| Background and purposeColorectal cancer (CRC) is the most common malignant tumors, and operation is the main treatment. But the result is poor,specially for the latter period. Metastasis is the main cause of the death of CRC patients. Many patients have occurred micro-metastasis before the operation, and metastasis is the direct cause of recurrence. Now there is lake of an efficient and specific way to detect the micro-metastasis, so it is necessary to find the efficient and specific metastasis-related genes and to further investigate its function.miRNAs are single-stranded RNAs of19-25nucleotides that negatively regulate genes by triggering either mRNAs. Recently, we have found miRNAs as suppressing genes play an important role in the process of tumor development. Now the research of miRNA in the tumor has became the hot spot. Lots of papers reported there many abnormal expression of miRNAs in tumor, for example, the breast cancers and colorectal cancers. miRNA is a break-through discovery in the21st Century. It regulates the post-transcriptional expression of targeted gene. miRNA targeting its target gene plays the role of oncogene or anti-oncogene and is related with the transformation development and EMT. Each miRNA has many targets mRNA, and each mRNA can be regulated by many miRNAs. Thus mRNA and miRNA form a complex and interconnected regulatory network.In this study,we screened differently expressed miRNA between CRC tissues with metastasis and tissues without metastasis. We chose miR-223as our research target. The main content of this research is as the following:1.Screening and confirming the miRNAs related with metastasis.2. Predicting and confirming the targets of miR-223, and identifying the role targets of miR-223in the invasion and metastasis of colorectal cancer.3. Predicting and confirming the regulators of miR-223, approaching the relationship between them.The aim this research is to reveal an new regulating mechanism of miR-223, approaching the role of miR-223in the development of CRC, and provide an new miRNA and target gene for the clinical application.Methods1. Screening and identifying the differential expression miRNA(1)Microarray analysis was performed to obtain the miRNA expression Profile in CRC tissues. miR-223expression in CRC tissues was examined by quantitative QPCR analysis.2. Prediction and Identification of miR-223target.(1) Five commonly databases including MiRanda, TargetScan, DIANA-microT, Pictar, and RNAhybrid. miRNAs were used to forecast the targets with the search terms of miR-223.(2)FBXO8with high predictive values was selected as candidate targets. Identifying the FBXO8as target of miR-223by the Luciferase reporter system, QPCR and western blot.3. The function of miR-223in CRC invasion and metastasis(1) The miR-223inhibitor was transfected transiently into CRC cell lines SW480and SW620. Western blot was performed and detect the expression of FBX08.(2) The miR-223lentiviral vector was designed and established. After viral packaging, it was infected into CRC cell lines SW480and HT29. The stable miRNA-expressing CRC cell lines were obtained by FCM(flow cytometry) method, and then detected the expression of FBXO8by Western blot.(3) The targeted miR-223lentiviral vector and the FBX08lentiviral vector without3’UTR region were co-transfected into CRC cell lines HT29and SW480. The stable miRNA and FBXO8-expressing CRC cell lines were obtained by FCM method, and then detected the expression of FBX08by Western blot.(4) To detect cell proliferation, invasion abilities in vitro after transfection of targeted miR-223inhibitor, we carried out TT method and in vitro invasion assay.(5) MTT method, colony formation assay and in vitro invasion assay were carried out to detect cell proliferation, invasion abilities in vitro after transfection of miR-223and co-transfection of miR-223/FBXO8(without3’UTR).(6) In a whole-body visualizing animal model, the effects of miR-223or co-transfection of targeted miRNAs and FBXO8were observed on the growth of subcutaneous tumors abilities in vivo.(7) Detecting the expression of BX08in the203CRC tissues with paired nomal tissues and cancer tissues by immunohistochemistry(IHC).4. Predicting and confirming the transcription factor of miR-223.(1)Finding the promoter sequence of miR-223, and predicting the transcription factor of miR-223.(2) Confirming the relationship between them by Chromatin Immunoprecipitation (Chip) and Luciferase activity assay.Results1. Screening and identification of miRNA with metastasis in CRC tissues(1)Result of microarray Within the four arrays, there are42miRNAs with high expression in the cancer without metastasis and cancer with metastasis, including miR-223.(2) Expression of miR-223in the CRC tissues In62cases of paired fresh CRC tissues, result indicated the expression of miR-223in CRC tissue was significantly higher than in normal tissues (t=4.498, P<0.001).By the further study, there are statistically differences between the cancer tissues without metastasis and tissues with metastasis (F=2.730, P=0.009)2. Prediction and validation of miR-223targets(1) Results showed that FBXO8(predicted by five common databases) and RhoB (predicted by four common databases) had the high predictive values. So we choose the FBXO8and RhoB as miR-223targets.(2) In HEK293A and SW480cell lines, Luciferase reporter system showed that the luciferase activity was decreased significantly respectively in miR-223group compared to Blank group or NC group (F=85.465, P<0.001, F=41.04, P<0.001; F=32.283, P<0.001,F=208.981, P<0.001) after transfection of wild-type FBXO8and RhoB3’UTR plasmid. The above results indicate that miR-223inhibits the activity of luciferase through the binding sites of FBXO83’UTR and RhoB3’UTR region.(3)The result of expression of miR-223in the CRC cell lines showed that there is significant difference among them(F=84.937, P<0.001). The expression of miR-223in SW480cell line was higher than in SW620. Lovo、LS174t and HT29cell lines(P=0.001, P=0.004, P=0.011, P=0.006).(4)The expression of FBXO8and RhoB protein was on the contrary. The expressions of FBXO8and miR-223had significant negative correlation (r=-1.000, P<0.001) in4(SW620、SW480、HT29and LS174T)CRC cell lines by Spearman correlation analysis. The expressions of RhoB and miR-223had significant negative correlation SW620、SW480、HT29and LS174T (r=-1.000, P<0.001).3. Effect of miR-223inhibitor on Biological behavior of the CRC cell lines(1)Western Blot results showed that the expression of FBXO8was significantly up-regulated after transfecting miR-223inhibitor into SW480and SW620cells compared to NC group and blank group.(2)MTT assay showed that compared to blank or NC group, the proliferation abilities of sw620and SW480cells were not changed among these groups (F=1.719P=0.194; F=1.863, P=0.17)(3) In vitro invasion assay results showed that invasive abilities of SW620and SW480cells were markedly decreased (F=37.945, P<.001; F=30.545, P<0.001) after transfection of miR-223inhibitor.(4) The result of FCM detecting the apoptotic cells of SW480and SW620transfected with miR-223inhibitor indicate that it is no statistical differences between the miR-223inhibitor gourp and NC group (t=0.359, P=0.737; t=1.083, P=0.340)4.The effects of miR-223and miR-223/FBXO8(without3’UTR) on CRC cell behaviors in vitro and in vivo(1) Real-time PCR results showed that after transfection of miR-223expression vector into HT29and SW480respectively, miR-223expression was significantly higher than blank group or NC group (F=13.719, P=0.006; F=27.865, P=0.001). Western Blot results showed that the expression of FBXO8was significantly down-regulated in HT29and SW480cells after transfection of miR-223expression vector.(2) After co-transfection of miR-223expression vector and FBX08lentivirus vector into HT29and SW480cells, QPCR results showed that there were no significant differences of miR-223expression between miR-223group and miR-223/FBXO8group (P=0.902). Western Blot results showed that FBXO8expression was up-regulated again in miR-223/FBXO8group compared to miR-223group.(3) MTT assay showed that the proliferative abilities of HT29and SW480cells were not changed in miR-223group compared to blank or nc group (P=0.266, P=0.873, P=0.204; P=0.242, P=0.133, P=0.202). The proliferation abilities of HT29and SW480cells were significantly reduced in miR-223/FBXO8group than in miR-223group (P<0.001; P<0.001).(4) Colony formation assay showed that the colony formation ability was no significant differences among the four groups in the HT29and SW480cell lines (F=0.568, P>0.05; F=0.613, P>0.05).(5) In vitro invasion assay results showed that HT29and SW480cells have significantly increased invasive abilities in miR-223group (F=51.054, P<0.001; F=104.515, P<0.001)(6)The result of FCM detecting the apoptotic cells of HT29and SW480transfected with miR-223indicate that it is no statistical differences between the miR-223inhibitor group and NC group (P=0.948, P=0.288)(7) The result of western blot detecting the EMT relating proteins show that there is no relationship between them.(8) The growth of subcutaneous tumors in nude mice in miR-223group were markedly increased compared to blank, NC group and miR-223/FBXO8(P<0.001, P<0.001, P<0.001).(9) Expression of BX08in CRC tissues by IHC FBXO8expression was lower in203CRC tissues than in adjacent normal tissues (P<0.001, z=-9.236). Its expression was positively correlated with patients ages and tumor differentiation (P=0.002; P=0.001). Patients with lower FBXO8expression had shorter overall survival time (P=0.032).5. Predicting and confirming the transcription factor of miR-223(1)Finding the promotor sequence of miR-223.And then by using the online TRED and Consite, we considered transcription factor MEF2A as a putative upstream regulator of miR-223.(2) In HEK293A and SW480cell lines, Luciferase reporter system showed that the luciferase activity was increased significantly respectively in miR-223-MEF2A group compared to the other group (P<0.001; P<0.001).(3) The result of Chip assay indicate there are2binding sites between the miR-223promoter and MEF2A.(4)By real time PCR, expression of miR-223were increased markedly in480cell line and SW620cell lines after transected with ME2A (t=10.737, P=0.009; t=52.218, P<0.001).While expression of FBXO8protein were decreased.Conclusion1. We have gained115miRNA related with metastasis, and miR-223is high expressed in the CRC tissues,especially in the tissues with metastasis;2. miR-223can promote the ability of invasion and metastasis of CRC cells by targeting FBXO8; 3、MEF2A promotes the expression of miR-223,and inhibits the expression of FBXO8;4. MEF2A/miR-223/FBXO8model may be an new regulating model, and miR-223promote the invasion and metastasis by this model and ATK/mTOR signal pathway.Innovation points of this paper:1. miR-223is an miRNA related with metastasis, and it is screened by microarray. By now, there is no report about the relation between the miR-223and the metastasis of CRC.2. We have raised an new regulation mechanism of miR-223, and this research is with character of primary innovation.3. We have present the mechanism of the role of miR-223/BXO8in the CRC metastasis and invasion, and provide an new gene and miRNA for the clinical application of the tumor metastasis. |
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