Is Dicer A Tumor Suppressor Gene In Colorectal Cancer?

BACKGROUND&OBJECTIVEColorectal cancer(CRC)is one of the most common malignant tumors in the world.Metastasis is the leading cause influencing the outcomes and mortality of the CRC patient.Therefore,it is critical to clarify the molecular mechanism of metastasis of CRC.Our previous study showed that overexpression of SATB2 inhibited proliferation and invasiveness of colorectal cancer cells.microRNA profile expression showed that 20 miRNAs were up-regulated while 2 miRNAs were down-regulated when SATB2 was over-expressed in HT29 cells.MiR-202,which was found to be significantly down-regulated by SATB2,was selected for further studied.Interestingly,Dicer,one of the key components for miRNA biogenesis was predicted as one of the main targets of miR202.MiRNAs,a class of small noncoding RNAs,play the key roles in gene regulation in multicellular organisms.In mammals,Dicer,one of the key enzymes in the miRNA biogenesis,consists of two RNase III domains,RNase ?a and RNase ?b,which are responsible for the biogenesis of short interfering RNAs and miRNAs.Karube et al found that Dicer protein is down-regulated in areas of invasion and advanced carcinomas in non-small cell lung carcinoma(NSCLC).And,the reduced mRNA expression is significantly associated with poorer survival of NSCLC patients.Similar results about expression of Dicer mRNA were found in breast cancer and ovarian cancer.Differently,Differently,Dicer was found to be up-regulated in 81%of patients with prostate cancer.And increased Dicer expression is related to advanced stages of patients with prostate cancer.A recent study showed that that high Dicer expression is related to poorer survival of CRC patients.These studies suggested that the roles of Dicer in different types of cancer may depend on expression pattern of Dicer and its related miRNAs.Methods1.Correlation analysis of clinicopathological features and expression levels of Dicer in tumor tissue of CRC.(1)Expressions of Dicer mRNA in 68 cases of clinical samples of CRC were detected by reverse transcriptional real-time PCR.The relationship between clinicopathological features of CRC patients and Dicer mRNA levels was analyzed.And,the relationship between mRNA levels of genes related to stenness(Nanog,Oct4,Lin28B,Axin,CD24,SATB2)and Dicer was also analyzed(2)Expression of Dicer protein in CRC tumor tissue and their corresponding counterparts was detected by Western blotting.The localization of Dicer in CRC tumor tissue was analyzed by immunoflorescent assay and confocal imaging.2.Effects of knockdown or knockout of Dicer on the biological behaviors of human CRC cells(1)siRNAs targeting Dicer(http://cgap.nci.nih.gov/RNAi)was used to knockdown expression of Dicer in SW480 adnHCT116 cells.Three human colorectal cancer cells(DLD1,HCT116 and RKO),in which Dicer was knocked out,were kindly gifted from Dr.Bert Vogelstein in The Sidney Kimmel Comprehensive Cancer Center and Howard Hughes Medical Institute of John Hopkins University Medical School.(2)Effects of knockdown or knockout of Dicer on proliferation,migration and adhesion of the CRC cells were detected by CCK-8 assay,plate colony formation assay,transwell chamber assay,and cell adhesion assay.(3)In vivo effects of Dicer knockout on tumor growth of CRC cells were analyzed by subcutaneous tumorigenesis assay in nude mouse.3.Generation of mouse models with intestinal specific knockout of Dicer and related CRC models induced by AOM.(1)Intestinal specific mutant mice for Dicer deficiency were generated by crossing Dicerloxp/loxp mice with Villin-Cre mice.(2)Body size and survival of Dicerflox/flox&Villin-Cre,Dicerloxp/loxp&Villin-Cre and wild types mice were observed.Ten milligram of AOM per kg body weight was administrated into the mice according to the protocol described previously to induce the colon tumor.Result:1.Correlation analysis of clinicopathological features and expression levels of Dicer in tumor tissue of CRC.1.1 Expression levels of Dicer in the CRC is significantly lower than that in their paired counterpart normal mucosa by reverse transcriptional real-time PCR(t=-4.043,P<0.01).However,no significant association were found between Dicer mRNA expression and age,gender,tumor size,tumor site,tumor differentiation,lymph node status and Dukes classification(P>0.05)of CRC patients.Interestingly,expression levels of Dicer mRNA is correlated with mRNA levels of Nanog,Oct4,Lin28B,Axin,CD24 and SATB2 by Bivariate analyses(r=0.584,P<0.01;r=0.44,P<0.01;r=0.362,P=0.003;r=0.664,P<0.01;r=0.444,P<0.01).1.2 Western blotting results showed that expression levels of Dicer protein in the tumor tissues of CRC were significantly lower than that in their paired counterparts.And,low expression of Dicer was most likely found in poor differentiated tumor tissue of CRC.Immunofluorescent staining and immunohistochemiscal staining showed that Dicer was mainly localized in cytoplasm of CRC cells.2.Effects of knockdown or knockout of Dicer on the biological behaviors of human CRC cells2.1 Knockdown of Dicer by siRNA targeting Dicer led to decreased proliferation of SW480 cells by in vitro CCK-8 assay(HCT116/HCT116siDicer:F=1.321 P=0.255;SW480/SW480siDicer:F=14.766 P<0.01).Similar to the above results,knockout of Dicer also resulted in inhibitory proliferation of HCT116,DLDl and RKO cells.(DLD1/DLD1 Dicer-/-:F=55.416 P=0.026;HCT116/HCT116 Dicer-/-:F=153.658 P<0.01;RKO/RKO Dicer-/-:F=47.298 P<0.01)2.2 Both Knockdown of Dicer by siRNAs in SW480 and HCT116 cells and knockout of Dicer in HCT116,DLD1 and RKO cells also exhibited inhibitory colony formation compared with that in controls(SW480/SW480 siDicer:t= 2.802 P=0.049;HCT116/HCT116 siDicer:t=14.186 P<0.01;DLD1/DLD1 Dicer-/-:t=4.117 P=0.001;HCT116/HCT116 Dicer-/-:t=4.632 P=0.01;RKO/RKO Dicer-/-:t=10.669 P<0.01)2.3 Both Knockdown of Dicer by siRNAs in SW480 and HCT116 cells and knockout of Dicer in HCT116,DLD1 and RKO cells demonstrated impaired invasive ability by invasive chamber experiment.(SW480/SW480siDicer:t= 2.839 P=0.011;HCT116/HCT116siDicer:t=5.297 P<0.01;DLD1/DLD1 Dicer-/-:t=6.297 P=<0.01;HCT116/HCT116 Dicer-/-..t=6.307 P<0.01;RKO/RKO Dicer-/-:t=5.031 P<0.01)2.4 Both Knockdown of Dicer by siRNAs in SW480 and HCT116 cells and knockout of Dicer in HCT116,DLD1 and RKO cells showed reduced fibronectin adhesive ability.(SW480/SW480siDicer:t= 4.333 P=0.001;HCT116/HCT116 siDicer:t=4.445 P=0.001;DLD1/DLD1 Dicer-/-:t=11.228 P<0.01;HCT116/HCT116 Dicer-/-t=2.825 P=0.01;RKO/RKO Dicer-/-:t=34.189 P<0.01)2.5 Knockout of Dicer led to a pronouncedly decreased tumor growth of CRC cells subcutaneously in nude mice compared with that of controls.(DLD1/DLD1 Dicer-/-:F=17.293 P<0.01;HCT116/HCT116 Dicer-/-:F=14.812 P=0.001 and RKO/RKO Dicer-/-:F=8.906 P=0.005)3.Generation of mouse models with intestinal specific knockout of Dicer and related CRC models induced by AOM.3.1 Identification of different genotypes for Dicer and Cre gene by genomic polymerase chain reaction(Dicer:Mutant=420bp,Heterozygote=351bp and 420 bp,Wild type=351 bp;Villin-Cre:Mutant=350 bp).The genotype distributions of all procedures involving mice were in accordance with Hardy Weinberg equilibrium.3.2 DicerlloxP/+&Villin-Cre mice seemed to display no phenotype compared with that of wild type controls.DicerlloxP/loxP&Villin-Cre mutants appeared normal at birth and were born in the expected Mendelian ratio.DicerlloxP/+&Villin-Cre mice fed normally but were significantly smaller than their littermate controls(F=144.748,P<0.01).In contrast,there was no significant difference of weight between DicerlloxP/+&Villin-Cre mice and wild type controls(P=0.694).Additionally,DicerlloxP/loxP&Villin-Cre mice had shorter survival time than DicerlloxP/+&Villin-Cre mice and wild type mice(?2= 26.401,P<0.01;x2=46.509,P<0.01).However,no significant difference of lifetime was found between DicerlloxP/+&Villin-Cre mice and wild type mice(?2=0.033,P=0.856).In Dicerloxp/loxp&Villin-Cre at the age of 9 months,the normal structure of colon epithelium was destroyed and the normal lamina propria has been replaced by fibrosis and increased infiltration of inflammatory cells in some area of colon.Similarly,even in Dicerloxp/loxp&Villin-Cre at the age of 5 days,ulceration of small intestine and increased infiltration of inflammatory cells in the lamina propria were found.3.3 Most Dicerloxp/loxp&Villin-Cre mice died early Only One Dicerloxp/loxp&Villin-Cre mouse was successfully induced by AOM for six times.However,the mouse died during about one month and showed that severe inflammatory reaction in most areas of large intestine.Amazingly,invasive adenocarcinoma of colon was induced by AOM for six months in a Dicerloxp/+&Villin-Cre mouse.?Conclusions:1.Dicer was down-regulated in the tumor tissues of CRC.And,low expression ofDicer was most likely found in poor differentiated tumor tissue of CRC.2.Both knockdown and knockout of Dicer led to impaired effects on proliferation,adhesion,migration in vitro and tumor growth in vivo.3.Mice with intestinal specific knockout of Dicer showed inhibitory growth anddevelopment and severe inflammatory reaction in small intestine and large intestine,4.Invasive adenocarcinoma of colon could be induced by AOM inDicerloxp/+&Villin-Cre mouse.And,Dicer was suggested as an important haploinsufficient tumor suppressor gene in colorectal cancer.