| Background and purposeColorectal cancer is the forth of the most common malignant tumor, the treatment of colorectal cancer with surgery is given priority to radiation and chemotherapy, but the 5-year survival rate is still not high. Metastasis is the most important biological characteristics of malignant tumor,10-60% of patients with early malignant tumor have tumor cells in the blood before metastasis. Even a radical resection was made in colorectal cancer, there are still 30-50% patients have metastasis in 5-10 years, which is the leading cause of death in colon cancer. Cellular and molecular basis of tumor metastasis, however, still not clarified, thus to explore mechanism of tumor metastasis, early blocking cancer metastasis and improve the therapeutic efficacy of patients with metastasis, for the prevention and treatment of colorectal cancer has the very vital significance.Tumor immune gene therapy as a treatment for one of the methods for tumor has received lots of attentions, the mechanism is mainly has two aspects:1, through a variety of methods to increase the corresponding cytokines to help the body eliminate tumor cells;2, improve the tumor cell immune prototype, make its can identify and eliminate by the body’s immune system. Gamma interferon (IFN-y) as an important immune cytokine, in human and disease struggle played important roles, IFN-gamma are synthesis and secretion by T cells and NK cells in the body, which have stronger immune regulation and the antitumor effect of many sided, especially to stimulate and enhance host antitumor immune response is most important. IFN-gamma is activator of NK cell activity, it can promote NK cell development, differentiation, and can activate the cytotoxic activity of NK cells. Research has shown that IFN-gamma can also direct inhibit tumor cell division, in the study of tumor gene therapy has a certain research value. IFN-gamma, TNF alpha, interleukin, etc can induce IRF1 (interferon regulatory factor 1) the activation of transcription factors, and the different reaction sensitivity of IRF1 to IFN-gamma of significantly predicted different clinical outcomes. But why there are different response activity for tumor cells to IFN-gamma is still unknown, under IFN-gamma IRF1 after elevated levels of different why can lead to different outcomes, increase the reactivity of colorectal cancer in IFN-gamma can targeted colorectal cancer metastasis is unclear, clarifying the mechanism of this phenomenon, could make us have deeper understanding for the complex relationship between inflammation and tumor, thus developing new strategies for colon cancer metastasis treatment, which has important clinical application prospect and significance.Small RNA (miRNA) is a kind of non-coding small RNA molecules exist in organism can regulate gene expression of endogenous. Mature miRNA are found in animal cells and target gene mRNA 3’untranslated region (3’UTR) complementary sequence of bases, mature miRNA can combine with target gene 3’UTR, and formed under the Argonaute proteins involved in RNA induce silence compounds (RNA-induced silencing complex, RISC), through the degradation of the target gene mRNA to stop their translation, in order to regulate target gene expression.It is reported that all human miRNA genes account for about 3% of the total gene, may regulate the human cell about 30% of protein-coding genes. KotaJ etc recovery miR-26a expression of liver cancer cells by adeno-associated virus transfection. and make the loss of migrate and metastasis ability with non-toxic reaction.This important results are published in the journal Cell. Therefore Rossi J make comments on Cell:miRNA genes has more efficient regulation function, which has the effect of kill two birds with one stone.IFN-y signals through the STAT pathway trigger the subsequent activation of IRF1, which then binds to the promoter region of miR-29b and initiates its transcription. IFN-y-induced and IRF1-dependent up-regulation of miR-29b causes a down-regulation of IGF1, which plays a crucial role in apoptosis and migration. IRF1 could also be induced by miR-29b in human CRC cells, thus forming a positive feedback loop, and enhancing IRF1 or miR-29b expression may sensitize CRC cells to IFN-y treatment.This topic using micro-array of colorectal cancer tissues to choose miR-29b as one of candidate microRNAs to contact IFN inflammatory and colorectal cancer, the role of miR-29b in CRC invasion and metastasis has been clear. The main contents are as follows:1. Screening and identifying the miRNAs related with CRC and IFN-y.2. Confirming the role of miR-29b in the anti-tumor effect of IFN-y.3. Predicting and confirming the targets of miR-29b, and identifying the role of targets in the invasion and metastasis of colorectal cancer.The aim of this research is to reveal an new regulating mechanism of miR-29b, approaching the role of miR-29b in the development of CRC, and provide an new miRNA and target gene for the clinical application. Targeting miR-29b may provide a strategy for blocking CRC growth and metastasis.Methods1.Analysis of miR-29b which is IFN-y targeted and colorectal cancer relatedThrough the bioinformatics and literature in colorectal cancer to screen significantly altered the miRNAs and obviously up-regulated miRNAs after IFN-gamma stimulation, and then verify miR-29b expression by using the fluorescent quantitative PCR. Detecting miR-29b expression in 41 paired CRC tissues.2.Research the mechanism of miR-29b induced by IFN-yTo determine the the upstream regulatory factor IRF1 of miR-29b and to discusses the regulate relationship between them, we used the luciferase report assay to detect transcriptional activity between transcription factors IRF1 and miR-29b promoter, the chromatin immune coprecipitation (ChIP) was used to verify the binding sites between them; Change the expression of transcription factors in cells and then detection miR-29b expression level.3. Effects of miR-29b on colorectal cancer cell biology behavior.(1) through leti-virus vector, anti-miR-29b was transfected to colorectal cancer cell lines HT29 and HCT116, stabe cell lines were established by FCM. Western blot was used to detect the change of related indicators.(2) designed and established the leti-virus vector to overexpress miR-29b,then infected colorectal cancer cell line SW480 and SW620, stabe cell lines were established by FCM. Western blot was used to detect the change of related indicators.(3) transfect the miR-29band IGF 1-CDS vector without 3’UTR to CRC cells SW480 and SW620, using western blot and QPCR to detect related protein expression.(4) using CCK8, plate colony formation assay, cell apoptosis, transwell assay and subcutaneous assay, to test the change of proliferation, apoptosis, and the influence of the invasive ability induced by miR-29b in colorectal cancer cell.4. The prediction and identification of target genes of miR-29b.(1) searched the term miR-29b, using database Targetscan and Pictar, Miranda to estimate the target genes of miR-29b, choose the genes which was more intersection by 3 software and have forecast higher, using dual luciferase report assay to identify the binding between them, finally using quantitative PCR, western blot to detect the expression of miR-29b and target genes, then analyze the correlation between them.(2) used CCK8, plate colony formation assay, cell apoptosis, transwell assay and subcutaneous assay, to test the change of proliferation, apoptosis, and the influence of the invasive ability induced by the co-transfection of miR-29b and IGF1-CDS in colorectal cancer cell. Western blot was used to detect the expression of relative protein in pathway signaling..5. Regulation between IRF1 and IGF1 in colorectal cancer(1) Correlation analysis between IRF1 and IGF1 expression in colorectal cancer organizations.(2) western blot was used to detect the expression of IRFl and IGF1 after cells was stimulating by IFN-gamma.(3) over-expressed transcription factor IRF1 in colorectal cancer cells and detect the expression of IGF 1 by western blot.(4) Gives IFN-gamma stimulation to up-regulate or down-regulate miR-29b cells group, then observed changes between IRF1 and IGF1 expression.Results1. Analysis of miR-29b which is IFN-y targeted and colorectal cancer related(1) Through the bioinformatics and literature in colorectal cancer to screen significantly altered the miRNAs and obviously up-regulated miRNAs after IFN-gamma stimulation, and then verify miR-29b expression by using the fluorescent quantitative PCR. While another group of microarray data found that miR-29 b expression in colorectal cancer tissue was significantly lower than normal tissue.IFN-gamma play a role mainly through make STAT1 phosphorylation and promote IRFl into nuclear and has a GAS (gamma activated sequence) structure of promoter sequences.(2)To examine whether IFN-y affected miR-29b in CRC,5 CRC cells were treated with IFN-y (10ng/ml for 24 h), then miR-29b was determined by quantitative real-time PCR (qPCR). MiR-29b expression was obviously upregulated in all cells after IFN-gamma treating (P=0.029; P=0.001; P<0.001; P=0.013; P=0.010). SW480 and SW620 cells were treated with IFN-y (10ng/ml) for different time periods. IFN-y induced miR-29b expression within 4 or 8 hours after IFN-y stimulation and reached the maximum level between 8 or 16 hours.(3)To determine the potential clinicopathological implications of miR-29b expression, we investigated the expression levels of miR-29b in 41 CRC tissues (T) and adjacent normal tissues (N) by qRT-PCR. MiR-29b was significantly decreased in 68.3%(28/41) of the CRC tissues (T/N<0.5-fold) compared to the matched adjacent normal tissues(t=5.593, P<0.001).2. Research the mechanism of miR-29b induced by IFN-γ(1) We analyzed the miR-29b promoter which was previously identified, and found the present of three IRF1-binding sites with 701 to 712,1483 to 1494,and 1531 to 1542 regions in the promoter of miR-29b.(2) The miR-29b promoter was subcloned into PGL3-basic vector, a dual-luciferase report assay was performed to study the functionality of interaction between IRF1 and miR-29b. IRF1 increased the activity of miR-29b promoter report compare to blank and control group (P=016; P=0.045), and mutating the IRF1 binding sites weaked the IRF1-mediated reporter induction, demonstrating that these sites are necessary and functional(P= 0.001; P= 0.005; P< 0.001).(3) Then the ChIP assay also proved it, because 1483 to 1494 and 1531 to 1542 regions in the promoter of miR-29b were clear, the interactions of IRF1 within the region of 701 to712 (sitel) and 1483 to 1542 (site2) were examined, and IRF1 bind to both of 2 sites. IFN-γ induced significant increased binding between IRF1 to the sites of miR-29b promoter in SW480.(4) Immunofluorescent staining was performed to detect the expressions of IRF1, results showed a significantly increased IRF1 expression in 8 h in nuclear compared with controls in SW480 cells. However, IFN-γ did not induce miR-29b expression in HCT116 cells transfected with shIRF1.(5) Transient expression of IRF1 led to increased expression of miR-29b in SW480 and SW620 cells. miR-29b in SW480 and SW620 are obviously upregulated (P=0.028; P=0.008), that means IRF1 could upregulate miR-29b.3.Effect of miR-29b on biological behavior of CRC cells.(1) Inhibition of miR-29b accelerates CRC cell proliferation and migration,establish stably miR-29b-knockdown HCT116 and HT29 cell, tested cell proliferation ability in vitro after miR-29b was inhibited, and draw the growth curve, factorial analysis of variance results showed that when knocked miR-29b, HCT116 and HT29 cell grow faster.Clone formation experiment shows:plate colony formation rate increases after miR-29b was knockdown, There were significant differences between HCT116 cell group (t= 12.333, P= 0.007). Significant difference also be observed between the HT29 cell groups (t= 23.463, P= 0.001).Transwell experiments showed:migrate ability was enhancd in HCT116 cells and HT29 cell (t= 8.6820, P<0.001) after miR-29b was downregulated (t= 4.597, P =0.008).Wound healing experiments showed:healing ability was enhancd in HCT116 cells and HT29 cell (t=-7.150, P<0.001) after miR-29b was downregulated (t= 14.947, P <0.001).The growth of subcutaneous tumors in nude mice:five nude mice was respectively injected to the negative control cells and miR-29b-knockdown HCT116 cells into the lower right, lower left back side of the nude mice. Mice were euthanized at 23 days before subcutaneous tumor removed.(P< 0.001).(2) MiR-29b inhibits CRC cell proliferation, migration and induced apoptosis.Establish stably miR-29b-overexpressing SW480 and SW620 cells, tested cell proliferation ability in vitro after miR-29b was overexpressed, and draw the growth curve, factorial analysis of variance results showed that when overexpress miR-29b, SW480 and SW620 cell grow slower.Clone formation experiment shows:plate colony formation rate decreased after miR-29b was upregulated. There were significant differences between SW480 and SW620 cell group (t=10.875, P<0.001). Significant difference also be observed between the SW620 cell groups (t=6.579, P=0.003).Transwell experiments showed:migrate ability was decreased in SW480 cells and SW620 cell (t= 8.972, P<0.001) after miR-29b was downregulated (t= 6.806, P =0.006).The growth of subcutaneous tumors in nude mice:five nude mice was respectively injected to the negative control cells and miR-29b-overexpressed SW620 cells into the lower right, lower left back side of the nude mice. Mice were euthanized at 23 days before subcutaneous tumor removed, and the tumor grow slower in cells overexpressed miR-29b.(P< 0.001).Cell Apoptosis:flow cytometry instrument were used to detect cell apoptosis, according to the results, after overexpressing miR-29b in SW620 cell apoptosis rate increased (P< 0.01).Western blot was used to detect apoptosis related proteins caspase-3 and caspase-9, in SW480 and SW620 cell lines in which miR-29b were upregulated, pro-aspase-3 and pro-caspase-9 protein expression decreased, cleaved caspase-3 and cleaved-caspase-9 elevated protein expression increased.Cell cycle:flow cytometry instrument were used to detect the cell cycle, according to the results, after overexpressing miR-29b in SW480 and SW620, there was no significant difference in cell cycle.Orthotopic implantation:The subcutaneous tumors were cut into small pieces and embedded into the mesentery at the tail end of caecum. Seven weeks later, the mice were killed and all organs were removed for examination. Hepatic and intestinal metastases were detected by HE staining and quantified by counting metastatic lesions.The results showed intestinal and hepatic metastasis. In the group of mice injected with S W620-PLV-miR29b, none of the mice had hepatic metastasis, and only 40%(2/5) had intestinal metastasis. However, in the SW620-PLVTHM group,60%(3/5) and 100%(5/5) of mice had hepatic and intestinal metastasis, respectively.4.Prediction and validation of miR-29b targets(1) Bioinformatic prediction of miR-29b associated target genes:we search target genes in microRNA.org, TargetScan and Pictar and miRanda four database, forecasting results of four database in intersection, which get higher reliability and accuracy of target genes.We picked out the IGF1, AKAP13, YY1 mRNA for further analysis.(2) Designed construction of IGF 1 3’UTR expression vector and Mut-IGF1-3’UTR mutation of the vector, we use luciferase report assay to assess the binding between miR-29b and IGF13’UTR, as the result showed, in the group of transfected with miR-29b or IGF1-3’UTR or IGF1-3’UTR-MUT, one-way ANOVA showed there was significant differents(F=117.348, P<0.001) The transient transfection of the wild IGF 1-SITE 1-WT reporter with miR-29b mimics into HEK293A cells led to a significant decrease in luciferase activity compared with the control analyzed by LSD compare(P=0.006), and when the miR-29b binding sites were mutated, we observed a dramatic relief in luciferase activity, which prove that miR-29b could combine with IGF13’UTR, thus inhibiting the enzyme activity of fluorescein. There was no significant difference between In transfection miR-29b mimics and IGF1 3’UTR mutation plasmid compared to the experimental group and the control group, there was no significant difference between groups WT+NC group and MUT+NC group, WT+NCgroup and MUT+miR-29b group, MUT+NC group and MUT+miR-29b group (P=0.902; P=0.279; P=0.332), whcih prove that miR-29 b can’t combine with IGF1 3’ UTR mutation vector.(3)We detect the IGF1 expression in stably overexpress miR-29b SW480 and SW620 cells and stably knockdown miR-29b HCT116 and HT29 cell by Western Blot.Result showed thant IGF1 gene expression changes after the transfection, in HCT116-miR-29bsh and HT29-miR-29bsh, IGF1 expression was lower than HCT116-NC and HT29-nc. In SW480-PLV-miR-29b and SW620-PLV-miR-29b, IGF1 expression was obviously decrease compare to control cells.IGF1 interact with GF1-R located in the cell membrane, and play important roles in cell biology behavior after activation cytoplasmic protein such as PI3K, PI3K leads to AKT ser473 after activation or thr308 phosphorylation sites, so as to activate AKT (p-AKT). Western blot results showed that expression of p-PI3K and p-Akt were increased in miR-29b-knockdown HCT116 and HT29 cells, and decreased in SW480 and SW620 cells after miR-29b was overexpressed. That means miR-29 b can promote the activation of PI3K/Akt pathway.(4) Re-transfect IGF1-CDS to the cells overexpressed miR-29b in SW480 and SW620. Observe whether there is rescue effect in miR-29b induced change in CRC cells.Repression of IGF1 activates PI3K/AKt signaling and plays an essential role in miR-29b-induced apoptosis and the decrease in the proliferation and migration of CRC cells. Further analyses revealed that the re-overexpression of IGF1 significantly promoted the migration ability of SW480-PLV-miR-29b and SW620-PLV-miR-29b cells in transwell assays. Flow cytometry analysis showed that re-overexpressing of IGF1 in miR-29b-upregulated CRC cells decreased the percentage of apoptotic cells. Anchorage-independent colony formation assays and CCK8 assays indicated that the overexpression of IGF1 significantly promoted proliferation ability. In addition, the overexpression of IGF1 dramatically activated the miR-29b-negative regulation of PI3K/AKt.5.Relationship of IFN, miR-29b and IRF1 in CRC.(1) Sixty-one pairs of CRC tissues and adjacent normal tissues were immunostained for IRF1 and IGF1. Results showed that IRF1 expression in cancer is higher than in normal tissue (P= 0.004);IGF1 expression in cancer was lower than that in normal tissue (P< 0.001),More importantly, there was a significant inverse correlation between IRF1 and IGF1 expression levels (r=-0.336, p< 0.001).(2) Transient expression of IRF1 led to a decreased expression of IGF1 protein.We also performed western blotting for IRF1 and IGF1 protein in CRC cells treated with IFN-y for different time periods. IFN-y treatment caused increased the expression of IRF-1 in a time-dependent manner, and the amount of IGF1 protein decreased after 8 or 16 hours of stimulation in parallel to an increase in miR-29b levels.(3) Overexpress transcriptional factor IRF1 in CRC cells, and analyze IRF1 and IGF1 expression. Results showed that, IRF1 inhibited the IGF1 expression.(4) The expression of miR-29b and IRF1 was detected in 12 matched-paired clinical CRC tissues. The results of qRT-PCR showed that there was a positive relationship between IRFl and miR-29b expression levels by Spearman correction analysis (r= 0.6467, p< 0.01).(5) Based on the result that miR-29b can increase IRF1 expression, we assessed the impact of miR-29b on the IFN-induced IRF1 expression in HCT1 16 cells, we found that miR-29b inhibition resulted in a decrease in both basal and IFN-induced IRF1 expression, and this result was confirmed with immunofluorescent staining. Additionally, in miR-29b-overexpressing cells, the response of IRF1 to IFN-y was obviously increased. IRF1 enhances miR-29b transcription, and miR-29b could upregulate IRF1 expression in turn; thus, the coregulation between them constituted a positive feedback loop.(6) We next assessed the role of miR-29b in IFN-induced apoptosis. in HCT1 16 cells transfected with miR-29b inhibitor and control sequences treated with IFN-y, IFN-y induced 8.23% apoptosis in HCT116 cells, and litter apoptosis in anti-miR-29b HCT116 cells was observed after IFN-y treating. Therefore, we reasoned that the IRF1/miR-29b positive feedback loop may sensitize CRC cells to IFN-induced apoptosis.Conclusion1. IFN-y induces miR-29b in CRC cells through recruiting IRF1 to the binding sites in miR-29b promoter.2. Decreased miR-29b expression in CRC is associated with advanced clinical stage, and lymph node metastasis.3. MiR-29b inhibits CRC cell proliferation, migration and induces apoptosis.4. MiR-29b directly targets IGF1 and inhibits PI3K/AKt pathway in CRC cells.5. IRF1 and miR-29b constitutes a positive feedback loop, which could sensitize CRC cells to IFN-induced apoptosis.Innovation point of this paper1. MiR-29b is a miRNA related with CRC and IFN-gamma.2. We have raised a new regulation mechanism of miR-29b, and this research is with character of primary innovation.3. We have present the mechanism of the role of miR-29b/IGF1 in the CRC metastasis and invasion, and provide an new gene and miRNA for the clinical application of the tumor metastasis... |
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