| Objective: We investigated the role of1,25(OH)2D3on proliferation and migrationof human ovarian cancer SKOV-3cell, and its molecular mechanism, providing a newdeliberation for prevention and treatment of ovarian cancer.Methods:(1) The effect of1,25(OH)2D3on proliferation of SKOV-3cell:SKOV-3cell was treated with1,25(OH)2D3(1、10、100nM). Cell proliferation wasdetected by CCK-8kit, cell cycle was analyzed by flow cytometry. Cell apoptosis wasassayed by TUNEL. The expressions and location of VDR and β–catenin weredetermined by immunofluorescence (IF). The expression level of VDR, β-catenin,CCND1and FRAT1were analyzed by Western blotting quantitatively.(2) The role of1,25(OH)2D3on migration of SKOV-3cell: Firstly, SKOV-3cell was treated with1,25(OH)2D3(1、10、100nM). Cell migration was determined by wound healing assayand Live cell Imaging System. The expressions of E-cadherin and Vimentin weredetected by IF and Western blotting. Then, the inhibition effect of1,25(OH)2D3onTGF-β-induced epithelial-mesenchymal transition (EMT) was studied. The cells weredivided into four groups: control, TGF-β, VD and VD+TGF-β. And they weretreated with the solvent control, TGF-β (10ng/ml),1,25(OH)2D3(100nM), TGF-β (10ng/ml)+1,25(OH)2D3(100nM) respectively. Cell migration was determined by woundhealing assay and Live cell Imaging System. The capability of invasion was detected bytranswell invasion assay. The expressions of VDR, Slug, Snail, β-catenin, E-cadherinand Vimentin were determined by IF and Western blotting.(3) microarray wasapplied to compare the microRNAs expression profile between control cells andSKOV-3cells treated by1,25(OH)2D3. Comparative microRNAs expression profilinganalyses were carried out on the SKOV-3cells treated by ethanol and100nM1,25(OH)2D3. Bioinformatics programs (TargetScan, miRDB, GSEA database and GO)were used to analyze the microRNAs and their potential target genes. Result:(1) The effect of1,25(OH)2D3on proliferation of SKOV-3cell: The resultsshowed that1,25(OH)2D3inhibited proliferation of SKOV-3cells by a way ofdose-dependent and time-dependent (P <0.01). Cell cycle analysis suggested thatG0/G1phase of SKOV-3cells were arrested by1,25(OH)2D3(P<0.05). The cellapoptosis was increased with the treatment of1,25(OH)2D3(P<0.05). After treatment of1,25(OH)2D3, the location of β-catenin was transferred from the nucleus to thecytoplasm, the expression of VDR in the nucleus was also increased. Western blottingshowed that the expression of VDR was increased, while the levels of β-catenin,CCND1and FRAT1were decreased with the treatment of1,25(OH)2D3,compared withcontrol cells. A dose-dependent relationship between these proteins level andconcentration of1,25(OH)2D3was observed (P<0.05).(2) The role of1,25(OH)2D3on migration of SKOV-3cell: The results showedthat1,25(OH)2D3inhibited the migration of SKVO-3cells (P<0.05). The IF showedthat the distribution of E-cadherin and Vimentin was not changed in SKOV-3cellstreated by1,25(OH)2D3. However, Western blotting suggested that the expression ofE-cadherin was increased with the treatment of1,25(OH)2D3, meanwhile the level ofVimentin was decreasedby a way of dose-dependent (P<0.05). After treated by TGF-β,the SKOV-3cells, polygonal-like epithelial appearance, converted to spindle-mesenchyme morphology. The migration of SKOV-3cells was stimulated by TGF-β.Slug, an EMT transcription factor, was also significantly increased with treatment ofTGF-β. Meanwhile, E-cadherin, an epithelial marker, was decreased, andVimentin, amesenchymal markers, was increased. These results indicated that TGF-β inducedEMT of SKOV-3cells. However, the migration and invasion assay showed that thepromotion of migration by TGF-β could be blocked by1,25(OH)2D3(P<0.05). The IFshowed that location of VDR mainly expressed in the cytoplasm in control group, and itgathered to nucleus in VD group and VD+TGF-β group. Compared with expression ofSlug, Snail and β-catenin in the cytoplasm of control cells, they gathered to nucleus inSKOV-3cells treated by TGF-β. Compared with SKOV-3cells treated by TGF-β, thelevel of Vimentin and EMT-inducing transcription, such as Snail, Slug and β-catenin,was markedly decreased, while E-cadherin was increased in cells treated by1,25(OH)2D3. These results indicated that TGF-β-inducing EMT in SKOV-3cells couldbe redressed by1,25(OH)2D3(P<0.05). (3) Analysis of microRNAs expression profiling suggested that expressions of8microRNAs were found significantly different between control cells and SKOV-3cellstreated by1,25(OH)2D3. Among them,6microRNAs were down-regulated, includingmiR-1224-3p, miR-206, miR-4723-5p, miR-5194, miR-634and miR-644b-5p; while2were up-regulated, for example, miR-1260a and miR-1280. Among them, miR-206,miR-1280, miR-634and miR-1224-3p may be involved in cell proliferation and cancerinvasion. Bioinformatic analysis suggested2086target genes were enriched in a numberof pathways, specifically pathway in cancer, MAPK signaling pathway, Wnt signalingpathway, adherens junction and regulation of actin cytoskeleton.Conclusion:(1)1,25(OH)2D3inhibited proliferation of SKOV-3cells by a way ofdose-dependent and time-dependet effect. It achieves this by induced-arrest in G0/G1phase, increasing apoptosis and blocking Wnt/β-catenin signal pathway.(2)1,25(OH)2D3reduced the migration and invasion of SKOV-3cells bydown-regulated Snail、Slug、β-catenin and Vimentin, up-regulated E-cadherin, whichwas associated with the induction of EMT by TGF-β.(3)1,25(OH)2D3-inducing mircroRNAs in SKOV-3cells and potential targetgenes were enriched in a number of pathways, specifically pathway in cancer, MAPKsignaling pathway, Wnt signaling pathway, adherens junction and regulation of actincytoskeleton. |
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