed,the test was homogeneity of variance(F = 2.086,P = 0.167);The differences in groups were statistically significant(F = 356.380,P<0.001);Compared with NC group,the ability of migration were significantly higher in overexpression of miR-106a/20a groups(P<0.001,P<0.001)by one-way ANOVA.The experimental results suggested that overexpression of miR-106a/20a promoted the ability of migration in HCT116 cell lines.(3)The effect of miRNAs overexpressed in vivoNC,miR-20a/106a groups of HCT116 cell line were inoculated subcutaneously into nude mice.the fifth day after inoculation visible subcutaneous tumors were identified,and the obvious green fluorescence was observed.23 days later,the mice were sacrificed and subcutaneous tumors were cut.According to the formula v =ab2/2,the volume of subcutaneous tumor was calculated.The differences of growth time level in 3 groups were statistically significant(F = 62.088,P<0.001);the proliferative capacity differences between groups were statistically significant(F=33.760,P<0.001);and the difference of interaction effect between the time of subcutaneous tumor cell group was statistically significant(F =3.662,P<0.001).The measured data of subcutaneous tumor volume in 3 groups in the same day,except five days(P = 2.828,P = 0.111),the differences were statistically significant(P<0.05).The tumor volume differences in different days between groups were statistically significant(P<0.05)by one-way ANOVA.4.The effect of WTX inactivation regulated by miRNAs on downstream signaling pathways in colorectal cancerBy two-dimensional electrophoresis and mass spectrometry,50 differentially expressed proteins were screened in overexpression the WTX stable cell lines SW620 and control cells.Rho GDIa was selected to verify his relationship with WTX as an up-regulated protein.The expression of Rho GDIa down-regulated in colorectal cancer tissues,and upregulated in normal colorectal mucosa by western blot.Rho GDIa was selected to verify his relationship with WTX as an up-regulated protein.Rho GDIa expression increased in LS174t and SW620 cells transfected with miR-106a and miR-20a inhibitor;and reduced in HCT116 cells with over-expression of miR-106a and miR-20a.Immunofluorescence staining showed that WTX protein expressed in the nucleus and cytoplasm,and Rho GDIa protein expressed in the cytoplasm of SW620 EGFP-WTX cells,moreover Rho GDI? and WTX protein co-localization in cytoplasm of SW620W+ cells,and the expression intensity was higher than SW620N1 cells.Anti-WTX antibody was co-immunoprecipited with SW620W+total protein,complexes were collected and separated by SDS-PAGE.Compared with the control group,the difference band of Rho GDIa was detected in vicinity of 35KDs by Coomassie blue staining.Conclusions1.The inactivation of WTX gene regulated by miR-106a and miR-20a in colorectal cancer.2.The inactivation of WTX suppressed Rho GDIa expression.3.MiR-106a/20a-WTX/Rho GDIa signal pathway promoted proliferation and invasion in colorectal cancer cells in vitro and in vivo.Innovations of this study1.Firstly proposed the inactivation of tumor suppressor gene WTX regulated by miR-106a/20a in colorectal cancer.2.Proposed a new mechinism that the inactivation of WTX protein by impact on the Rho GDIa downstream signaling pathways promoted the progress of colorectal cancer. |
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