MiR-103a-3p antagonism attenuates pneumonia via inactivating the PI3K/AKT/NF-κB signaling pathway by targeting PTEN

Pneumonia accounts for approximately 15% mortalities in adolescents worldwide. MicroRNAs (miRNAs) regulate numerous diseases including pneumonia. miRNA and mRNA expression levels were detected by real time polymerase chain reaction (RT-qPCR). Protein expression levels were determined by enzyme-linked immunosorbent assay (ELISA) and western blot. The interaction between phosphatase and tensin homolog on chromosome ten (PTEN) and miR-103a-3p was explored by dual luciferase reporter assay. Cell viability and cell apoptosis were detected by cell Counting Kit-8 (CCK-8) and flow cytometry. Herein, we discovered that PTEN was decreased and miR-103a-3p was overexpressed in Ana-1 cells of in vitro pneumonia model. miR-103a-3p downregulated the expression levels of PTEN. AntagomiR-103a-3p reversed the increased cell apoptosis and decreased cell viability and inflammatory cytokine expression levels (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6) induced by LPS in Ana-1 cells by PTEN. AntagomiR-103a-3p inhibited the activation of PTEN/PI3K/AKT/NF-κB signaling pathway induced by LPS in Ana-1 cells. Taken together, our findings exhibited that miR-103a-3p attenuated LPS induced pneumonia by blocking the activation of PTEN/PI3K/AKT/NF-κB signaling pathway and the following cell apoptosis as well as release of proinflammatory cytokines, suggesting that miR-103a-3p might serve as a novel therapeutic target for the treatment of pneumonia.


Background
In adolescents, pneumonia brings about 15% mortalities globally (1). In 2013, there were approximately 950,000 children who were less than 5 years old suffered with pneumonia (2).
Vaccines against viruses, for instance, Haemophilus influenzae type b and Streptococcus pneumoniae, are introduced in a number of countries; whereas, in developing countries, it remains challenging to achieve effective protection against the viruses (2). Therefore, it is urgent to investigate novel therapeutic methods for patients with pneumonia.
MiRNAs are a type of small, non-coding RNAs which regulate mRNA expression at the transcriptional and post-transcriptional levels by targeting their 3'UTR (3,4). MiRNAs function in numerous biological processes (5,6). Additionally, miRNAs regulate genes which are involved in the immune system (7), 3 including macrophages (8); and dysfunction of miRNAs contributes to pulmonary diseases (9).
PTEN was reported to be related with pneumonia, for instance, lipoxin A4 promotes the activation of alveolar epithelial sodium channel γ by miR-21/PTEN (13); miR-371b-5p induces cell proliferation in lung alveolar progenitor type II cells through targeting PTEN (14); inhibition of miR-92a inhibits LPSinduced pulmonary inflammation by targeting PTEN (15). Whether there are other miRNAs that can target PTEN are left to be investigated in the present study.
The current study demonstrated that, in LPS induced in vitro model of pneumonia, miR-103a-3p attenuated inflammation by inactivating PTEN/PI3K/AKT/NF-κB signaling pathway, indicating that miR-103a-3p has a potential to be a novel therapeutic target for the treatment of pneumonia.

PTEN was downregulated in pneumonia
After the establishment of the in vitro pneumonia model by LPS in Ana-1 murine macrophages, the PTEN mRNA and protein levels were measured by RT-qPCR and western blot, respectively. We found that, in comparison with the control group, PTEN mRNA ( Fig. 1A) and protein levels ( Figure 1B&C) were significantly downregulated by LPS.
Afterwards, LPS treated Ana-1 murine macrophages were applied for the following experiments.

miR-103a-3p inhibited PTEN expression
After the establishment of the in vitro pneumonia model by LPS in Ana-1 murine macrophages, the miR-103a-3p level was measured by RT-qPCR. The results exhibited that, in comparison with the control group, miR-103a-3p was significantly upregulated by LPS (Fig. 3A).
To verify the successful transfection of antagomiR-103a-3p into the Ana-1 murine macrophages, the difference of miR-103a-3p level between antagomiR-NC group and antagomiR-103a-3p group was detected by RT-qPCR. Results exhibited that, in comparison with the antagomiR-NC group, there was significantly downregulated miR-103a-3p level in the antagomiR-103a-3p group (Fig. 3B). Afterwards, the effects of antagomiR-103a-3p on PTEN mRNA and protein levels were measured by western blot and RT-qPCR, respectively. We found that, in comparison with the antagomiR-NC group, there were significantly upregulated mRNA and protein levels of PTEN in the antagomiR-103a-3p group ( Fig. 3C-E).

miR-103a-3p was correlated with PTEN mRNA in the serum of patients with pneumonia
PTEN mRNA level was significantly decreased in the serum of in patients with pneumonia compared to the healthy volunteers (Fig. 4A). miR-103a-3p level was significantly increased in the serum of in patients with pneumonia compared to the healthy volunteers (Fig. 4B). In the serum of in patients with pneumonia, miR-103a-3p was negatively correlated with PTEN mRNA level (Fig. 4C).

AntagomiR-103a-3p rescued LPS-induced decrease of cell viability in Ana-1 murine macrophages by targeting PTEN
To explore the function of miR-103a-3p on cell viability of Ana-1 murine macrophages, cell viability was measured by CCK-8. In comparison with the siRNA group, siRNA1 PTEN and siRNA2 PTEN significantly decreased the mRNA (Fig. 5A) and protein level (Fig. 5B&C) of PTEN. On account of the more important inhibitory effects on PTEN expression, siRNA2-PTEN was used in the subsequent experiments, and named as siRNA PTEN. Compared to control group, there was significantly downregulated cell viability of Ana-1 murine macrophages in LPS group, which was significantly rescued by antagomiR-103a-3p but not antagomiR-103a-3p+siRNA PTEN (Fig. 5D).

AntagomiR-103a-3p rescued LPS-induced increase of TNF-α, IL-1β and IL-6 levels by targeting PTEN
To explore the function of miR-103a-3p in inflammation of pneumonia, RT-qPCR and ELISA were applied for the measurement of mRNA and protein level of inflammatory cytokines, including TNF-α, IL-1β and IL-6. As for mRNA level, RT-qPCR results exhibited that, in comparison with the control group, there were significantly upregulated mRNA levels of TNF-α, IL-1β and IL-6 in the LPS group, which were significantly downregulated by antagomiR-103a-3p but not antagomiR-103a-3p+siRNA PTEN (Fig. 9A). As for protein level, ELISA results exhibited that, in comparison with the control group, there were significantly upregulated protein concentration of TNF-α, IL-1β and IL-6 in the LPS group, which was significantly downregulated by antagomiR-103a-3p but not siRNA PTEN+antagomiR-103a-3p (Fig. 9B).

Discussion
PTEN plays a pivotal role in various cellular processes, for instance, inflammation (16). miRNAs function by targeting mRNAs (3,4). In the present study, PTEN 3'UTR was found to be targeted by miR-103a-3p; in addition, PTEN was downregulated while by miR-103a-3p was upregulated by LPS, indicating the involvement of miR-103a-3p in pneumonia. As previously reported, miR-103a-3p plays a role in numerous cancers, for instance, miR-103a-3p regulated the progression of human gastric 6 cancer (17) and glioma (18). Currently, we further enriched the function of miR-103a-3p in human diseases, we also demonstrated the potential role of miR-103a-3p in pneumonia. Interestingly, during our conduction of the study, there was also a novel report showing the increased level of miR-103a-3p in the serum of children with pneumonia (19), which further verified our findings in pneumonia regarding the role of miR-103a-3p.
Cell apoptosis is involved in the process of pneumonia. Inhibition of cell apoptosis suppresses the progression of pulmonary fibrosis (20) and LPS-induced acute pneumonia (21,22). Herein, we found that, LPS induced cell apoptosis in Ana-1 murine macrophages was reduced by miR-103a-3p/PTEN axis, with the potential molecules and/or signaling pathways unelucidated.
PTEN inhibits the activation of PI3K/AKT signaling pathway, which was also related with cell apoptosis (17). Meanwhile, PTEN also regulates the activation of PI3K/AKT signaling pathway in pneumonia.
Such as, PTEN regulated AKT pathway in LPS-induced inflammatory lung injury (13); controlled PI3K/AKT pathway in lung alveolar progenitor type II cells (14); inactivated AKT/NF-κB pathway in pulmonary inflammation (15). Moreover, mounting studies are showing the involvement of PI3K/AKT signaling pathway in pneumonia, such as, knockdown of HAGLROS inhibits cell apoptosis and inactivates PI3K/AKT signaling pathway (22); inhibition of PI3K/AKT signaling pathway attenuates the reinfection of streptococcus pneumoniae (23). Consistently, the p-PI3K and p-AKT levels were measured in the present study, LPS induced upregulation of p-PI3K and p-AKT, which was reduced by antagomiR-103a-3p.
Furthermore, studies also have indicated the involvement of AKT/NF-κB pathway in LPS-induced inflammatory responses in murine macrophages (15,25). In addition, the activation of NF-κB is a prerequisite for production of various inflammatory cytokines, including TNF-α, IL-1 and IL-6 (26), generating a much more severe inflammatory response in stimulated macrophages (27). Expression levels of proinflammatory cytokines including IL-6, TNF-α and IL-1β are correlated with pneumonia (28), which are also reported to be induced by LPS in the in vitro pneumonia model in A549 cells or monocytes cells (29,30). Consequently, NF-κB p-p65, IL-6, TNF-α and IL-1β expression levels were measured in the present in vitro pneumonia model. miR-103a-3p was found to attenuate pneumonia, evidenced by the downregulation of cytokine release induced by LPS including TNF-α, IL-1β and IL-6 as well as the inactivation of NF-κB p-p65 induced by LPS from Ana-1 cells following antagomiR-103a-3p.
Conclusions miR-103a-3p suppressed cell apoptosis and inflammation in pneumonia by inhibiting the activation of PTEN/PI3K/AKT/NF-κB signaling pathway induced by LPS in macrophages.

Serum samples
The blood samples were obtained from 29 healthy children (16 males and 13 females, aged from 3 to

Cell apoptosis assay
Cell apoptosis was detected by Annexin-V/Dead Cell Apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were then diluted in 100 µl 1X Annexin-binding buffer to 1x10 6 cells/ml. Annexin V (5 µl) and propidium iodide (1 µl) were added to the above cell suspension. Cells were placed at room temperature for 15 min. Afterwards, annexin-binding buffer (400 µl) was added. Stained cells were analyzed by BD FACSCalibur flow cytometer (BD Biosciences).

ELISA
The Ana-1 murine macrophages (5x10 5 cells/well) in 4 groups were plated in their corresponding 24well plate, followed by incubation in 95% humidified atmosphere and 5% CO 2 at 37˚C overnight.

RT-qPCR
TRIzol (Invitrogen, Carlsbad, CA, USA) and mirVana kit (Applied Biosystems, Thermo Fisher Scientific, CA, USA) were used for the extraction of RNA in the detection of RNA and miRNA, respectively.
TaqMan Gene Expression Assays kit and TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific, CA, USA) were applied for the reverse transcription of RNA and miRNA, respectively. RT-qPCR reactions were carried out with the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). The expression level of miR-103a-3p and the mRNA levels of IL-6, TNF-α and IL-1β were normalized to U6 and GAPDH, respectively. The data was analyzed with the quantification 2 -ΔΔCq method (32).

Statistical analysis
Data were analyzed by GraphPad Prism software version 5.04 (San Diego, CA, USA). The differences between two groups were analyzed by student's t-test, while the differences among 4 groups were analyzed by one-way analysis of variance followed by Bonferroni's post hoc test. Data were presented as the mean ± standard error of the mean. Experiments were performed at least 3 times. P<0.05 indicated a statistically significant difference.

Consent for publication
All the parents or the legal guardians of the participants provided written informed consent prior to the conduction of the present study.

Availability of data and materials
The data are available from the corresponding author upon request.

Competing interests
There was not any type of conflict of interests in current study.     Inhibition of miR-103a-3p reversed LPS-induced increase of TNF-α, IL-1β and IL-6 levels by PTEN AntagomiR-103a-3p significantly rescued LPS induced up-regulation in mRNA (A) and protein levels (B) of TNF-α, IL-1β and IL-6, which were reversed by siRNA PTEN. ** p<0.01,