Regulation Of Colorectal Carcinoma Stemness, Growth And Metastasis By An MiR-200c-Sox2-negative Feedback Loop Mechanism

Background and ObjectiveCancer cells are considered to be capable of unlimited proliferation and self-renewal, similar to stem cells. Moreover, a small number of cancer cells express stem cell markers and possess the stem cell-like ability to sustain tumour growth metastasis and recurrence. Previous studies suggested that carcinoma cells most likely transform into cancer stem cell (CSC)-like cells due to genetic alterations or microenvironment changes. Sternness-associated genes such as Sox2, KLF4, Bmil and Oct4are involved in the maintenance of the stemness of embryonic stem cells, and more recent studies have demonstrated their expression in some human tumours, governing tumorigenesis and metastases. However, the functions of stemness-associated genes in control of CRC tumorigenesis and metastases remain elusive.MicroRNA (miRNA) is a class of small molecule RNA size of about19-22nucleotides. MiRNAs are mostly transcribed from a hairpin intermediate of about70-90nucleotides called pre-miRNA, and undergo further processing by the ribonucleases Dicer. The mature miRNA targets the3’untranslated region (3’UTR) of its target mRNA, and induces either translational repression or mRNA degradation. Growing evidence indicates that miRNAs are aberrantly expressed in many human cancers and involved in the initiation, growth and metastasis of cancers. Several miRNAs are emerging as important regulators of the proliferation and metastases of CSCs. Recent findings have noted a connection between miRNAs and CSCs in CRC. MiR-93is down-regulated during the course of colon CSC differentiation into colon cancer cells and depresses the proliferation of colon CSCs. MiR-328maintains a CSC-like SP phenotype in CRC. MiR-200c is the predominant member of the miR-200family, which suppresses epithelial-mesenchymal transition (EMT), and the down-regulation of miR-200family members in some tumours promotes invasion and metastasis. The significance of miR-200c in cancer biological processes is becoming apparent. Moreover, there has been no published data regarding the role of miR-200c in CRC sternness. Therefore, we examined its expression and roles on sternness sustainment and metastasis in CRC.In addition, we identified Sox2as a novel target of miR-200c. Together with Oct4and NANOG, Sox2encodes homeodomain proteins that are required for the early development and propagation of undifferentiated embryonic stem cells. Sox2is involved in the proliferation or initiation of several cancers such as glioblastoma, gastric and breast cancer. Sox2is essential for the transformation of foregut basal progenitor cells to the oesophagus and forestomach and cooperates with inflammatory signalling pathways to induce the formation of carcinomas.In summary, miR-200c and its target may be involved in tumorigenesis and CSC-like cell transformation, but there is no direct evidence that the network including miR-200c and Sox2is implicated in CRC sternness and metastasis. We therefore attempted to focus on the miR-200c-Sox2-related mechanism that transforms CRC sternness and modulates proliferation and metastasis.Methods1. miR-200c expression in CRC tissues and cells was detected using quantitative RT-PCRQuantitative reverse transcription-PCR (qRT-PCR) was employed to detect the expression of miR-200c in34specimens from fresh colorectal cancer biopsies and colorectal cancer cell lines with different metastatic potentials including SW480, SW620, HT29, Lovo and HCT116.2. Prediction and Identification of miR-200c targets(1) Potential molecular targets of miR-200c were analysed by bioinformatic algorithms in the three commonly used databases including TargetScan, Pictar and microRNA.org.(2) The predicted targets Sox2, Bmil and KLF4were examined in colorectal cancer cell lines by qRT-PCR, and the correlation was analysis. Sox2, the target gene of miR-200c, was examined in colorectal cancer biopsies by qRT-PCR and Western blotting. The expression of Sox2was analysed in colorectal cancer cell lines by Western blotting.(3) The Sox23’UTR was amplified by PCR and then cloned downstream of the Renilla luciferase open reading frame of the psi-CHECK2vector (Promega) as Wt Sox23’UTR; the predicted target site for miR-200c was mutated using the KOD Plus Mutagenesis Kit (Toyobo), generating Mut Sox23’UTR. To test whether the Sox2was a target of miR-200c, we performed a luciferase reporter assay.3. The function of miR-200c on CRC growth and metastasis in vitro and in vivo(1) Lentiviral constructs expressing (Lenti-miRTM microRNA precursor clone collection; System Biosciences) or repressing (miRZipsTM lentiviral-based microRNA inhibition; System Biosciences) miR-200c were packaged using the pPACKHl lentivector packaging kit (System Biosciences). Pseudovirus particles were subsequently used to infect CRC cells. SW480and SW620cell lines constitutively expressing miR-200c and HCT116and SW480cell lines stably suppressing miR-200c were generated. Then, the miR-200c-over-expressing cells were transfected with Sox2vectors not containing its3’UTR.(2) The CCK-8cell proliferation assays, plate colony formation assays, soft agar assays and Transwell migration and invasion assays were carried out to detect cell proliferation, plate colony formation, migration and invasion in vitro after transfection of miR-200c and co-transfection of miR-200c/Sox2(not containing its3’UTR) or suppression of miR-200c.(3) Xenograft tumours were generated by subcutaneous injection to assess the effect of miR-200c and miR-200c/Sox2(not containing its3’UTR) on tumour growth in vivo. A orthotopic transplantation assay, in which tiny tumour masses of subcutaneous tumorstumours were transplanted into the mouse caecal subserosa, was employed to evaluate the effect of miR-200c and miR-200c/Sox2(not containing its3’UTR) on the metastasis of CRC cells in vivo.4. The effect of miR-200c on CRC sternnessWe observed the morphology, and assessed stem cell sphere-forming capacity and expression of CRC stem cell markers in miR-200c suppression cells.5. The verification of miR-200c-Sox2negative feedback loop(1) Finding the promoter sequence of miR-200c, and predicting the transcription factor of miR-200c using UCSC, TESS and TFSEARCH.(2) To generate a miR-200c promoter vector, a667bp fragment containing the two binding sites of Sox2was PCR amplified and inserted into a PGL3-luciferase reporter vector (Promega). Additionally, some mutation vectors related to the Sox2binding site were constructed. These PGL3-derived vectors, the control pRL-TK Renilla plasmids (Promega) and Sox2-expressing vectors were co-transfected into HEK293or SW480cells using Lipofectamine2000Reagent (Invitrogen). The luciferase activity of PGL3-derived vectors was determined using the Dual Luciferase Reporter Assay Kit (Promega).(3)The direct association of Sox2with miR-200c promoter was validated by Chromatin Immunoprecipitation (ChIP) analyses in CRC cells.6. The relational colorectal cancer signal pathway was detectedThe expression of T-PI3K, T-AKT, p-PI3K and p-AKT was assessed by Western blotting after transfection of miR-200c, co-transfection of miR-200c/Sox2, suppression of miR-200c or suppression of miR-200c followed by the treatment of LY294002.7. Statistical analysisSPSS13.0software was used for statistical analysis. Quantitative values of all experiments are expressed as the mean±standard deviation (SD). Relative quantification value(2-△△Ct) of qRT-PCR in cells were analysed by One-way ANOBA,with the SNK,LSD or Dunnett T3tests for multiple comparisons.It also analysed through two-tailed independent-samples t-test. The data of colony formation assay, Transwell migration and invasion assay and relative luciferase activities were analysed through One-way ANOVA. The data of CCK8assay was analysed by Factorial design analysis of variance. Relationships between miR-200c expression and clinicopathologic characteristics were tested using Fisher’s exact test. Differences were considered significant if P<0.05.Results1. miR-200c downregulation in CRC correlates with metastatic status, tumour grade and growth(1) miR-200c was downregulation in CRC tissues. The average expression level of miR-200c was significantly decreased in30of34CRC specimens (t=-13.758, P<0.001) compared to their normal counterparts, with a9.35-fold decrease in the colorectal cancer tissue samples. Furthermore, to investigate the clinicopathologic significance of miR-200c expression in CRC patients, the median relative expression level of miR-200c in the34CRC samples was recommended as the cut-off point for dividing miR-200c level into a low expression group and a high expression group. Correlation analysis showed that the miR-200c expression level was reversely correlated to tumour size (P=0.042), serosal invasion (P=0.016), lymph metastasis (P=0.013) and TNM classification (P=0.002).(2) miR-200c expression was detected in colorectal cancer cell lines.We further examined miR-200c expression in the CRC cell lines with different metastatic potentials by qRT-PCR. Relative miR-200c expression was significantly was significant difference between the CRC cell lines (F=370.078, P<0.001) The Dunnett T3multiple comparison indicated the expression of miR-200c in HCT116and HT29cells was higher than that of the SW480cells (P=0.011; P=0.008), while the expression of miR-200c in SW620and Lovo cells was lower than that of the SW480cells (P=0.007; P<0.001). We reasoned that expression of miR-200c is negatively correlated with the metastatic potential of CRC and that miR-200c may suppress CRC growth.2. Sox2is a novel target for miR-200c(1) Potential targets of miR-200c were analysed by bioinformatic algorithms. Sox2, Bmil and KLF4were the predicted targets of miR-200c in TargetScan, Pictar and microRNA.org in unison.(2) The basal expression of miR-200c was inversely correlated with Sox2mRNA in the CRC cell lines (P<0.001, r=-0.914), whereas the Bmil and KLF4 mRNAs were not inversely correlated with miR-200c in these cells (P=0.144, r=-0.396; P=0.692, r=-0.112). Accordingly, we chose Sox2as a preferential, predicted target of miR-200c.(3) Sox2expression in colorectal cancer (91.2%,31of34) was significantly more frequent than that in the corresponding normal tissue. Spearman’s correlation analysis demonstrated that Sox2and miR-200c were again inversely related in expression (P<0.001, r=-0.870).(4) There was scarcely any expression of miR-200c in the HEK293cells. First, we co-transfected the luciferase plasmids Wt Sox23’UTR or Mut Sox23’UTR, and pre-miR-200c into the HEK293cells and compared these cells with cells transfected with negative control pre-miRNA. We observed that the exogenous expression of miR-200c significantly decreased the luciferase activity of the Wt Sox23’UTR but not the Mut Sox23’UTR (t=10.721, P<0.001). To determine if endogenous miR-200c levels regulated Sox2expression, SW620and HCT116cells were transfected with the Wt Sox23’UTR or Mut Sox23’UTR. The luciferase activity of Wt Sox23’UTR was lower in the HCT116cells (t=-48.772,P<0.001),but the luciferase activity of Mut Sox23’UTR had no significant difference in HCT116and SW620cells. Therefore, Sox2is a direct target of miR-200c, and the negative regulation of Sox2by miR-200c is due to the miR-200c-binding sites in the Sox23’UTR.3. miR-200c represses Sox2to suppress CRC growth and invasion in vitro and in vivo(1) The miR-200c expression lentiviruses were stably transduced into SW480and SW620cells generating sub-cell lines SW480/miR-200c and SW620/miR-200c, which persistently overexpressed mature miR-200c (P=0.010; P=0.016); their controls were infected with lentiviruses containing empty vectors. Meanwhile, miR-200c was permanently knocked down in SW480and HCT116by the anti-miR-200c lentiviruses producing the SW480/zip-200c and HCT116/zip-200c sub-cell lines (P=0.001; P=0.039), and Scramble was used as a control. The co-transfection of miR-200c/Sox2(not containing its3’UTR) rescued Sox2expression (P=0.010; P=0.049). We transfected Sox2into HCT116and SW480cells, and the increased Sox2expression in Sox2-transfected cells was confirmed by Western blotting.(2) We observed an inverse change in Sox2mRNA and protein expression when the level of the miR-200c was altered. The expression of Sox2was reduced in miR-200c-overexpressing cells (P=0.016; P=0.034) and increased in miR-200c suppressing cells (P=0.002; P=0.005) compared to their controls respectively.(3) Sox2over-expression increased the cell proliferation (F=347.819, P<0.001; F=421.909, P<0.001), colony formation ability (t=-7.303, P=0.002; t=-12.520, P<0.001), migration (t=-23.433, P<0.001; t=-15.905P<0.001) and invasion (t=-14.053, P<0.001; t=-17.529, P<0.001) of HCT116and SW480cells.(4) CCK-8assay showed that the up-regulation of miR-200c restrained cell proliferation of SW480and SW620cells compared with control cells (P=0.018; P=0.011). The proliferative abilities of SW480and SW620cells were significantly higher in miR-200c/Sox2group than in miR-200c group (P=0.033; P=0.040). However, the suppression of miR-200c increased the proliferative abilities of HCT116and SW480cells (P=0.012; P=0.041). The plate colony formation assays yielded the similar effect.(5) The soft agar assays showed that over-expression of miR-200c caused a marked reduction of anchorage-independent growth ability, as indicated by the reduction in colony number and volume in soft agar (P<0.001; P<0.001). Re-transfection of Sox2(not containing its3’UTR) could enhance the ability again (P<0.001; P<0.001).Inversely, the clonogenicity in soft agar was observably increased in SW480and HCT116cells depleted of endogenous miR-200c in contrast with scramble-transfected cells (P<0.001; P<0.001).(6) Migration assays showed that miR-200c over-expression markedly repressed the motility of SW480and SW620cells as compared with their control cells (P<0.001; P<0.001).Re-transfection of Sox2(not containing its3’UTR) could enhance the motility ability than before (P<0.001; P<0.001).However, the knockdown of miR-200c significantly enhanced the migration of HCT116and SW480cells (P<0.001; P<0.001),compared to scramble-transfected cells. The invasion assays yielded the similar effect.(7) Subcutaneous tumour growth in the SW620/miR-200c group was slower than that in the SW620/Control (P<0.001). Tumours in the SW620/miR-200c/Sox2group was larger than that in the SW620/miR-200c group (P<0.001). Immunostaining confirmed that the cell proliferation index Ki-67and the CSC biomarkers β-catenin, CD133and CD44were down-regulated by miR-200c (P<0.001; P=0.013;P<0.001; P<0.001;P<0.001) and restored by Sox2(P<0.001; P<0.001;P<0.001; P<0.001;P<0.001).(8) Orthotopic cecum implantation animal model showed that sixty percent (3/5) of mice in the SW620/Control group or the SW620/miR-200c/Sox2group had hepatic metastatic lesions, and the hepatic metastatic lesions in the two groups showed no significant differences. However, no mice in the SW620/miR-200c group had hepatic metastatic lesions. The number of hepatic metastatic lesions in mice of the three groups was significant differences (F=55.300, P<0.001). No metastatic nodules were discovered in the other organs in any group.4. miR-200c depletion increases the stemness of colorectal carcinoma cellsLoss of miR-200c results in the acquisition of stemness, which is characterised by an increased stem cell sphere-forming capacity and an increased expression of CRC stem cell markers CD166, CD133and β-catenin in SW480(P<0.001; P<0.001; P<0.001) and HCT116(P<0.001; P<0.001;P<0.001) cells. Most essentially, loss of miR-200c causes SW480and HCT116cells to reverse differentiate to CSC-like cells, represented by the cells transitioning from spindle-shaped cells to round cells.5. miR-200c and Sox2reciprocally control their expression through a feedback loop(1) Over-expressing Sox2in SW480and HCT116cells accompanied a decrease of miR-200c (t=10.335, P=0.009; t=29.643, P=0.001).(2) While analysing a2kb region upstream of the transcription start site (TSS) of miR-200c using UCSC, TESS and TFSEARCH, we noted that there were two Sox2transcription factor binding sites (TFBSs) located within the miR-200c promoter. For convenience, the two TFBSs were named A and B.(3) A miR-200c promoter vector, containing the two binding sites of Sox2was PCR amplified and inserted into a PGL3-luciferase reporter vector. Additionally, some mutation vectors related to the Sox2binding site were constructed. These PGL3-derived vectors, the control pRL-TK Renilla plasmidsand Sox2-expressing vectors were co-transfected into HEK293or SW480cells. A reduction of the wild-type miR-200c promoter luciferase activity was observed upon up-regulation of Sox2in the HEK293and SW480cell lines (P<0.001; P<0.001), and a similar effect was observed when B was mutated alone (P<0.001; P<0.001). However, Sox2over-expression did not result in further reduction of the luciferase activity when A was mutated alone or A and B were mutated together. The ChIP analyses revealed that Sox2is most significantly bound to TFBS A within the miR-200c promoter indicating a direct association of Sox2with miR-200c promoter.6. miR-200c is a PI3K-AKT signalling pathway regulator in CRC (1) The up-regulation of miR-200c restrained the phosphorylation of PI3K and AKT (p-PI3K and p-AKT) compared to the Control and Blank cells; the restrained of p-PI3K and p-AKT by miR-200c was restored by Sox2.(2) The down-regulation of miR-200c caused the opposite effect. Treatment with the PI3K-Akt inhibitor LY294002could abrogate the activities of p-PI3K and p-AKT by inhibiting miR-200c.Conclusion1. miR-200c down-regulation in CRC correlates with metastatic status, tumour grade and growth. Moreover, relative miR-200c expression was significantly lower in highly metastatic CRC cell lines. We reasoned that expression of miR-200c is negatively correlated with the metastatic potential of CRC and that miR-200c may suppress CRC growth.2. Sox2was validated as a target for miR-200c. MiR-200c inhibits tumour growth and metastasis by down-regulating Sox2in vitro and in vivo.3. miR-200c depletion increases the sternness of colorectal carcinoma cells.4. miR-200c and Sox2reciprocally control their expression through a feedback loop.5. miR-200c is a PI3K-AKT signalling pathway regulator in CRC The PI3K-AKT pathway has been considered relevant to the maintenance of CRC sternness, growth and metastasis.