| BackgroundErectile dysfunction (ED), one of the most common men’s sexual dysfunction, is a troubling problem. With the increased pace of life and pressure, the prevalence rate of ED gradually increased. Although ED is a benign disorder, it is closely related to people’s physical and mental health, quality of life for patients and their families. Thus, ED have a significant negative effects for men and their partners.It is reported that 52% of the men aged between 40-70 in the world are having ED in various degrees, and by the year of 2025,332 million males are estimated to suffer ED. According to a recent epiderniologic study, the prevalence of ED was 1%-10% in men younger than 40 years,2%-9% among men between 40 and 49 years, and it increased to 20%-40% among men between 60-69 years, reaching the highest rate in men older than 70 years (50%-100%). Although there are many methods of the treatment of ED. Oral medications (5 phosphodiesterasetype 5 inhibitors, PDE5i) are considered as the first line therapy for management of ED. If oral agents cannot be used or have insufficient efficacy despite appropriate dosing and education, second-line treatments should be addressed. When there is lack of efficacy or when there is dissatisfaction with other modalities, penile prostheses are often the best alternative for ED and are considered as the third line therapy for ED. However, a large part of ED patients are not sensitive to first line drugs, and unable or unwilling to accept the second-and third-line therapy. Therefore, searching a more preferably way for management of ED is imminent.ED is a multi-factor, complicated disease. The specific pathogenesis is not very clear. Therefore, to clarify the pathogenesis of ED is very important to find effective treatment measures. The penis is composed of trabeculae of corpora cavernosa and trabecular space. The cavernous trabecular is composed of a large number of smooth muscle cells, which is the main ingredients of penis contraction and relaxation. The corpus cavernosum smooth muscle tissue is the terminal tissue of penis erection process. They has always been in an important position in penis erection.As is known corpus cavernosum smooth muscle cell (CCSMC) has an importance impact during the process of erection. It makes the most of the contraction and relaxation of the corpora cavernosa penis, being the fundamental of penis’s normal erection, as well as the basics of terminal end of the impacts via vessels, nerves and internal secretion incretion. Some scholars have proposed that decreased number significantly, ultrastructure pathological changes and damaged systolic and diastolic function of CCSM cells casued by diabetes mellitus is an important cause of ED. Our previous study has confirmed that the expression of smooth muscle cell (SMC) marker proteins and contractility-associated genes, such as a-smooth muscle actin (a-SMA), smooth muscle myosin heavy chain (SMMHC), smoothelin and calponin, are significantly reduced in the tissue and cells of CCSM of diabetic rats with ED, and its amount is decresead and function is damaged. Therefore, the incidence of ED would closely associated with pathological changes and deletions CCSM cells. At present, there is no ideal way to improve these pathological states fundamentally.The studies showed that when the number or the function of the CCSMC changes, the function of erection will also be greatly affected. The amount of CCSMC and ED has a sharp direct correlation. When the amount of CCSMC goes down, the fibrosis of extracellular matrix increases, the trabeculae of corpora cavernosa penis can not maintain the normal function of contraction and relaxation of penis, the arterial blood thus could not flow into the sinusoidal organization sufficiently, which eventually cause of insufficient pressure in the sinusoidal of the corpora cavernosa penis to cut the vain blood net under the tunica, which then lead to ED. Apoptosis, called programmed death of cells, refers to the course of programmed death of cells in relative gene conditionings when the cells of some organism are activated to death paths which is caused by certain internal and external signals. Apoptosis mainly emphasizes the morphological transformations, relating to chromatin agglutination and peripheralization, the reduction of cytoplasm, fragmentation of cell nucleus, densification of cytoplasm, the disconnection with the peripheral cells, the fusion of endoplasmic reticulum and cell membrane, and finally the apoptotic bodies would be formed as a result of the fragmentation of cells, and then replaced by other cells. Along with the development of the cell biological technology, we now have a much clear understanding on the course of many sorts of apoptosis. However, the exact pathogenesis of the apoptosis is still not very well studied so far. The cell proliferation, the process of cell production by cell division, is a major feature of organisms, the foundation of reproduction of creatures, as well as a key need to keep the balance of the amount of cells and the basic functions of organisms. The apoptosis and proliferation of cells are closed connected, and both all along the beginning and the end of our life.Unlike myocardium and skeletal muscle cells which are terminally differentiated, CCSMCs maintain plasticity in cellular phenotype and can change from a contractile (differentiated) state to a synthetic (dedifferentiated) state in response to extracellular cues. The main function of contraction CCSMCs is systolic and diastolic, and maintaining the normal morphology and function. The main function of synthetic type of CCSMCs is cell proliferation, migration, secretion and degradation of extracellular proteins. CCSMCs have bidirectional differentiation, can remodel its phenotype under the stimulators in the local microenvironment. CCSMCs shift from a contractile phenotype to a synthetic phenotype, which is called as phenotypic modulation. The CCSMCs is similar to vascular smooth muscle cells. Our previous study has confirmed that CCSMCs possess the ability to modulate phenotype from a contractile to a proliferative state under hyperglycaemia condition. Our study also shows that CCSMCs switched their phenotype from a contractile to a proliferative state under the stimuli of PDGF-BB. It is reported that the phenotype transformation of CCSMCs is happened in animal models of hypertension and diabetes ED. The changes may play a key role in the pathogenesis of ED. Thus, it is important to study the phenotype transformation of CCSMCs.MicroRNAs (miRNAs) are family of small, non-coding RNA that exists in various chromosomes. These miRNAs only account for 2% of the total number of human genes but regulate more than 30% of the human gene expression. They play an important role in the regulation of cells, especially the occurrence of tumor, organ growth, and cell proliferation and differentiation. Several studies found that miR-145 is mainly expressed in vascular smooth muscle cells. It participated in transformation of smooth muscle cell phenotype, which is an important regulator of vascular smooth muscle phenotype. The expression of miR-145 is decreased in vascular injury and atherosclerosis. The phenotype would be reversed when the expression was inhibited. Many studies have found that miR-145 plays an important role in the phenotype of vascular smooth muscle cell transformation. However, the effect of miR-145 on the phenotypic transformation have been poorly defined. In our previous study, we observed that the expression of miR-145 is decreased in diabetic rats, and promoted phenotypic modulation of CCSMCs from a proliferative to a contractile state. Thus, We speculate that miR-145 also plays an important role in the phenotype transformation of CCSMCs.ObjectiveStable expression systems of silencing and overexpression of miR-145 can be built stably and efficiently by lentiviral vector. The effect of miRNA-145 on the phenotypic transformation of rat CCSMCs was further explored in cell experiments and animal model. This study aimed to provide a theoretical basis for the pathogenesis and treatment of ED.Methods1. The construction and identification of miR-145 silencing and over-expression of lentivirus vector.1) Construction of miR-145 silencing and overexpression of lentivirus vector2) The primary cultured and identification of rat CCSMCs3) RT-PCR was used to detect the infection efficiency2. The effect of miR-145 on proliferation and phenotype of rat CCSMCs in vitro.1) MTS was used to detect the effect of miR-145 on cell proliferation2) Flow cytometry was used to detect the effect of miR-145 on cell cycle3) Wound healing assay were performed to detect the PDGF-BB-induced CCSMCs migration4) The expressions of phenotypy-associated genes was determined by RT-PCR5) The expressions of phenotypy-associated genes was determined by Western blot3. The effect of miR-145 on erectile function and phenotype of rat CCSMCs in vivo.1) The effect of silencing miR-145 on erectile function in rats2) Immunohistochemical assay was used to detect the effect of silencing miR-145 on expression of phenotype marker in rat CCSMCs3) RT-PCR was used to detect the effect of silencing miR-145 on the mRNA expression of phenotype marker in rat CCSMCs4) Western blot was used to detect the effect of silencing miR-145 on the protein expression of phenotype marker in rat CCSMCsResults1. The construction and identification of miR-145 silencing and overexpression of lentivirus vector.1) The silencing and over-expression lentivirus vector of miR-145 were successfully constructed.2) We isolated and passaged primary cultures of cells from rat penile cavernous tissue fragments. The microscope showed that primary cultured cells showed smooth muscle cell growth characteristics. The immunofluorescence staining of a-SMA showed that the distribution of green fluorescence was in the nucleus and cytoplasm, the rate of positive cell was more than 95%.3) The RT-PCR analysis indicated that compared with the control group, the expression of miR-145 in the anti-NC group was decrease by 8%±3%. The difference was not statistically significant. However, the expression of miR-145 was decrease by 76%±5% in the anti-miR-145. The difference was statistically significant (P<0.05). Compared with the control group, the expression of miR-145 in the miR-NC group was increased by 1.52±0.5 fold. The difference was not statistically significant. The expression of miR-145 group cells in the miR-145 group was increased 135.5±9 times, the difference was statistical significance (P<0.05).2. The effect of miR-145 on proliferation and phenotype of rat CCSMCs in vitro.1) The results of MTS analysis showed that the proliferation ability in the anti-miR-145 group was increased, compared with the control group and anti-NC group, and the difference was statistically significant (P<0.05). However, the proliferation ability is weakened in the miR-145 group. Compared with the control group, the proliferation ability is increased in the PDGF-BB group. The proliferation was lower in the PDGF-BB+miR-145 group than the PDGF-BB group, but was higher compared with the miR-145 group. The differences between each group all have statistical significance (P<0.05).2) Flow cytometry showed that the percentage of cells in G0/G1 phase cells was 90.3%±1.94% in the control group. And the anti-NC group was 89.5%±2.16, the anti-miR-145 group was 71.6%±3.28%. There was not significant difference between the anti-NC group and the control group. However, there was significant difference between anti-miR-145 group and anti-NC group (P<0.05). The percentage of cells in S phase cells was 6.2%±0.87% in the control group. And the anti-NC group was 7.3%±1.13%, the anti-miR-145 group was 18.2%±1.16%. There was not significant difference between the anti-NC group and the control group. However, there was significant difference between anti-miR-145 group and anti-NC group (P<0.05). The percentage of cells in G2/M phase cells was 3.5%±0.75% in the control group. And the anti-NC group was 3.2%±1.15%, the anti-miR-145 group was 10.2%±3.15%. There was not significant difference between the anti-NC group and the control group. However, there was significant difference between anti-miR-145 group and anti-NC group (P<0.05).3) Wound-healing assay showed that the migration of CCSMCs in the anti-miR-145 group were notably increased in comparison with the control and anti-NC group. And the migration is decreased. The migration ability was significantly increased in PDGF-BB group than the control group. The migration ability in PDGF-BB+miR-145 group was decreased in PDGF-BB group, but is increased compared with the miR-145 group.4) The results of RT-PCR indicated that compared with the control group, the expressions of contractility-associated genes a-SMA, SMMHC and calponin was markedly decreased in the anti-miR-145 group (P<0.05). There is no different between the anti-NC group and control group. However, the expressions of contractility-associated genes a-SMA, SMMHC and calponin was markedly increased in the miR-145 group (P<O.05). The mRNA expressions of a-SMA, SMMHC and calponin was markedly decreased in the PDGF-BB group compared with the control group (P<0.05). But the mRNA expressions is increased in the PDGF-BB+miR-145 group (P<0.05). There is no different between the miR-NC group and control group.5) Western blot analysis showed that the protein expression of SMA and calponin was decreased in the anti-miR-145 group compared with the control group and anti-NC group. And they were increased in the miR-145 group. PDGF-BB inhibited the expressions of a-SMA and calponin. However, the expressions were increased in the PDGF-BB+miR-145 group.3. The effect of miR-145 on erectile function and phenotype of rat CCSMCs in vivo.1) There was no significant difference in MAP among the four groups. The ICP and ICP/MAP ratio were significantly lower in the anti-miR-145 group than the sham group, anti-NC group, and DMEM group (P<0.05).2) Immunohistochemical assay showed that the expression of a-SMA and calponin were downregulated in the anti-miR-145 group.3) The results of qRT-PCR showed that compared with the control group, anti-NC group, and DMEM group, the mRNA levels of a-SMA, SMMHC, and calponin were significantly decreased in the anti-miR-145 group (P<0.05). There was no significant difference among the other groups (P>0.05).4) The results of western blot showed that the anti-miR-145 group had a marked reduction in the protein level of a-SMA, SMMHC, and calponin as compared to the control group, anti-NC group and the DMEM group.Conclusions1. The silencing and overexpression lentivirus vector of miR-145 was successfully constructed.2. MiR-145 can inhibit the proliferation and migration of CCSMCs.3. MiR-145 can inhibit CCSMCs phenotype from contractile to a synthetic phenotype, called as phenotype modulation.4. The silencing expression of miR-145 attenuated erectile function of rats. It provided the theoretical basis for further research on the etiology and treatment of ED. |
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