The Molecular Mechanism Of IRF1 In The Inhibition Of Colorectal Cancer Proliferation And Metastasis

Background and ObjectiveColorectal cancer(CRC)is one of the most common malignant tumor,The incidence of colorectal cancer is increasing along with the change of environmental factors,diet habits and lifestyle.Therapeutic effect and survival rate are distinctly difference between the patients with or without metastasis.Therefore,to clarify the molecular mechanism of colorectal cancer metastasis related and find out the effective clinical treatment targets is an important topic in the research of colorectal cancer.In molecular biology and genetics,a transcription factor(sometimes called a sequence-specific DNA-binding factor)is a protein that binding to specific DNA sequences,to control the rate of transcription of genetic information from DNA to messenger RNA.Transcription factors perform this function alone or with other proteins in a complex,by promoting(as an activator),or blocking(as a repressor)the recruitment of RNA polymerase(the enzyme that performs the transcription of genetic information from DNA to RNA)to specific genes.A defining feature of transcription factors is that they contain one or more DNA-binding domains(DBDs),which binded to specific sequences DNA on the promotor of the genes that they regulated.Additional proteins such as coactivators,chromatin remodelers,histone acetylases,deacetylases,kinases,and methylases,while also playing crucial roles in gene regulation,but lack of DNA-binding domains,and,therefore,are not classified as transcription factors.Transcription factors are essential for the regulation of gene expression,found in all living organisms.There are approximately 2600 proteins in the human genome that contain DNA-binding domains,and most of which are presumed to function as transcription factors.Therefore,approximately 10%of genes in the genome code for transcription factors,which makes this family the single largest family of human proteins.Furthermore,genes are often flanked by several binding sites for distinct transcription factors,and efficient expression of each of these genes requires the cooperative action of several different transcription factors(for example,hepatocyte nuclear factors).Hence,the combinatorial use of a subset of the approximately 200。human transcription factors easily accounts for the unique regulation of each gene in the human genome during development.Transcription factors bind to either enhancer or promoter regions of DNA adjacent to the genes that they regulated.Depending on the transcription factor,the transcription of the gene is either up-or down-regulated.Transcription factors use a variety of mechanisms for the regulation of gene expression.Interferon regulatory factor 1(IRF1)is a protein that encoded by the IRF1 gene in humans.Interferon regulatory factor 1 is the first member of the interferon regulatory transcription factor(IRF)family initially described as a transcription factor which can activate the expression of the cytokine Interferon beta.IRF-1 was subsequently shown to function as a transcriptional activator or repressor of a variety of target genes.IRF-1 regulates expression of target genes by binding to an interferon stimulated response element(ISRE)in their promoters.The IRF-1 protein binds to the ISRE via an N-terminal helix-turn-helix DNA binding domain,which was highly conserved among all IRF family proteins.IRF-1 has been shown to trans-activate the tumour suppressor protein p53 through the recruitment of its co-factor p300.IRF-1 has been repoeted to play the roles in the immune response,apoptosis,DNA damage and tumor suppression.It has been shown that the extreme C-terminus of IRF-1 regulates its ability to activate transcription,and the nanobodies targeting this domain(MF1)are able to increase IRF-1 activity.The function of a transcription factor has a closely related to its target genes.The literature showed that RASSF5 is a target gene of IRF1 regulating through Chip-Chip screening,but the experiment have not confirmed.Ras association domain －containing protein 5(RASSF5)was generally accepted that RASSF5 is a tumor-suppressor factor,it could inhibit the activity of ERK signaling pathway to inhibit the proliferation of tumor cells,also could promote cell apoptosis in lung cancer.RASSF5 was identified in a yeast two-hybrid screen as a putative Ras effector.Activated Ras proteins induce a wide variety of biological phenotypes associated with the loss of normal growth control.These include reduced requirement for growth factors,enhanced invasiveness,resistance to apoptosis,and tumorigenic transformation.However,activated Ras may also induce senescence,differentiation,cell cycle arrest,or apoptosis.The ability of Ras to produce such a range of apparently contradictory biological effects may be derived from its ability to interact with a diverse array of effector proteins.It has been well documented that IRP1 inhibits cell proliferation and promote tumor cell apoptosis,and increase the sensitivity of tumor cells to chemotherapy drugs.For example,IRF-1 expression induces apoptosis and inhibits tumor growth in mouse mammary cancer cells in vitro and in vivo.IRF1 suppresses Ki-67 promoter activity in renal cell carcinoma.Howerver,the biological functions of IRF1 in the control of colorectal metastasis and tumor progression have not been well characterized.Therefore,we sought to investigate the potential role and mechanism of IRF1 in cell proliferation and metastasis in colorectal cancer and provide experimental basis for colorectal cancer growth and metastasis.Methods1.IRF1 expression in CRC tissues and cells was detected using quantitative RT-PCR and Western blotting.Quantitative reverse transcription-PCR(qRT-PCR)was employed to detect the expression of IRF1 in 39 cases of fresh colorectal cancer tissue and paired normal tissue and colorectal cancer cell lines including HCT116,HT29,SW480,SW620,Lovo,LS174T.At the same time,Western blotting was employed to detect the expression of IRF1 in 7 cases of colorectal cancer tissue and paired normal tissue,and the colorectal cancer cell lines above.Immunohistochemical was used to detect the expression of IRF1 in 61 cases paraffin of colorectal cancer specimens.2.The effect of IRF1 on colorectal cancer cells biological characteristics in vitro(1)PubMed database was used to find basic information about IRF1,and confirm the purpose sequence.Then CDS region of IRF1 was amplified by PCR,followed through the steps of restriction enzyme,purification,connection,transformation,which make CDS region of IRF1 connected with the plasmid vector pcDNA3.0.Sequencing was proved that the recombinant vector was successfully constructed.SW480 and SW620 cells were transfected with pcDNA3.0/IRF1,and screened by G418 for 2 weeks.qRT-PCR and Western blotting were used to test the expression level of IRF1 in SW480 and SW620 cells.At the same time,the purchased shRNA-IRF1 which can intervene the expression of IRF1,was transfected into HT29 and LS174T cells.The efficiency of transfection was observed by fluorescence microscope and Western blotting in HT29 and LS174T cells.(2)CCK8 cells proliferation assays,plate colony formation assays,Transwell migration assays,and flow cytometry were carried out to detect the influence of IRF1 on colorectal cancer cell proliferation,apopptosis,clone forming ability,migration ability in vitro after IRF1 was up-regulated or down-regulated.(3)Nude mice subcutaneously tumor experimental used to detect the effects of IRF1 in colorectal cancer in vivo.3.The molecular mechanism of IRF1 on proliferation and invasion in CRCThe gene regulated by IRF1 transcription factors were analysed through references and biological information database,such as TFSEARCH,CONSITE.ChIP experiment was performed to test the binding of transcription factor IRF1 and RASSF5 promoter.Then western blotting was used to detect the variation of RASSF5,RAS,Racl,p21 and P53 protein after over-expression and interference of IRF1 in colorectal cancer cell lines.4.The relationship between the expression level of IRF1 and clinicopathological characteristic.The results of qRT-PCR was analysed the relationship of the expression of IRF1 in CRC and Clinicopathological characteristic;statistical methods was used to analyse the relationship between IRF1 and the factors including age,gender,clinical stage,tumor differentiation,metastasis,TNM stage.5.Statistical analyzeData were analyzed by using SPSS 19.0 statistical software.Comparing 2-△△Ct value based on cell qRT-PCR results was analysed by single factor analysis of variance(One-Way ANOVA).Welch approximate variance analysis was used when it is not homogeneity of variance;multiple homogeneity of variance was analysed with LSD method,SNK(Student-Newman-Keuls)method,Dunnett ’s T3 was used when it is not homogeneity of variance.The two groups were analysed by two independent samples t test(Independent-Samples t text),or approximate t test when it is not homogeneity of variance.Plate clone formation assay s,migration of Transwell assays were analysed by One-Way ANOVA.The CCK8 cell proliferation assays were analysed by factorial design and single factor variance analysis.The relationship between IRF1 and clinicopathological characteristic were tested using the Fisher exact probability test.Differences were considered significant if P<0.05.Results1.IRFl downregulation in CRC correlates with metastasis,classification and growth of colorectal cancer.IRF1 was downregulation in colorectal cancer tissue.The average expression level of IRF1 was significantly decreased in 34 of 39 CRC specimens(t=3.290,P=0.002)compared to their normal counterparts,with a 1.56-fold decrease in the colorectal cancer tissue samples.Furthermore,to investigate the clinicopathologic significance of IRF1 expression in CRC patients,the median relative expression level of IRF1 in the 39 CRC samples was recommended as the cut-off point for dividng IRF1 level into a low expression group and a high expression group.Correlation analysis showed that the IRF1 expression level was reversely correlated to serosal invasion(P=0.002),lymph metastasis(P=0.002)and TNM classification(P=0.043).We further examined IRF1 expression in the CRC cell lines with different metastatic potentials by qRT-PCR and Western blotting.Relative IRF1 expression was significantly was significant difference between the CRC cell lines(F=30.791,P<0.001).With SW480 as a reference,the Dunnett ’s T3 multiple comparison indicated that expression of IRF1 in HT29,LS174T cells was higher than SW480 cells(P<0.001,P=0.004).But the expression of IRF1 in SW620,HCT116 and LOVO cells was no significant difference when compared with SW480(P=0.389,P=0.367,P=0.197).The expression of IRF1 in normal tissues was higher than that in cancer tissues in 6 cases of the 7 paired clinical samples by western blotting.The expression of IRF1 was higher in HT29 and LS174T cells;while lower in SW480,SW620,HCT116 and LOVO by western blotting,and differences of the 4 kinds of cell which lower expression IRF1 were not remarkable.And the results of western blotting were similar to qRT-PCR.2.IRF1 was stablely overexpressed and transiently inhibited in colorectal cancer cells.IRF1 was up-regulated in SW480 and SW620 cells(P=0.002,P=0.01)after the transfection of pcDNA3.0-IRF1 by qRT-PCR and western blotting.The expression of IRF1 in HT29 and LS174T cells was determined by western blotting after the transfection of 4 shRNA-IRF1 fragments respectively.It was showed that the expression level of IRF1 was obviously down-regulated after the transfection sh1-IRF1.sh1-IRF1 was used in the following experiments.3.Overexpression of IRF1 inhibits colorectal cancer cell proliferation and migration in vitro.CCK-8 cell proliferation assay showed that the up-regulation of IRF1 restrained cell proliferation of SW480 and SW620 cells compared with vector cells(P<0.001,P<0.001).Correspondingly,the suppression of IRF1 increased the proliferative abilities of HT29 and 1s174T cells(P<0.001,P<0.001).The plate colony formation assays yielded the similar effect(P=0.007,P=0.014).Transwell migration assay showed that IRF1 over-expression markedly repressed the motility of SW480 and SW620 cells as compared with vector cells(P<0.001,P=0.002).The knockdown of IRFl significantly enhanced the migration of HT29 and LS174T cells(P<0.001,P<0.001),compared to NC cells.Cell cycle and apoptosis experiments showed that IRF1 over-expression SW480 and SW620 cells arrest in G1 phase as compared with vector cells(P = 0.02;P ＝0.002),we got the opposite result after interference expression of IRF1(P<0.001;P =0.014).But there was no statistically significance of apoptosis among differences groups(P=0.438,P=0.742,P=0.547,P=0.222).Nude mice subcutaneously tumor experiment showed that the subcutaneous tumor growed slowly significantly after injected SW620/IRF1 than SW620/Vector(P<0.001).To sum up,our study showed that IRF1 can inhibit colorectal cancer cell proliferation and migration in vitro and in vivo.4.The signaling pathway targeted by IRFl involved in colorectal cancer.Based on the results of cell cycle and apoptosis,we speculated that IRF1regulation proliferation through the cell cycle pathways.Western blotting results showed that the overexpression of IRF1 enable increase the expression of P21 and P53.RASSF5 was one of the IRF1 transcription factors regulate genes through Chip-Chip sequencing.The binding sites of IRF1 transcription factor in RASSF5 promoter was analyzed by UCSC,TFSEARCH,CONSITE bioinformatics database and confirmed by Chip assays.Further western blotting experiment verified that the expression of RASSF5 was up-regulation,and RAS and Racl expression level were down-regulation when IRF1 was overexpressed.In summary,IRF1 inhibited colorectal cancer cell proliferation through activating cell cycle regulators P53 and P21,and suppressed colorectal cancer cell metastasis by blocking RAS-Racl pathways.Conclusion1.IRF1 down-regulation in CRC correlates with metastatic status,tumour grade and growth.We reasoned that expression of IRF1 is negatively correlated with the metastatic potential of CRC and that IRF1 may suppress CRC growth.2.IRF1 inhibit the ability of proliferation and migration of colorectal cancer cells.3.IRF1 inhibited colorectal cancer cell proliferation through activating cell cycle regulators P53 and P21,and suppressed colorectal cancer cell metastasis by blocking RAS-Racl pathways.