Cell proliferation assays indicated that miR expression affected cell cycle Keywords: choriocarcinoma, microRNA, Sox2, proliferation, invasion. Go to: . Jun 20, The TRIM11 knockdown significantly suppressed proliferation and inhibited . The Transwell assay of cell invasion was with 8-µm-pore filters. Jul 26, We have examined these cells in vitro for proliferation, migration, branching . In in vitro invasion assays in 3D Matrigel, DB-P cells were most.
and invasion assays Proliferation
Recently, it has been reported that TRIM11 is upregulated and play an oncogenic function in glioma 8 , lung cancer 9 and colon cancer However, the expression, and functions of TRIM11 in ovarian cancer have been insufficiently characterized. The present study concluded that the level of TRIM11 expression is higher in ovarian cancer tissues than in matched adjacent non-cancerous tissues.
We then investigated the biological functions of TRIM11 by knocking down its expression in ovarian cancer cell lines. The TRIM11 knockdown significantly suppressed proliferation and inhibited invasion of cells, and induced apoptosis.
TRIM11 is a potent oncogene in ovarian cancer. A total of 40 patients median age, 53 years; range, 30—68 years with ovarian cancer who underwent surgical resection at Clinical Laboratory, the Womens Hospital, Zhejiang University School of Medicine were registered in this project. Ovarian cancer tissues and matched non-cancerous tissues were obtained from the above 40 patients after written informed consent was obtained.
GAPDH served as an internal control. The sequences of PCR primers are as follows: Protein concentrations depend on Bradford assay. Rabbit polyclonal TRIM11 antibody dilution, 1: Subsequently, the membranes were applied with secondary goat anti-rabbit HRP IgG antibody dilution, 1: GAPDH was used as loading control. Band intensities were assessed on ImageJ software version 1. At 48 h post-transfection, by real-time PCR and western blot analysis, the knockdown efficiency was assessed.
At nm, the absorbance was measured on a microplate reader Perlong, Beijing, China with a reference wave length at nm. Using an inverted microscope, cells in 5-random fields were counted. One-way analysis of variance was used to test the statistical differences. As shown in Fig. Positive log2 value indicated increased upregulation of TRIM11 in cancer tissues while negative log2 indicated downregulation of TRIM11 in cancer tissues.
TRIM11, tripartite motif-containing Knockdown of TRIM11 reduced ovarian cancer cell proliferation. Cell proliferation was detected by CCK-8 assay at 0, 24, 48 and 72 h after transfection. Suppressing TRIM11 expression induces ovarian cancer cell apoptosis. Knockdown of TRIM11 inhibits ovarian cancer cell invasion. The protein levels of cell apoptosis and invasion related proteins by western blot analysis Fig. Dysregulated expression of several members of TRIM proteins has been found in ovarian cancer tissues 11 — The diagnostic and prognostic values of TRIM11 have been reported in gliomas 8 , lung cancer 9 and colon cancer Mesothelioma cell lines were incubated overnight with 5 pmol miRNA inhibitors in collagen-coated well plates.
The area of wound in percentage was determined using Scratch software The number of adherent cells was determined using Cell Titer-Glo 2. Afterwards, membranes were incubated with the secondary antibody [rabbit 1: A MicroRNA in situ hybridization of human mesothelioma tissues shows miR and miR expression in the cytoplasm and nuclei of mesothelioma cells. U6 probe is the positive control, and scramble probe is the negative control.
Upper panel shows representative images of epithelioid mesothelioma. Lower panel shows representative images of sarcomatoid mesothelioma. B The specificity of miR and miR probes is confirmed by in situ hybridization of lung cancer tissues showing both expression of miR and miR in lung squamous cell carcinoma upper panel and no expression in lung adenocarcinoma lower panel.
Mesothelioma cells transfected with miR or miR inhibitors showed reduced cell proliferation rates compared to cells treated with negative control inhibitor. A Cell proliferation analysis. Upper panel figures show representative inverted fluorescence images acquired using CellProfiler Image analysis software. Upper panel figures are representative images acquired using an inverted phase contrast microscope. D Cell Adhesion assay. In invasion assay, inhibition of miR significantly reduced cell invasion.
Although ACC-Meso1 transfected with miR inhibitor did not show significant difference, CRL transfected with miR inhibitor showed lower invasion compared to cells transfected with negative control inhibitor. Furthermore, cells co-transfected with both miR and miR inhibitor showed the least invasion Figure 2B.
Moreover, mesothelioma cells transfected with miR or miR inhibitor showed lower cell migration abilities than cells treated with negative control inhibitor. Cells transfected with both inhibitors showed the lowest cell migration abilities Figure 2C.
This result indicated that miR and miR may help mesothelioma cells metastasize by promoting adhesion of cells to other places. We investigated the targets of miR and miR to further understand their biological roles in mesothelioma.
Analysis using TarBase v7. Furthermore, cells treated with both miR and miR inhibitors showed significant upregulation of FOXO1 expression than cells treated with miR or miR inhibitor alone.
The transfection of mesothelioma cells with miR and miR inhibitors also upregulated the protein expression levels of p21 and p Malignant pleural mesothelioma is fatal cancer that is mostly caused by asbestos exposure. Malignant pleural mesothelioma has poor prognosis and is associated with survival periods ranging from 5 to Pemetrexed and platinum combination chemotherapy is the standard and first line of treatment for inoperable malignant mesothelioma. However, the applicability of this treatment for mesothelioma remains to be established.
Therefore, there is an urgent need to develop novel therapeutic regimes for advanced mesothelioma, which can facilitate the management of the predicted peak incidence of malignant mesothelioma. The present study focused on microRNAs as novel therapeutic targets for the treatment of malignant mesothelioma.
In fact, certain miRNAs have entered the preclinical and clinical stages as therapeutic targets, and these recently developed treatments are expected to be available in the market. Anti-miRb has been investigated as a therapeutic target for glioblastoma and is currently at the preclinical stage. The use of a miR mimic for the treatment of liver cancer is currently undergoing phase 1 clinical trials.
The use of anti-miR has been studied for its protective effects against HCV infection and is currently undergoing phase 2a clinical trials MiR and miR have been recognized as potential diagnostic markers, and miR-let-7c-5p and miRa-5p have been identified as potential prognostic markers for mesothelioma In a previous study, we performed comprehensive analysis of microRNA expression in mesothelioma cell lines.
In the present study, we identified the novel roles of miR and miR as onco-microRNAs in mesothelioma cell lines. We showed the association of miR and miR with proliferations of mesothelioma cells. We also found the association of miR and miR with invasion, migration, and adhesion of mesothelioma cells by conventional assays.
However, it needs to be clarified whether invasion, migration, and adhesion are influenced by the proliferation due to miRNAs of mesothelioma cells by more precise experiments like real-time cell tracking analysis.
Furthermore, we investigated the experimentally validated targets of miR and miR in mesothelioma cells. FOXO1 is an experimentally validated target of miR in breast cancer 28 and a target of miR in non-small cell lung cancer The phosphatidylinositolkinase PI3K pathway is known to play an important role in many biological functions, including cell proliferation FOXO1 is a transcription factor that regulates various biological events, including gluconeogenesis, differentiation, cell growth, and apoptosis Tripartite motif containing 37 TRIM37 , a member of the tripartite motif TRIM family, has been involved in the development and progression of several tumors.
Knockdown of TRIM37 obviously inhibited the proliferation in vitro and xenografted tumor growth in vivo. The article was received on 29 Jul , accepted on 22 Oct and first published on 31 Oct Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material and it is not used for commercial purposes. Information about reproducing material from RSC articles with different licences is available on our Permission Requests page.
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Proliferation and invasion of ovarian cancer cells are suppressed by knockdown of TRIM11
sessing proliferation and invasion properties of tumor cells both in vitro and in vivo is especially transwell invasive assay have the disadvantages of caus-. Sep 26, TFPI-2 suppresses breast cancer cell proliferation and invasion through Immunoprecipitation assays identified myosin-9 and actinin-4 as. Jan 18, NOX4 knockout cell lines showed reduced cell proliferation with an increase of Invasion assays were performed as described [22–24]. Briefly.