miR-195-5p Regulates the Phenotype Switch of CCSM Cells by Targeting Smad7

ABSTRACTIntroduction

Phenotype switch refers to the process in which smooth muscle cells change from contractile type to synthetic type and acquire the ability of proliferation. Phenotypic transformation involves many changes of cell function, such as collagen deposition and fibrosis, which affect the normal erectile function of penis.

Aim

To investigate the role of miR-195-5p in regulating the Phenotype switch of the corpus cavernosum smooth muscle (CCSM) cells.

Methods

A small mother against decapentaplegic 7(Smad7) virus vector and a miR-195-5p mimics or an si-Smad7 viral vector and a miR-195-5p inhibitor were transfected into CCSM cells. The cells were obtained by primary culture of rat corpus cavernosum smooth muscle tissue. Real-time polymerase chain reaction (PCR) experiments, Western blotting, hematoxylin-eosin (HE) staining, transwell experiments, MTT assays, and flow cytometry were used to detect miR-195-5p, Smad7, phenotype switch markers of CCSM cells and related protein expression, as well as changes in cell morphology, migration, proliferation and apoptosis.

Main Outcome Measure

To study the regulation of miR-195-5p in CCSM cells by overexpression and silencing strategies.

Results

Overexpressed miR-195-5p promoted the transformation of CCSM cells from a contractile type to a synthetic type. Meanwhile, the migration ability and proliferation ability of CCSM cells increased, and the apoptosis rate decreased. The expression-silencing of miR-195-5p gave rise to the opposite effect. The results of the rescue experiment demonstrated that overexpressed Smad7 rescued the inhibitory of the switch of the CCSM cell phenotype from the contractile type to the synthesis type caused by overexpression of miR-195-5p alone. Moreover, the enhancement effect of the migration ability and proliferation ability of CCSM cells was also eliminated, and the apoptosis rate was increased. Silencing miR-195-5p and Smad7 at the same time resulted in the opposite effect.

Conclusion

miR-195-5p may regulate the phenotype switch of CCSM cells by targeting Smad7.

Zhang J, Zhang X, Zhang J, et al. miR-195-5p Regulates the Phenotype Switch of CCSM Cells by Targeting Smad7. Sex Med 2021;9:100349.

INTRODUCTIONErectile dysfunction (ED) is defined inability to achieve and maintain a sufficient penile erection to successfully engage in sexual intercourse. ED is a common disease that is a serious problem for men. There are more than 100 million ED patients in the world. It is estimated that by 2025, there will be more than 300 million ED patients worldwide.The worldwide prevalence and epidemiology of erectile dysfunction. The data show that the total prevalence of ED in China is 26.1%, while that in men over 40 years old is as high as 40.2%. ED seriously affects quality of life in men and is an important hidden danger to social stability. At present, the treatment of ED is insufficient, which is largely due to the complex pathogenesis of ED.The pathogenesis of ED is complex and has not been fully elucidated. Research shows that the causes of ED mainly include vascular disease, hypogonadism, increased oxygen free radicals, and autonomic and peripheral nerve damage.Maiorino M Bellastella G Esposito K. Diabetes and sexual dysfunction: current perspectives.Thorve VS Kshirsagar AD Vyawahare NS et al.Diabetes-induced erectile dysfunction: epidemiology, pathophysiology and management.Pathophysiology and treatment of diabetic erectile dysfunction. The main tissues exhibiting pathology are the penile vascular endothelium and cavernous smooth muscle. Pathology observed in patients with ED included a decrease in nitric oxide synthesis, impaired vascular endothelial function, a decrease in smooth muscle cells and an accumulation of collagen fibers.Thorve VS Kshirsagar AD Vyawahare NS et al.Diabetes-induced erectile dysfunction: epidemiology, pathophysiology and management.Pathophysiology and treatment of diabetic erectile dysfunction.Decaluwé K Pauwels B Boydens C et al.Treatment of erectile dysfunction: new targets and strategies from recent research. There are many molecular mechanisms involved in ED, such as disruption of the endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase G (PKG) pathway, upregulation of the transforming growth factor-β1(TGF-β1)/ Smad pathway, and upregulation of the RhoA pathway.Bivalacqua TJ Champion HC Usta MF et al.RhoA/Rho-kinase suppresses endothelial nitric oxide synthase in the penis: a mechanism for diabetes-associated erectile dysfunction. However, these findings have not completely solved clinical problems. It is still of great significance to further explore the pathogenesis of ED and find new therapeutic targets.

In the past, studies on the pathogenesis of ED have mainly focused on changes in the vascular endothelium and vasoactive factors. It is well known that a normal penile erection depends on a certain number of cavernous smooth muscle cells with diastolic function. It is worth noting that in most cases, ED eventually leads to changes in the structure of the cavernous body of the penis and to changes in specific functions, including different degrees of fibrosis, decreased cavernous smooth muscle cell number, and the loss of normal diastolic function in the cavernous body. In other words, collagen deposition and cavernous fibrosis are the common pathological outcomes of ED with different causes.

In recent years, the study of smooth muscle phenotype switching in cardiovascular disease has increased in depth. Vascular smooth muscle cells (VSMCs) have bidirectional differentiation functions. According to the differences in structure and function, VSMCs can be divided into contractile and synthetic phenotypes. The function of contractile cells is contraction, while the main functions of synthetic cells are proliferation, migration, and the regulation and secretion of extracellular matrix.Owens GK Kumar MS Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease.,Rzucidlo EM Martin KA Powell RJ. Regulation of vascular smooth muscle cell differentiation. When blood vessels are damaged or VSMCs cultured in vitro are stimulated by certain factors, VSMCs can transform from the contractile phenotype to the synthetic phenotype and acquire the proliferative ability. This process of morphological, structural and functional changes is called phenotypic transformation. During the phenotypic switch, contractile phenotype markers such as α-smooth muscle actin (a-SMA), smooth muscle myosin heavy chain (SMMHC), smoothelin, smooth muscle 22α (SM22α) and calponin 1 are downregulated. Corpus cavernosum is considered a special vascular tissue. The smooth muscle cells in the trabeculae of the corpus cavernosum are similar to vascular smooth muscle cells (VSMCs) in function.Goldstein AM Meehan JP Zakhary R et al.New observations on microarchitecture of corpora cavernosa in man and possible relationship to mechanism of erection. Cavernous smooth muscle (CCSM) is the structural basis of cavernous sinus space relaxation and penile erection, accounting for 40%-50% of the total composition of cavernous tissue. CCSM plays a key role in the hemodynamic changes of normal penile erections. Moreover, CCSM is the terminal tissue of various factors and plays an important role in the core position. When the number of CCSM cells is reduced or phenotypic transformation is induced by various causes, hemodynamic changes in the corpus cavernosum will be caused, and then the normal erectile function of the penis will be affected.Burchardt T Burchardt M Karden J et al.Reduction of endothelial and smooth muscle density in the corpora cavernosa of the streptozotocin induced diabetic rat.Kovanecz I Nolazco G Ferrini MG et al.Early onset of fibrosis within the arterial media in a rat model of type 2 diabetes mellitus with erectile dysfunction.Simopoulos DN Gibbons SJ Malysz J et al.Corporeal structural and vascular micro architecture with X-ray micro computerized tomography in normal and diabetic rabbits: histopathological correlation. Previous studies have confirmed that in penile tissue, upregulation of the TGF-β1/Smad signaling pathway can promote the switching of the corpus cavernosum smooth muscle (CCSM) cell phenotype from a systolic type to a synthetic phenotype.Leungwattanakij S Bivalacqua TJ Usta MF et al.Cavernous neurotomy causes hypoxia and fibrosis in rat corpus cavernosum. The increase in synthetic cells leads to an increase in collagen fibers, which leads to fibrosis of the penile cavernous body.Leungwattanakij S Bivalacqua TJ Usta MF et al.Cavernous neurotomy causes hypoxia and fibrosis in rat corpus cavernosum. Smad7 belongs to the Smad family, which is an intermediary signaling molecule that helps TGF-β1 bind with its receptor and then transmit a signal from the cytoplasm to the nucleus. Smad7 is an inhibitory Smad protein and has been well established as a key negative regulator of TGF-β1/Smad signaling. Smad7 can competitively and tightly bind to the TGF-β1 receptor, thus blocking the TGF-β1 signal transduction pathway.Negative regulation of TGF-beta receptor/Smad signal transduction.,Smad7: not only a regulator, but also a cross-talk mediator of TGF-β signalling.MicroRNAs (miRNAs) are highly conserved in structure and have offered a new point for the study of disease mechanisms. According to previous studies, miRNAs are closely associated with the onset of diseaseand mainly regulate target genes at the posttranscriptional level.The emerging role of microRNAs in the regulation of gene expression by nutrients.,The role of microRNAs in regulating neuronal connectivity. Recently, functional research on miRNAs has been performed in many areas of life science, such as cardiovascular disease, nervous system diseases, and cancer.Jingcao H Hui L Jianxiang W et al.MicroRNA regulation and therapeutic targeting of survivin in cancer.,The role of microRNAs in neural stem cells and neurogenesis. miR-195-5p, a less well-studied miRNA, plays crucial roles in many diseases, including cancersDu W Liu T Zhang Y et al.MiR-195-5p is a potential factor responsible for CPNE1 differential expression between subtypes of non-small cell lung cancer.Leng W Liu Q Zhang S et al.LncRNA AFAP1-AS1 modulates the sensitivity of paclitaxel-resistant prostate cancer cells to paclitaxel via miR-195-5p/FKBP1A axis.miR-195-5p suppresses lung cancer cell proliferation, migration, and invasion via FOXK1., acute kidney injuryXu Y Jiang W Zhong L et al.miR-195-5p alleviates acute kidney injury through repression of inflammation and oxidative stress by targeting vascular endothelial growth factor A. and gestational diabetes.Wang J Pan Y Dai F et al.Serum miR-195-5p is upregulated in gestational diabetes mellitus. Only a few targets of miR-195-5p are known, such as FKBP1ALeng W Liu Q Zhang S et al.LncRNA AFAP1-AS1 modulates the sensitivity of paclitaxel-resistant prostate cancer cells to paclitaxel via miR-195-5p/FKBP1A axis., MACC1Wan T Zheng J Yao R et al.LncRNA DDX11-AS1 accelerates hepatocellular carcinoma progression via the miR-195-5p/MACC1 pathway. and VEGFAWang H Niu X Jiang H et al.Long non-coding RNA DLX6-AS1 facilitates bladder cancer progression through modulating miR-195-5p/VEGFA signaling pathway.. It has been reported that miR-195-5p plays an important regulatory role in smooth muscle cells,Anti-apoptosis endothelial cell-secreted microRNA-195-5p promotes pulmonary arterial smooth muscle cell proliferation and migration in pulmonary arterial hypertension.Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p.Zampetaki A Attia R Mayr U et al.Role of miR-195 in aortic aneurysmal disease. and smooth muscle is an important effector tissue of ED. However, no study has reported the role of miR-195-5p in ED. In this work, we carried out functional studies of miR-195-5p in the phenotypic switching of CCSM cells. We also identified Smad7 as a novel target of miR-195-5p in CCSM. Taken together, these data indicate that miR-195-5p may serve as a promising therapeutic target for the treatment of ED.MATERIALS AND METHODS Separating and Identifying Corpus Cavernosum Smooth Muscle (CCSM) CellsNanjing Medical University Animal Laboratory (Nanjing, China) provided us with male Sprague-Dawley rats. Then, following protocols from other authors, explant cell cultures were prepared.Ouyang X Han X Chen Z et al.MSC-derived exosomes ameliorate erectile dysfunction by alleviation of corpus cavernosum smooth muscle apoptosis in a rat model of cavernous nerve injury.,Characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction. The Ethics Committee of Nanjing Maternal and Child Health Hospital approved this study. Experimental Grouping and Cell Transfection

The Smad7 overexpression vector, which was used to induce Smad7 overexpression (rescue experiment), and miR-195-5p mimics were used to establish 5 groups: miR-195-5p mimics (mimics), Smad7 overexpression (op-Smad7), miR-195-5p mimics+Smad7 overexpression (MO), blank control (BC), and negative control (NC) groups. Then real-time PCR was used to determine whether mature miR-195-5p and Smad7 were overexpressed in the cells.

A Smad7 expression-silencing vector was constructed. Smad7 was silenced by miR-195-5p-mediated overexpression (rescue experiment), and 5 groups were established: the miR-195-5p (inhibitor), Smad7 expression-silencing (si-Smad7), miR-195-5p inhibitor +Smad7 expression-silencing (IS), blank control (BC), and negative control (NC) groups. Then, real-time PCR was applied to determine whether mature miR-195-5p and Smad7 expression in the cells was silenced.

 Histopathological Evaluation

HE staining was performed on second-passage CCSM cells. Eighty percent of the cells were fused in the 6-well plate. The culture medium was removed. After washing them twice with PBS, 4% of the cells were fixed with POM. After 24 hours, hematoxylin and eosin staining was performed, and then the cells were observed under a microscope (Olympus; Tokyo, Japan).

 MTT Assay of Cell Viability

IPEC-1 cells in the logarithmic growth phase were inoculated into 96-well plates at a density of 1.0 × 104 cells/well. 5% CO2 was used to incubate the cells at 37°C for 24 hour and Se-AOS was exploited to treat them for 24 hours. Then, 0.5 mg/mL MTT solution replaced the medium. Incubation for 4 hour later, the 96-well plates were centrifuged at 4000 r/min for 10 minutes. Then we discarded the liquid in each well, added 150 μL of DMSO, and measured the OD values of each well at a wavelength of 570 nm.

 Isolation of RNA and Quantitative Real-Time PCR

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to collect CCSM cells. According to the manufacturer's protocol, the total RNA was extracted and purified by RNeasy Mini Kit (Qiagen, Hilden, Germany). Then, an M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) was used to perform reverse transcription for miRNA analysis. SYBR Premix Ex Taq Reagent test kit (TaKaRa, Foster City, CA, USA) on a StepOne Real-Time PCR System (Life Technologies) were leveraged for performing qPCRs. Expression levels of miR-195-5p or mRNAs were standardized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or the levels of U6 expression. The results were analyzed using the ΔΔCT method. The primer sequences were as follows: mir-195-5p RT- CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCCAATAT; mir-195-5p F- CGCAGCACAGAAATATTGGC; mir-195-5p R- CTCAACTGGTGTCGTGGAGTC; Smad7 F- GGGCTTTCAGATTCCCAACTT; Smad7 R- GCCGATCTTGCTCCTCACTT; GAPDHF- CGCTAACATCAAATGGGGTG; GAPDH R- TTGCTGACAATCTTGAGGGA G; hU6 F- CCTGCTTCGGCAGCACAT; h-U6 R- AACGCTTCACGAATTTGCGT; R- OPN sense: AGCACACAAGCAGACGTTTTG; R- OPN antisense: GCAACTGGGATGACCTTGATAG; R- Calponin1 sense: AGCAGG AGCTGAGAGAGTGGAT; and R- Calponin1 antisense: TTCTC CAGCTGGTGCCAGTT.

 Western Blotting

RIPA lysis buffer (ASPEN, NanTong, China) containing phenylmethylsulfonyl fluoride (Beyotime) and protease inhibitor cocktail (ASPEN, Pleasanton, CA, USA) were used to extract proteins. Then, the proteins were carried on a 10% SDS-PAGE gel via electrophoresis and transferred onto a polyvinylidene difluoride membrane. Five percent of skim milk was used to block the membrane for 2 hours and then together with an anti-Smad7 antibody were incubated (ab15116) for 24 hours and with secondary antibodies for 30 minutes. A developing agent was added to the membrane, and the results were recorded after X-ray exposure.

 Apoptosis Induction and Cell Viability Assay

H2O2 was added to the culture medium at a final concentration of 200 μM to induce apoptosis in CCSMs. The cells were placed in a 6-well plate with 2 × 105 cells/cm2 density, cultivated for 24 hours, and then the cell survival rate was determined. After rinsing the cells, they were cultivated with exosome-depleted media containing FBS with or without exosomes at 10 μg/mL or 20 μg/mL for 6 hours and then treated with H2O2 for 18 hours to induce apoptosis. Soon afterward, PBS was explored to collect and wash the cells twice, which was treated with Annexin-V-propidium iodide double staining. Cell viability was detected by a flow cytometer (BD FACS Canto, Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

 Statistical Analysis

The mean ± standard deviation (SD) was utilized to express the data. Comparison between 2 groups were analyzed with Student's 2-tailed t-tests, and comparisons among 3 or more groups were analyzed with 1-way ANOVA. P < .05 was considered statistically significant and is indicated in the figures.

DISCUSSIONErectile dysfunction is a serious problem for male patients. Because the pathogenesis of ED is very complex, there are many shortcomings in the current clinical treatment methods. For example, phosphodiesterase 5 (PDE5) inhibitors are commonly used in the clinical treatment of ED and ease the suffering of some patients.Mitidieri E Cirino G d'Emmanuele di Villa Bianca R et al.Pharmacology and perspectives in erectile dysfunction in man. However, for ED related to senility, diabetes, and nerve-damage, such treatments are not effective,Cai Z Song X Zhang J et al.Practical approaches to treat ED in PDE5i nonresponders.,Erectile dysfunction management for the future. and the cost is high. PDE5-Is are mainly used to alleviate symptoms and cannot cure the condition, and their long-term safety is not fully understood. Studies have found that PDE5-Is may be related to hearing impairment.Phosphodiesterase type 5 inhibitor use and hearing impairment. Other methods currently used for the clinical treatment of ED, such as testosterone replacement therapy, sexual psychotherapy, and lifestyle improvement, also have limitations.Hatzichristou D Rosen RC Derogatis LR et al.Recommendations for the clinical evaluation of men and women with sexual dysfunction., Therefore, exploring new mechanisms of ED pathogenesis and finding a more effective treatment and a cure for ED has been a popular area of research in the field of andrology.Past research on the pathogenesis of ED has focused on changes in the vascular endothelium and vasoactive factors. However, normal penile erections require a specific number of CCSM cells with diastolic function. Changes in the structure of the cavernous body of the penis, which can occur via fibrosis and reduction of cavernous smooth muscle cells, will cause the cavernous body to lose its normal diastolic function, resulting in ED. ED is closely related to phenotypic transformation of the CCSM. CCSM accounts for 40%-50% of the entire penile corpus cavernosum tissue composition. The phenotypic transformation of CCSM cells refers to the change in CCSM cells from "contractile" to "synthetic" or "proliferative." Smooth muscle myosin heavy chain (SMMHC), osteopontin (OPN), calponin, α-smooth muscle actin (α-SMA), smoothelin, and desmin can be used as molecular markers of contractile CCSM cells; vimentinThe role of osteopontin in kidney diseases.,Calcium in vascular smooth muscle cell elasticity and adhesion: novel insights into the mechanism of action. and collagen I can be used as molecular markers of synthetic or proliferative CCSM cells. The 2 phenotypes of CCSM cells have an important effect on penile erectile function. The proper proportion of CCSM cells of different phenotypes must be maintained, or the conversion from the contractile phenotype to the synthetic phenotype will lead to ED.Since the sex organs are situated on the superficial part of the body with relatively slow blood circulation, they may be a target for gene therapy.Recently, investigators found that miRNAs play a significant role during the onset of reproductive system diseases, cardiovascular diseases, cancer, and so on.The role of microRNAs in regulating neuronal connectivity.,Jingcao H Hui L Jianxiang W et al.MicroRNA regulation and therapeutic targeting of survivin in cancer. Therefore, determining the role of miRNAs in the diastolic function of CCSM cells is necessary.

In addition, miR-195-5p plays an essential role in the phenotypic transformation of CCSM cells, and Smad7 was confirmed to target miR-195-5p via prediction and detection. Therefore, this study explored the effect of miR-195-5p on Smad7 expression levels and the CCSM cell phenotype transformation via overexpression and inhibition experiments. Moreover, rescue experiments were performed to demonstrate the mechanism by which miR-195-5p regulates CCSM phenotypic phenotype transformation through Smad7.

Our research found that after overexpressing miR-195-5p suggested that the CCSM contractile markers Calponin 1, α-SMA, caldesmon, and Smad7 protein expression levels were significantly lower than those of the control group, while the CCSM synthetic markers OPN and collagen1 protein expression levels have opposite effects. The results of miR-195-5p silencing were contrary to the above. The above findings indicated that overexpressed miR-195-5p can affect the phenotypic transformation of CCSM cells. Overexpressed miR-195-5p can lead to the transformation from contractile CCSM cells to syngeneic CCSM cells, increase the synthesis of the CCSM, and increase collagen, which weakens or eliminates the diastolic function of the cavernous body, thus regulating the process of ED by remodeling the cavernous tissue structure. The increase in the number of synthetic cells leads to proliferation, migration, secretion and degradation of extracellular proteins, which is consistent with our experimental results.

In the rescue experiment, we found that miR-195-5p overexpression significantly downregulated Smad7 expression at the protein level. When miR-195-5p and Smad7 were overexpressed at the same time, the expression level of Smad7 protein was increased in the double overexpression group compared to the control group but was still lower than that in the Smad7 overexpression group. When miR-195-5p and Smad7 were simultaneously silenced, the protein expression level of Smad7 in the double silencing group was significantly lower than that in the control group but was higher than that in the Smad7 silencing expression group. These results suggest that miR-195-5p downregulates Smad7 expression at the protein level. In the rescue experiment, detection of Calponin 1, OPN and proteins related to CCSM phenotype transformation also showed that simultaneous overexpression of miR-195-5p and Smad7 could rescue the increase in synthetic CCSM caused by miR-195-5p overexpression alone, and the number of contractile CCSM cells increased. Moreover, the proliferation capacity of CCSM cells was also reduced, and the rate of apoptosis increased. Under normal homeostatic conditions found in healthy vascular physiology, smooth muscle cells (SMCs) ordinarily have low baseline levels of proliferationRegulation of differentiation of vascular smooth muscle cells.The role of apoptosis in vascular disease.Muto A Fitzgerald TN Pimiento JM et al.Smooth muscle cell signal transduction: implications of vascular biology for vascular surgeons.. SMCs with increased proliferation and decreased apoptosis may indicate a phenotypic switch from the contractile to the synthetic phenotype.In addition, the results of silencing miR-195-5p and Smad7 were the opposite. These results indicatethat miR-195-5p can regulate the phenotype switch of CCSM through Smad7.Previous studies have confirmed that Smad7 belongs to the Smad family. At present, 8 kinds of Smad proteins have been found in mammals, and they can be divided into 3 categories: (i) receptor-regulated Smads (R-smads), including Smad l, Smad2, Smad3, Smad5 and Smad8; (ii) common Smads (co-Smads), including only Smad4 in mammals; and (iii) inhibitory Smads (I-Smads), including Smad6 and Smad7.TGF beta-related pathways. Roles in Caenorhabditis Elegans development.,Itoh S Itoh F Goumans MJ Dijke PT. Signaling of transforming growth factor-β family members through Smad proteins. Smad proteins are signal-mediating molecules that can transmit signals from the cytoplasm to the nucleus after binding to TGF-β1 and its receptor. Smad7 is an inhibitory Smad protein and one of the main negative regulators of the TGF-β1/Smad signaling pathway. It can competitively and tightly bind to the TGF-β1 receptor, thus blocking the TGF-β1 signal transduction pathway.Negative regulation of TGF-beta receptor/Smad signal transduction.,Smad7: not only a regulator, but also a cross-talk mediator of TGF-β signalling. TGF-β is a multifunctional mediator that regulates proliferation, differentiation, migration, adhesion and apoptosis in different cell types, such as macrophages, activated T and B cells, dendritic cells, and so on.TGFβ pathway inhibition in the treatment of non-small cell lung cancer.,Nickel J Dijke PT Mueller TD. TGF-β family co-receptor function and signaling, Acta Biochim. A great number of studies have indicated that disorder of the TGF-β1/Smad pathway can lead to tissue fibrosis, which is an essential pathogenic mechanism. As 2 major downstream regulators of TGF-β1 signaling, Smad2 and Smad3 promote TGF-β1-mediated tissue fibrosis, while Smad7 plays a protective role against TGF-β1-mediated fibrosis as a negative feedback regulator of the TGF-β1/Smad pathway.Chen L Yang T Lu D-W et al.Central role of dysregulation of TGF-β/Smad in CKD progression and potential targets of its treatment.,Walton KL Johnson KE Harrison CA. Targeting TGF-β mediated SMAD signaling for the prevention of fibrosis.

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