| Metastasis, one of the six initial cancer hallmarks, is still the major cause ofCRC mortality. The formation of cancer metastases is a multi-step biologicalprocess, wherein the cancer cells spread from primary site to secondary tissues.To successfully and effectively prevent tumor metastasis, a better understandingof the key molecular components involved in this process is necessary. So far,however, little is known about the exact mechanisms underlying in themetastatic process of cancer.Epithelial-mesenchymal transition (EMT) is one of the critical steps duringtumor metastasis and plays a central role in tumor progression. And notably,EMT is recently considered as an essential early step in tumor metastasis,characterized by loss of the epithelial marker, increase in the mesenchymalmarkers, and an increase in the migratory and invasive potential. EMT endowscancer cells with invasive potential and stem cell properties which are relatedwith the capacity of cancer cells to initiate metastasis. Most recently, EMT was reported as the dominant program in human cancer. Therefore, the identificationof key molecules associated with EMT is helpful to clarify the potentialmechanism of cancer metastasis. Understanding the roles of key factors in EMTis crucial to the development and optimal use of novel anticancer therapies.MiRNAs are regulatory, non-coding single-stranded small RNAs that exerta posttranscriptional regulation on protein expression by base-pairing with the3’untranslated region (UTR) of the target mRNA, which results in either mRNAtranslation repression or RNA degradation. Several studies have showed thatmiRNAs play an important regulation role in the metastasis of a variety ofhuman cancers. Recently, some studies highlighted the importance of miRNA inthe regulation of EMT. However, the information about the role of miRNA inCRC EMT is limited and still need to be explored.【Aims】To explore the role and mechanism of miRNA in EMT-related metastasis incolorectal cancer, open a new avenue for the therapy of CRC.【Methods】1、Using immunofluorescence, real-time RT-PCR and Western Blotting forthe identification of EMT phenotypes of a variety of colon cancer cell lines,screening for EMT cell model of CRC;2、Using miRNA microarray for screening CRC EMT-related miRNAs, andusing real-time RT-PCR to test the chip results;3、Collecting CRC tissue specimens, and using real-time RT-PCR to detectcandidate miRNA expression, and examined the association between itsexpression level and clinical parameters;4、Transwell assays for the detection of invasion and migration ability of candidate miRNA in CRC cell, Immunofluorescence, real-time RT-PCR andWestern Blotting for detecting influence of candidate miRNA on cytoskeletonand EMT marker molecule expression;5、Using bioinformatics to predict potential target genes of candidatemiRNA, and using western blotting, qRT-PCR and dual luciferase report geneexperiments for further validation;6、Using immunohistochemical to detect the expression of target genes incolorectal cancer specimens, and to explore correlation between its level andclinical parameters.7、Using transwell, immunofluorescence, western blotting and real-timeRT-PCR technical to validate the effects of target gene on EMT phenotypic inCRC cells.【Results】1. Highly invasive SW620cells displayed fibroblastic morphologywhen comparing to SW480cells showing epithelial-like appearance.Immunocytochemical staining showed apparent differences in cellmorphology and cytoskeletal element between SW480and SW620cell lines.SW480cells displayed well spread with flattened morphology and the stressfiber was well organized, while SW620cells presented shrinked morphologyand the stress fiber was disorganized. In accordance with these findings,immunofluorescence assay showed that E-cadherin, the epithelial marker,localized clearly to the cell-cell junction of almost all SW480cells, while thelevel of E-cadherin expression markedly decreased in SW620cells. Especially,few SW620cells were found the E-cadherin fluorescence signal exhibiting onthe cell-cell junction. The result of western blotting also showed a decreasedexpression of E-cadherin in SW620. At the same time, a mesenchymal marker, vimentin, was also detected by western blotting. In contrast, expression ofVimentin was significantly increased in SW620cells.2. miR-185screened by miRNA microarry was down-regulated inCRC and correlated with the progression of CRC.MiR-185was identified by miRNA microarray to be the most significantlydownregulated microRNA in SW620compared to SW480cell lines. Weexamined the endogenous miR-185expression level by qRT-PCR in SW480andSW620cells. MiR-185was significantly lower in high STIM1expressingSW620cells, showing that miR-185level inversely correlated with STIM1expression in CRC cells. To investigate the clinical relevance of miR-185,expression of miR-185was analyzed in90CRC samples by qRT-PCR. Theassociation of miR-185with clinicopathologic factorswas examined. In contrastto STIM1, lower level of miR-185was significantly associated with advancedclinical TNM stage and lymph node metastasis. MiR-185expression in CRCpatients did not correlate with age, gender, tumor size or cell differentiation.3. miR-185inhibited invasion and migration, and reversed EMTphenotype of CRC cells.To investigate the role of miR-185in migration and invasion potential of CRCcells, SW620cell line was transiently transfected with miR-185mimics.Successful up-regulation of miR-185expression was confirmed by qRT-PCR.Increasing expression of miR-185resulted in a significantly inhibition ofmigration and invasion in SW620cells. Correspondingly, to determine whetherthe loss of miR-185would promote the migration or invasion of CRC cells, wesilenced miR-185with an antisense oligonucleotide inhibitor in SW480cell line.Inhibition of miR-185significantly promoted cell migration and invasion inSW480cells. The effect of ectopic expression of miR-185on cytoskeletal element and EMT markers was investigated in SW620cells.Immunocytochemical staining showed that ectopic expression of miR-185inSW620cells induced the rearrangement of F-actin distribution, which resultedin the change of stress-fibre from a mesenchymal to an epithelial pattern.Furthermore, western blotting showed that transfection of miR-185induced aloss of the mesenchymal phenotype by restoring epithelial marker E-cadherinand reducing mesenchymal marker Vimentin expression.4．STIM1was a direct function target gene of miR-185.We investigated the binding site of miR-185in the3’-UTR of STIM1andperformed luciferase reporter assay. STIM13’UTR reporter (Luc-STIM1),STIM13’UTR mutant reporter (Luc-STIM1-mu) or control reporter (Luc-Ctrl)were co-transfected with miR-185or control miRNA into SW620cells. Asexpected, transfection with miR-185dramatically attenuated STIM13’-UTRluciferase activity. Interestingly, the STIM13’-UTR luciferase activity inmiR-185-transfected SW620cells was significantly lower when comparing withcontrol miRNA-transfected cells, whereas mutation of miR-185recognition siterescued the luciferase activity. To confirm whether the targeting of STIM13’UTR by miR-185was functional, SW620cells transfected with miR-185mimic and SW480cells transfected with miR-185inhibitor were subjected toqRT-PCR and Western blotting analysis. It was found that both mRNA andprotein levels of STIM1were effectively down-regulated in miR-185mimictransfected SW620cells, while miR-185inhibitor transfected SW480cellsshowed an apparent upregulation of STIM1mRNA and protein expression. Tofurther corroborate this hypothesis, we performed a ‘rescue’ experiment byco-transfecting the SW620cells with miR-185mimic and a pcDNA3.1vectorcarrying STIM1(pcDNA3.1-STIM1) expression cassette with mutated seed sequences for miR-185(STIM1-mu) at its3’-UTR. Western blottingdemonstrated that co-transfection of SW620cells with miR-185mimic andSTIM1-mu retrieved STIM1protein expression, while co-transfection withpcDNA3.1-STIM1could not recover STIM1expression silenced by miR-185mimic (Figure5a). Furthermore, recovering STIM1expression by introducingSTIM1-mu in SW620cells transfected with miR-185mimic inhibited theprotein level of E-cadherin and increased that of vimenti. In addition, retrievingSTIM1expression in the presence of miR-185mimic enhanced the invasion andmigration potential of SW620cells.5．STIM1was overexpressed in CRC tissues and positively correlatedwith the progression of CRC.Immunohistochemistry was used to investigate the expression of STIM1in90primary CRC samples and corresponding non-tumor tissues. STIM1waspredominantly located in the cytoplasm and membrane of CRC cells and it had ahigher expression in CRC tissues than adjacent nontumorous tissues. Moreover,we compared the expression of STIM1in primary sites from patients enduringnonmetastatic CRC with those from patients enduring metastatic CRC. It wasfound that expression of STIM1in metastatic primary sites was apparentlyhigher than in nonmetastatic primary sites. To investigate if the STIM1expression might be associated with the progression of CRC, the expressionlevels of STIM1and the clinicopathologic characteristics of CRC patients werecompared. It is apparent that STIM1expression did not correlate with Age,Gender, Tumor size or degree of differentiation with a statistic P＞0.05in eachparameter, while it was significantly associated with TNM stage and Lymphnode status with a statistic P＜0.01in each parameter. These results providedevidence that expression level of STIM1positively correlated with the progression of CRC.6．STIM1promoted adhesive, migration and invasive abilities of CRCcells in vitro. Silencing of STIM1expression reversed EMT phenotype ofCRC cells.Western blot and qRT-PCR analysis showed that both the mRNA andprotein levels in SW620cells were markedly higher compared to that in SW480cells. To down-regulate the expression of STIM1, a STIM1-specific siRNA or acontrol siRNA was transfected into SW620cells. Western blotting and qRT-PCRshowed that the the siRNA could down-regulate the expression of STIM1inSW620effectively. To evaluate the effect of STIM1on cell adhesion ability,adhesive assay was performed. The result showed that all the cells bound tomatrigel with a time-dependent manner. Down-regulation of STIM1in SW620decreased their adhesive ability to matrigel. To evaluate the effect of STIM1oncell migration and invasion potential, in vitro migration and invasion assay wasperformed and showed that STIM1siRNA transfection produced a markedinhibition of migration and invasion of SW620cells. These results suggestedthat STIM1had a potential to promote metastasis of CRC.Immunocytochemical staining showed apparent differences in cellmorphology and cytoskeletal element between SW480and SW620cell lines.SW480cells displayed well spread with flattened morphology and the stressfiber was well organized, while SW620cells presented shrinked morphologyand the stress fiber was disorganized. In accordance with these findings,immunofluorescence assay showed that E-cadherin, the epithelial marker,localized clearly to the cell-cell junction of almost all SW480cells, while thelevel of E-cadherin expression markedly decreased in SW620cells. Especially,few SW620cells were found the E-cadherin fluorescence signal exhibiting on the cell-cell junction. The result of western blotting also showed a decreasedexpression of E-cadherin in SW620. At the same time, a mesenchymal marker,vimentin, was also detected by western blotting. In contrast, expression ofVimentin was significantly increased in SW620cells. To further validatewhether down-regulation of STIM1expression affects EMT of CRC cells,STIM1siRNA or the control siRNA was transfected into SW620cells. Then,cytoskeletal element and expression of E-cadherin in SW620cells wereexamined by immunocytochemical staining. It was showed that stress fiber cellsbecame well organized and expression of E-cadherin was up-regulated markedlyin SW620cells transfected with STIM1siRNA. At the same time, westernblotting also showed an increased expression of E-cadherin and a decreasedexpression of Vimentin in these cells.【Conclusions】In the present study, we showed that STIM1was overexpressed in cancertissues and positively correlated with progression of cancer. Then we found thatsilencing of STIM1promoted adhesion, migration and invasive abilities andreversed EMT phenotype of cancer cells. We further demonstrated that STIM1is a direct functional target of miR-185, a miRNA was found for the first time toinhibit EMT in cancer cells. The same as STIM1silence, the ectopic expressionof miR-185inhibited cell migration and invasion and reversed EMT phenotype.Furthermore, the restoration of STIM1by using an expression vector clonedwithout the3’UTR eliminated the effects of miR-185-induced phenotypes.Moreover, an inverse correlation between STIM1and miR-185was alsoidentified in cancer tissues. Thus, we demonstrated a critical function of STIM1in promoting cancer metastasis via regulating EMT and we revealed a new regulatory mechanism of STIM1expression in cancer by miR-185. These resultsmake the miR-185-STIM1pathway-mediated signaling an attractive target fortherapeutic intervention in cancer metastasis. |
PDF Full Text Request