| MicroRNAs (miRNAs) are short (21-24-nt) non-coding RNAs generated from longed transcripts, termed pri-miRNAs and pre-miRNAs. They act as post-transcriptional regulators of gene expression and induce translational repression. Recently, miRNAs have been discovered to play important roles in tumourigenesis. By targeting 3'untranslated regions (3'UTRs) of cognate mRNAs, miRNAs are involved in diverse processes, including cell differentiation, proliferation, apoptosis and metastasis. Understanding the specific functions of individual miRNAs may contribute to our knowledge of the molecular properties of cancers and may provide new biomarkers for cancer diagnosis and therapy.The Rho family of small GTPases, of which the best studied members are Rho, Racl and CDC42, are involved in regulation of a variety of cellular processes includ-ing reorganization of the actin cytoskeleton, cell motility and invasive phenotypes. Rho-associated serine-threonine protein kinase (Rock), one of the best characterized downstream effectors of Rho, is activated when it selectively binds to the active GTP-bound form of Rho. As with Rho, Rock has been implicated in altering cell migration and invasion during tumor cell metastasis.Tumor invasion and metastasis are the critical steps in determining the death of cancer patients. Recently, miRNA have been discovered to have a role in invasion and metastasis. Emerging studies have indicated that miRNAs participate in the signaling of the Rho pathway. Among the tested miRNAs, miR-155 may play an important role in TGF-β-induced epithelial-mesenchymal transition (EMT) and cell migration and invasion by targeting RhoA. During tumor progression, TWIST 1 induces miR-10b, which in turn indirectly increases RhoC levels by direct down-regulation of its transcriptional repressor HOXD10. Studies on the association of Rho with miRNAs could highlight the importance of miRNAs in tumorigenesis and lead to the development of novel therapeutic strategies for patients with metastases.MiR-125a is one of the many miRNAs that remain to be fully characterized. Using miRNA microarray analysis, Yanainhara and colleagues found that miR-125a, specifically the hsa-miR-125a-5p mature miRNA, is located at 19q13.41 and that its expression is downregulated in NSCLC. Recently, a new member of the mature miR-125a family has been identified and named hsa-miR-125a-3p. Unfortunately, the expression and function of hsa-miR-125a-3p are currently unknown. In the present study, we investigated the relationships between hsa-miR-125a-3p/5p expression and lymph node metastasis in NSCLC tissues, and explored the impact of expression of these miRNAs on invasive and migratory capabilities of lung cancer cells. In add-ition, the effects of hsa-miR-125a-3p/5p on the invasive and migratory capabilities of lung cancer cells were studied by investigating its effects on Rho/Rock pathway. These results provide basis that hsa-miR-125a-3p and hsa-miR-125a-5p maybe very important regulators in Rho/Rock pathway.Materials and MethodsSamples preparation:We analyzed 52 pairs of non-small cell lung cancer specimens and corresponding normal lung tissues (LAC) collected at the time of surgery and prior to chemotherapy. Specimens were obtained from patients at the First Affiliated Hospital of China Medical University from 1 January 2006 to 1 December 2007 with informed consent. For the majority of samples, clinical and biological information was available. The study has been approved by the Hospitals'Ethical Review Committee.Cell culture: The HBE (Human Bronchiolar Epithelium) cell line and the human lung cancer cell lines A549 and SPC-A-1 (adenocarcinoma), LH7 and NCI-H460 (large cell cancinoma) were propagated in RPMI1640 (Gibco). The A549 (adenoca-rcinoma) cell line was propagated in Dulbecco's Modifed Eagle Medium (Gibco). In both cases, the medium was supplemented with 10% fetal bovine serum (FBS),100 U/ml penicillin, and 100 U/ml streptomycin. Cells were cultured at 37℃in 5% CO2 until they reached a confluency of 75%.Transfection:Depletion of hsa-miR-125a-3p/5p in the A549 and SPC-A-1 cell lines was achieved through transfection with antisense 2'-O-methyl oligonucleotides directed against hsa-miR-125a-3p/5p. Cells were transfected using LipofetamineTM 2000 (Invitrogen) according to the manufacturer's protocol. Briefly, complexes con-taining the oligonucleotides were prepared according to the recommended protocol and added directly to cells at a final oligonucleo tide concentration of 0.4 nmol/ml. Oligonucleotides composed entirely of 2'-O-methyl bases were chemically synthe-sized by Integrated DNA Technologies (GeneChem) and were comprised of the following sequences:2'-O-Me-sense-3p:5'-ACA GGU GAG GUU CUU GGG AGCC-3'2'-O-Me-antisense-3p:5'-GGC UCC CAA GAA CC U CAC CUGU-3', 2'-O-Me-scramble-3p:5'-GGU CGG UGC UCG AUG CAG GUAA-3',2'-O-Me-sense-5p:5'-UCC CUG AGA CCC UUU AAC CUG UGA-3',2'-O-Me-antisense-5p:5'-UCA CAG GUU AAA GGG UCU CAG GGA-3',2'-O-Me-scramble-5p: 5'-GGA CG G CGA UCA GAU AAG AGU UCU-3'. Cells were divided into four groups:an untreated group incubated only in the normal media (untreated), a group transfected with the scrambled 2'-O-methyl oligonucleotide (scramble); a group transfected with the sense 2'-O-methyl oligonucleotide (sense) and a group transfected with the antisense 2'-O-methyl oligonucleotide (antisense). The untreated and scramble groups served as negative controls.Quantitative real-time polymerase chain reaction (qRT-PCR):Expression of mature miRNAs was assayed using the TaqMan MicroRNA Assay in accordance with the manufacturer's instructions (Applied Biosystems). All reactions, including no-template controls and RT-minus controls, were run in an ABI Prism 7900HT Sequence detection system (Applied Biosystems). Specific RT primers and TaqMan probes were used to quantify the expression of hsa-miR-125a-3p (PN:4395310) and hsa-miR-125a-5p (PN:4395309). Samples were normalized to RNU6B (PN:4373381) or U18 (PN:4380904) as indicated. For quantification of tissue samples, RT-PCR analysis was performed in two independent experiments, each using two independent samples. For quantification of cell samples, RT-PCR analysis was performed in three independent experiments, each using three independent samples. MiRNA expression data is presented as fold difference relative to either RNU6B or U18 based on the following equation:RQ=2-△△Ct.Semi-quantitative RT-PCR: Total RNA was prepared using Trizol (Invitrogen, USA). cDNA was generated by reverse transcription of 500ng total RNA using PrimeScript RT reagent Kit (TaKaRa, Japan). Primer sequences of RhoA were 5'-TAT GTG CCC ACA GTG TTT GA-3'(forward) and 5'-CAT TCC GAA GAT CCT CCT TCT TATT-3'(reverse); Rockl primer sequences were 5'-GCA ACT ATG ATG TGC CTG AA-3'(forward) and 5'-CAC CAT TTC GCC CTA ACC-3'(reverse), and that ofβ-actin were 5'-AAA TCG TGC GTG ACA TTA A-3'(forward) and 5'-CTC GTC ATA CTC CTG CTTG-3'(reverse). The lengths of RhoA, Rockl andβ-actin PCR products were 270 bp,489 bp and 513 bp respectively. PCR was carried out by an initial denaturation step at 94℃for 5min, followed by 35 cycles of denaturation at 94℃for 40s, annealing at 56℃(RhoA) or 57.2℃(Rock1) for 40s, and elongation at 72℃for 40s. Cycling was completed by a final elongation step at 72℃for 5min. The PCR forβ-actin was performed in the same manner as above, except that 30 cycles of annealing at 50℃was used. The EC3 Imaging system (UVP Inc.) was used to catch up the specific bands, and the optical density of each band was measured using the Image J software.Western blot: Cells were washed twice with ice-cold phosphate buffered saline (PBS) and lysed in M-PER reagent (Pierce Biotechnology) containing 1 mM PMSF and phosphatase inhibitors for 1h at 4℃. The supernatants were centrifuged at 12 OOO×g for 30 min at 4℃. The supernatant containing total protein was harvested. Aliquots containing 80μg of proteins were separated on 12% (RhoA) or 8% (Rockl) or 10%(β-actin) SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk, respectively incubated with mouse anti-RhoA, mouse anti-Rockl and rabbit anti-β-actin (1:200, Santa Cruz Biotechnology, USA) at 4℃overnight, and followed by each corresponding second antibody (1:4000, Chemicon, USA) at room temperature for 2 h. Immunoreactive straps were identified using Super ECL reagent (Pierce Biotechnology) according to the manufacturer's protocol. Specific bands for RhoA, Rockl andβ-actin were identified by pre-stained protein molecular weight marker (SM0441, MBI Fermentas). The EC3 Imaging System (UVP Inc.) was used to catch up the specific bands, and the optical density of each band was measured using the Image J software.Cell migration and invasion assays:For the migration assay,5×104 cells were trypsinised, washed, resuspended in serum-free RPMI1640 or DMEM, and placed in the top portion of the chamber. The lower portion of the chamber contained 10% FBS as a chemo-attractant. The chambers were incubated at 37℃,5% CO2 for 12 h, and then cells on the membrane were washed with PBS, and fixed in 100% methanol, stained with Haematoxylin, photographed, and counted. For the invasion assay, pre-cooled serum-free RPMI1640 or DMEM was mixed with Matrigel (1:7 dilution, BD Biosciences, USA). The upper compartments were filled with 100μl of the mixture, and the Matrigel was allowed to solidify at room temperature for 4h. Other procedures followed the migration protocol, except that the chambers were incubated for 24h. Five random fields for each chamber were counted. Chambers were conducted in duplicate in three separate experiments.Statistical analysis:The statistical package SPSS 13.0 was used for all analyses. For real-time PCR data, the statistical analysis of hsa-miR-125a-3p expression level between NSCLC tissues and corresponding LAC tissues was log2 transformed. Paired-Samples T Test was used to analyze the significant differences of hsa-miR-125a-3p expression between NSCLCs and LACs. All values were expressed as mean±SD. The Chi-square test and Two-sided fisher's exact test were used to determine the relationship between hsa-miR-125a-3p expression and clinicopathological factors. Mann-whitney test was used for pathological grade and pathological stage ranked data analysis, and Spearman correlation analysis was determined the correlation between hsa-miR-125a-3p expression and clinical stage and lymph node metastasis status. Other results were analyzed using Independent-Samples T Test. Results were considered statistically significant at the values of p<0.05.Results1,Hsa-miR-125a-3p and hsa-miR-125a-5p are downregulated in non-small cell lung cancer and have inverse effects on invasion and migration of lung cancer cells.(1) Downregulation of hsa-miR-125a-3p/-5p expression in NSCLC tissues:The results, the relative expression of hsa-miR-125a-3p and hsa-miR-125a-5p in 52 NSCLCs and paired LACs by real-time PCR, showed that the expressions of hsa-miR-125a-3p and hsa-miR-125a-5p decreased in NSCLCs in comparison to matched LACs (p<0.05). The mean expression level of hsa-miR-125a-3p in NSCLCs was decreased about 4.5 folds (p<0.001) and hsa-miR-125a-5p was reduced about 6 folds (p<0.001) in NSCLCs of that in LACs.(2) Correlation between hsa-miR-125a-3p/5p expression and clinicopathological variables of NSCLC:To determine the effects of hsa-miR-125a-3p/5p expression on tumor initiation and progression, lung cancer patients were divided into two groups based on the mean level of hsa-miR-125a-3p and hsa-miR-125a-5p expression in 52 NSCLCs. The two groups were defined as follows:hsa-miR-125a-3p low expression and high expression (1.2352 of the log2 value) and hsa-miR-125a-5p low expression and high expression (1.8594 of the log2 value). The results indicated that statistically significant associations between hsa-miR-125a-3p and hsa-miR-125a-5p expression and pathological stage were observed (p=0.012 and p=0.002, respectively). Changes in expression of hsa-miR-125a-3p and hsa-miR-125a-5p were also statistically significantly associated with lymph node metastasis in lung cancer (p=0.034 and p=0.042, respectively).No correlation was observed between hsa-miR-125a-3p/5p expression and gender, histology type, or pathological grade. However, the hsa-miR-125a-3p expression level was correlated with age (p=0.031), in contrast to that of hsa-miR-125a-5p. Spearman correlation test results showed a negative correlation between hsa-miR-125a-3p expression and pathological stage (r=-0.352, p=0.011), as well as lymph node metastasis (r=-0.326, p=0.018). However the correlations between hsa-miR-125a-5p expression and pathological stage (r=0.439, p=0.001) and lymph node metastasis (r=0.300, p=0.031) were positive.2,Hsa-miR-125a-3p and hsa-miR-125a-5p regulate migration and invasion in lung cancer cell lines by Rho/Rockl pathway.(1) Downregulation of hsa-miR-125a-3p/5p expression in NSCLC cell lines:In order to identify suitable cell lines for further studies, we examined the relative expression levels of hsa-miR-125a-3p/5p in four lung cancer cell lines (LH7, A549, SPC-A-1, and NCI-H460). Expression of hsa-miR-125a-3p/5p in the lung cancer cell lines was normalized to that of a control human bronchiolar epithelium (HBE) cell line. U18 was used as an internal standard for real-time PCR. We found that the expression levels of hsa-miR-125a-3p and hsa-miR-125a-5p were lower in each of the four lung cancer cell lines than in the HBE cell line (p<0.01). The mean level of hsa-miR-125a-3p expression was moderately decreased in A549 cells (p<0.001), and the mean level of hsa-miR-125a-5p expression was moderately decreased in SPC-A-1 cells (p<0.001).(2) Effects of 2'-O-methyl oligonucleotides on the expressions of hsa-miR-125a-3p and hsa-miR-125a-5p:Real-time PCR results ensured that transfection with sense 2'-O-methyl oligonucleotides increased hsa-miR-125a-3p (p<0.001) and hsa-miR-125a-5p expressions (p<0.001), and antisense 2'-O-methyl oligonucleotides decreased hsa-miR-125a-3p (p<0.001) and hsa-miR-125a-5p expressions after 24 hours (p<0.001). Expression in transfected cells was normalized to that of untreated cells, and U18 expression was used as an internal standard.(3) Effects of hsa-miR-125a-3p on migratory and invasive capabilities of A549 cells:For analysis of hsa-miR-125a-3p, the number of A549 cells in the untreated group that migrated through a microporous membrane was 34.40±2.47. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (34.40±2.38, p=1.000). However, the number of migrating cells was significantly decreased when cells were transfected with the sense-3p oligonucleotide (25.33±3.15, p<0.001), and the number of migrating cells was significantly increased when cells were transfected with the antisense-3p oligonucleotide(48.80±2.65, p<0.001). In the hsa-miR-125a-3p invasion analysis, the number of A549 cells in the untreated group that invaded through the Matrigel was 24.33±2.06. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (24.00±2.54, p=0.696). The number of invading cells decreased significantly when cells were transfected with the sense-3p oligonucleotide (11.13±1.60, p<0.001), and the number of invading cells was significantly increased when cells were transfected with the antisense-3p oligonucleotide (34.80±2.88, p<0.001).(4) Effects of hsa-miR-125a-5p on migratory and invasive capabilities of SPC-A-1:For analysis of hsa-miR-125a-5p, the number of A549 cells in the untreated group that migrated through a microporous membrane was 24.20±1.78. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (28.80±2.70, p=0.445). However, the number of migrating cells was significantly increased when cells were transfected with the sense-3p oligonucleotide (45.27±2.71, p<0.001), and the number of migrating cells was significantly decreased when cells were transfected with the antisense-3p oligonucleotide (16.93±3.24, p<0.001). In the hsa-miR-125a-5p invasion analysis, the number of A549 cells in the untreated group that invaded through the Matrigel was 24.20±1.78. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (23.80±2.00, p=0.568). The number of invading cells increased significantly when cells were transfected with the sense-3p oligonucleotide (35.80±1.74, p<0.001), and the number of invading cells was significantly decreased when cells were transfected with the antisense-3p oligonucleotide (17.47±2.07, p<0.001).(5) Predicted and confirmed hsa-miR-125a-3p target:We proceeded to identify potential targets known to play a role in cell mobility and invasion by using microrna.org and TargetScanHuman 5.1 webservers. Among the candidates surveyed, we found that the 3'UTR of the RhoA gene contains two highly conserved regions that may serve as a binding site for hsa-miR-125a-3p as determined by the miRanda and TargetScan algorithm.(6) Hsa-miR-125a-3p inhibited migration and invasion by targeting RhoA: Western blotting and RT-PCR analyses revealed that RhoA protein (p=0.001) but not RhoA mRNA (p=0.482) was considerably decreased in sense-3p transfected cells. As expected, Rockl protein (p=0.001) and mRNA (p=0.003) were also reduced clearly. Transfection with 20μM of antisense-3p resulted in an increase in RhoA protein measured by Western blotting (p<0.001), however, RhoA mRNA remained unchanged (p=0.374). As expected, Rockl mRNA and protein were also increased obviously (p<0.001). These results indicated that hsa-miR-125a-3p could post-transcriptionally regulate RhoA mRNA and repressed its translation and contribute to mediate Rockl.To further demonstrate that RhoA is a potential target of hsa-miR-125a-3p, we generated luciferase reporters that contained the full length of the RhoA gene 3'UTR. Results from triplicate experiments showed that reporter activity was reduced by the ectopic expression of hsa-miR-125a-3p (p<0.001). Taken together, these results indicated that hsa-miR-125a-3p inhibited cell migration and invasion in SPC-A-1 cells by targeting RhoA.To further validate whether inhibitor of hsa-miR-125a-3p regulate migration and invasion via the Rho pathway, we blocked Rho with Rho inhibitor CT04 at a final concentration of 2.0μg/ml according to the previous studies of our laboratory in A549 cell line. The number of migratory cells and invasive cells were decreased clearly after treatment with CT04 when compared with the untreated group (p<0.001). The number of migratory cells and invasive cells were not significantly decreased in cells transfected with sense-3p (p>0.05), and also not increased obviously in cells transefected with antisense-3p (p>0.05), when compared with CT04 group. These results suggest that hsa-miR-125a-3p regulate migration and invasion in Rho-dependent manner.(7) Hsa-miR-125a-5p enhanced migration and invasion by targeting RhoA: Western blotting and RT-PCR analyses revealed that RhoA protein (p=0.001) and mRNA (p=0.263) remained unchanged (p=0.146). Interestingly, RhoC protein and mRNA were considerably increased in sense-3p transfected cells (p<0.001). As expected, Rockl protein (p<0.001) and mRNA (p<0.001) were also increased clearly. Transfection with 20μM of antisense-3p resulted in a decrease in RhoC protein measured by Western blotting (p<0.001), however, RhoA mRNA (p=0.087) and protein (p=0.147) remained unchanged. As expected, Rockl mRNA and protein were also decreased obviously (p<0.001).To further validate whether inhibitor of hsa-miR-125a-5p induce migration and invasion via the Rho pathway, we blocked Rho with Rho inhibitor CT04 in SPC-A-1 cell line. The number of migratory cells and invasive cells were decreased clearly after treatment with CT04 when compared with the untreated group (p<0.001). The number of migratory cells and invasive cells were not significantly decreased in cells transfected with antisense-5p (p>0.05), and also not increased obviously in cells transefected with sense-5p (p>0.05), when compared with CT04 group. These results suggest that hsa-miR-125a-5p regulate migration and invasion in Rho-dependent manner.Conclusions1,Hsa-miR-125a-3p and hsa-miR-125a-5p play distinct roles in regulation of invasive and metastatic capabilities of lung cancer cells, consistent with the opposing correlations between the expression of these miRNAs and lymph node metastasis in NSCLC.2,Hsa-miR-125a-3p inhibited migration and invasion by RhoA/Rockl pathway.3,Hsa-miR-125a-5p enhanced migration and invasion by RhoC/Rockl pathway. |
PDF Full Text Request