3.1 CIH exposure significantly down-regulates miR-301a-3p in patients with ED and in rats induced with ED
ADSCs obtained from adipose tissues of SD rats displayed a typical fibroblastic-like morphology under the microscope (Fig. 1A). Oil Red O staining confirmed that they were undergoing adipogenesis (Fig. 1B). To confirm the identity of ADSCs, they were incubated with conjugated monoclonal antibodies against CD29, CD44, CD90, CD105, CD34 and vWF with isotype-identical antibodies (PharMingen) being used as controls. Immunofluorescence and flow cytometer results showed that ADSCs were positive for the mesenchymal stem cell (MSC) markers CD29, CD34, CD44, CD90 and CD 105 (Fig. 1C and D). The sequences between hsa-miR-301a-3p (human) and rno-miR-301a-3p (rat) were same (obtained from http://www.mirbase.org/) (Fig. 1E) and RT-qPCR analysis of serum samples collected from 30 ED patients showed that expression levels of hsa-miR-301a-3p in ED patients were significantly lower than those in healthy patients (p<0.001) (Fig. 1F). To further determine the expression of rno-miR-301a-3p, CIH exposure was done on SD rats. Results showed that rno-miR-301a-3p expression was inhibited at gene level in CIH exposed rats compared to control group (n=8, p<0.01) (Fig. 1G).
ADSC-derived exosomes were isolated and confirmed under a microscope (Fig. 1H) with transmission electron microscope (TEM) analysis showing that the diameter of most exosomes was approximately 100mm (Fig. 1I). Examination of ADSCs and exosomes using Western blot resulted in exosomes testing positive against exosome markers TSG101, CD9 and CD63 while ADSCs tested negative (Fig. 1J). After transfection with miR-301a-3p-overexpressed mimic, ADSCs and ADSC-derived exosomes were analyzed using RT-qPCR. Results confirmed the overexpression of miR-301a-3p in both ADSCs and ADSC-derived exosomes, compared to control and negative control groups (Fig. 1J). The results indicated that miR-301a-3p was significantly down-regulated in ED patients and CIH exposure rats. In addition, miR-301a-3p mimic had a good transcription efficiency in ADSCs.
3.2 CIH exposure negatively influences erectile function while miR-301a-3p-enriched exosomes treatment repairs the damage in SD rats
Masson trichrome staining of actin and collagen was done for each group where smooth muscle and connective tissue in the corpus cavernosum stained red and blue, respectively. Results indicated a decrease in the proportion of smooth muscle when CIH exposure rats were compared with the sham group after (p<0.001). When compared with CIH exposure group, miR-301a-3p-enriched exosomes treatment significantly promoted the proportion of smooth muscle indicating that exosomes treatment had therapeutic effects on the repair of smooth muscle (p<0.001) (Fig. 2A and B). Results obtained after Phalloidin staining indicated that CIH exposure destroyed F-actin. Significantly more stained cytoskeleton area was observed after normal exosomes treatment and miR-301a-3p-enriched exosomes treatment with the effects of the latter being more pronounced (Fig. 2C and D).
The ratio of ICP/RT-AP was used to assess erectile function (Fig. 2E) with results showing that CIH exposure significantly inhibited erectile function in SD rats (Fig. 2G). Normal exosomes and miR-301a-3p-enriched exosomes treatments had significant effects on recover ICP/RT-AP when compared to CIH exposure group (p<0.001) with miR-301a-3p-enriched exosomes having more pronounced effects (Fig. 2G). Western blot analysis was done to measure the level of myofibroblast formation with results indicating that α-SMA was down-regulated after CIH exposure. However, exosome treatment significantly increased the expression of α-SMA when compared with CIH exposure group (Fig. 2F).
To determine the level of nNOS in DNP of the penis, harvested tissues were prepared for immunofluorescence staining and Western blot analysis. Results showed no significant changes in the ratio of nNOS-positive nerve counts/DAPI in all areas of CIH exposure groups when compared with Sham groups indicating that CIH exposure did not alter NO release from peripheral nerve endings (Fig.3 A-C). This was confirmed by the results of Western blot analysis which indicated that CIH exposure had no effect on the expression of nNOS. Interestingly, CIH exposure stimulated the expression of iNOS while miR-301a-3p-enriched exosomes reduced its expression (Fig. 3D and E). Immunofluorescence staining of endothelial cells showed that eNOS expression decreased significantly after CIH exposure (p<0.00) when compared to Sham group. Exosomes treatment had positive effects on recovering the expression level of eNOS with miR-301a-3p-enriched exosomes treatment having significantly better results (Fig. 3F-H). Results indicate that CIH exposure negatively affected erectile function while miR-301a-3p-enriched exosomes treatment had significant remediation effects on SD rats, including ICP/RT-AP and expression levels of α-SMA and eNOS.
3.3 miR-301a-3p suppressed the level of PTEN and TLR4 in vivo
DNP tissue were collected from SD rats in all groups (Sham, CIH, CIH+EXO, CIH+EXO-301a) and analyzed using RT-qPCR to determine the signaling pathway used by miR-301a-3p to influence erectile function. RT-qPCR results showed that the expression level of miR-301a-3p in rat DNP tissue significantly decreased in CIH exposure group when compared to Sham group (p<0.001). There was no significant difference between CIH exposure group and CIH+EXO group, while miR-301a-3p was significantly overexpressed in CIH+EXO-301a group (Fig. 4A). Furthermore, the results showed a significant increase of PTEN and TLR4 gene levels in CIH and CIH+EXO groups (Fig. 4B and D). Treatment with miR-301a-3p reversed the expression of PTEN and TLR4 leading to a decrease in PTEN and TLR4 levels (Fig. 4B and D). Protein levels of PTEN and TLR4 in rat DNP tissue in each group were confirmed using Western blot analysis (Fig. 4C and E).
Results obtained after Western blot analysis in rat DNP tissue showed that CIH exposure directly induced overexpression of LC3I/II and p65 in the nucleus (Fig. 4C and E) indicating that the level of autophagy was up-regulated by CIH exposure. Up-regulated autophagy was also confirmed by the inhibited expression of p62 (Fig. 4C). Exosomes treatment increased the level of autophagy through overexpressing LC3I/II and p65 while the levels of p62 decreased with miR-301a-3p enriched exosomes having a more pronounced effect (Fig. 4C and E). These results suggest that PTEN and TLR4 can be directly targeted by miR-301a-3p.
Bioinformatics was used to predict the possible targets in the determination of the potential association between miR-301a-3p and PTEN/TLR4 with results showing that both PTEN and TLR4 could be possible targets of miR-301a-3p (Fig. 5A and C). Dual-luciferase reporter assay results showed that overexpression of miR-301a-3p reduced the intensity of fluorescence in CCSMCs transfected with TLR4-WT and PTEN-WT vectors while having no effect on CCSMCs transfected with TLR4-MUT and PTEN-MUT vectors (Fig. 5B and D). RT-qPCR and Western blot results further confirmed that both TLR4 and PTEN were inhibited at mRNA and protein level after cells were transfected with miR-301a-3p (Fig. 5E and F). Combining both sets of results made a clear indication that both PTEN and TLR4 are direct targets of miR-301a-3p.
3.4 miR-301a-3p-enriched exosomes inhibits CIH-induced apoptosis and up-regulates CIH-induced overexpression of autophagy in CCSMCs
For CIH exposure, CCSMCs were exposed to 5 s of 14% to 15% O2 during every 60 s cycle for 24 h. All cells were cultured for 24h and Co-culturing with miR-301a-3p-enriched exosomes for 48 h. Results obtained after CIH exposure of CCSMCs indicated that α-SMA was down-regulated at protein level. On the other hand, exosome treatment increased the level of α-SMA after CIH exposure with miR-301a-3p-enriched exosomes treatment having a more significant effect than normal exosomes treatment (Fig. 6A). Flow cytometry with Annexin V- FITC staining was used to assess the apoptosis rate with results showing that CIH exposure directly led to a significant increase in the apoptosis rate. However, exosomes treatment inhibited apoptosis with miR-301a-3p-enriched exosomes treatment having a significantly higher CIH-induced apoptosis rate inhibition than normal exosomes treatment (p<0.001) (Fig. 6 B and C). Levels of miR-301a-3p, PTEN, and TLR4 were analyzed using RT-qPCR. As we had hypothesized, results indicated that miR-301a-3p levels decreased after CIH exposure (p<0.01) when compared to control group. There was no significant difference between CIH group and CIH+EXO group (Fig. 6D). However, miR-301a-3p-enriched exosomes treatment led to a significant overexpression of miR-301a-3p levels in CCSMCS after CIH exposure (Fig. 6D). Both PTEN and TLR4 levels increased significantly after CIH exposure while miR-301a-3p-enriched exosomes treatment significantly decreased the mRNA expression level of PTEN and TLR4 (Fig. 6D). Results obtained after Western blot analysis confirmed the expression levels of PTEN and TLR4 (Fig. 6G and H).
In addition, Western blot results showed that CIH exposure directly induced overexpression of LC3I/II and p65 in the nucleus indicating that the level of autophagy was up-regulated by CIH exposure (Fig. 5G and H). Increased autophagy was confirmed by the inhibited expression of p62 (Fig. 5G). The level of autophagy was further increased by exosomes treatment, especially treatment with miR-301a-3p-enriched exosomes (Fig. 5G and H). Autophagic flux analysis was further done where CCSMCs were transfected with mRFP-GFP-LC3 with results showing that the quantity of autophagosomes, autolysosomes, and autophagic vacuoles increased significantly after CIH exposure (Fig. 5 I-L). There was no significant difference between CIH group and CIH+EXO group, while miR-301a-3p led to a significant increase of autophagosomes, autolysosomes, and autophagic vacuoles in CCSMCs ((Fig. 5 I-L)). Our findings suggest that miR-301a-3p-enriched exosomes treatment inhibits CIH-induced apoptosis and up-regulates CIH-induced overexpression of autophagy in CCSMCs.
3.5 PTEN or TLR4 overexpression significantly suppresses exo-301a-3p-induced positive effect on autophagy and inhibitory effect on apoptosis
PTEN-overexpression (PTEN-OE) and TLR4-overexpression (TLR4-OE) vectors were constructed to determine whether miR-301a-3p/PTEN/TLR4 signaling pathways were involved in the progression of apoptosis and autophagy. After transfection, overexpression of PTEN was detected using RT-qPCR and Western blot analysis (Fig. 7A and B). Western blot results confirmed that miR-301a-3p reversed the CIH-induced suppressive effects on α-SMA while PTEN-OE inhibited the expression level of α-SMA in CIH+EXO-301a+PTEN-OE group (Fig. 7C). On the other hand, miR-301a-3p-enriched exosomes treatment resulted in CIH-induced increase of HIF-1α and LC3I/II levels while at the same time inhibiting the expression of p62 (Fig. 7G). All miR-301a-3p-induced effects on levels of HIF-1α, LC3I/II, and p62 were reversed by PTEN-OE (Fig. 7G). Flow cytometry results indicated that CIH exposure led to a significantly high apoptosis rate with the effects promoting apoptosis being suppressed by miR-301a-3p. However, PTEN-OE reversed the miR-301a-3p-induced inhibitory effects on apoptosis (Fig. 7D and E). In addition, autophagic flux analysis confirmed that CIH-induced increase of autophagosomes, autolysosomes, and autophagic vacuoles after miR-301a-3p-enriched exosomes treatment (Fig. 7H-K). However, transfection with PTEN-OE significantly decreased the quantity of autolysosomes, and autophagic vacuoles in CCSMCs.
Potential roles of TLR4 were also determined where the effectiveness of TLR4-OE was checked at protein and gene level (Fig. 7L and M). Flow cytometry results showed that the high CIH-induced apoptosis rate was inhibited by miR-301a-3p. TLR4-OE significantly suppressed miR-301a-induced inhibitory effects thereby promoting increased apoptosis (Fig. 7N and O). Similar to results of CIH+EXO-301a+PTEN-OE group, miR-301a-induced expression of high α-SMA levels was significantly suppressed by TLR4-OE vectors (Fig. 7P). In addition, Western blot analysis confirmed that TLR4-OE reversed the miR-301a-3p-inhibted expression level of p62 in CCSMCs (Fig. 7Q and R). When combined, the results suggest that both PTEN-OE and TLR4-OE significantly suppressed miR-301a-3p-induced positive effects on autophagy and inhibitory effects on apoptosis.