| As the third leading culprit in cancer incidence worldwide, CRC continues to pose significant diagnostic, prognostic and therapeutic tribulations for clinicians. The American Joint Committee on Cancer(AJCC) TNM staging system is currently the only prognostic classification used in clinical practice to select patients for adjuvant chemotherapy. However, the AJCC stage fails to predict recurrence accurately in many patients undergoing curative surgery for localized CRC. This highlights the need for new biomarkers for a more precise prediction of high-risk patients with CRC recurrence and consequently improved personalized cancer care.mi RNAs are a novel class of small noncoding RNAs that typically inhibit the translation and stability of messenger RNAs(m RNAs) by binding to the 3’-untranslated regions(3’-UTR) of their target m RNAs. mi RNAs have nucleotides and are found in all multi-cellular eukaryotic cells. mi RNAs have important roles in various biological and pathological processes, such as development, cell proliferation, differentiation, apoptosis, inflammation, stress response and migration. Increasing evidences have suggested that mi RNAs are deregulated or upregulated in all types of cancers, acting either as tumor suppressors(e.g. mi R-34, mi R-15/16, let-7, mi R 200 family) or as oncogenes(e.g. mi R-155, mi R-222/221, mi R-17-5p, mi R-21), in which the mi RNAs play key roles in important aspects of tumorigenesis, such as cancer initiation, differentiation, growth and progression, mainly by interfering with the expression of target genes involved in cell cycle, apoptosis, cell migration and invasion, angiogenesis.In this study, we investigated the role of mi R-300 in regulating cell proliferation and invasion of colorectal cancer cells. Micro RNA and protein expression patterns were compared between colorectal cancer tissue and normal tissue and between two different prognostic groups. The up-regulation of mi R-300 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in Sw620 colorectal cancer cells. We observed that mi R-300 expression was frequently and dramatically up-regulated in human colorectal cancer tissues and cell lines compared with the matched adjacent normal tissues and cells. We further showed that transient and stable over-expression of mi R-300 could promote cell proliferation and cell cycle progression. Moreover, p53, a key inhibitor of cell cycle, was verified as a direct target of mi R-300, suggesting that mi R-300 might promote colorectal cancer cell proliferation and invasion by increasing p53 expression.This study was to determine the expression of mi R-300 and association with colorectal cancer formation, progression and the underlying mechanisms. We found that mi R-300 was significantly reduced in human colorectal cancer tissues and in colorectal cancer cell lines. Using the approaches of mi RNA array, systemic biology, in vitro manipulating expression of mi R-300 and in vivo tumor-bearing mouse model, we found that mi R-300 acted as a tumor promoter in colorectal cancer, which was through targeting p53.Material and methodsLo Vo, Caco2 and Sw620 cell lines were purchased from American Type Culture Collection(Rockville, MD, USA). Cells were maintained in RPMI-1640 in 5% CO2 at 37?C. NS si RNA and scrambled si RNA were purchased from Invitrogen Life Technologies and transfected using Lipofectamine® 2000(Invitrogen Life Technologies) according to the manufacturer’s instructions.Sw620 were harvested at specific times after treatment with regents as indicated in each experiment. Cells were mixed with loading buffer and subject to electrophoresis. After electrophoresis, proteins were transferred to polyvinyl difluoride membranes(Pall Filtron) using a semidry blotting apparatus(Pharmacia) and probed with mouse m Abs, followed by incubation with peroxidase-labeled secondary antibodies.Cell viability was assessed using an MTT assay. Following transfection, cells were plated in 96-well plates and incubated for 24, 48 and 72 h. A total of 20 μl 5 mg/ml MTT(Sigma-Aldrich) was added to each corresponding test well and incubated for 4 h at 37?C. The supernatant was then discarded and 200 μl dimethyl sulfoxide was added to each well to dissolve the formazan. Optical density was assessed by measuring the absorbance of each well at 490 nm using a spectrophotometer(Spectra Max Plus384; Molecular Devices, Sunnyvale, CA, USA). All experiments were performed in triplicate.DNA contents of cells were analyzed using flow cytometry as described previously. Control and transfected cells were harvested and washed twice with PBS(Phosphate Buffer Saline), fixed in 70% ethanol and kept at-20 °C until analysis. Then the cells were stained with 20 μg/ml PI containing 20 μg/ml RNase(DNase free) for 2 h. The stained cells were analyzed by flow cytometry(Partec Pas, Germany). The population of G0/G1, S, G2/M and sub-G1 cells was determined using Mulicycle Cell Cycle Software. The results are expressed as percentage of the cells in each phase.ResultsResults of the human colorectal cancer tissues compared with paired adjacent noncancerous(normal) colorectal tissues, the mi R-300 was up-regulated. The results are displayed on a log scale. The statistical differences between samples were analyzed with the wilcoxon signed-rank test. The expression of mi R-300 in 3 colorectal cancer cell lines and one immortalized normal colorectal mucosal epithelial cell line(NS) was carried out by real-time PCR and normalized to RNU6 B.The upregulation of mi R-300 promoted Sw620(human colorectal cancer cells) cell growth. Cell viability was measured using a WST-1 kit at indicated time points. Data are presented as mean±SD from three independent experiments performed in sextuple; mi R-300 over-expression induced colony formation of Sw620 cells. 103 cells were mixed with agarose and seeded in 9-well plates for two weeks; The expression levels of mi R-300 in the parental Sw620 and the Sw620/mi R-300 stable cell line. Data are presented as mean ± SD. *P < 0.05 vs control. Scratch wound-healing assay was conducted in mi R-300-transfected Sw620 cells and control cells. Migration distance was measured at 0, 24, 48 hours after cells were scratched. Values represent mean±SD. n=3. *P<0.05.Cells stably expressing mi R-300 or mi R-NC were incubated in Dulbecco’s modified Eagle’s medium and subcutaneously injected into each side of the posterior flank of nude mice(n=24). Thirty-three days after injection, mice were sacrificed and tumors were removed.Tumor volume at 33 d; Tumor volumes were detected every three days from the time they were obvious; Average tumor weights. aDenoted statistical significance between the two groups of mi R-300 and control. aP < 0.05 vs control.Expression of mi R-300 and p53 were positively correlated(r=0.712; P < 0.001). p53 expression level in colorectal cancer vs non-cancer tissues and in the C1 vs the C2 group. Results are presented as means with 95% CI; Kaplan-Meier plot of overall survival according to the expression level of mi R-300 and p53. The possible mi R-300 binding site in CDNK1B(p53) m RNA 3’UTR was predicted by bioinformatic analyses. The mutant seed sequence was underlined. p53 had a single predicted binding site in m RNA 3’UTR that was highly conserved in mammals, and in chicken(the complementary sequences of seed sequence of mi R-300 were highlighted). The numbers of viable Sw620 cells were determined by cell count 72 hours after cotransfection with mi R-300 mimic and CMV-HA-p53 vector. Values represent mean ± SD. n=3. **P<0.01ConclusionThe finding that mi R-300 was up-regulated in metastatic CRC is intriguing, as decreased mi R-300 levels have been found in solid tumors, indicating that the loss of mi R-300 may be a common event in tumorigenesis.We focused on the effect of mi R-300 on CRC metastasis and demonstrated that mi R-300 acts as a tumor promoter in CRC metastasis. Restoration of mi R-300 induced cell migration and invasion in vitro and tumor metastasis in vivo.To obtain stable cell lines that over-expressed mi R-300, we transfected Sw620-M cells with mi R-300 plasmids and screened by G418. We selected twelve cell colonies in the mi R-300-transfected group and found 10 out of 12 colonies exhibited remarkably uniform, stable and high-level expression of mi R-300. Furthermore, three randomly chosen monoclonal cell lines exhibited similar reduction in invasive ablility.Changes in mi RNA expression have been found to contribute to the initiation and progression of cancer. The relationship between mi RNAs and tumors has suggested that mi RNAs may be altered by treatment in patients with CRC. In the present study, we focused on the effect of mi R-300 on CRC metastasis and found that mi R-300 was a tumor promoter in CRC metastasis.As part of our research on how the increase of mi R-300 affects CRC metastasis, we demonstrated that p53 was a critical downstream target of mi R-300. In the present study, mi R-300 and P53 were co-activated in colorectal cancer samples, especially in the poor-prognosis group. mi R-300 and P53 are also closely related to the evelopment and progression of hematopoietic stem cells. This suggests that colorectal cancer co-activated by mi R-300 and P53 might share the same pathogenesis as leukemia. |
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