| Background and Aims:Colorectal cancer (CRC) is the fourth most common cancer and the second leadingcause of cancer death worldwide. The standard care in patients with CRC isrepresented by a combination of chemotherapy, radiotherapy and surgery. However,there are still approximately30-40%of patients develop metastasis that is mainreason for the low patient survival account（s50%）. More and more evidence supportscolorectal cancer stem-like cells (CSLCs)or cancer stem cells (CSCs) may play acrucial role in the progression and metastasis of CRC.In recent years, researches indicated that inhibitor of differentiation or DNA bindingprotein (Id1) is overexpressed in various types of cancer, including breast, prostate,lung, gastric, esophageal, andCRC， and thus Id1is considered to be a newtumor-related gene. Studies demonstrated that Id1could promote tumor cells growth,invasion and metastasis. And Id1isalso been found to be essential for themaintenance of self-renewal of neural and embryonic stem cells; however, its roles inhuman colorectal CSCsare still elusive. Although it a few studies have investigatedthat Id1promoted CRC haematogenous metastasis at early steps and regulatedcolorectal CSCs self-renewal capacities, it is essentially to confirm Id1’s role in CRCliver metastasis and colorectal stem-like cell features and to explore the potentialmolecular mechanisms.In the present study, we first aimed to found whether Id1was related to the stem cellmaker CD133expression in CRC patients. Next, we intended to evaluate the effectsof Id1silence on proliferation and CSCs features and Wnt/β-catenin signal pathwayin HCT116cellsin vitro, and to detect the effects of Id1silence on tumor growth andinitiation capacity in vivo. And in the third part of our study, we explored the effectsof Id1silence on epithelial-mesenchymal transition (EMT) and invasivenessfeaturesin vitro, liver metastasis in vivo. Materials and Methods:1. The messenger RNA (mRNA) and protein levels of Id1and CD133were detectedby reverse transcription quantitative PCR in30fresh tissue sampleswithCRC andmatched normal mucosa.Immunohistochemical analysis was used to detect Id1andCD133protein expression in20normal mucosa and75primary CRC specimens.Both the Id1and CD133expression with clinical features, and the relationshipbetween them by spearman analysis were evaluated.2. A lentiviral vector overexpressing short hairpin RNA (shRNA) targeting the Id1gene was used in this study, and stable silence of Id1in human CRC cell linesHCT116was achieved. The silence efficiency of Id1was routinely detected byWestern blot and RT-PCR The cell proliferation was evaluated by MTS assay andcolony forming assay. Apoptosis was assessed by AnnexinV-PE and7-AAD doublestaining assays. Serum-free spheres forming assay was performed to evaluateself-renewal capacity in vitro. Colon CSCs markers were evaluated by qRT-PCR,Western blot and FACS assays. The proliferative maker (PCNA) and cell survivinwere detected by RT-PCR and Western blot. CSCs and TCF/LEF promoter activitywere assessed after Id1silenced in HCT116cells. Different dose of cells wereinjected into BABL/C null mice subcutaneously to assess tumor growth andinitiationcapacity in vivo.3. Cell morphology was observed under a microscope. Migrationand invasionpotentials were individually evaluated by wound healing assayand transwellmigration/invasion assays.EMTand invasion relative makers(MMPs, TIMPs, CXCR4,snail, twist and E-cadherin) were detected by RT-PCR and Western blot.Additionally,in vivo properties of cells were observed in a mouse model of livermetastasis.Results:1. Id1mRNA expression was significantly higher than the matched normal mucosa(0.391±0.095vs.0.21±0.039, p<0.05) and the CD133mRNA expression was alsosignificantly higher than the matched normal mucosa (0.207±0.036vs.0.105±0.028, p<0.05). The Id1mRNA expression was significantly related to CD133mRNAexpression (R=0.401，P=0.031). In CRC samples, high expression of Id1was found in35cases (46.7%), and the Id1protein expression was significantly related to CRCdifferentiation status; however, Id1was not observed in the stained normal mucosatissue. For CD133,18cases showed negative expression and two cases showed weakexpression of in adjacent normal mucosa. However, twenty-two cases (29.3%) hadhigh expression of CD133. CD133expression was positively related to T categoriesin75CRC patients.Id1expression was significantly and positively correlated withCD133expression at protein levels (R=0.319, p=0.005).2.(1) Id1silence resulted in a decreased ability of HCT116cells to proliferate butinducedapoptosis rate compared to the control cells. The mRNA and protein levels ofPCNA and survivin were decreased by Id1silence. Silenced Id1decreased the size andnumber of spheres in compared to the control HCT116cells. Stem cell makersincluding CD24, CD133, EZH2, EpCAM, OCT4andβ-catenin but not CD44,CD166and Lgr5proteins were remarkably reduced in Id1silenced HCT116cellscompared to the control cells. The IC50value of5-FUwas5.2±0.3μM and8.4±0.4μM （p<0.05）for the Id1silenced and control HCT116cells, respectively.Silenced Id1decreased nuclear and cytoplasmic β-catenin and decreased TCF/LEFpromoter activity. Target genes of canonical Wnt signaling such as CyclinD1andsurvivin was downregulated mRNA and protein levels after Id1silenced.(2) HCT116cells with Id1silenced grew into smaller tumors than control cells in asubcutaneous tumor model.A total of1×105cells were sufficient to initiate tumor forcontrol cells. In contrast,5×105cells were minimal number needed for tumorformation with Id1silenced cells. Silenced Id1led to decrease about5-folds intumor-initiating ability in nude mice compared to the control.3.(1) Cells with Id1silenced underwent EMT reversal, as indicated by the changes incell morphology a tightly packed, cuboid, epithelial-like appearance. Id1silenceup-regulated E-cadherin and down-regulated snail and twist expression, which are the typical markers of EMT. Id1silenced cells migrated through the wound scratch moreslowly than the control cells. We also found a significant decrease in number ofmigration/invasion cells with Id1silence. Q-PCR and Western blot revealed thatMMP2, MMP9and CXCR4,were significantly decreased at both themRNA andprotein levels, however, TIMP1and TIMP2were increasedafter Id1silencedinHCT116cells. Functionally, overexpression of CXCR4protected HCT116cells fromId1silence reduced migration and invasionin vitro.(2) We implanted HCT116cells into the spleen leading to tumor growth in the spleenand to metastasis to the liver. All animals in the control group developed livermetastases (10/10). In contrast, only6/10animals in Id1silence group developed livermetastases. As compared to the control, Id1significantly suppressed liver weight inmice.Conclusions:Id1expression was positively related to the CSC maker CD133expression in CRC.Id1silence can suppress the CRCs self-renewal, possibly through Wntpathway.Inactivation of Id1can reverse EMT features and inhibits migrationin CRCcells lines in vitro andreduces liver metastases of CRC in vivo. Importantly, Id1functions to govern CRC migration and invasion partly through Id1/CXCR4axis. |
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