CircRNA-012091/PPP1R13B-mediated lung fibrotic response in silicosis via ER stress and autophagy
Running title: circRNA/PPP1R13B in SiO2-induced pulmonary fibrosis
Yusi Cheng1,2,3, Wei Luo1, Zhuang Li1, Mumin Cao1, Zixin Zhu1, Chen Han1, Xiaoniu Dai1, Wei Zhang1, Jing Wang1, Honghong Yao4, Jie Chao1,2,3#
1Department of Physiology, School of Medicine, Southeast University, Nanjing, Jiangsu, 210009, China
2Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu, 210009, China
3Department of Respiration, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu, 210009, China
4Department of Pharmacology, School of Medicine, Southeast University,
Nanjing, Jiangsu, 210009, China
# Corresponding author: Jie Chao, Ph.D. Jie Chao
ORCID ID orcid.org/0000-0002-7800-3557
Department of Physiology, School of Medicine, Southeast University 87 Dingjiaqiao Rd
Nanjing, Jiangsu, 210009, China Phone: +86-25-83272312
Fax: +86-25-83272312
Email: [email protected]
Abstract
Silicosis is a progressive fibrotic disease of lung tissue caused by long-term inhalation of SiO2. However, relatively few studies on the direct effects of SiO2 on lung fibroblasts have been performed. PPP1R13B is a major member of the apoptosis-stimulating proteins of the p53 family (ASPPs), but its role in pulmonary fibrosis is unclear. To elucidate the role of PPP1R13B in the pathological process of silicosis, this study explored the molecular mechanisms related to PPP1R13B and the functional effects of proliferation and migration of fibroblasts. Through lentivirus transfection, western blotting and fluorescent in situ hybridization experiments, we found that SiO2 downregulated ciR-012091 expression in lung fibroblasts and induced upregulation of downstream PPP1R13B. Transfection of L929 cells with PPP1R13B CRISPR NIC plasmid inhibited the upregulation of endoplasmic reticulum stress (ERS) and autophagy-related protein expression in lung fibroblasts treated with SiO2 and induced decreases in cell proliferation, migration, and viability. Transfection of L929 cells with the PPP1R13B CRISPR ACT plasmid induced increases in cell proliferation, migration, and viability. In addition, the ERS inhibitor salubrinal and the autophagy inhibitor 3-MA inhibited the increased migration of L929 cells transfected with the PPP1R13B CRISPR ACT plasmid. These results suggest that PPP1R13B regulated by ciR-012091 promotes the proliferation and migration of lung fibroblasts through ERS and autophagy and plays a crucial role in the development of pulmonary fibrosis in silicosis.
Keywords
silicosis, ciR-012091, PPP1R13B, endoplasmic reticulum stress, autophagy
Introduction
Silicosis is one of the most serious occupational diseases worldwide (1). Due to the lack of effective early diagnosis and monitoring methods for silicosis patients and exposed populations, the process of pulmonary fibrosis is irreversible once a patient is diagnosed with silicosis by chest X-ray (2-4). Therefore, identification of biomarkers for the diagnosis of silicosis and evaluation of the pulmonary fibrosis process have become urgent problems in research. The proliferation and migration of lung fibroblasts are important factors in pulmonary fibrosis (5-8), but the molecular mechanism underlying these activities has not yet been fully elucidated. PPP1R13B is a member of the apoptosis-stimulating proteins of the p53 family (ASPPs) (9). Previous studies have reported that the PPP1R13B-p53 complex can act on the promoter of pro-apoptotic genes by specifically enhancing the ability of p53 to bind DNA, thus promoting p53-induced apoptosis and regulating apoptosis (10, 11). Conversely, some studies have suggested that PPP1R13B can inhibit apoptosis in other systems (12). Our previous study found that SiO2 activated macrophages to promote the proliferation and migration of fibroblasts through the p53-mediated autophagy pathway (8), but the role of the p53-related protein PPP1R13B in pulmonary fibrosis is still unclear. Cell repair and apoptosis are related to endoplasmic reticulum stress (ERS) (13, 14). ERS is triggered by various stress stimuli, such as ischemia-reperfusion injury, oxidative stress and calcium homeostasis imbalance (15-17). ERS can induce an unfolded protein response (UPR) by inducing misfolded or unfolded protein aggregation in the endoplasmic reticulum (ER) (18). Appropriate ERS can induce upregulation of protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) and glucose-regulating protein 78 (GRP78 or Bip), which can enhance the ability of the ER to manage erroneous and unfolded proteins and to exert a cytoprotective effect. Excessive and persistent ERS can induce apoptosis through ER-related apoptosis molecules, such as GADD153/C/EBP homologous protein (CHOP), c-Jun N-terminal kinase (JNK) and caspase-12 (19). Our group reported that activated ERS was induced in HPF-a cells stimulated by SiO2 (20). PPP1R13B has also been reported to be associated with apoptosis or anti-apoptosis (12, 21). Therefore, we explored the relationship between PPP1R13B and ERS.
The main function of autophagy is to degrade damaged cellular structures, aging organelles, and unnecessary biological macromolecules. A
utophagy not only plays a role in degradation but also provides raw materials for reconstructing organelles in cells via recycling of cell structures. Imbalanced autophagy will lead to cell abnormalities and even cell death (22). Previous studies in our laboratory showed that autophagy was involved in the pathological process of silicosis (8, 23). Nonetheless, the role of PPP1R13B in autophagy remains to be explored.
Circular RNA (circRNA) is a new type of noncoding RNA. Its 3’ and 5’ ends form a closed loop through covalent bonds, showing different characteristics from linear RNA. CircRNA is not easily degraded by RNase and is more stable than linear RNA in vivo; therefore, it has potential application value as a clinical diagnostic and prognostic marker. Previous studies have reported that circRNA plays an important role in cardiovascular diseases, tumors, respiratory diseases etc. (24-26). CircRNA, which is ubiquitous in organisms, is likely a new class of important post-transcriptional regulators that regulate other kinds of RNA and proteins (27). Studies in our laboratory using microarray analysis revealed that 120 circRNAs were differentially expressed in SiO2-treated model mice compared with control mice. We found 73 upregulated and 47 downregulated circRNAs in a silicosis model (28-30). ciR-012091 is one of the downregulated circRNAs. The host gene of ciR-012091 is ppp1r13b, indicating that PPP1R13B may be involved in the pathological process of silicosis in mice. Therefore, we explored the roles of ciR-012091 and PPP1R13B in silicosis.
We hypothesized that the ciR-012091/PPP1R13B pathway is a key target for the treatment of fibrosis in silicosis. SiO2 regulates the expression of ciR-012091 in lung fibroblasts, induces abnormal expression of PPP1R13B downstream, and further induces activated ERS and autophagy, thereby regulating the proliferation and migration of fibroblasts and leading to pulmonary fibrosis. This study will reveal the mechanism of fibrosis in silicosisat the molecular and genetic levels and provide a new strategy for the prevention and treatment of silicosis.
Methods Cell culture
Mouse pulmonary fibroblasts (L929) and human pulmonary fibroblasts from adults (HPF-a) (ScienCell, Carlsbad, CA, USA) were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA). When the cells were 80% confluent, they were digested and passaged with 0.25% trypsin. Passages 3–10 (P3-10) were used in this experiment.
Western blotting
RIPA lysis buffer (MCL1-1KT, Sigma-Aldrich, St. Louis, MO, USA) was used to extract proteins from cells. After SDS-PAGE electrophoresis, the proteins were transferred to PVDF membranes (Millipore, Bedford, MS, USA). Then, the membranes were incubated with diluted primary antibodies against PPP1R13B and LC3b (Sigma-Aldrich, St. Louis, MO, USA), GAPDH and BECN (Santa Cruz, CA, USA), Bax, BCL2, PERK and P62 (Cell Signaling Tech., MA, USA), Bip (Abcam, Cambridge, UK), ATF6 (Absin, Shanghai, China) and alkaline phosphatase-conjugated secondary antibodies (ZSGB-BIO, Beijing, China).
Immunocytochemistry
Immunocytochemistry was performed as previously described (23).
Fluorescent in situ hybridization (FISH)
Cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100. Biotin-labeled DNA oligo probes (Invitrogen, Carlsbad, CA, USA) were hybridized with samples on coverslips in hybridization buffer (HB) solution overnight at 37 °C. After incubation in blocking buffer, the samples were incubated with anti-biotin HRP antibody (1:200). Then, the samples were restained with DAPI and observed under a fluorescence microscope (Olympus BX53, Olympus America, Inc., Center Valley, PA, USA).
Quantitative real-time PCR qRT-PCR was conducted as previously described (20). The primers are listed in Table 1.
In vitro scratch assa An in vitro scratch assay was conducted to evaluate cell migration as previously described (8). Digital images of the scratches were captured at different times. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay kits (Solarbio, Beijing, China) according to the manufacturer’s instructions.
Bromodeoxyuridine (BrdU) labeling
BrdU labeling was employed to examine cell proliferation as previously described (20). The signals were observed under a fluorescence microscope (Olympus BX53, Olympus America, Inc., USA).
CRISPR/Cas9 plasmid transfection
Approximately 5 × 104/ml cells were seeded into 24-well plates. After the cells were 40-80% confluent, the old medium was replaced with 200 μL of fresh medium without antibiotics. The transfection reagent (Santa Cruz, CA, USA) and transfection plasmid (Santa Cruz, CA, USA) were mixed evenly and incubated for 20 min. The mixed solution was dropped into the 24-well plates and mixed to render the medium homogeneous. After 12 h of transfection, fresh medium was supplemented with FBS. After 24-72 h of transfection, samples were collected for analysis.
Lentiviral transfection
Cells were transfected with circRNA-012091 by a lentivirus (HANBIO Inc., Shanghai, China) using a previously described method (23). The purity of culture was estimated using a fluorescence microscope (EVOS FL fluorescence microscope, Thermo Fisher Scientific Inc, Waltham, MA, USA). The details of the methods can be found in the supplementary information.
Statistical analysis
The data are expressed as the mean±SD. Unpaired numerical data were compared using an unpaired t-test (two groups) or analysis of variance (more than two groups). P < 0.05 was considered statistically significant.
Results
SiO2 induced upregulation of PPP1R13B in human tissue and HPF-a and L929 cells
Although PPP1R13B has been reported to be associated with cell survival and apoptosis, its role in fibrosis induced by SiO2 is not well understood (12, 31). In this study, the immunohistochemical expression of PPP1R13B in lung tissues from healthy and silicosis patients was detected. The results showed that the expression of PPP1R13B was upregulated in silicosis patients' lung tissues, which was accompanied by upregulation of vimentin (Fig. 1A). Moreover, the protein expression of PPP1R13B was upregulated in a time-dependent manner in HPF-a and L929 cells after SiO2 stimulation, with a peak response at 6 h (Fig. 1B-E). The immunocytochemical expression of PPP1R13B in L929 cells was also detected. SiO2 stimulation could induce upregulation of PPP1R13B (Fig. 1F). These results suggested that abnormal expression of PPP1R13B occurred in the pathological process of silicosis (Fig. 1).
PPP1R13B mediates the anti-apoptosis effect in L929 cells stimulated by SiO2 To elucidate whether the abnormal expression of PPP1R13B promoted apoptosis or survival in the process of fibrosis in silicosis, we transfected L929 cells with PPP1R13B CRISPR NIC plasmid and detected cell viability- an apoptosis-related proteins. First, western blot and real-time PCR were used to detect whether transfection was successful. The results showed that L929 cells transfected with PPP1R13B CRISPR NIC plasmid exhibited decreased mRNA expression of ppp1r13b and protein expression of PPP1R13B but no change in the expression of circ-012091 (Fig. 2A, B; Supp. Fig. S1A, B). In addition, the viability of L929 cells stimulated by SiO2 was significantly higher than that of control cells, while the viability of L929 cells transfected with PPP1R13B CRISPR NIC plasmid was significantly lower than that of the Con-NIC group (Fig. 2C). To determine whether PPP1R13B affected L929 cell survival via an anti-apoptotic mechanism, the expression levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins were measured using western blotting. The Bcl-2/Bax ratio was increased in a time-dependent manner in L929 cells stimulated by SiO2, suggesting that SiO2 can promote anti-apoptotic effects in fibroblasts (Fig. 2D, E). After transfection of the PPP1R13B CRISPR NIC plasmid, the Bcl-2/Bax ratio was decreased (Fig. 2F, G), suggesting that PPP1R13B promoted the anti-apoptotic response rather than apoptosis of L929 cells treated with SiO2.
PPP1R13B is involved in SiO2-induced L929 cell proliferation and migration
Accumulating evidence has shown that fibroblast proliferation and migration play an important role in pulmonary fibrosis in silicosis (5-7). We explored whether PPP1R13B is involved in the proliferation and migration processes of L929 cells stimulated by SiO2. The BrdU labeling assay showed that the ratio of positive nuclei in L929 cells transfected with the PPP1R13B CRISPR NIC plasmid was significantly lower than that in the Con-NIC group, indicating that PPP1R13B was involved in L929 cell proliferation (Fig. 3A, B). The 2D cell scratch assay showed that the scratch width of L929 cells transfected with the PPP1R13B CRISPR NIC plasmid was significantly wider than that of the Con-NIC group after SiO2 stimulation for 24 h, which further indicated that PPP1R13B was involved in the migration of L929 cells stimulated by SiO2 (Fig. 3C, D).
To further prove that PPP1R13B promoted the proliferation and migration of L929 cells stimulated by SiO2, the PPP1R13B CRISPR ACT plasmid was transfected into L929 cells. First, western blotting was used to detect whether transfection was successful. The expression of PPP1R13B protein in L929 cells transfected with PPP1R13B CRISPR ACT plasmid was significantly upregulated compared with that in the Con-ACT group (Fig. S2A, B), which was accompanied by increased cell viability as detected by MTT assay (Fig. S2C). A BrdU labeling assay showed that the ratio of positive nuclei of L929 cells transfected with PPP1R13B CRISPR ACT plasmid was significantly higher than that of the Con-ACT group (Fig. S2D, E). Cell scratch assays showed that the scratch width of L929 cells transfected with the PPP1R13B CRISPR ACT plasmid was significantly narrower than that of the Con-ACT group (Fig. S2F, G). These results further suggested that PPP1R13B promoted the proliferation and migration of L929 cells stimulated by SiO2 (Fig. S2).
PPP1R13B mediates ERS and autophagy in response to SiO2 in L929 cells
ERS and autophagy have been reported to be associated with cell repair or apoptosis and play important roles in respiratory diseases (32-34). However, the relationship between PPP1R13B and ERS and autophagy is unclear. First, we examined the expression of ERS- and autophagy-related proteins. The expression levels of the ERS markers PERK, Bip and ATF6 were upregulated in a time-dependent manner after SiO2 stimulation, which was accompanied by upregulation of the autophagy markers LC3b, BECN and P62 (Fig. 4A-D) as well as the ratio of LC3 II/I (Fig. S3A, B). In addition, the results showed that ERS and autophagy were involved in the SiO2-induced increase in L929 cell viability and migration according to interventions with inhibitors or inducers of ERS and autophagy to L929 cells (Supp. Fig. S4A-C). Moreover, the inhibitor of ER stress, salubrinal, was found to inhibit the upregulation of autophagy-related proteins LC3b and BECN induced by SiO2 (Supp. Fig. S5A-C). These findings suggested that ERS and autophagy were activated during SiO2-induced L929 cell proliferation and migration, and ERS can further activate autophagy. The immunohistochemical expression of PPP1R13B and PERK in L929 cells transfected with the PPP1R13B CRISPR NIC plasmid was decreased compared with that in the Con-NIC group (Fig. 4E; Supp. Fig. S6).
Additionally, the protein expression levels of PPP1R13B, BECN and LC3b (Fig. 4F, G) as well as the ratio of LC3 II/I (Fig. S3C, D) were downregulated after transfection of the PPP1R13B CRISPR NIC plasmid. The expression levels of the ERS-related proteins PERK, Bip, and ATF6 were also downregulated compared with those in the Con-NIC group (Fig. 4F, H). These results indicated that PPP1R13B is an upstream molecule related to ERS and autophagy.
PPP1R13B mediates L929 cell proliferation and migration via ERS and autophagy Next, we explored the molecular mechanism by which PPP1R13B promotes the proliferation and migration of L929 cells. The PPP1R13B CRISPR ACT plasmid was transfected into L929 cells. The viability of L929 cells transfected with the PPP1R13B CRISPR ACT plasmid was significantly higher than that of the Con-ACT group and was accompanied by an increase in cell migration. This effect was significantly reversed by the ERS inhibitor salubrinal (Sigma-Aldrich, St. Louis, MO, USA) and the autophagy inhibitor 3-methyladenine (3-MA) (Sigma-Aldrich, St. Louis, MO, USA) (Fig. S7A-D). These results indicate that ERS and autophagy are involved in PPP1R13B-mediated L929 cell proliferation and migration.
The expression of ppp1r13b mRNA and ciR-012091 in L929 cells in response to SiO2
Abnormal expression of circRNA is involved in the pathological process of many diseases. However, the role of circRNA in silicosis fibrosis has not yet been clearly elucidated. In this study, the results showed that the expression of ciR-012091 was downregulated in a time-dependent manner in L929 cells induced by SiO2, with no changes in the mRNA expression of ppp1r13b (Fig. 5A, B). This result is consistent with the microarray analysis data of the mouse silicosis model. The expression of ciR-012091 was downregulated in mouse lung tissue in the silicosis model group (SiO2) compared with that in the normal saline-treated group (NS) (Fig. 5C). The results of in situ hybridization also showed that the expression of ciR-012091 was significantly downregulated in L929 cells treated with SiO2 (Fig. 5D, Fig. S7E).
ciR-012091 mediates the protein expression of PPP1R13B in L929 cells exposed to SiO2
To further explore whether a regulatory relationship exists between circ-012091 and PPP1R13B, ciR-012091 lentivirus was transfected into L929 cells. The results showed that the expression of ciR-012091 increased 26.30-times after transfection of ciR-012091 lentivirus in L929 cells, while the mRNA expression of ppp1r13b did not change significantly after transfection (Fig. 6A, B). Conversely, the protein expression level of PPP1R13B was significantly lower than that of the Lv-con group after transfection (Fig. 6C, D). Combined with immunocytochemistry and in situ hybridization assays, the results showed that the expression of PPP1R13B decreased in the ciR-012091 lentivirus transfection group (Fig. 6E).
SiO2 induces activation of ERS and autophagy in HPF-a cells
Activated ERS and autophagy in HPF-a cells were also discovered. After stimulation with SiO2 in HPF-a cells, the expression levels of the ERS-related proteins PERK and Bip and autophagy-related proteins P62, BECN and LC3b, as well as the ratio of LC3 II/I, were upregulated in a time-dependent manner (Fig. S8A-E). At the same time, SiO2 promoted the migration of HPF-a cells, which was inhibited by the autophagy inhibitor 3-MA and enhanced by the autophagy inducer rapamycin (Sigma-Aldrich, St. Louis, MO, USA) (Fig. S8F, G).
Discussion
Silicosis is caused by long-term inhalation of a large amount of free silicon dioxide. The main manifestations of silicosis are diffuse alveolitis, disorder of the alveolar unit structure and pulmonary fibrosis, which eventually lead to severely damaged lung function (35, 36). Once silicosis occurs, it develops progressively, endangering the health of people in contact with silica and reducing their labor capacity and quality of life. Recent studies have shown that the proliferation and migration of pulmonary fibroblasts play a key role in the occurrence and development of pulmonary fibrosis (37, 38). Under stimulation by various injury factors, pulmonary fibroblasts are activated and migrate to the site of alveolar injury through the gaps in the epithelial basement membrane, where they proliferate and secrete a large amount of extracellularmatrix, initiate the repair process and eventually lead to pulmonary fibrosis. Therefore, blocking the proliferation and migration of pulmonary fibroblasts is an important factor to delay the progression of pulmonary fibrosis. This study focused on the molecular mechanism of fibroblast proliferation and migration induced by SiO2, providing new targets for the clinical diagnosis and treatment of pulmonary fibrosis.
Previous studies have shown that PPP1R13B regulates p53 and promotes p53-mediated apoptosis (31). Yamashita M et al. found that PPP1R13B (ASPP1) caused apoptosis in damaged hematopoietic stem cells (HSCs) and coordinated with p53 to limit HSC self-renewal (21). Double knockout of PPP1R13B and p53 in HSCs resulted in hematological malignancy in vivo (21). However, contrary to the tumor-suppressive activity of PPP1R13B, Vigneron et al. reported that cytoplasmic PPP1R13B inhibited apoptosis through control of Yes-associated protein (YAP) (12). He also reported that PPP1R13B had p53-independent activities (12). In the present study, upregulation of PPP1R13B expression was found in vivo and in vitro with SiO2 stimulation. We wanted to clarify which role of PPP1R13B plays: promotion of proliferation or induction of apoptosis. We found that transfection of L929 cells with PPP1R13B CRISPR NIC plasmid could induce a decrease in cell proliferation, migration, and viability. L929 cells transfected with the PPP1R13B CRISPR ACT plasmid showed increases in cell proliferation, migration, and viability. These results suggest that PPP1R13B promotes the proliferation and migration of L929 cells and plays a crucial role in the development of pulmonary fibrosis.
ERS is prevalent in human diseases. Factors that interfere with the function of the ER (e.g., hypoxia, nutritional deficiency, viral infection) can cause accumulation of unfolded proteins in the ER and lead to ERS. Several studies have reported that ERS is important in the progression of idiopathic pulmonary fibrosis (IPF) (39, 40). ERS induced tissue fibrosis through apoptosis, epithelial-mesenchymal transition (EMT), and activation of inflammatory responses (41, 42). Moreover, ERS can affect the differentiation of fibroblasts into myofibroblasts and then produce large amounts of collagen and other extracellular matrix components. As an inhibitor of ERS, 4-phenylbutyric acid (4-PBA) reduced TGFβ1-induced myofibroblast differentiation and collagen production in IPF patients (43). Upstream factors that induce ERS in IPF remain unknown. In this study, we found that ERS was activated in L929 and HPF-a cells stimulated by SiO2, which increased the viability of fibroblasts and promoted cell migration. L929 cells transfected with PPP1R13B CRISPR NIC plasmid inhibited the upregulation of ER stress-related protein expression in L929 cells treated with SiO2. The ERS inhibitor salubrinal inhibited the increased migration of L929 cells transfected with the PPP1R13B CRISPR ACT plasmid.
These results indicate that PPP1R13B is the upstream regulator of ERS in fibroblasts stimulated by SiO2. ERS is an important cause of autophagy because autophagy is highly sensitive to changes in intracellular and extracellular environments (44, 45). Autophagy is involved in protein metabolism, organelle renewal, cell differentiation, cell survival and death. Inhibition or promotion of autophagy may lead to the occurrence of many diseases. Due to the important role of autophagy in cell metabolism and structural remodeling, the function of autophagy in fibrotic diseases (such as liver fibrosis, pulmonary fibrosis, renal fibrosis, etc.) has attracted more attention in recent years (46, 47). Leptin accelerated the epithelial-mesenchymal transition (EMT) of A549 cells by decreasing the level of autophagy (48). Reduced autophagy is an important risk factor for the development of IPF (49). Previous studies in our laboratory found that autophagy was increased in fibroblasts cultured with the conditioned medium from macrophages exposed to SiO2, and the inhibition of autophagy reduced the proliferation and migration of fibroblasts (23). In the present study, we also found that SiO2 stimulation in L929 cells induced an increased level of autophagy, and the autophagy inhibitor 3-MA can inhibit the migration of fibroblasts and reduce cell viability. However, what is the upstream factor regulating autophagy in L929 cells under the stimulation of SiO2? In this study, L929 cells transfected with the PPP1R13B CRISPR NIC plasmid inhibited the upregulation of autophagy-related protein expression in L929 cells treated with SiO2.
The autophagy inhibitor 3-MA inhibited the migration of L929 cells transfected with the PPP1R13B CRISPR ACT plasmid. These results suggested that PPP1R13B was a key regulatory molecule of autophagy in SiO2-stimulated fibroblasts and induced the proliferation and migration of fibroblasts.
Circular RNA has been reported to be a novel potential biomarker and therapeutic target in respiratory diseases (20, 30). In this study, downregulated ciR-012091 and upregulation of its downstream target protein PPP1R13B were found in SiO2-stimulated L929 cells. Additionally, a certain regulatory relationship was discovered between them through lentivirus transfection experiments. CircRNA represents a new class of important post-transcriptional regulators. While competing endogenous RNA (ceRNA) was a main mechanism of circRNA (27), ciR-012091 may regulate PPP1R13B by another mechanism. Interestingly, ciR-012091 was formed from its host gene ppp1r13b, indicating that the balance of its circular form and linear form may be interrupted by SiO2. Another explanation is that ciR-012091 may increase the expression of PPP1R13B by regulating RNA-binding proteins (RBPs) (50). The specific regulatory mechanism of ciR-012091/PPP1R13B in the development of silicosis fibrosis remains to be further explored.
Conclusion
In summary, our study demonstrates that PPP1R13B promotes SiO2-mediated proliferation and migration of fibroblasts by activating ERS and autophagy. In addition, this study suggests that the activation of PPP1R13B can be regulated by decreased ciR-012091 in response to SiO2, providing a new strategy for the diagnosis and treatment of silicosis (Fig. 7).
Declarations Ethics approval
All animal procedures were performed in strict accordance with the ARRIVE guidelines, and the animal protocols were approved by the Institutional Animal Care and Use Committee of Southeast University. Human tissue samples were used in accordance with the approved guidelines of the Research and Development Committee of Nanjing Chest Hospital (2016-KL002-01) and with the Declaration of Helsinki.
Availability of data and materials
All relevant raw data and materials are freely available to any scientist wishing to use them.
Conflict of interest
The authors declare no competing financial interests.
Funding
The National Natural Science Foundation of China (nos. 81773796 and 81473263).
National Key R&D Program of China (2017YFA0104303).
Fundamental Research Funds for the Central Universities (No.
2242018K40028).
Author contributions
Y.C. performed the experiments, interpreted the data, prepared the figures and wrote the manuscript. W.L., X.D., W.Z. and J.W. performed the experiments and interpreted the data. Z.L., M.C., Z.Z. and C.H. performed the experiments. H.Y. and J.C. provided laboratory space and funding, designed the experiments, interpreted the data, wrote the manuscript and directed the project. All the authors read, discussed and approved the final manuscript. Acknowledgments
This study is the result of work that was partially supported by the resources and facilities of the core laboratory at the Medical School of Southeast University.
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