MiR-218 Inhibits EMT, Invasion, Migration And Its Mechanism In Cervical Cancer Cells

BackgroundCervical cancer is one of the most common malignant tumor among women worldwide, cervical cancer is the second leading cause of cancer mortality in the world, just after breast cancer. More than 85% of the cervical cancer patients are in developing countries. Cervical cancer is one malignant tumor which can seriously damage health among women. The leading cause of treatment failure is cancer recurrence and metastasis. EMT, a process in which epithelial cells lose their polarity and are converted into a mesenchymal phenotype, is regarded as a critical event during tumour metastasis. Morphologically, EMT is characterized by the loss of tight cell-cell junctions and accompanied by reorganization of the actin cytoskeleton resulting in spindle shaped mesenchymal-like cells, which are capable of migrating and invading other tissues. A number of distinct molecular processes are engaged in order to initiate an EMT and enable it to reach completion. These include activation of transcription factors, expression of specific cell-surface proteins, reorganization and expression of cytoskeletal proteins, production of ECM-degrading enzymes, and changes in the expression of specific microRNAs. In many cases, the involved factors are also used as biomarkers to demonstrate the passage of a cell through an EMT. The loss of E-cadherin expression is considered a crucial step in the progression of papilloma to invasive carcinoma, and it is also a fundamental event in EMT.microRNA (miRNA) function as 20-25-nucleotide (nt) guides that regulate the expression of mRNAs containing complementary sequences. Owing to their imperfect complementation with their target sites, one miRNA can often target multiple genes. They are diverse not only in the messenger RNAs (mRNA) they target, but in their production; the same hairpin RNA structure can generate mature products from each strand, termed 5p and 3p, that can bind different mRNAs. Studies have documented the role of miRNAs in the processes involved in metastasis, including cellular proliferation, migration and invasion, and the epithelial-to-mesenchymal transition (EMT). Given that many miRNAs are deregulated in cancers but have not yet been further studied, it is expected that more miRNAs will emerge as players in the etiology and progression of cancer.Human SFMBT1 is a PcG protein, also belong to the MBT (malignant brain tumor) protein family, plays an important role in maintaining transcriptional silencing. It is reported that SFMBT1 can indirectly promotes HSC/progenitor cell activity, playing important role in the maintenance of stem characteristics in hematopoietic stem cells. SFMBT1 can regulate MyoD mediated transcription silence to stabilize the undifferentiated state of precursor cells muscle from, and can promote cell proliferation. SFMBT1 participates the inhibition of transcription induced by Snail, thus inhibits cell epithelial characteristic, and induces epithelial to mesenchymal transformation in breast cancer. SFMBT1 is an essential factor in TGF-β inhibition of lung cancer epithelial gene expression and in the EMT process of lung cancer. LSD1-SFMBT1-Snail induced chromatin regulation program can induce the EMT of cancer cells and promote the progression and metastasis of cancer. In short, through its function on transcription inhibition, SFMBTlplays an important role in maintaining stem cell characteristicscells, promoting cell proliferation, promoting the EMT and invasion of cancer cells, can promote cell development and metastasis, and is associated with poor prognosis of tumor.Our findings showed that SFMBT1 is a direct and functional target for miR-218, which can directly suppress SFMBT1 expression. What’s more, we found that downregulation of SFMBT1 inhibits EMT, which was first reported in cervical cancer in this research. Taken together, downregulation of SFMBT1 by miR-218 inhibits cell motility and invasiveness, as well as inhibits EMT, contribute to inhibition of metastasis formation in cancer ultimately.SCCRO/DCUN1D1/DCN1 is a oncogenic gene. Studies show that DCUN1D1 overexpression exists in over than 50% of head and neck cancer 55% of lung squamous carcinoma,70% of ovarian cystadenocarcinoma,34% of cervical cancer and endometrial carcinoma,26% of lung adenocarcinoma, and 15% of malignant glioma. DCUN1D1 is associated with T stage and tumor clinical stage of lung cancer. The overexpression of DCUN1D1 can promote local lymph node metastasis and brain metastasis of lung squamous carcinoma, reduce the survival of lung squamous carcinoma patients; can enhance local invasion of bronchioalveolar carcinoma cells; can participate in the formation of glioma, promote the progress of malignant glioma. In addition, the expression of DCUN1D1 is associated with vascular endothelial growth factor alpha (VEGF-a) expression, can promote angiogenesis. Excessive drinking or smoking also causes abnormal chromatin, promotes DCUN1D1 expression, and promotes cancer development, such as, the head and neck squamous cell carcinomas, lung cancer.In the current study, we discovered that DCUN1D1 was downregulated and targeted by miR-218 in human cervical cancer. Furthermore, transfection of mimics of miR-218 and knockdown of DCUN1D1 by a DCUN1D1-specific siRNA significantly decreased the migration of cervical cancer cells. Considering our own and these previous published findings, it is tempting to speculate that miR218-DCUN1D1-dependent changes in DCUN1D1 could inhibit the invasion and metastasis of cervical cancer cells.A growing number of studies have found that miR-218 is abnormally expressed in a variety of tumors. The expression of miR-218 is downregulated in many cancers, such as, lung cancer, breast cancer, thyroid cancer, pancreatic cancer, colon cancer, gastric cancer, prostate cancer, malignant glioma, bladder cancer and cervical cancer and its expression is lowered in malignant tumor than that in benign tumor. The downregulated expression of miR-218 is in closely related to tumor migration and poor prognosis. These findings suggest that miR-218 may play an important role in tumorigenesis and metastasis of those tumors. Despite the tumor suppresive role of miR-218 has been identified by a number of previous studies, the molecular mechanisms by which miR-218 regulated metastasis are still elusive.Methods1 Cell cultureHuman cervical cancer cell lines SiHa and HeLa were maintained in Dulbecco’s Modified Eagle Medium, and supplemented with 10% FBS and incubated at 37℃ in 5% CO2. They had been passed for less than 6 months in culture when the experiments were carried out. Cell lines were characterized using DNA analysis by short tandem repeat fingerprinting.2 Invasion and motility assaysFor motility assays,1.0×105 cells were placed in the top chamber of each insert with 8.0-mm pores; for invasion assays,2.0x105 cells were seeded in a Matrigel-coated chamber. Cells were seeded in serum free media and translocated toward complete growth media. After 24 hours of incubation at 37℃, cells that had migrated or invaded were fixed and stained in dye solution containing 20% methanol violet and 0.1% crystal. The cells that had migrated or invaded were imaged using a BH-2 inverted microscope.3 ImmunoblotsCells were lysed in 2×SDS sample buffer on ice and total proteins were further analyzed by SDS-PAGE and transferred to a polyvinylidene difluoride membrane, and probed with antibodies against β-actin, SFMBT1, DCUN1D1, N-cadherin, E-cadherin, and Flag. After incubation with primary antibodies, the membranes were washed with TBS/0.05% Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Proteins were detected by enhanced chemiluminescence substrates.4 Luciferase assaysA total of 5.0×104 SiHa cells were cotransfected with 50 ng of the indicated pMIR-REPORT luciferase construct and 50 ng of a pMR-REPORT β-gal normalization control. All cells were also transfected with miR-218 mimics or negative control. Lysates were collected 36 hours after transfection, and β-gal and firefly luciferase activities were measured with β-gal and the Luciferase Reporter System.5 miRNA detectionTotal RN A, inclusive of the small RNA fraction, was extracted from cultured cells with a Tissue Total RNA Extraction Kit (GenePharma). Reverse transcription reactions were carried out using M-MLV reverse transcriptase. Real-time PCR was performed on an Applied Biosystem StepOnePlus,using a SYBR Green I Real-Time PCR Kit for miR-218. The relative expression levels of miRNAs in each sample were calculated and quantified using the2"△△Ctmethod after normalization for expression of the positive control.6 In situ hybridization and immunohistochemistry of human TMAsCervical cancer tissue microarrays were constructed with 104 formalin-fixed, paraffin-embedded cervical cancer tissues and their corresponding adjacent cervical tissues. The protocol for detection of miRNAs by in situ hybridization (ISH) has been previously published. Immunohistochemical staining for proteins was carried out as previously described. The intensity of miR-218 and proteins staining in epithelial cells of the 104 cervical cancer samples was scored using a semi quantitative scale as previously described. Shortly, immunostaining was defined as "high" if the immunoreactivity was observed in 10% or more of the cells in paraffin sections; tumors with lower percentages of immunoreactive cells showed "low" immunostaining.7 Statistical analysisStatistical analysis was conducted using the SPSS statistical software program (Version 13.0; SPSS Inc.). The association between miR-218 and protein expression was analyzed by the χ2 test. Differences between groups were analyzed using the Student t test and one-way ANOVA, or if the data violated anormal distribution, by the nonparametric Mann-Whitney test, and a level of P<0.05 was considered statistically significant. Data are presented as mean±SEM unless otherwise indicated. Correlations were performed using Pearson correlation analysis.Results1 miR-218 is decreased in cervical cancer and inhibits EMT, invasion and migration in cervical cancer(1) Correlation of miR-218 with tumor grade and metastatic status in cervical cancersWe analyzed the correlation between the miR-218 expression and the clinicopathological parameters among the patients with cervical cancer based on the results from the tissue microarray analysis. A in situ hybridization TMA was used in this study, which was constructed with 104 formalin-fixed, paraffin-embedded cervical cancer tissues and their corresponding adjacent cervical tissues. The results indicated that the level of miR-218 in cervical cancer tissues was markedly decreased as compared with that in the adjacent tissues. Furthermore, miR-218 was significantly down-regulated in malignant as compared with benign tumors.(2) miR-218 inhibits EMT of cervical cancer cellsTo identify the role of miR-218 in cervical cancer EMT, we transfected cervical cancer cell SiHa or HeLa with miR-218 mimcs or miR-218 inhibitor.48 hours after transfection with miR-218 inhibitor, SiHa cells turned from round into a spindle-like mesenchymal phenotype. In addition, We visualized the actin cytoskeleton by phalloidin staining. The result demonstrated that F-actin distribution was rearranged in these cells from a cortical to a stress-fibre pattern, a hallmark of the mesenchymal phenotype. Immunofluorescent staining of these cells for N-cadherin revealed expression was increased, typical a marker of EMT. Inhibiting miR-218 also significantly reduced the E-cadherin, a hallmark of the epithelial phenotype.(3) miR-218 significantly inhibits cervical cancer cell migration and invasion.To identify the role of miR-218 in cervical cancer migration and invasion, we transfected cervical cancer cell SiHa or HeLa with miR-218 mimcs or miR-218 inhibitor.48 hours after transfection with miR-218 mimics or inhibitor, We found that miR-218 mimics decreased cervical cancer cell migration and invasion. Furthermore, miR-218 inhibitors led to a significant increase in cervical cancer cell migration and invasion.2 The mechanism of miR-218 inhibits EMT, migration and invasion(1) miR-218 directly targets to the SFMBT1 and DCUN1D13-UTR.miR-218’s ability to inhibit multiple steps of the invasion-metastasis cascade might derive from its ability to regulate genes involved in diverse aspects of metastatic dissemination. We employed two strategies to identify the effectors of miR-218. We applied three algorithms that predict the mRNA target. Among these candidates, seven genes (HAPLN1、SFMBTl、RNF38、UGT8、ARID4B、BCAT1、 CTNND2 and DCUN1D1) were involved in the promotion of cancer cell migration or invasion. SFMBT1 and DCUNID1 emerged as the most strongly downregulated proteins.To determine whether miR-218 targets these genes directly, We cloned the 3’ UTRs of 8 putative miR-218 targets into a luciferase construct. Reporter assays using miR-218-expressing SiHa cells revealed that miR-218 repressed two of the UTRs:SFMBT1, DCUNID1. Mutation of the putative miR-218 site(s) in these two 3’UTRs abrogated responsiveness to miR-218.In concord with these results, we observed a clear decrease in endogenous SFMBT1 and DCUNIDI protein in SiHa and HeLa cells with miR-218 over-expression. Furthermore, over-expression of miR-218, significantly decreased the expression levels of SFMBT1 and DCUNIDI protein in SiHa cells. Inhibition of miR-218 significantly increased the expression levels of SFMBT1 and DCUNIDI protein in SiHa cells. Taken together, these results suggest that miR-218 can downregulate SFMBT1 and DCUN1D1 expression by directly targeting its 3’UTR.(2) Decrease of SFMBTl and DCUN1D1 is required for miR-218-mediated inhibition of motility and invasiveness in cervical cancer.We found that knockdown of SFMBT1 and DCUNIDI produced similar changes in invasion assay as miR-218 overexpression. To determine whether these effects depend specifically on SFMBT1 or DCUN1D1 suppression, we employed an expression construct that encodes the entire SFMBT1 or DCUNIDI coding sequence but lacks the 3’UTR, yielding an mRNA resistant to miRNA-mediated suppression. Ectopic expression of DCUN1D1 with this construct completely rescued miR-218-mediated invasion surppresiveness in miR-218-overexpressing cells. Re-expression of SFMBT1 also completely rescued miR-218-mediated invasion surppresiveness. This suggests that, after miR-218 overexpression, a decrease in SFMBT1 is required for cells to show decreased motility and invasiveness.(3) miR-218 inhibits cancer cell EMT by inhibiting SFMBTlSiHa cells turned from round into a spindle-like mesenchymal phenotype after overexpression of SFMBT1, which mimic the effect of miR-218 inhibitor on EMT, while DCUN1D1 did not. What’s more, overexpression of SFMBT1 also significantly reduced the E-cadherin, but increased the N-cadherin levels. Given that SFMBT1 may connects miR-218, we ask if SFMBT1 connects to miR-218 and EMT. Taken together, these data indicated that miR-218’s ability to inhibit metastasis is attributable, in significant part, to its capacity to inhibit SFMBT1.(4) HPV16 E6 promotes EMT and invasion ability by repressing miR-218 expression in cervical cancerIn order to verify the effect of HPV on miR-218, we tested the expression of miR-218 in cervical cancer cells with different HPV status. We verified the direct repression of HPV16 E6 on miR-218 in cervical cancer cell. We verified the interaction between HPV 16 E6 and miR-218 in EMT and invasion ability in cervical cancer. The result showed that HPV16 E6 promotes EMT and invasion ability in cervical cancer, and miR-218 can reversally inhibits the oncogenetic effect. In a word, HPV16 E6 promotes EMT and invasion ability in cervical cancer by inhibiting miR-218, promoting the metastasis of cervical cancer in further.(5) Correlation of miR-218 with SFMBTl and DCUN1D1 in cervical cancer tissuesIn order to study the relationship between miR-218 with the two target genes, SFMBT1 and DCUN1D1, we detected the expression of SFMBT1 and DCUN1D1 in cervical cancer tissues with immunohistochemical method and analyzed the correlation between miR-218 and the two genes. Results showed that the spatial distribution of the DCUN1D1 signal in cervical cancer tissues is opposite to the spatial distribution of the miR-218 signal. Statistical analysis found that DCUN1D1 signal strength in 104 cases of cervical cancer and cancer adjacent tissues showed negative correlation (P< 0.05) with miR-218 signal strength.This study failed to detect SFMBT1 expression in cervical cancer and its normal cervical tissues adjacent to carcinoma. This may be associated with SFMBT1 speciality. At present, SFMBT1 protein expression and orientation in tissue has not been reported successfully. It is repored that SFMBT1 expressed in a particular of cell lines originated from human, such as cells of hematopoietic system, brain cells, etc. This study is the first one to successfully detect SFMBTl expression in cervical cancer cells, and discover its relationship with the EMT, invasion and migration of cervical cancer.ConclusionHere, for the first time, we reveal the mechanism of miR-218 role in metastasis in cervical cancer. Here we show that miR-218 was downregulated in cervical cancer, and moreover, its expression levels were lower in malignant than benign tumors. We found that miR-218, inhibits EMT and cervical cancer metastasis through direct targeting of SFMBT1 and DCUN1D1 in vivo. What’s more, we found that the inhibition of miR-218 on EMT, invasion and migration in cervical cancer is controlled by HPV16 E6. These findings provide new insights into the molecular functions of miR-218 as well as the role of SFMBT1 and DCUN1D1 in metastasis. Distant metastases are responsible for patient mortality in the vast majority of human carcinomas, therefore, miR-218’s ability to impede metastasis may prove to be clinically useful.