CLIC4 Mediates Tumour-associated Fibroblast Transdifferentiation In Ovarian Cancer Stroma And MiRNAs Are Involved In The Cell Differentiation

BackgroundEpithelial ovarian cancer is the most lethal disease among gynaecologic malignancies.For years,it has been dubbed as a "silent killer"--causing no symptom till it is too advanced at the time of diagnosis.5-year survival for patients with advanced ovarian cancer improved little even with the treatment of optimal cytoreductive surgery and systemic combination chemotherapy.Increasing evidence has indicated that cancer development is facilitated by interaction between tumour cells and activated stromal cells.The tumour stroma(also referred to as "reactive stroma") is characterized by marked alterations in the phenotype and expression profile of fibroblast-like cells.These cells commonly expressα-SMA and thus are termed as myofibroblasts.Stromal fibroblasts are located at the tumour border near the invasion front,and play a crucial role in tumour progression.Myofibroblasts are recruited from different sources,including local fibroblast population,distant fibroblasts,EMT (epithelial-mesenchymal transition) and bone marrow progenitor cells or stem cells,during cancer development and the invasion progression. Although it has been shown that the conversion from fibroblasts to myofibroblasts constitutes the major source of myofibrblasts in tumour stroma,the molecular mechanism underlying fibroblast-to-myofibroblast transdifferentiation is still not fully understood.Chloride intracellular channel 4(CLIC4),a chloride channel of intracellular organelles,regulates intracellular pH and cell volume. Besides its presence on the organelle membrane,CLIC4 exists in soluble form in the cytoplasm and nucleus acting as signalling protein or channel regulator.Transcription level of CLIC4 is up-regulated when fibroblasts transdifferentiate into myofibroblasts induced by TGF-β1,and more importantly CLIC4 is highly expressed in myofibroblasts of breast cancer. Hence,we hypothesized that CLIC4 might invlove in myofibroblast transdifferentiation in ovarian cancer stroma,but the pathway in which CLIC4 participated during the fibroblast-to-myofibroblast transition induced by TGF-β1 from ovarian cancer cells is still unknown.MicroRNAs(miRNAs) are a class of endogenous,short (20-25-nucleotide),noncoding single-stranded RNA molecules, which negatively regulate gene expression through mRNAs degradation or translational inhibition of their target gene.miRNAs are involved in regulators of gene expression that control diverse physiological and pathological processes,such as development,cell differentiation, migration,proliferation and apoptosis,miRNAs represent a new layer of gene expression regulators at the transcript and translational levels,but the involvement of miRNAs and the roles of these miRNAs in TGF-β1-induced myofibroblasts differentiation in tumor-stroma interaction are uncertain. PartⅠCLIC4 mediares TGF-β1-induced fibroblast-to-myofibroblast transdifferentiation in ovarian cancer【Objective】Stromal myofibroblasts,activated by crosstalk signaling between the tumour and stroma,play a critical role in tumour development and progression.Chloride intracellular channel 4(CLIC4) may be functionally import for tumour stromal fibroblast-to-myofibroblast transdifferentiaton,but the molecular mechanism of the process has not been addressed.This study was to investigate the role of CLIC4 in the myofibroblast transdifferentiaton of ovarian cancer stroma.【Methods】1.The expression of CLIC4 in ovarian cancer tissues was analyzed by immunohischemistry,2.A cell model of fibroblast-to-myofibroblast transdifferentiaton was constructed,where ovarian primary fibroblasts or human fibroblasts MRC-5 cells were cultured with conditioned medium(CM) from ovarian cancer cells or transforming growth factor-β1(TGF-β1).Cellular morphologic change and the upregulation ofα-SMA expression were used to identify the differentiated myofibroblasts.3.The cell model of myofibroblast transdifferentiaton was used to demonstrate whether CLIC4 and ROS participated in the fibroblast-to-myofibroblast transdifferentiation.4.Specific CLIC4 siRNA was used to knockdown the CLIC4 expression, then to investigate the role of CLIC4 in the myofibroblast transdifferentiaton.5.The agents,specific CLIC4 siRNA,chloride channel inhibitor and antioxidant,were used to investigate the roles in suppression of the functional gene expression related with the myofibroblasts conversion.【Results】1.The expression of CLIC4 in 97.22%of ovarian cancer stroma and correlated with the up-regulation of myofibroblast markerα-SMA,which was detected in 94.44%of patients(P＜0.05).The immunohischemistry data in different pathologic groups showed that the protein expression level of CLIC4 in ovarian cancer stroma which was in mid- or low-differentiated and accompanied by lymphatic metastasis was obviously higher than that which was in high-differentiated and nonaccompanied by lymphatic metastasis(P＜0.05).2.In the presence of CM^SKOV3 or TGF-β1,the fibroblasts,which displayed typical small spindle shape under serum-free condition, changed into bigger and polygonal cells,andα-SMA transcript and translational expression increased(P＜0.05),indicating fibroblast-to-myofibroblast conversion.3.The expression of CLIC4 in the activated fibroblasts increased significantly in the presence of CM^SKOV3 or 10 ng/ml TGF-β1 for 48 h both at the transcription(P＜0.05) and protein levels compared with that in serum-free medium control.4.When cells were pretreated with the antioxidant NAC,which inhibited the generation of intracellular ROS,the up-regulation ofα-SMA and CLIC4 expression stimulated by TGF-β1 or CM^SKOV3 was significantly suppressed(P＜0.05).5.The up-regulation ofα-SMA expression induced by TGF-β1 or CM^SKOV3 was significantly blocked in CLIC4 siRNA-transfected fibroblasts(P＜0.05).6.Pre-treatment with CLIC4 siRNA to silence CLIC4 expression, chloride channel inhibitor IAA-94 to block function of CLIC4 or NAC to inhibit the generation of ROS lowered VEGF and HGF expression levels in the activated fibroblasts by 29-80%(P＜0.05).【Conclusion】These results suggest that ROS-initiated CLIC4 up-regulation is required for TGF-β1-induced fibroblast-to-myofibroblast transdifferentiaton in ovarian cancer,indicating inhibiting CLIC4 or ROS might have therapeutic potential targeting tumour stroma.PartⅡMicroRNA-21 participates in TGF-β1-induced myofibroblasts differentiation in tumor stroma【Objectire】Stromal fibroblast-to-myofibroblast transdifferentiation induced by TGF-β1 in the microenvironment of tumor and stroma interaction via expression changes of multiple phenotypic and functional genes plays a critical role in the tumor progression.Up to now,the involvement of microRNAs(miRNAs) and the roles of these miRNAs in TGF-β1-induced myofibroblasts differentiation in tumor-stroma interaction are unclear.【Methods】 1.Quantitative real time RT-PCR was used to measure the expression change of a range of microRNAs during the fibroblast-to-myofibroblast transdifferentiation induced by TGF-β1 or CM from cancer cells.2.To determine the potential roles of miR-21 in TGF-β1-mediated gene regulation during myofibroblast conversion,miR-21 expression was regulated by miR-21 inhibitor and miR-21 mimic,respectively.3.Computer analysis,luciferase report construction including the fragment of 3'-UTR of PDCD4(programmed cell death 4) mRNA with the putative miR-21 binding sequence and loss-of-function and gain-of-function experiments using miR-21 inhibitor and miR-21 mimic were used to predict and detect the target genes of miR-21 during the fibroblast-to-myofibroblasts transdifferentiation in ovarian cancer stroma.【Results】1.miR-21 was up-regulated in active fibroblasts after treatment with TGF-β1 or CM from ovarian cancer cells in time-dependent and dose-dependent manner(P＜0.05).2.The expression of miR-21 was downregulated by miR-21 inhibitor and upregulated by miR-21 mimic in fibroblasts MRC-5.TGF-β1-induced myofibroblasts differentiation was inhibited by mir-21 inhibitor and promoted by miR-21 mimic(P＜0.05).3.Computer analysis shows that PDCD4 might be a potential target gene of miR-21 based on existing the "seed sequence" of miR-21 in the PDCD4 3'-UTR.4.The fragment of 3'-UTR of PDCD4 mRNA with the putative miR-21 binding sequence was cloned into a pGL3 vector at the downstream of the luciferase gene and co-transfected the pGL3 construct with miR-21 mimic or miR-21 inhibitor into MRC-5 fibroblasts using Lipofectamine 2000. The miR-21 mimic increased miR-21 expression and inhibited luciferase activity.On the contrary,the miR-21 inhibitor down-regulated miR-21 and sequentially up-regulated luciferase activity(P＜0.05).5.Reduction of miR-21 by miR-21 inhibitor and enhanced expression of miR-21 by the miR-21 mimic increased or decreased PDCD4 protein expression,respectively.TGF-β1 stimulated miR-21 expression,then reduced PDCD4 expression in the translation level,however miR-21 inhibitor abolished the effect.【Conclusion】miR-21 participated in TGF-β1-induced gene regulation and functional modulation by targeting PDCD4 in stomal myofibroblasts differentiation.