| Background:Colorectal cancer(CRC) is one of the most common gastrointestinal cancer. According to the site of its occurrence, colorectal cancer can be divided into colon cancer and rectal cancer. In China, the incidence of CRC is rising year by year with the improvement of living standards and structural changes in diet and lifestyle. In recent years, despite the continuous improvement of the diagnosis and treatment of CRC, the 5-year survival rate has not improved significantly. The reason is that the exact molecular mechanism of the development of colorectal cancer has not yet been fully elucidated. Therefore, to elucidate the exact molecular mechanisms of CRC is the current research focus. microRNAs(miRNAs, miRNA) are a class of small single-strand non-coding 18-to-25-nucleotide-long endogenous RNAs, which are new research focus in the field of human molecular oncology. miRNAs can regulate gene expression mainly through binding to the 3’-untranslated region (3’UTR) of target messenger mRNA at the post-transcriptional level to cause translational repression and/or mRNA degradation. Recent studies have demonstrated that abnormally high or low expression of many miRNAs in colorectal cancer. It may play as tumor suppressors and/or oncogenes in the development of CRC by regulating the expression of multiple target genes. Astrocyte elevated gene-1 (AEG-1), also known as metadherin(MTDH), was initially found upregulated expression in a human immunodeficiency virus type-1(HIV-1)-infected and tumor necrosis factor-α(TNF-α)-treated primary fetal astrocytes. It was cloned through flash subtractive hybridization by Su initially, which is located on human chromosome 8q22, a site that significantly correlated with a variety of malignancies. AEG-1, overexpressed in many cancers including CRC, has been reported to be closely associated to the carcinogenesis. In our previous study, we has reported that AEG-1 can promote malignant behaviors of cancer cells including the proliferation, migration and invasion, chemoresistance and neovascularization and so on. Recent studies have found that miR-375, which is a member of the microRNAs family, may participate in the development of liver cancer and head and neck squamous cell carcinoma by regulating the expression of AEG-1 gene. But the relationship between miR-375 and AEG-1 in colorectal cancer has not yet been confirmed. In this study we transfected the colon cancer cells with the miR-375 mimics to upregulate the expression of miR-375 in HCT116. The aim of this study was to find a new theoretical basis for the occurrence and development of colon cancer by studying the effects of miR-375 on the biological behavior of HCT116.Aim:In this study, we transfected the miR-375 mimics into HCT116 cells by Lipofectamine TM 2000. The expression of miR-375 and AEG-1 mRNA level were detected by real-time quantitative-PCR. The HCT116 cell activity was detected by MTT method. The changes of apoptosis and cell cycle distribution were analyzed by flow cytometry. The aim of our study was to find a new theoretical basis for the occurrence and development of colon cancer by studying the effects of miR-375 on the biological function of HCT116.Materials and methods:1. Material:Human colorectal cancer cell lines Caco2, HCT116, SW480, SW620(Chinese Type Culture Collection, Beijing, China); DMEM, RPMI-1640 and fetal bovine serum (Gibco, AUS); trypsin (0.25%EDTA, Genom, Hangzhou). miR-375 mimics and mimics negative control(RiboBio, Guangzhou). Lipofectmanine TM 2000 and TRIzol reagents (Invitrogen, USA); MTT(Weijia, Guangzhou); DMSO (Sigma, USA); Cell cycle staining buffer and Annexin V/PI apoptosis kit(LiankeBio, Hangzhou); RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA); SYBR Premix Ex Taq Ii (Tli RNaseH Plus, TaKaRa).2. Cell culture:CRC cell lines Caco2,HCT116 were maintained in DMEM supplemented with 10% FBS, while SW480, SW620 were maintained in RPMI-1640 medium with 10% FBS in an atmosphere of 5% CO2 concentrations at 37℃ with humidity. Culture medium was changed every one to two days according to the cell growth. When the cells covering the majority of the culture flask, we passage the cells or collect the cells.3. The expression levels of miR-375 in human colorectal cancer cell lines including Caco2, HCT116, SW480 and SW620 cells detected by Real-time quantitative-PCR(qRT-PCR). The cells were collected and the total RNA was extracted according to the instructions of TRIzol reagent and stocked in -80℃. The expression of miR-375 was measured by qRT-PCR, while U6 was as an internal control. Primers were purchased from Guangzhou RiboBio.The relative expression of miR-375 was calculated using the 2-ΔCt.ΔCt=Ave Ct(target gene)-Ave Ct(reference gene).4. miRNA transfection:The miR-375mimics and mimics negative control were designed and chemically synthesized by Guangzhou RiboBio. Twenty-four hours prior to transfection, HCT116 cells were seeded into 24-well plates with approximately 4×104～5×104 cells per well and cultured in 400μL medium without antibiotics. When the cell density reached about 30% to 50%, transfection was performed by using Lipofectamine TM 2000 and Opti-MEM without serum medium (Gibco) according to the manufacturer’s instructions.5μL miR-375 mimics or negative control (20μmol/L) was diluted in 50μL Opti-MEM; mixed gently and incubated at room temperature for 5 min.1.5μL Lipofectmanine TM 2000 was mixed in 50μL Opti-MEM in the same way; mixed gently and incubated for 5 minutes at room temperature. After the 5 minute incubation, the diluted miR-375 mimics or negative control were combined with diluted Lipofectmanine TM 2000; mixed gently and incubated for 20 minutes at room temperature. 100uL of complexes was added to each well containing cells and medium. Cultures were maintained at 37℃ in the 5% CO2, humidified atmosphere. The medium was changed for fresh medium after 6 hours. Incubate cells at 37℃ and 5% CO2 for 24h hours prior to testing.5. The expression levels of miR-375 and AEG-1 mRNA in human colon cancer HCT116 cells after transfection detected by qRT-PCR. Cells were harvested 48h after transfection and the total RNA was extracted according to the instructions of TRIzol reagent and stocked in -80℃.The expression of miR-375 was detected by qRT-PCR and U6 was as an internal reference. The expression of AEG-1 mRNA was detected in the same method. GAPDH was as an internal control. The primers were purchased from TaKaRa Company. The relative expression of miR-375 and AEG-1 mRNA was calculated using the 2-ΔΔCt. ACt=Ave Ct(target gene)-Ave Ct(reference gene).6. The effect of miR-375 over-expression on the cell activity of HCT116 cells detected by MTT assay. Cells were trypsinized and plated in 96-well plates with 3000 cells in 100uL of the medium and incubated at 37℃ in the 5% CO2, humidified atmosphere for 4h,24h,48h,72h respectively after transfection.20uL of 5 mg/mL MTT was added into each test well and incubated for 4 h. The supernatant was then discarded and 150uL of DMSO was added to each well to dissolve the formazan. Optical density (OD) was measured using a Microplate Reader with a test wave length of 490 nm. The cell proliferation curve was plotted and all experiments were performed in triplicate.7. The cell cycle distribution of HCT116 cells 48h after transfection detected by flow cytometry. Cells were harvested to a single cell suspension 48h after transfection. The supernatant was discarded after centrifugating for 5 min(1200r/ min). The cells were washed twice with PBS. PI staining was performed acording to the producer’s manual(add 1000uL staining buffer (A) and 10uL reagent B per well) Flow cytometry was performed immediately after staining in the dark at room temperature after 30 min.8. The effect of miR-375 over-expression on the apoptosis of HCT116 cells detected by flow cytometry. The transfected cells were harvested to a single cell suspension after 48h. The supernatant was discarded after centrifugating for 5 min(1200r/min). The cells were washed twice with PBS and were added 500uL 1× binding buffer,5uL FITC-annexin-V and lOuL PI. Flow cytometry was performed immediately after staining in the dark at room temperature after 5 min.9. Statistical analyses were performed using the SPSS 20.0 statistical software. All experiments were repeated three times. All measurement datas were expressed as mean±SD. The two-tailed Student’s t test, ANOVA, or Dunnett T3 were used to analyse.Statistically significance was defined as P-value<0.05.Results:1. The total RNA was extracted from colorectal cancer cell lines including Caco2, HCT116, SW480 and SW620 cells. The expression levels of miR-375 in human were detected by qRT-PCR. U6 served as an internal control. The relative expression level of miR-375 was calculated by 2-ΔCt. The expression level of miR-375 in HCT116 was the lowest, so we choose the HCT116 as the object of our research.2. The expression levels of miR-375 and AEG-1 mRNA in human colon cancer HCT116 cells after transfection detected by qRT-PCR. After transfection for 48h, qRT-PCR was used for detecting the expression levels of miR-375 and AEG-1 mRNA. Results showed that miR-375 mimics upregulated the expression of miR-375 significantly about 2000 times comparing to negative control group (P<0.01), while overexpression of miR-375 resulted in downregulation of AEG-1 mRNA (P<0.05). These results suggest that miR-375 mimics can upregulate the expression of miR-375 and the upregulation of miR-375 can inhibit the expression of AEG-1 mRNA simultaneously.3. The effect of miR-375 over-expression on the cell activity of HCT116 cells detected by MTT assay. OD values at 24h,48h and 72h in miR-375 mimics group were significantly lower than that in negative control group, and the difference was statistically significant (P<0.01). That is the cell activity was inhibited after transfection with miR-375 mimics.4. The cell cycle distribution of HCT116 cells 48h after transfection detected by flow cytometry. We evaluated cell cycle distribution by flow cytometry after transfection for 48h. The percentage of G1, S and G2 phase of cell was (68.323±2.975)% vs (54.973±3.056)%, (16.443±0.422)% vs (27.767±3.636)%, (14.200±0.943)% vs (17.26±2.268)% respectively in miR-375 mimics group and negative control group. The results showed that overexpression of miR-375 significantly increased the proportion of G1-phase cells (P<0.01)and decreased the proportion of S-phase (P<0.05).5. The effect of miR-375 over-expression on the apoptosis of HCT116 cells detected by flow cytometry. We evaluated apoptotic cell fraction by flow cytometry after transfection for 48h. Results showed that the apoptotic rate was (8.468±1.546)% and (6.252±1.201)% respectively in miR-375 mimics and negative control group. The apoptotic rate was increased in miR-375 mimics group comparing to the negative control group. The difference was statistically significant (P<0.05).Conclusion:1. The expression of miR-375 was significantly increased in miR-375mimics group compared to the negative control group. miR-375 inhibits the cell activity, mediates the cell cycle arrest and promotes the apoptosis of colon cancer HCT116 cells.2. Overexpressed miR-375 significantly inhibited the expression of AEG-1 mRNA. miR-375 may play a role in the development of colon cancer as a tumor suppressor by inhibiting the expression of AEG-1. |
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