| Backgroud and ObjectiveColorectal cancer is one of the most prevalent carcinomas throughout the world. Every year, more than1million individuals will develop colorectal cancer, and the disease-specific mortality is nearly33%in the developed world. In our country, with the improvement of living standards and dietary changes, colorectal cancer has been increasing gradually year by year. With the change of dietary habits and structure, the morbidity and mortality were significantly increased. And younger, familial aggregation, genetic susceptibility are the characteristics of the disease in our country.Colorectal cancer, like other tumor types, is a disease full of complex gene expression abnormalities, including abnormal and expression of coding and noncoding gene structure. Then the the molecular biological mechanism which controls node specific gene expression and gene expression in the process of development of the disease is need to study.In recent years, non-coding RNA (non.coding RNAs), especially the microRNA (microRNA, miRNA) can regulate gene expression in the world, which offers a new perspective to solve this problem. MicroRNA (miRNA) is a class of small molecule RNA size of about19-22nucleotides. The processing of miRNA ripening process is divided into two steps:pri-miRNA Drosha enzyme processing into the precursor in the nucleus is called pre-miRNA. Pre-miRNA translocation to the cytosol in Dicer is under the action of the enzyme, the formation of two mature sequence by editing processing, which are called miRNA/miRNA*. And now the abundance of miRNAs does not be decided through experiments, are named miR-5p (5’arm) and miR-3p (3’ arm). More recent literatures show that:5p and3p can be applied to the same or different target genes, play the same or different function.Human pre-miR-339has two different sequences of the splicing:miR-339-5p and miR-339-3p. What is their expression in colon cancer? How do they control the biological cells process? There are no reports about it. To discuss these issues, we examined the expression level of miR-339-5p in human colon cancer cells and cancer tissues using qRT-PCR, and tested its effects on cell growth, cell-cycle distribution, and colony formation and invasion capacity in vitro using RNA interference technology and construction of miRNAs expression by lentiviral vector. We administered miR-339-5p precursor to a mouse colon cancer tumor xenograft model and further demonstrated that it could suppress colon tumor growth in vivo. We determined the target gene of miR-339-5p according to the prediction of biological information, real-time quantitative PCR, protein electrophoresis, luciferase reporter experiment. The solution of questions would indicate the function of miR-339-5p/3p in colorectal cancer growth and metastasis of colorectal cancer. These results indicate that miR-339-5p/3p can function as a tumor suppressor, regulating the maintenance and progression of cancers, providing the clue and thinking for exploring new therapeutic targets.Methods1. Mature miR-339-5p/3p expression in tissues and cells was detected using Real-time RT-PCR Mature miR-339-5p/3p expression in tissues and cells was detected using Real-time RT-PCR. The specimens include samples of colorectal cancer tissue and matched normal colonic mucosa of30patients. While colorectal cancer six human colonic carcinoma cell lines include HCT116, HT29, LS174T, SW480, SW620and LOVO.2. Effect of miR-339-5p/3p on colorectal cancer cells in vitro and in vivo biological characteristics was examined(1) Using the cationic liposome method, the miR-339-5p/3p mimics was transiently transfected into colorectal cancer cell line SW620. The cell proliferation, invasion and migration ability was evaluated using CCK8method, cell cycle and transwell migration, invasion assay.(2) Using the cationic liposome method, the miR-339-5p/3p inhibitor was transiently transfected into colorectal cancer cell line HCT116. The cell proliferation, invasion and migration ability was evaluated using CCK8method, cell cycle and transwell migration, invasion assay.(3) A lentiviral expression vector pLVTHM-pre-miR-339was designed^and constructed. SW620cells were transduced with vector supernatant and subsequently FACS-sorted for green fluorescent protein (GFP). Confirmation of stable transfection of the plasmids was obtained using the miR qRT-PCR assay. The colorectal cancer cell line was called SW620-p1VTHM/pre-miR-339. The cell proliferation, invasion and migration ability was evaluated using CCK8method, cell cycle, clone formation assay and transwell migration, invasion assay. The effect of tumorigenicity in nude mice subcutaneous was observed using the whole visualization animal model.3. miR-339-5p/3p target genes were predicted, related pathways were verified.(1) Three commonly used databases including TargetScan, Pictar and microRNA.org were used to forecast the target genes with the search terms of miR-339-5p/3p.(2) PRL-1, the target gene of miR-339-5p, mRNA was detected in different colorectal cancer cell lines by qRT-PCR, and the correlation was analysis. The expression of PRL-1mRNA and protein was detected in SW620overexpressing miR-339-5p/3p and in HCT116transfected with miR-339-5p/3p inhibitor.(3) The sequence containing the PRL-1gene3’UTR seed region fragment3’UTR seed region was subcloned into psiCHECK-2vector, and the recombinant vector psiCHECK-2-wt-PRL-1an psiCHECK-2-mut-PRL-1vector site-directed mutations were constructed. Combination of PRL-1and miR-339-5p/3p were detected by luciferase reporter system in order to determine whether miR-339-5p/3p targeted PRL-1gene directly.(4) The relational colorectal cancer signal pathway was detected.ERK1/2, p-ERK1/2protein expression was detected in SW620overexpressing by miR-339-5p/3p and in HCT116transfected with miR-339-5p/3p inhibitor using Western Blot.4. Statistical analysisSPSS13.0sorftware was used for statistical analysis. Data were presented as Mean±SEM of at least3independent experiments. Shapiro-Wilk test was used to verify the clinical samples’distribution. Differences were analyzed using the nonparametric test Mann-Whitney-Wilcoxon. Relative quantification value(2-ΔΔCt) of QPCR in cells were analyzed through One-way ANOVA, with the SNK, LSD or Dunnett’s T3tests for multiple comparisons. It also analyzed through two-tailed Student’s t test. Colony formation assay and transwell in vitro invasion assay were analyzed through two-tailed Student’s t test. And it also was used for comparisons of relative luciferase activities experiments. The results of CCK8assay was analyzed by Factorial design analysis of variance. P values of <0.05were considered statistically significant.Result1. miR-339-5p/3p expression was detected in colorectal cancer tissue and cells.(1) The expression of miR-339-5p/3p were detected in colorectal cancer tissues.In30fresh colorectal cancer paired tissues, fluorescence quantitative PCR test results show that the expression of miR-339-5p/3p in colorectal cancer tissues was significantly lower than that in normal mucosa (z=-5.671, P<0.001)(z=-5.597, P<0.001). The result also showed significant difference between miR-339-5p/3p node metastasis in colorectal cancer group with lymph and without lymph node metastasis in colorectal cancer group (t=2.063, P=0.041). Clinical and pathological analysis showed that miR-339-5p/3p in30cases of colorectal carcinoma tissues has no relationship with matched with age, sex, the degree of tumor differentiation (P=0.853, P=0.179, P=0.397).(2) miR-339-5p/3p expression was detected in colorectal cancer cell lines with different metastatic potential.qRT-PCR results showed that there was significant difference between the6kinds of cells expressing miR-339-5p/3p (F=66.554, P<0.001)(F-74.603, P<0.001). The Dunnett’s T3multiple comparison indicated the expression of miR-339-5p in HCT116cells was higher than that of the other6cells (P=0.03, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001). The expression of miR-339-3p in HT-29cells was higher than that of the other6cells (P<0.001, P=0.112, P<0.001, P<0.001, P<0.001).2. Effect of miR-339-5p/3p on colorectal cancer cells in vitro and in vivo biological characteristics was examined.a. The biological behavior of of SW620cell lines was performed transfected with miR-339-5p/3p mimics. (1) Compared with miR mimics control, increased miR-339-5p/3p expression levels were measured in SW620cells after transfected with miR-339-5p/3p mimics. The difference was statistically significant (t=-5.780, P=0.029)(t=-7.020, P=0.002) showed significant moderating effect.(2) Compared with miR mimics control, decreased proliferation properties of SW620transfected with the miR-339-5p/3p mimics were also detected by CCK-8assay. The difference was statistically significant (F=60.735, P<0.001)(F=7.175, P<0.001) showed significant moderating effect.(3) By analyzing the distribution of cell cycle by flow cytometry, compared with miR mimics control, miR-339-5p overexpression in SW620cells after S cells were decreased (t=8.516, P<0.001), and the corresponding G1phase increased (t=-13.178, P<0.001). miR-339-3p overexpression has no influence on SW620cells, S cells were not changed (t=-5.220, P=0.629), and the corresponding G1phase was not changed (t=0.508, P=0.6)(4) The number of migratory SW620cells transfected with the miR-339-5p mimics was fewer than miR mimics control, the difference was statistically significant (t=14.219, P<0.001). The number of migratory SW620cells transfected with the miR-339-3p mimics was fewer than miR mimics control, the difference was statistically significant (t=10.862, P<0.001).(5) The number of invasive SW620cells transfected with the miR-339-5p mimics was fewer than miR mimics control, the difference was statistically significant (t=8.817, P<0.001). The number of invasive SW620cells transfected with the miR-339-3p mimics was fewer than miR mimics control, the difference was statistically significant (t=9.014, P<0.001).b. The biological behavior of of HCT116cell lines was performed transfected with miR-339-5p/3p inhibitor. (1) Compared with miR inhibitor control, decreased miR-339-5p/3p expression level was measured in HCT116cells after transfected with miR-339-5p/3p inhibitor. The difference was statistically significant showed significant moderating effect (t=-36.847, P=0.001)(t=-6.924, P=0.02)(2) Compared with miR inhibitor control, increased proliferation properties of HCT116transfected with the miR-339-5p/3p inhibitor were also detected by CCK-8assay. The difference was statistically significant showed significant moderating effect (F=33.604, P<0.001)(F=18.204, P<0.001)(3) By analyzing the distribution of cell cycle by flow cytometry, compared with miR inhibitor control, inhibition of miR-339-5p expression in HCT116cells after S cells were increased (t=-8.335, P=0.001), and the corresponding G1phase decreased (t=7.393, P=0.002). Inhibition of miR-339-3p expression in HCT116cells, S cells were not changed (t=-0.518, P=0.632), and the corresponding G1phase was not changed (t=-13.917, P<0.001).(4) The number of migratory HCT116cells transfected with the miR-339-5p inhibitor was more than miR inhibitor control, the difference was statistically significant (t=-17.882, P<0.001). The number of migratory HCT116cells transfected with the miR-339-3p inhibitor were more than miR inhibitor control, the difference was statistically significant (t=-13.917, P<0.001).(5) The number of invasive HCT116cells transfected with the miR-339-5p inhibitor were more than miR inhibitor control, the difference was statistically significant (t=-7.178, P<0.001). The number of migratory HCT116cells transfected with the miR-339-3p inhibitor was more than miR inhibitor control, the difference was statistically significant (t=-8.243, P<0.001).c. The biological behavior of of SW620lines was performed transfected with pLVTHM/pre-miR-339. (1) The pLVTHM/pre-miR-339lentiviral expression vector was constructed.(2) The established stable cell line SW620expressing of miR-339-5p and miR-339-3p was named SW620/pLVTHM-pre-miR-339.(3) The expression of miR-339-5p and miR-339-3p in SW620/pLVTHM-pre-miR-339group was higher than that of SW620/group pLVTHM-NC (P=0.046, P=0.046). Compared with SW620/pLVTHM-NC, increased miR-339-5p/3p expression levels were measured in SW620/pLVTHM-pre-miR-339.The difference was statistically significant (t=-7.621, P=0.02)(t=-5.638, P=0.03) showed significant moderating effect.(4) Compared with SW620/pLVTHM-NC, decreased proliferation properties of SW620transfected with the SW620/pLVTHM-pre-miR-339were also detected by CCK-8assay (F=12.189, P=0.001). The difference was statistically significant showed significant moderating effect.(5) By analyzing the distribution of cell cycle by flow cytometry, compared with/pLVTHM-NC, S cells in SW620/pLVTHM-pre-miR-339were decreased (t=5.056, P=0.007), and the corresponding G1phase increased (t=-3.669, P=0.021).(6) Colony formation assay showed that the ability of SW620cells to form colony were decreased after pLVTHM-pre-miR-339(t=11.841, P<0.001).(7) The number of migratory SW620cells transfected with pLVTHM-pre-miR-339were fewer than with the control, the difference was statistically significant (t=11.841, P<0.001).(8) The number of invasive SW620cells transfected with pLVTHM-pre-miR-339were fewer than with the control, the difference was statistically significant (t=7.606, P<0.001).(9) SW620/pLVTHM-pre-miR-339cells or SW620/pLVTHM cells were injected subcutaneously to the blank of nude mice, respectively, into nude mice. The tumour growth rate of SW620/pLVTHM-pre-miR-339cells was slower than that of SW620/pLVTHM cells the difference was statistically significant (F=5.024, P=0.008).4. Bioinformatics analysis of target gene of miR-339-5p and miR-339-3p function, detection of miR-339-5p target gene identification and the downstream signal molecules(1) The bioinformatics prediction of target genes for miR-339-5p/3p was perdicted by software TargetScan, PicTar, MICRORNA.ORG. Considering the development and metastasis related genes of tumor, preliminary candidate PRL-1was studied as a target gene miR-339-5p. The miR-339-3p target gene was predicted, the final results will be performed in subsequent experiments certification.(2) qRT-PCR detection in6kinds colon cell lines indicated the highest expression level in LOVO, and lowest expression level in HCT116(F=7.432, P=0.002). In6colorectal cancer cell lines, expression of miR-339-5p and PRL-1in the opposite trend.(3) Compared with the miR mimics control, in SW620cells transfected with miR-339-5p mimics, mRNA and protein expression level of PRL-1decreased significantly (t=26.858,/P=0.001). However, compared with miR mimics control, in SW620cells transfected with miR-339-3p mimics, mRNA and protein expression level of PRL-1had no change (t=0.452, P=0.675). Also in the stable transfection experiments, compared with the SW620/group pLVTHM-NC, mRNA and PRL-1protein expression in SW620/pLVTHM-pre-miR-339cells was decreased. QRT-PCR and Western blot results showed that compared with miR inhibitor control, transfection of miR-339-5p inhibitor in HCT116cells, mRNA and protein expression level of PRL-1was increased (t=-9.036, P=0.012). Compared with miR inhibitor control, transfection of miR-339-3p inhibitor in HCT116cells, mRNA and protein expression level of PRL-1had no change (t=-1.501, P=0.272).(4) After constructing psiCHECK-2/PRL-13’UTR vector and its mutation carriers. PsiCHECK-2/WT-3’UTR and miR-339-5p mimics, miR mimics control after transfection,293FT cells and HCT116cells luciferase activity was decreased respectively; by the statistical analysis, the differences were statistically significant (P<0.001, P=0.002). PsiCHECK-2/MUT-3’UTR and miR-339-5p mutant plasmid mimics, miR mimics control cotransfected luciferase activity were not changed (P=0.069, P=0.453), showed that miR-339-5p and PRL-13’UTR can specifically bind. PsiCHECK-2/WT-3’UTR and miR-339-3p mimics/miR mimics control after transfection,293FT cells and HCT116cells can not be changed in luciferase activity (P=0.466, P=0.243). The mutation plasmid psiCHECK-2/MUT-3’UTR and miR-339-3p mimics/miR mimics control were transfected into cells also did not change the luciferase activity (P=0.379, P=0.953), showed that miR-339-3p did not act on PRL-13’UTR.(5) The Western blot results showed that compared with pLVTHM-NC/SW620, in SW620cells transfected with pLVTHM-pre-miR-339, ERK1/2expression levels did not change, and the decreased expression of p-ERK1/2was detected. Compared with miR inhibitor control, transfection of miR-339-5p inhibitor in HCT116cells following ERK1/2expression levels did not change, and the expression of p-ERKl/2increased. MiR-339-5p may inhibit the activity of p-ERK1/2.Conclusion1MiR-339-5p regulates the growth, colony formation and metastasis of colorectal cancer cells by targeting PRL-12MiR-339-3p inhibits colorectal cancer cell proliferation, invasion and metastasis ability, the identification of target genes will be carried out in a follow-up experiment3MiR-339-5p may affect the activation of ERK signaling pathway. |
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