| Currently, colorectal cancer is a common malignancy in the world, its incidence and mortality rise. Treatment of colorectal cancer with surgery, combined with chemotherapy, but poor efficacy, and colorectal cancer treatment in advanced colorectal worse. Elucidate the molecular mechanisms of colorectal cancer metastasis and provide theoretical and experimental basis for clinical treatment is very important. microRNA is a class of small non-coding RNA molecules with a regulatory function, usually containing 19-24 nucleotides. Mature miRNA is processed through a series of nucleic acid cleavage reaction is formed by a long pre-microRNAs, by way of complementary base pair to identify target genes 3’UTR region of non-coding mRNA, thereby combining with the target mRNA degradation or repression of target mRNA translation. miRNAs through different target genes involved in the regulation of a variety of biological processes, such as tumor development and progression, hematopoietic organ, tissue development and differentiation, cell proliferation, apoptosis and basal metabolism and so on; while miRNAs can also be a variety of biological basics signaling pathways play a regulatory role. Based on this, miRNA potential for tumor diagnosis, treatment and prognosis of the value of research in recent year has become a hot spot.In this study, we screened differently expressed miRNAs in CRC tissues and cells, and determined miR-214, which was associated with metastasis and radiotherapy resistance of CRC as our research candidate by means of miRNA microarray. We demonstrated the role of miR-214 in invasion, metastasis, and radiotherapy resistance of CRC. The main contents of this research are as the following:(1) Screening and identifying miRNA associated with metastasis and radiotherapy resistance of CRC.(2) Predicting and identifying target genesof miR-214, and demonstrating their roles in invasion, metastasis, and radiotherapy resistance of CRC.(3) Confirming the upstream transcription factor of miR-214, and investigating the regulation relationship between miR-214 and transcription factors, and then demonstrating the roles of transcription factor in invasion, metastasis, and radiotherapy resistance of CRC.This research aims to reveal a new regulating mechanism of miR-214, approaching the role of miR-214 in the development of CRC, and provide a new miRNA and target gene for clinical therapy of invasion, metastasis and radiotherapy resistance of CRC.Methods1. Screening and identifying differently expressed miRNAs in tissues and cellsof CRCMicroarray analysis was performed in tissues and cells ofCRC to preliminarily screen differently expressed miRNAs associated with metastasis and radiotherapy resistance of CRC. miR-214 expression in tissues and cells of CRC was investigated by real-time PCR.2. Prediction and identification of miR-214 target.Four public databases including miRanda、TargetScan、Pictar and RNAhybrid were used to predict the targets with the search terms of miR-214. MED 19 and CDC25A with high predictive values were selected as candidate targets. Identifying MED19 and CDC25A as target genes of miR-214 by the Luciferase reporter system. Detecting the expression of miR-214 and its target genes by real-time PCR and Western blot, and investigating correlations between them.3. Effects of miR-214 on cell biological behaviors of CRC(1) miR-214 lentivirus-expressing vectorwas successfully designed and established. After viral packaging, it was transfected into SW480 and HCT116cells lines. The stable miRNA-expressing CRC cell lines were obtained by flow cytometry, and then detected the expression of miR-214 by real-time PCR.(2) MiR-214 lentivirus-expressing vector and target genes (MED19 and CDC25A) lentivirus-expressing vectors without 3’UTR on coding areas were co-transfected into SW480 and HCT116cell lines to establish stable expressing cell lines. The expressions of MED 19 and CDC25A were detected by Western blot.(3) Transfecting miR-214 expressing vector, or miR-214 and target genes without 3’UTR co-expressing vector into SW480 and HCT116cells lines, and investigating their effects on cell proliferation, invasion, and radiotherapy by CCK-8, plate clone, matrigel invasion assay, Western blot and immunofluorescence.(4)In a whole-body visualizing animal model, the effects of miR-214 or co-transfection of miR-214 and target genes (without 3’UTR) were observed on the growth of subcutaneous tumors abilities and metastatic abilities of tail vein injection in vivo.4. Predicting and confirming the upstream transcription factors of miR-214, and investigating their functions in vivo and in vitro.(1) Finding the promoter sequence of miR-214, and then predicting the transcription factor of miR-214. Luciferase reporter system was used to detect the effect of transcription factor on the transcription activity of miR-214 promoter. ChIP assay was adopted to determine the binding site of transcription factor andmiR-214 promoter. Real-time PCR was employed to investigate the expression of miR-214 after overexpression of transcription factor.(2) Real-time PCR and Western blot were used to detect the expression of FOXD3 in six CRC cell lines. FOXD3 lentivirus-expressing vectorwas successfully designed and established. ShFOXD3 lentivirus-expressing vector or shFOXD3 and miR-214 lentivirus-co-expressing vector were transfected into CRC cell lines SW480 and SW620. The stable CRC cell lines were obtained by flow cytometry, and then detected the expression of FOXD3 and miR-214 by Western blot and real-time PCR.(3) Transfecting shFOXD3 lentivirus-expressing vector, orshFOXD3 and miR-214 co-expressing vector into SW480 and SW620 cells, and investigating their effects on cell proliferation, invasion, and radiotherapy by CCK-8, plate clone, matrigel invasion assay, Western blot and immunofluorescence.(4)In a whole-body visualizing animal model, the effects of shFOXD3 or co-transfection of shFOXD3 and miR-214 were observed on the growth of subcutaneous tumors abilities and metastatic abilities of tail vein injection in vivo.Results1. Screening and identification of miRNA associated with metastasis and radiotherapy resistance of CRC.(1) Three miRNA microarray results showed that 42 miRNAs were downregulated in primary CRC tissues anddistant metastatic CRC tissues, including miRNA-214, which was significantly downregulated in metastatic CRC tissues. Then, we also detected the expression of miRNA by means of microRNA microarray in CRC cell line SW837 pre- and post-radiotherapy at a dose of 8Gy. And the result showed that miR-214 expression of SW837 cell with radiotherapy was significantly decreased in comparison with that of SW837 cell without radiotherapy, indicating miR-214 was correlated with radiotherapy.(2) Detecting the expression of miR-214 in 30 matched fresh CRC tissues and corresponding normal mucosa by real-time PCR, and the value of 2-△△Ct was analyzed with paired t test and one-way ANOVA analysis. The expression of miR-214 in CRC tissues was significantly lower than that of normal mucosa(t=2.285, P<0.001).The expression of miR-214 in CRC tissues with metastasis was significantly lower than that of CRC without metasatasis(t=2.175, P<0.001). MiR-214 expression was also detected in six CRC cell lines by real-time PCR, which was analyzed with one-way ANOVA analysis. The statistical analysis showed that miR-214 expression difference was significant among five CRC cell lines (F=114.632, P<0.001). Pairwise comparison analysis indicated that miR-214 expression level was highest in HT29 cell, which was significantly higher than that of LOVO, SW620, SW480, and HCT116 (P=0.020, P=0.019, P=0.022, P=0.038). Real-time PCR was used to detect miR-214 expression of SW480 and HCT116 cells after 24 hours radiotherapy with a dosage of OGy,2Gy,4Gy,6Gy, and 8Gy, respectively. The result showed that with the increase of radiotherapy dosage, miR-214 was gradually decreased in SW480 and HCT116 cells, and differences were statistically significant(F=1029.532, P<0.001; F=618.172, P<0.001).2. Prediction and validation of miR-214 targets(1) Results showed that MED 19 (predicted by four common databases) was associated with proliferation and metastasis, and CDC25A (predicted by two common databases) was associated with radiotherapy. So we chose MED19 and CDC25A as miR-214 targets.(2) In HEK293A, SW480, and HCT116 cells, luciferase reporter system showed that the luciferase activity was decreased significantly respectively in miR-214/MED19 group compared to mock group or MED 19 group (F=15.426, P<0.005; F=59.820, P<0.001; F=73.500, P<0.001) after transfection of MED19 3’UTR plasmid. The above results indicate that miR-214 inhibits the activity of luciferase throuth the binding sites of MED19 3’UTR region. Luciferase activity of miR-214/CD25A group was significantly decreased compared to mock group or CDC25A group (F=35.618, P<0.010; F=313.848; P<0.001; F=100.236, P<0.001), indicating that miR-214 inhibits the activity of luciferase through the binding sites of CDC25A 3’UTR region.(3) Detecting the expression of MED19 and CDC25A in six CRC cell lines by Western blot, the results showed that MED19 expression level was highest in HCT116 cells, and was gradually decreased in SW480, SW620, LOVO, LS174t, and HT29 cells; CDC25A expression level was highest in HCT116 cell, and was gradually decreased in SW480, LS174t, SW620, LOVO and HT29 cells.(4) MED19 and CDC25A protein levels were quantitatively analyzed using Quantity One software, which were made correlations with real-time PCR result of miR-214 using Spearman analysis. The results showed that miR-214 was significantly negatively associated with MED19 in six CRC cell lines (r=-0.920,P<0.001). Similarly, miR-214 was also negatively associated with CDC25A in six CRC cell lines (r=-0.829, P=0.042).3. Effects of miR-214 overexpression and target genesre-expression on biological behaviors of CRC.(1) Detecting miR-214 expression in miR-214 overexpression group, mock group, miR-214/MED19 co-expression group, and miR-214/CDC25A co-expression group by real-time PCR. One-way ANOVA analysis was used to analyze differences of miR-214 expression between groups. Results showed that differences of miR-214 expression were significant among these four groups both in SW480 and HCT116 cells (F=47219.146,P<0.001; F=17177.032, P<0.001). Pairwise comparison in SW480 showed that miR-214 expression of miR-214 transfection group was significantly higher than that of mock group(P<0.001), while no significant differences were found between miR-214 transfection groupand miR-214/MED19 group or miR-214/CDC25A group, respectively (P=0.946; P=0.594). Pairwise comparison in HCT116 cell showed that miR-214 expression of miR-214 transfection group was significantly higher than that of mock group(P<0.001), while no significant differences were found between miR-214 transfection groupand miR-214/MED19 group or miR-214/CDC25A group, respectively (P=0.747; P=0.749). The above results showed that miR-214 was successfully transfected into CRC cells, and re-expression of MED19 and CDC25A made no effects on miR-214 expression.(2) Detecting expression levels of MED 19 and CDC25A proteins in mock group, miR-214 overexpression group, miR-214/MED19 co-expression group, and miR-214/CDC25A co-expression group in SW480 and HCT116 cells by Western blot. Results showed that expression levels MED 19 and CDC25A proteins were both reduced in miR-214 overexpression group compared with mock group, but their expressions increased after re-transfection of MED19 and CDC25A. The above results indicated that miR-214 transfection could decrease expressions of MED 19 and CDC25A proteins, while miR-214/MED19 co-expression and miR-214/CDC25A co-expression could effectively reverse expression levelsof MED19 and CDC25 A proteins.(3) Investigating cell proliferation of mock group, miR-214 overexpression group, miR-214/MED19 co-expression group, and miR-214/CDC25A co-expression group in SW480 and HCT116 cells using CCK-8 method, and drawing cell growth curves. Factorial design one-way ANOVA analysis of MED 19 showed that differences among three groups were significant at growth time level (F=2607.833, P<0.001; F=35.355, P<0.001). Cell proliferation differences among three groups were significant (F=271.281, P<0.001; F=4.376, P<0.001). Interaction effect between time and group was significant (F=52.77,P<0.001; F=62.274, P<0.001). Multiple comparisons results showed that cell proliferation of miR-214 group was significantly decreased compared with that of mock group, indicating that miR-214 overexpression could decrease cell proliferation in vitro. Cell proliferation of miR-214/MED19 group was enhanced compared with that of miR-214 overexpression, indicating that MED 19 could reverse the inhibiting effect of miR-214 on cell proliferation in vitro.Factorial design one-way ANOVA analysis of MED 19 showed that differences among three groups were significant at growth time level (F=2170.620, P<0.001; F=1501.804, P<0.001). Cell proliferation differences among groups were significant (F=227.617, P<0.001; F=173.087, P<0.037), interaction effect between time and group was significant (F=43.861, P<0.001; F=39.225, P<0.001). Multiple comparisons indicating that cell proliferation of miR-214 overexpression group was significantly decreased compared with mock group, indicating that miR-214 overexpression could decrease cell proliferation in vitro. Cell proliferation of miR-214/CDC25A group was enhanced compared with miR-214 overexpression group, indicating that CDC25A could reverse inhibiting effect of miR-214 on cell proliferation in vitro. The above results indicated that miR-214 could inhibit cell proliferation in vitro.(4) Investigating colony forming ability of mock group, miR-214 overexpression group, miR-214/MED19 co-expression group, and miR-214/CDC25A co-expression group in SW480 and HCT116 cells using plate clone formation assay. Statistical analysis showed that colony forming ability of miR-214 overexpression group was significantly decreased compared with that of mock group (P<0.001; P<0.001), while the colony forming ability of both miR-214/MED19 and miR-214/CDC25A group was increased compared with that of miR-214 overexpression group (P<0.001; P<0.001), the above results indicating that miR-214 could inhibit cell proliferation in vitro.(5)Investigating colony forming ability of SW480 and HCT116 treated with radiotherapy at different dosage. Results showed that with an increase of dosage, radiotherapy sensitivity of cells in miR-214 overexpression group was significantly decreased while colony forming ability was increased compared with that of mock group (P<0.001). Radiotherapy sensitivity of miR-214/CDC25A group was increased and colony forming ability was decreased compared with that of miR-214 group (P<0.001).(6) Investigating cell invading ability of mock group, miR-214 overexpression group, miR-214/MED19 co-expression group in SW480 and HCT116 cells using Boyden Chamber assay. Differences of cell invasion among three groups in SW480 and HCT116 were significant (F=110.710, P<0.001; F=95.564, P<0.001). LSD pairwise comparison showed that invading cell number of miR-214 was significantly decreased compared with that of mock group, while invading cell number of miR-214/MED19 was significantly increased compared with that of miR-214 overexpressing group (P<0.001), indicating that miR-214 could inhibit cell invasion of CRC in vitro.(7) Immunofluorescence results showed that γ-H2AX foci of miR-214 group were significantly decreased compared with those of mock group after 12 hours radiotherapy at a dosage of 4Gy (P<0.001), while γ-H2AX foci of miR-214/CDC25A co-expression group were significantly increased compared with those of miR-214 group (P<0.001). Western blot results showed that y-H2AX protein of miR-214 group was significantly decreased compared with that of mock group, while y-H2AXprotein of miR-214/CDC25A group was significantly increased compared with that of miR-214 group. The above results indicated that miR-214 could increase radiotherapy resistance of CRC, while its target gene CDC25A could partially reverse miR-214 induced radiotherapy resistance of CRC.(9) Cells of SW480/Mock,SW480/miR-214, and SW480/miR-214/MED19 co-expression group were injected subcutaneously to form tumor, or injected into tail vein to form lung metastasis in vivo. Subcutaneous tumorigenicity in nude mice was analyzed using repeated measures analysis of variance. Results showed that differences of subcutaneous tumorigenicity ability among SW480/Mock group,SW480/miR-214 group, and SW480/miR-214/MED19 group were significant (F=153.593,P<0.001; F=45.355, P<0.001; F=53.981, P<0.001). The growth speed of subcutaneous tumor of miR-214 group was significantly decreased compared with that of mock group (P<0.001), the growth speed of subcutaneous tumor of miR-214/MED19 group was significantly increased compared with that of miR-214 group (P<0.001). Immunohistochemistry staining of Ki-67 showed that positivity of mock group, miR-214 group, and miR-214/MED19 group was 87.84%,19.47%, and 59.58%, respectively. Tail vein injection results showed that SW480/Mock group,SW480/miR-214 group, and SW480/miR-214/MED19 group induced obvious metastasis on lung surface. The lung metastatic rate of SW480/mock,SW480/miR-214, andmiR-214/MED19 group in nude mice was 100% (6/6),33.33%(2/6), and 66.67%(4/6), respectively. The above results indicated that miR-214 could inhibit tumorigenicity and metastasis of CRC cell lines in vivo, while MED 19 could partially reverse the inhibiting effect of tumorigenicity and metastasis ability of miR-214 on CRC cell lines.4.Predicting and confirming the transcription factor of miR-214and then demonstrating the roles of transcription factor in invasion, metastasis, and radiotherapy resistance of CRC.(1) Finding the promoter sequence of miR-214 by using the online Ensembl database.(2) By using the online ConsiteandTFsearch, we considered FOXD3 as a putativetranscription factor.(3) Luciferase reporter system was used to detect the luciferase activity of miR-214 and FOXD3. Results showed that luciferase activity of HEK293A and SW480 transfected with PGL3-miR-214 plasmid was enhanced compared with that of blank group(P<0.001; P<0.001), indicating that miR-214 promoter sequence has transcription activity. The luciferase activity of HEK293A and SW480 co-transfected with PGL3-miR-214 and pEGFP-FOXD3 was significantly increased compared with that of miR-214-Luc(P<0.001; P<0.001), indicating that FOXD3 could significantly promote transcription activity of miR-214 promoter.(4) CHIP was adopted to detect the binding sites between miR-214 promoter and transcription factor FOXD3, and results showed that there was a direct binding site between miR-214promoter and FOXD3.(5) The expression of miR-214 was detected in SW480 and SW620 transfected with FOXD3 using real-time PCR, and results showed that FOXD3 was significantly increased in these two cell lines transfected with FOXD3 overexpression vector (t=-18.038, P<0.001; t=-13.877, P<0.001), indicating transfection was successful. Moreover, miR-214 expression were significantly increased in SW480 and SW620 cells transfected with FOXD3 (t=-31.409,P<0.001; t=-17.720, P<0.001), indicating FOXD3 could promote miR-214 expression.(6) FOXD3 expression was detected in six CRC cell lines by real-time PCR and Western blot, results showed that the expression of FOXD3 was highest in HT29 cell, and was gradually decreased in SW620, LS174t, SW480, LOVO, and HCT116 cells. To further investigate the positive regulation between FOXD3 and miR-214, SW620 and SW480 cell lines were chosen to knockdown FOXD3 expression in the following experiment.(7) Three FOXD3 siRNAs were detected to determine interference rate in SW480 and SW620 cell lines by Western blot, results showed that of siRNA3 has highestinterference rate in SW480 and SW620 cell lines, so we chose siRNA3 to establish FOXD3 interference lentivirus and construct stable expressing cell lines. Detecting FOXD3 protein in NC group, shFOXD3 group, and shFOXD3/miR-214 group in SW480 and SW620 cell lines by Western blot, and results showed that FOXD3 expression in shFOXD3 group was significantly decreased compared with that of NC group, while re-expression of miR-214 had barely effect on FOXD3 expression. The above results indicated that FOXD3 stable interference cell lines were successfully establish in SW480 and SW620.(8) Investigating cell proliferation of NC group, shFOXD3 group, shFOXD3/miR-214 co-expression group in SW480 and SW620 cells using CCK-8 method, and drawing cell growth curves. Factorial design one-way ANOVA analysis showed that differences of cell proliferation were different at growth time level (F=5395.231, P<0.001; F=16869.718, P<0.001). Differences of cell proliferation among different groups were significant (F=330.935,P<0.001; F=614.063, P<0.001). The interaction effect between time and group was significant (F=59.307,P<0.001; F=89.725, P<0.001). Multiple comparisons showed that cell proliferation of shFOXD3 group was significantly increased compared with NC group, indicating that FOXD3 could enhance cell proliferation in vitro. Meanwhile, cell proliferation of shFOXD3/miR-214 group was significantly decreased compared with that of shFOXD3 group, indicating that FOXD3 could inhibit cell proliferation in vitro, and miR-214 could reverse the inhibiting effect of FOXD3 on cell proliferation.(9) Detecting colony forming ability of NC group, shFOXD3 group, shFOXD3/miR-214 co-expression group in SW480 and SW620 cells using plate clone formation assay, and statistical analysis revealed that colony forming ability of shFOXD3 was significantly increased compared with that of NC group (P<0.001; P<0.001). Meanwhile, the colony forming ability of shFOXD3/miR-214 co-expression group was significantly decreased compared with that of shFOXD3 group (P<0.001; P<0.001). The above results showed that FOXD3 could inhibit cell proliferation in vitro.(10) Detecting cell invasion of NC group, shFOXD3 group, shFOXD3/miR-214 co-expression group in SW480 and SW620 cells using Boyden Chamber assay, and results showed that differences of cell migration among groups in SW480 and SW620 cells were significant (F=91.221, P<0.001; F=89.463, P<0.001). LSD pairwise comparison showed that invading cell number of shFOXD3 group was significantly increased compared with that of NC group (P<0.001). Meanwhile, invading cell number of shFOXD3/miR-214 co-expression group was significantly decreased compared with that of shFOXD3 (P<0.001). The above results indicated that FOXD3 could inhibit cell invasion of CRC in vitro.(11) Detecting colony forming ability of NC group, shFOXD3 group, and shFOXD3/miR-214 co-expression group in SW480 and SW620 cells using plate clone formation assay. Results showed that no significant differences were found among three groups (P>0.005).(12) Immunofluorescence results showed that differences of y-H2AX foci number among three groups were found after 12 hours radiotherapy at a dosage of 4Gy (P>0.005). Western blot results showed that no differences of y-H2AX protein among three groups were found. The above results indicated that FOXD3 had no effect on radiotherapy resistance of CRC.(13) Cells of SW620/NC,SW620/shFOXD3, and SW620/shFOXD3/miR-214 co-expression group were injected subcutaneously to form tumor, or injected into tail vein to form metastasis in vivo. Subcutaneous tumorigenicity in nude mice was analyzed using repeated measures analysis of variance. Results showed that differences of subcutaneous tumorigenicity ability among SW620/NC,SW620/shFOXD3, and SW620/shFOXD3/miR-214 co-expression group were significant (F=402.994, P<0.001; F=256.014, P<0.001; F=41.996, P<0.001). Tumor growth of shFOXD3 group was significantly increased compared with that of NC group (P<0.001), and tumor growth of shFOXD3/miR-214 co-expression group was significantly decreased compared with that of shFOXD3 group (P<0.001). Immunohistochemistry staining of Ki-67 showed that the positive rate of Ki-67 in NC,shFOXD3, and shFOXD3/miR-214 co-expression group was 37.333%、81.000% and 52.000%, respectively. Results of tail vein injection showed that significant metastasison lung surface, and lung metastasis rate of NC,shFOXD3, and shFOXD3/miR-214 co-expression group was 16.667%(1/6), 83.333%(5/6),50.000%(3/6), respectively. The above results indicated that FOXD3 could inhibit tumorigenicity and metastasis of CRC cells in vivo, while miR-214 could partially reverse the inhibiting effect of FOXD3 on tumorigenicity and metastasis of CRC.Conclusions:1. Downregulation of miR-214 is associated with metastasis and radiotherapy of CRC.2. FOXD3/miR-214/MED19 axis inhibits proliferation, invasion and metastasis of CRC.3. miR-214 enhances radiotherapy resistance of CRC by target gene CDC25A.Innovation points of this paper:1. We have clarified the key role of FOXD3/miR-214/MED19 axis in the invasion and metastasis of colorectal cancer, which provides new ideas and therapeutic targets for cancer diagnosis and therapy.2. We have provided evidence for the potential predictive value of miR-214 in metastastic colorectal cancer patients prior to radiation therapy. |
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