| Backgroud and ObjectiveAccording to the data provided by American Cancer Society, the number of colorectal cancer (CRC) incidence, mortality in the United States is the third among various cancer. In China, colorectal cancer account for the forth most commonly diagnosed types of cancer. What’s more, the morbidity and mortality of CRC grow gradually with the development of economy and dietary changes. Although the overall prognosis of CRC has been maintained relative well among various types of cancer, metastatic disease is responsible for the main death cause of colorectal cancer. So, it’s very important for us to find some novel molecular biomarker which could be used to early diagnosis for CRC to improve the positive rate of early diagnosis and the prognosis of patients by application of modern tumor molecular biology.MicroRNA (miRNA) is one class of small molecular RNA size of about 19-22 nucleotides. MiRNA could inhibit its target expression by forming miRISC (miRNA-associated, multiprotein RNA induced-silencing complex) with target mRNA 3’-untranslated region (3’UTR). More and more researches show miRNA deregulated in many types of solid cancer and participation in many cellar life activities, such as:proliferation, migration, metastasis and metabolic ability. Meanwhile, likewise ordinary genes, mature miRNA is undergo further processing by the ribonucleases Dicer from pre-miRNA. Pre-miRNA is transcribed from corresponding gene, with the assistant of transcription factor (TF). Specially, with the technology progress, we can exam the expression of miRNAs in peripheral blood, which is hopeful to became gold standard of tumor clinical diagnosis, prognosis assessment and postoperative monitoring.RX Li et al reported that microRNA520d-3p inhibits gastric cancer cell proliferation, migration, and invasion by downregulating EphA2 expression, and miR-520d-3p could abrogate the expression of c-Myc and MMP9. P Tsukerman et al have reported that miR-520d-5p directly targets TWIST1 and downregulates the metastamiR miR-10b, which causes the up-regulation of E-cadherin. As for now, there is no more research and report about miR-520d how to play a role in colorectal cancer. So, we detected the expression of miR-520d-5p in colorectal cancer and cells firstly. Then, we researched the relevant mechanism.We validated Cthrcl abnormal up-regulation in colorectal cancer by application with gene-chip and Western Blot in our early work. We also proved that Cthrcl is a independent prognosis risk factor in peritoneal carcinomatosis colorectal cancer by analysis the expression of Cthrcl and clinicopathologic features. S Yamamoto et al have reported that Cthrcl selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex. Some researches have proved that Cthrcl could reinforce the expression of MMP9 by activiation of ERK1/2. We find Cthrcl could be a potential target of miR-520d-5p when we search on the relevant websites.Transcription factor Speciality Protein 1 (SP1) could promote or suppress the transcription of corresponding gene by bound to the promoter of gene. SP1 regulates progression of many types of cancer. WB Yang et al have reported that SP1 regulates lung cancer progression by bound to the promoter of miRl 82.Taken together, we figure that miR-520d-5p regulate the progression of CRC by targeting Cthrcl, and miR-520d-5p is under the transcription regulation of SP1. The aim of this study is to detect the expression of miR-520d-5p in CRC tussue and cell, identity its possible role in CRC tumorgenesis, and elucidate the molecular mechanism of its activities in CRC.Methods1. Quantitative RT-PCR was used to detect the expression of miR-520-5p in CRC tissues and cells.Quantitative RT-PCR was used to exam the expression of miR-520d-5p in 42 CRC and paired normal mucosa tissues and colorectal cancer cell lines with different metastatic potentials including SW480, SW620, HT29, LoVo and HCT116, DLD-1, LS174T.2. Predication and identification of miR-520d-5p targets(1) Potential molecular targets of miR-520d-5p were analysed by bioinformatic algorithms in the three commonly used databases including TargetScan, miRWalk and microRNA.org.(2) QRT-PCR was applied to detect the expression of Cthrcl mRNA in 52 paired biopsies from fresh CRC and paired mucosa tissues. Meanwhile, Western Blot was used to analyzed protein expression of Cthrcl in colorectal cancer tissues and cell lines including:SW480, SW620, HT29, LoVo and HCT116, DLD-1, LS174T.(3) The Cthrcl 3’UTR was amplified by PCR and then cloned downstream of the Renilla luciferase open reading frame of the pmirGLO vector (Promega) as Wt Cthrcl 3’UTR; the predicted target site for miR-520d-5p was mutated using the KOD Plus Mutagenesis Kit (Toyobo), generating Mut Cthrcl 3’UTR. A luciferase reporter assay was performed to test if the Cthrcl was a target of miR-520d-5p.3. The function of miR-520d-5p on CRC growth and metastasis in vitro and in vivo(1) Lentiviral constructs expressing miR-520d-5p was purchased from Soochow Genepharma bioscience company. MiR-520d-5p lentiviral and negative control vector was used to infect LoVo cells. LoVo cell line steady expressing miR-520d-5p was generated. Then, a series get-function assays were conducted in vitro.(2) The CCK8 cell proliferation assays, plate colony formation assays, Transwell migration and invasion assays were carried out to detect cell proliferation, plate colony formation, migration and invasion in vitro after transfection of miR-520d-5p mimic or inhibitor.(3) Xenograft tumours were generated by subcutaneous injection to assess the effect of miR-520d-5p on tumour growth in vivo.4. Predication and identification of transcription factor SP1 bound to promoter of miR-520d-5p(1) Finding the promoter sequence of miR-520d-5p, and predicting the transcription factor of miR-200c using UCSC, TFSEARCH, Promoter 2.0 Prediction Server and Mapper2.0.(2) QRT-PCR was applied to detect the expression of SP1 mRNA in 20 biopsies from fresh colorectal cancer and paired normal mucosa tissues. Spearman correlation analysis was conducted to find the potential correlation between miR-520d-5p and SP1.(3) QRT-PCR was applied to detect the expression of miR-520d-5p after trancfection of SP1 siRNA in SW620 and LoVo cell lines.(4) To generate a miR-520d-5p promoter vector, a 185 bp fragment containing the three binding sites of SP1 was PCR amplified and inserted into a pmirGLO-luciferase reporter vector (Promega). Additionally, some mutation vectors related to the SP1 binding site were constructed. Those pmirGLO vectors, the mock control pmirGLO plasmid (Promega) was transfected into HEK29T and LoVo cells by using LipofectaminTM3000 Reagent(Invitrogen) after a 24h prior transfection of SP1 siRNA. The luciferase activity of pmirGLO vectors was determined using the Dual Luciferase Reporter Assay Kit (Promega).5. The relational colorectal cancer signal pathway was detectedWestern Blot was conducted to asses the expression of T-ERK1/2, p-ERK1/2, E-cadherin, N-cadherin and vimentin after transfection of miR-520d-5p mimic or inhibitor.6. Statistical analysisSPSS 13.0 software was used for statistical analysis. Quantitative values of all experiments are expressed as the mean ± standard deviation (SD). Relative quantification value (2-ΔΔCt) of qRT-PCR in cells were analyzed by two-tailed independent-samples t-test. The data of colony formation assay, Transwell migration and invasion assay and relative luciferase activities were analysed through two-tailed independent-samples t-test. The data of CCK8 assay was analysed by Factorial design analysis of variance. Relationships between miR-520d-5p expression and clinicopathologic characteristics were tested using Fisher’s exact test. Differences were considered significant if P<0.05.Results1. MiR-520d-5p was down-regulated in colorectal cancer and cell lines.(1) MiR-520d-5p was down-expression in colorectal cancer tissuesThe average expression level of miR-520d-5p was significantly decreased in CRC specimens (t=-13.758, P<0.001) compared to their normal counterparts. Spearman correlation analysis was applied to find the relationship between miR-520d-5p expression and clinicopathologic significance. Correlation analysis showed that there is no significant correlation between the miR-520d-5p expression level and tumour size (P>0.05), or serosal invasion (P>0.05), lymph metastasis (P >0.05) and TNM classification (P>0.05).(2) MiR-520d-5p expression was detected in colorectal cancer cell lines.Seven colorectal cancer cell lines with different metastatic potentials was examined by qRT-PCR. Relative miR-520d-5p expression was significantly different between the CRC cell lines (F=370.078, P<0.001). Dunnett T3 multiple comparison indicated the expression of miR-520d-5p in HCT116 and DLD-1 cells was higher than that of the SW480 cells (P=0.011; P=0.008), while the expression of miR-520d-5p in SW620 and LoVo cells was lower than that of the SW480 cells (P =0.007; P< 0.001). We reasoned that miR-520d-5p expression is negatively correlated with the metastatic potential of colorectal cancer.2. Cthrcl is a novel target of miR-520d-5p(1) Cthrcl was predicted as a potential target of miR-520d-5p when we analysed it on TargetScan, miRWalk and microRNA.org in unison.(2) QRT-PCR and Western Blot was applied to detect the basal expression in CRC and corresponding counterpart. Cthrcl expression in colorectal cancer was significantly higher than its counterpart. Spearman correlation analysis shown that Cthrcl mRNA and miR-520d-5p was inversely related in expression (P<0.001, r=-0.555).(3) First, co-transfected the luciferase plasmids Wt Cthrcl 3’UTR or Mut Cthrcl 3’UTR, and miR-520d-5p mimic into the SW620 cells and compared these cells with cells transfected with negative control. We observed that the exogenous expression of miR-52Od-5p significantly decreased the luciferase activity of the Wt Cthrcl 3’UTR but not the Mut Cthrcl 3’UTR (1=10.721, P<0.001). Second, co-transfected the luciferase plasmids Wt Cthrcl 3’UTR or Mut Cthrcl 3’UTR or mock control pmirGLO plasmid, and 20pmol or 50 pmol miR-520d-5p mimic into the LoVo and HEK293T cells, we found that 50pmol mimic exerted more decrease of luciferase activity than 20pmol in the Wt Cthrcl 3’UTR(P<0.001) but not the Mut Cthrcl 3’UTR or mock control pmirGLO plasmid(P>0.05; P>0.05). Therefore, we validated that Cthrcl is a direct target of miR-520d-5p.3. MiR-520d-5p suppresses colorectal cancer cell proliferation and metastasis in vitro and in vivo by targeting Cthrcl(1) Lentivius was applied to infect LoVo cell lines, then LoVo/miR-520d-5p and LoVo/vector were generated. QRT-PCR and Western Blot was used to verify the miR-520d-5p and Cthrcl expression in LoVo/miR-520d-5p and LoVo/vector cells. T test shown that the miR-520d-5p expression in LoVo/miR-520d-5p was more higher than LoVo/vector (P< 0.01), meanwhile, Cthrcl expression was decreased in LoVo/miR-520d-5p than LoVo/vector.(2) QRT-PCR and Western Blot analysis shown that mimic up-regulated miR-520d-5p expression in SW480, SW620 and LoVo cells, inhibitor down-regulated miR-520d-5p expression in HCT116 cell; meanwhile, the Cthrcl expression in SW480, SW620, LoVo cells decreased and HCT116 cell increased, respectively.(3) MiR-520d-5p over-expression decreased the cell proliferation (P<0.05), colony formation ability (P<0.05), migration (P<0.05) and invasion (P<0.05) of LoVo cells.(4) CCK8 assay shown that miR-520d-5p mimic restrained cell proliferation of SW480, SW620 and LoVo cells compared with control cells (P<0.001; P<0.001; P<0.001); Meanwhile, miR-520d-5p inhibitor reinforced cell proliferation of HCT116 compared with inhibitor NC cells (P<0.001).(5) The plate colony formation assays demonstrated that miR-520d-5p mimic reduced cell colony formation ability of SW480, SW620 and LoVo cells compared with control cells (P<0.001; P=0.002; P=0.003); On the contrary, miR-520d-5p inhibitor enhanced cell colony formation ability of HCT116 compared with inhibitor NC cells (P=0.002).(6) Transwell migration assays showed that that miR-520d-5p mimic repressed cell migration ability of SW480, SW620 and LoVo cells compared with control cells (P< 0.001; P=0.010; P<0.001); Inversely, miR-520d-5p inhibitor strengthened cell migration ability of HCT116 compared with inhibitor NC cells (P=0.001). The invasion assays yielded the similar effect.(7) Subcutaneous tumour growth in the LoVo/miR-520d-5p group was slower than that in the LoVo/vector (P<0.05). QRT-PCR and Western Blot analysis shown that miR-520d-5p expression was significantly higher in athymic mouse subcutaneous tumour biopsy from LoVo/miR-520d-5p group than LoVo/vector. Contrarily, the Cthrcl expression in LoVo/miR-520d-5p group was lower than LoVo/vector. IHC assays validated that LoVo/miR-520d-5p group showed lower Ki-67 positive index than LoVo/vector.4. SP1 transactivatd miR-520d-5p via bound to its promoter.(1) We found the promoter by searching on the bioinformatics websites. Then, we found three potential SP1 binding sites on the promoter of miR-520d-5p via searching on the UCSC, Ensemble, TFSEARCH, Mapper 2.0 and Promoter 2.0 Prediction Server.(2) QRT-PCR analysis verified that SP1 mRNA expression lower in 20 CRC biopsies than the normal counterpart(P<0.05). Spearman correlation test showed a obviously positive correlation between SP1 mRNA and miR-520d-5p(P<0.01).(3) QRT-PCR and Western Blot validated that SP1 mRNA and protein expression apparently repressed via transfection of SP1 siRNA. Consistent with this, the expression of miR-520d-5p was also down-regulated(P<0.05).(4) A miR-520d-5p promoter vector, containing the three binding sites of SP1 was PCR amplified and inserted into a pmirGLO-luciferase reporter vector. Additionally, some mutation vectors related to the SP1 binding site were constructed. These pmirGLO-derived vectors, pmirGLO control plasmid were transfected into HEK293T and LoVo cells after another transfection of SP1 siRNA 24h prior. A reduced luciferase activity was observed in wild-type miR-520d-5p promoter in the HEK293T and LoVo cells(P<0.01),and a similar effect was observed when site C was mutated alone(P<0.05). However, there were no significant difference among mut-type A or B pmirGLO vector and the control pmirGLO plasmid.5. miR-520d-5p abrogate EMT via inactivation of p-ERK1/2(1) The transient up-regulation of miR-520d-5p repressed the phosphorylation of ERK1/2 and the secondary EMT (Epithelial Mesenchymal Transition) reversion via transfection of mimic compared to the control cells.(2) The transient down-regulation of miR-520d-5p induced the phosphorylation of ERK1/2 and the secondary EMT phenomena via transfection of inhibitor compared to the control cells.Conclusion1. MiR-520d-5p was down-regulated in colorectal cancer tissues and high metastatic potential CRC cell lines.2. Cthrcl was validated as a novel direct target of miR-520d-5p. MiR-520d-5p suppresses CRC tumor growth and metastasis via targeting Cthrcl in vitro and in vivo.3. SP1 regulates miR-520d-5p transcription by bound to its promoter.4. MiR-520d-5p abrogates EMT via inactivation of p-ERK1/2. |
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