IRF-1 Mediate Repression Of Let-7a Cluster And Reduce Metastasis Of Colorectal Cancer Cells In The Liver Of Inflammation

Background and Aims:Colorectal cancer(CRC)has become the third most common type of cancer worldwide and the fourth most common cause of cancer-related mortality.Tumor recurrence and colorectal liver metastasis(CRLM)are still the main causes of death in CRC patients.Clinically,the liver is the most common site of metastases in patients with colorectal cancer.Interestingly,CRC patients with pathological liver disease were noted to have a lower incidence of CRLM.Although numerous theories have been proposed in an effort to explain this clinical feature,there is still no consensus as to what molecular mechanisms underlie the inverse relationship between pathological liver diseases and the presentation of CRLM.Micro RNAs(mi RNA),one of the endogenous non-coding RNAs,regulate the expression of about 60% of human genes,especially oncogenes,by enhancing m RNA degradation.Mi RNA let-7,comprised of 12 members,has been reported as a tumor suppressor in many kinds of malignancies including CRC.Our previous work has demonstrated that the biogenesis of let-7a-1-5p,let-7d-5p,let-7f-1-5p(let-7a cluster)was strongly inhibited by stimulation of IFN-? in colorectal cancer cells HT29 and sensitized the cells to Fas-related apoptosis.In this study,we aimed to explore that IRF-1 mediated down-regulation of the let-7a cluster in the inflammatory microenvironment might prevent the circulating CRC cells from adhering to and colonizing the liver by enforcing the mesenchymal characteristics of the CRC cells.Methods: Male BALB/c mice(6 weeks old)were used to constructed hepatic inflammatory animal models by 40% CCl4 gavage and CRLM models by splentic injection of CRC cell line CT26.WT.The whole-body in vivo imaging system and Hematoxylin and Eosin(HE)stain were used to determine the modeling times and detected the incidence of CRLM.ELISA assay was used to observe the expression level of pro-inflammatory factors,such as IFN-?,TNF-? and interleukin(IL-1?)in liver specimens and corresponding serum sampled from the inflammatory and controlled mice.The receptor of IFN-?(IFNGR1) were knocked down by transfecting with lentiviral vectors(sh RNA-IRFGR1),and those sh RNA-IRFGR1-CT26 cells were injected into BALB/c mice with hepatitis to analyze the critical roles of IFN-? in the resistance of CRLM.Real-time PCR(q RT-PCR),western blot and immunohistochemical staining(IHC)were used to test the expression level of IRF-1/IRF-2 and let-7a cluster in inflammatory circumstance in vivo and in vitro.Searching the website of UCSC ENCODE Genome Browser,rescue assay,Chromatin immunoprecipitation(Ch IP)assay and Dual-Luciferase Reporter Assay System were performed to validate our hypotheses and explore the potential mechanism that IRF-1 mediated IFN-?/let-7a-1-5p pathway.Stable overexpression and knockdown of let-7a-1-5p in CT26.WT cells were established using lentiviral vectors(sh RNA-let-7a-1-5p and LV-let-7a-1-5p).The resistant roles of let-7a cluster in the metastatic process were detected using those reconstructed cell lines which were injected into the spleen of inflammatory and normal mice.In order to explore the potential role of EMT/MET in regulating of CRLM,EMT related markers,such as E-cadherin,N-cadherin and Vimentin,were investigated using q RT-PCR,western blot and IHC in vitro and in vivo.Results:The inflammatory liver microenvironment prevents colorectal liver metastasis.Liver inflammatory animal models were constructed by gavage of 40% CCl4.H&E stain results showed that the liver inflammatory animal models were constructed successfully by 5 weeks' gavage of 40%CCl4(6ml/kg,2 times per week).With seven weeks' gavage,typical pseudo-lobule formation was identified and cirrhosis models were built.CRLM animal models were constructed through splenic injection of CT26.WT cells into BALB/c mice.The hepatic metastatic foci could be observed as early as 6 days after model construction.By day 12,more than 80%(5/6)of mice exhibited signs of liver metastases.shRNA-NC-CT26.WT cells were injected into BALB/c mice with or without hepatitis to build the CRLM models.The result told that the metastatic rate in inflammatory group less than that in normal mice(2/6 vs.5/6).In addition,the powerful of total fluorescence(P<0.05),the maximum diameter of metastatic tumor(P<0.05)and the number of metastasis(P<0.05)were dramatically lower in the mice with hepatitis.IFN-? may play a role in determining the lower incidence of CRLM in inflammatory circumstance.ELISA was used to test the pro-inflammatory factors,such as IFN-?,TNF-? and interleukin(IL-1?),in liver specimens and corresponding serum of normal and inflammatory mice.Some slight expression differences,no statistical significance,were noticed in those three factors in liver specimens.However,the expression of IFN-?(P<0.01),TNF-?(P<0.05)and IL-1?(P<0.01)were significantly increased in the serum of mice with hepatitis,particularly IFN-?(Fold change=6.55).The reconstructed sh RNA-IRFGR1-CT26 cells were injected into BALB/c mice with hepatitis.The higher incidence of metastasis(8/10 vs.3/10),more powerful fluorescence(P<0.01),more metastatic foci(P<0.01)and larger maximum diameter of metastatic tumor(P<0.01)were detected in IRFGR1 depleted group.There was an inverse correlation between the expression of let-7a cluster and IRF-1 in the liver inflammatory environment.Numerous interferon regulatory factor(IRF-1 and IRF-2)predicted binding sites were found to be located at the upstream of the let-7a cluster transcription start site(TSS)by searching UCSC website.q RT-PCR and western blot were performed to test the expression of IRF-1 and IRF-2 in IFN-? treated CRC cell lines HT29 and CT26.WT.The results told that the expression of IRF-1,not IRF-2,was significantly increased following stimulations of IFN-? in both cell lines.Further immunohistochemical(IHC)analysis confirmed these studies.In addition,let-7a cluster was dramatically down-regulated in response to the treatment of IFN-? in two CRC cell lines.IRF-1 mediated IFN-?/let-7a-1-5p pathway.Rescue assay was performed in HT29 cell line,and we found that IFN-?-mediated repression of the let-7a cluster was significantly reduced in this cells in which IRF-1 had been knocked down(P<0.01).Marked down-regulation of let-7a-1-5p(P<0.05)and its precursor(P<0.05)were also detected in IFN-? treated HT29 cells,whereas the expression trends of let-7a-1-5p and its precursor were not statistically significant(P>0.05).Ch IP study revealed that the predicted binding site(chr9: 96,931,077-96,931,229),the most strongly enriched amplification with primer #4,was located in the upstream region of the TSS.The constructs of upstream of let-7a cluster TSS(879bp),contained the predicted binding site(chr9: 96,931,077-96,931,229),and its deletion mutation(718-721),was subsequently synthesized and cloned into the report plasmid.The results of luciferase activity test suggested that IRF-1 bound the upstream of TSS(718-721).Expression level of let-7a-1-5p was related to the colorectal liver metastases ability in vivo.sh RNA-let-7a-1-5p-CT26 and LV-let-7a-1-5p-CT26 were used to constructed CRLM models.Compared with the control group,depletion of let-7a-1-5p caused a lower incidence of CRLM(1/6 vs.5/6)with less powerful fluorescence(P=0.05),less metastatic foci(P<0.05)and smaller maximum diameter(P=0.05).A light higher incidence of CRLM(6/6 vs.5/6)with more powerful fluorescence(P<0.05),more metastatic foci(P<0.05)and larger maximum diameter(P<0.05)were detected in upregulated group.On the other hand,depletion of let-7a-1-5p resulted in lower incidence of CRLM(1/10 vs.5/10)in mice with hepatitis.And the total powerful of fluorescence,the number of metastatic foci and the maximum diameter of metastasis are also reached the statistical significance(All value P<0.05).Repression of the let-7a cluster maintained the mesenchymal phenotype of CRC cells and prevented their subsequent settlement in the liver.Some of HT29 cells were reversed from epithelial phenotype to mesenchymal-like phenotype in the process of modeling sh RNA-let-7a-1-5p-HT29 cell line.q RT-PCR and western blot were used to further detected the expression level of EMT related markers(E-cadherin,N-cadherin and Vimentin).The expression of N-cadherin was dramatically increased with stimulation of IFN-? and depletion of let-7a-1-5p in HT29,but the other two markers(N-cadherin and Vimentin)showed no significant differential expression trend.In the meantime,the IHC studies of the liver specimen revealed that N-cadherin was remarkably increased in inflammatory group and let-7a-1-5p down-regulated group.And the N-cadherin-positive structure was located mainly in the tumor area.Conclusion:1.CCl4 induced liver inflammatory microenvironment reduces the incidence of colorectal liver metastasis in BALB/c mice,and IFN-? may play an important role in this process.2.IFN-? induced down-regulation of let-7a cluster can be mediated by IRF-1 through binding to the upstream of let-7a cluster TSS.3.Repression of let-7a-1-5p reduced the metastatic ability of colorectal cancer in the liver of inflammation.4.Repression of the let-7a cluster trigger the epithelial-mesenchymal transition of CRC cells by upregulating the expression of N-cadherin and prevents their subsequent settlement in the liver.