Inhibition of circular RNA 006029 alleviates pancreatic β-cell injury through the AKT/mTOR signaling pathway

doi:10.21203/rs.3.rs-4851054/v1

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Inhibition of circular RNA 006029 alleviates pancreatic β-cell injury through the AKT/mTOR signaling pathway

https://doi.org/10.21203/rs.3.rs-4851054/v1

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Type 1 diabetes mellitus (T1DM) is characterized by the damage of pancreatic β-cells induced by autoimmune responses. Circular RNAs (circRNAs) serve important regulatory functions in the pathogenesis of T1DM, but the underlying mechanisms require more substantiation. This study focused on a novel circRNA circ006029 to investigate its regulations on β-cell damage. The potential involvement of circ006029 in β-cell proliferation, apoptosis, autophagy, and inflammatory responses was investigated via experiments such as CCK-8, qRT-PCR, and immunoblot. The utilization of a cytokine mixture, and specific molecular blockers Rapamycin and Capivasertib were applied to investigate the pathway by which circ006029 regulates in β-cell damage. Transcriptome sequencing and bioinformatics analysis were conducted to explore differentially expressed mRNAs related to circ006029 regulation. The expression of circ006029 was observed to increase in damaged MIN6 cells. The inhibition of circ006029 serves a protective role in MIN6 β-cells by promoting β-cell proliferation and attenuating apoptosis. circ006029-knockdown could augment β-cell autophagy and attenuate apoptosis through the AKT/mTOR signaling pathway. Moreover, circ006029 might be involved in the inflammatory response of MIN6 cells. These findings suggest that circ006029 may serve a detrimental role in β-cell damage, which provides new ideas for exploring the mechanism of β-cell damage in early insulitis in T1DM.

Biological sciences/Cell biology/Cell death/Apoptosis

Biological sciences/Cell biology/Cell death/Autophagy

Biological sciences/Molecular biology/Non coding rnas

Health sciences/Endocrinology/Endocrine system and metabolic diseases/Diabetes/Type 1 diabetes mellitus

β-cells

circ006029

autophagy

apoptosis

type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) has been driving scientists’ attention for decades, especially since the 1970s when Bottazzo, G F et al. and MacCuish, A C et al. successively proved that the presence of circulating islet cell-related antibodies is an important mechanism of the kind of insulin-dependent diabetes [1, 2]. Increasing evidence has been proven that the most fundamental trigger of T1DM is the imbalance of the immune system, which would result in severe insulin deficiency by loss of insulin-producing islet β-cells. As well known, autoreactive T cells can act as the most important effector cells to induce β-cell apoptosis [3]. In addition, islet-infiltrating immune cells could secrete many types of pro-inflammatory factors, including cytokines such as IFN-γ, IL-1β, TNF-α, and IL-6, and chemokines such as IL-8, Monocyte Chemoattractant Protein-1 (MCP-1), and C-C Motif Chemokine Ligand 5 (CCL5) [3, 4], which could cause an irreversible damage of pancreatic β-cells, ultimately leading to absolute inadequate insulin secretion and β-cell apoptosis. Autophagy is an important endogenous mechanism of reducing ROS and protecting β-cell against apoptosis [5]. Pancreatic β-cell autophagy has been reported to be impaired in T1DM, which contributes to β-cell apoptosis in T1DM [6]. However, the specific regulatory mechanisms of pancreatic β-cell autophagy dysfunction in T1DM have not been clarified yet.

Non-coding RNAs (ncRNAs) have been reported to show significant regulatory roles in T1DM, especially in the inflammatory process. Most of which were long non-coding RNAs (lncRNAs), mircoRNAs (miRNAs), etc. Motterle, Anna et al. revealed significant modulations of lncRNAs via different inflammatory pathways in injured β-cells in NOD mice undergoing insulitis [7]. From clinical patient specimens, lncRNA SRA was substantiated to act as an miR-146b sponge, closely associated with metabolic reprogramming and β-cell apoptosis [8]. Dysregulations of plasma-derived exosomal lncRNAs currently have been verified to favor β-cell injury and contribute to T1DM progression [9]. Roggli E et al. reported the miR-29 family as pivotal promoters of β-cell death via downregulating the antiapoptotic protein Mcl1 expression [10]. Interestingly, extrinsic miRNAs such as miR-142-3p/-5p, and miR-155, which were transported from T lymphocyte-derived exosome, were also found to favor the loss of β-cells [11]. Our previous findings also pointed that miR-101a and miR-30b not only affect insulin production but trigger β-cell failure upon the overexpression of these miRNAs [12].

Circular RNAs (circRNAs) are a class of ncRNAs characterized by a covalently closed single-stranded RNA loop with no 5’ and 3’ ends. Research on the interplay between circRNAs versus β-cell damage has been shown during the past few years. circHIPK3 and ciRS-7/CDR1as have been demonstrated to have protective potentials on insulin-secreting cells by preventing the occurrence of apoptosis [13]. Monocyte-specific circPPM1F was verified to be involved in MΦ activation, and the overexpression of circPPM1F exacerbated β-cell injury [14]. Our previous research demonstrated that prolonged exposure to inflammatory conditions led to β-cell failure and the dysregulation of circRNAs, including circRNA 006029 (upregulated), circRNA 000286 (downregulated), and 017277 (downregulated) in vitro, suggesting a potentially crucial role of circRNAs in β-cell injury during the progression of T1DM [15].

Herein, we primarily investigated the potential regulatory mechanism of circRNA 006029 in cytokine-induced β-cell injury. We demonstrated that circ006029 is involved in pancreatic β-cell injury by regulating the AKT/mTOR signaling pathway.

circ006029 is increased in cytokine-induced injured MIN6 cells

In our previous study, to figure out the potential functions of circRNAs in the initial stage of T1DM, we treated MIN6 cells with a cytokine cocktail containing IFN-γ, IL-1β, and TNF-α, and preliminarily screened certain dysregulated circRNAs through RNA sequencing. In the present study, our experimental result was in line with previous findings [15], demonstrating an upregulation of circ006029 when MIN6 cells underwent cytokine-induced inflammatory injury (Fig. 1A). Circ006029 (ID: mmu_circ_0000172; Position: chr10:62233602–62253790) belongs to exonic circRNA and is derived from the exons of CCAR1. circBase shows the specific junction site of circ006029 (Fig. 1B), and thus we designed specific primers for both circ006029 and CCAR1. Furthermore, according to qRT-PCR, circ006029 was demonstrated to exhibit a more stable characteristic compared to its linear counterpart CCAR1, either upon the digestion of RNase R or undergoing the interference of Actinomycin D (Fig. 1C and 1D). For the localization of circRNA, RNA fractionation assays were further performed. The cytoplasmic and nuclear RNA of MIN6 cells were separated and extracted, and qRT–PCR was performed to determine circ006029 expression in the cytoplasm and nucleus. Here, circ006029 was observed preferentially expressing in the cytoplasm (Fig. 1E). In brief, our preliminary data verified the increase of circ006029 in cytokine-induced damaged MIN6 cells and the stability of circ006029, revealing a potential role that circ006029 might serve in inflammatory cytokines-induced damaged pancreatic β-cells.

Knockdown of circ006029 facilitates proliferation and alleviates injury of MIN6 pancreatic β-cells

Owing to the upregulation of circ006029 in cytokine-induced pancreatic β-cell damage, we speculated a detrimentally regulatory role which circ006029 might play in β-cell destruction induced by inflammatory cytokines. Firstly, we verified the specific knockdown efficiency of circ006029 siRNA in MIN6 cells transfected with circ006029 or NC siRNA. As shown in Fig. 2A and Fig. 2B, the expression level of circ006029 was significantly decreased in MIN6 cells with circ006029 knockdown, while the mRNA and protein levels of CCAR1 displayed no significant change in the group with circ006029 knockdown (Fig. 2A and 2B). CCK-8 assay revealed that the inhibition of circ006029 could promote the proliferation of MIN6 β-cells (Fig. 2C). Meanwhile, circ006029 inhibition could increase the protein levels of PCNA and cyclin D2 (Fig. 2D). Next, we observed the regulatory effect of circ006029 on cytokines-induced β-cell damage. The flow cytometry data manifested a decreased apoptosis rate (Q2 + Q3) in MIN6 cells with circ006029 knockdown, and the necrosis rate (Q1) was concurrently reduced compared with the cells transfected with NC siRNA (Fig. 2E). The expression levels of apoptosis-related proteins, cleaved-caspase 3 and bax were observed to be declined in MIN6 cells with circ006029 knockdown, (Fig. 2F). These data demonstrated an important role of circ006029 in regulating β-cell proliferation and apoptosis.

Knockdown of circ006029 increases MIN6 cell autophagy through the AKT/mTOR signaling pathway

Autophagy is a protective mechanism maintaining cellular homeostasis and may protect cells from apoptosis [5]. Thus, we sought to explore whether circ006029 could improve β-cell survival through regulating β-cell autophagy. Preliminary immunoblotting results displayed a significant downregulation of p62 and an increased ratio of LC3 II/I in MIN6 cells with circ006029 inhibition, giving a hint of the participation of circ006029 in β-cell autophagy (Fig. 3A). Autophagy flux was monitored in MIN6 transfected with GFP-mCherry-LC3b plasmids, so we could measure the tandem fluorescence in different conditions. In a standard autophagy flux assay, GFP-mCherry-LC3b shows green (due to GFP) and red (due to mCherry) double fluorescence in autophagic vesicles (AVs) and only red fluorescence in autolysosomes (ALs), indicating spatiotemporal progress of formation and maturation of AVs. As shown in Fig. 3B, the increased autophagic flux was observed to occur in MIN6 cells transfected with circ006029 siRNA. The cells with circ006029 knockdown showed more apparent and brighter red granular AVs and ALs when compared to the control. Such observed results were more evident when cells were treated with Rapamycin, an autophagy inducer that promotes autophagy through inhibition of the nutrient-sensing molecule mammalian target of rapamycin (mTOR) [16] (Fig. 3B).

Based on these findings, we further confirmed whether circ006029 regulates β-cell autophagy through the AKT/mTOR pathway. As shown in Fig. 3C, the decreased phosphorylation of mTOR (p-mTOR) suggested an activated autophagy process in MIN6 cells with circ006029 knockdown. Then, we applied Rapamycin to observe the effect of circ006029 on autophagy activation. Decreased expression of p-mTOR implied enhanced autophagy in MIN6 cells with circ006029 knockdown. Furthermore, the lower level of p62 and the increased ratio of LC3II/I were observed in the cells transfected with circ006029 knockdown, either along with the treatment of rapamycin or not (Fig. 3D). As illustrated in Fig. 3C, the decreased phosphorylated AKT (p-AKT) suggested that the AKT participates in circ006029-regulated MIN6 cellular events as well. Thus, we further employed the pan-AKT kinase inhibitor Capivasertib (AZD5363) to determine whether AKT inhibition affected circ006029 regulation on β-cell autophagy. Capivasertib is a novel pyrrolopyrimidine-derived compound that can inhibit three isoforms AKT1, AKT2, and AKT3, preventing the phosphorylation of the AKT downstream substrates and resulting in an accumulated p-AKT level and an inhibited AKT kinase activity [17]. Immunoblotting results revealed that the levels of p-AKT, p-mTOR, and p62 were reduced in MIN6 transfected with circ006029 siRNA, while the ratio of LC3 II/I was increased (Fig. 3E), whether with the application of Capivasertib or not. The autophagy level was further augmented after using Capivasertib. These data suggested that circ006029 participated in autophagy regulation via the AKT/mTOR pathway.

Knockdown of circ006029 increases autophagy and concomitantly attenuates apoptosis of MIN6 cells

To investigate whether the altered autophagy affects MIN6 cell apoptosis, we further measured the expression of apoptosis-related proteins, such as bcl-2, bax, and cleaved-caspase 3 after treating cells with or without Rapamycin and Capivasertib respectively. We observed a dampened apoptosis level in MIN6 cells with circ006029 knockdown compared to cells transfected with NC siRNA, regardless of the Rapamycin or Capivasertib application. The dampened apoptosis process was supported by less expression of bax and cleaved-caspase 3 (Fig. 4A and 4B). To summarize, the data above underscore the inhibition of circ006029 increases autophagy and concomitantly attenuates apoptosis of MIN6 cells via the AKT/mTOR regulation.

Circ006029 may be involved in the inflammatory response of β-cells

Considering the increase of circ006029 in inflammatory cytokines-treated β-cells, we presumed that circ006029 may also be involved in inflammatory pathways such as NF-κB, an essential transcriptional factor contributing to inflammation and immune response [18]. Co-transfection of the NF-κB activity luciferase reporter plasmid and circ006029 siRNA could result in a significantly decreased activity of NF-κB (Fig. 5A). The following immunoblotting results also underscored the inactivation of the NF-κB signaling, which displayed a reduced level of iNOS and p-p65 (Fig. 5B). These results implied a regulatory role of circ006029 may serve in pancreatic islet inflammation.

Changed Expression Profile of mRNAs in circ006029-depleted MIN6 cells

To investigate the potential mechanisms of circ006029 participation in inflammatory cytokine-induced β-cell destruction, transcriptome sequencing was conducted to show various protein-coding genes that were differentially expressed under the condition of circ006029 knockdown. Hierarchical cluster analysis manifested 73 upregulated mRNAs and 59 downregulated mRNAs in terms of the criteria of fold change (FC) > |2.0| and p < 0.05, upon the knockdown of circ006029 (Fig. 6A and 6B). We selected 4 mRNAs (Lgals3, Pax4, Spink1, Ifitm10) from the sequencing data and validated their expression in MIN6 cells transfected with circ006029 siRNA. qRT-PCR results showed a general consistency with transcriptome sequencing that depleting circ006029 in MIN6 cells significantly upregulated the expressions of Lgals3, Spink1, but dampened the expressions of Pax4 (Fig. 6C).

To investigate the potential biological interplay of these mRNAs, we performed GO analysis based on three categories, including Biological Process, Cellular Component, and Molecular Function. Figure 7A and 7B displayed significant enrichment of putative gene clusters in the top 30 upregulated and downregulated GO Terms. Among these GO Terms, immune- or cell viability-related accounted for a notable portion, particularly, apoptosis and autophagy regulatory genes were observed in these enrichments. To illustrate, pro-apoptosis-related genes cytochrome c, testis (Cyct) and serine/threonine/tyrosine interacting-like 1 (Styxl1) were significantly downregulated after the knockdown of circ006029; Nucleotide-binding oligomerization domain containing 2 (Nod2), substantiated to protect cells from bacteria attacking through promoting autophagy in Crohn diseases [19], and Galectin-3 (Lgals3), verified to interact with autophagy-related proteins and facilitate autophagy [20], were both displayed an increased expression in circ006029-knockdown β-cells. Additionally, KEGG pathway analysis showed that the differentially up- or down-regulated genes were mainly enriched in the immune system, endocrine and metabolic disease, and signal transduction pathways (Fig. 7C and 7D). These pathways likewise involved genes, which were demonstrated to refer to apoptosis, such as lymphocyte-specific 1 (Lsp1) [21]. Thus, these data implied a potential regulatory role of circ006029 may serve in pancreatic β-cell injury.

To bring it all together, our investigation identified a regulatory role of circ006029 in pancreatic β-cells. Knockdown of circ006029 alleviates β-cell apoptosis and improves cell proliferation by promoting cell autophagy via the AKT/mTOR signaling pathway. It also enlightens us that circ006029 could become the potential intervening target during the early injury of pancreatic cells, due to its attenuating cell damage and rebuilding pancreatic cell functions.

T1DM is a chronic autoimmune disease characterized by the infiltration of immune cells and the destruction of pancreatic β-cells. One key aspect of β-cell damage is the mistaken attack by the immune system. At the early stage of insulitis, APCs, macrophages (MΦ), and dendritic cells (DCs) migrate to pancreatic lymph nodes, further recruiting and activating T cells. Activated T cells release a series of proinflammatory factors such as IFN-γ, IL-1β, IL-2, and TNF-α, and trigger a cascade of immune responses. Such inflammatory responses serve a crucial role in the process of β-cell apoptosis, ultimately resulting in a loss of β-cells and absolute insulin deficiency [3, 22]. Although treatments have been improved [23, 24], intensifying research into the mechanisms of β-cell damage so that to seek novel therapeutic strategies or early interventions is still important.

The involvement of ncRNAs in human disorders has been proved, among which, lncRNAs and miRNAs are currently the well-studied classes of ncRNAs. Identified lncRNAs and miRNAs contributing to T1DM may either directly regulate β-cell apoptosis or be expressed in other cells, giving rise to an indirect effect on β-cell survival [12, 25–27]. CircRNA is another class of the ncRNAs that have been verified to exert critical regulatory functions in the onset and progression of many diseases. Increasing data and certain functions of circRNAs have been elucidating. CircHIPK3 was found to regulate glucose transportation of β-cells and protect β-cells from apoptosis [13]. CircPPM1F could induce pancreatic β-cell injury by activating M1 macrophages [14].

Based on our preliminary studies [15, 28], we further investigated the possible functions of the increased circRNA, circ006029, in β-cell damage. In the present study, we yet again verified the upregulated circ006029 in mouse pancreatic β-cells (MIN6) upon the stimulation of cytokine cocktails of IFN-γ, IL-1β and TNF-α, confirming the potential involvement of circ006029 in β-cell injury (Fig. 1A). The knockdown of circ006029 was found to promote MIN6 proliferation, reduce the apoptotic ratio, and decrease the levels of apoptosis-related proteins (Fig. 2). Thus, we speculated that the presence of circ006029 may detrimentally regulate β-cell survival.

Intriguingly, increased autophagy in MIN6 cells with circ006029 knockdown was observed. Autophagy is an important cellular process responsible for maintaining cellular homeostasis [5]. It is an important mechanism protecting β-cells from apoptosis upon the initial of β-cell damage [29]. Moreover, increasing evidence has shown the importance and involvement of circRNA in cell autophagy [30, 31]. Our data substantiated an enhanced autophagy in circ006029-knockdown MIN6 cells (Fig. 3), manifested as the changed expression of autophagy-related proteins, and the altered displayed fluorescence in autophagic vesicles (AVs) and autolysosomes (ALs) respectively. As the evidence of ncRNA-involved cell autophagy via the AKT/mTOR pathway has been increasing [32, 33], we further traced potential associated molecules. Significant alterations of phosphorylated AKT/mTOR, apoptosis- and autophagy-related protein levels were found occurring in MIN6 cells with circ006029 knockdown. These data denoted that the regulation of circ006029 on β-cell survival might in all likelihood associated with the AKT/mTOR signaling pathway (Fig. 3). The AKT pathway has been elucidated to negatively regulate cell apoptosis [34]. Interestingly, we observed that the inhibition of circ006029 could decrease the phosphorylation of AKT but reduce MIN6 cell apoptosis (Fig. 3C). We also observed that inhibiting the AKT by using Capivasertib (AZD5363) could increase the level of apoptosis (Fig. 4B). Capivasertib has been proven to have a potent inhibition to the phosphorylation of the AKT downstream substrates [17]. We thus speculated that the effect of circ006029 knockdown on the AKT inhibition exists, but partially. This might explain the non-significant change in the bcl-2 level. Nevertheless, the protective role (i.e., the promotion of autophagy) of circ006029 knockdown on MIN6 β-cells was still unignorable.

Considering that the β-cell damage was mainly mediated by the proinflammatory factors, we additionally explored the NF-κB, a critical transcription factor regarded significant in the inflammatory response [18], to see whether circ006029 participates in β-cell inflammatory response. We observed a significant restraint of NF-κB activity and decreased protein levels of iNOS and p-p65 in β-cells with circ006029 knockdown (Fig. 5), further elucidating a potential involvement of circ006029 in β-cell inflammation.

Additionally, in this study, a series of differentially expressed mRNAs were identified in β-cells with circ006029 knockdown, among which, certain genes have been recently clarified to contribute to cell autophagy, such as Nod2 and Lgals3 [19, 20]. The related pathways were mainly focused on immune system, endocrine and metabolic disease, and signal transduction, implying possible roles of circ006029 in regulating inflammatory responses.

To summarize, in this study, the knockdown of circ006029 was demonstrated to alleviate β-cell inflammation and reduce cell apoptosis. The promotion of proliferation and heightened autophagy of β-cells also substantiated such protective effects of circ006029 silence. Furthermore, we also proved that circ006029 might contribute to autophagy via the AKT/mTOR signaling pathway. All the results implied that the presence of circ006029 may drive a detrimental regulatory role in pancreatic β-cells. This may provide valuable evidence that circ006029 might be a potential target for alleviating β-cell damage in T1DM and rebuilding β-cell function.

Cell culture. The murine insulin-secreting cell line MIN6 was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Laboratories, Grand Island, NY, USA), supplemented with 15% fetal bovine serum (FBS, Bovogen, Australia), and 70 mmol/L β-mercaptoethanol (Genview, China). Cells were cultured at 37℃ with a humidified atmosphere of 5% CO₂. To simulate MIN6 cells, cells were exposed to cytokine cocktails consisting of 25 ng/ml IFN-γ (PeproTech, USA), 2.5 ng/ml IL-1β (PeproTech, USA), and 2.5 ng/ml TNF-α (PeproTech, USA) for 24 h.

Cell transfection. For inhibition of circRNA 006029 (circ006029), cells were seeded one day before transfection with circ006029 siRNA and the corresponding negative control (NC), which were obtained from GenePharma (China). The transfection was performed with Lipofectamine™ RNAiMAX (Invitrogen, CA, USA) according to the manufacturer’s protocol.

RNA isolation and Quantitative Real-Time PCR. Total RNA was extracted from MIN6 cells using TRIzol reagent (Life Technologies, USA) and was quantified by using Nanodrop (ThermoScientific, USA), followed by reverse transcription of RNA into cDNA using Evo M-MLV RT Kit with gDNA Clean for qPCR Ⅱ (Accurate Biology, China) following the manufacturer’s instruction. The qPCR was carried out on Bio-Rad CFX Connect™ Real-Time System via SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, China). Reactions were proceeded in triplicate in a 20 ul PCR reaction in the following steps: Denaturation at 95°C for 3 min, followed by 40 cycles of amplification at 95°C for 5 s and annealing and extension at 60°C for 30 s. U6 and β-actin were used as internal control. Results were determined by using the 2^−ΔΔCT method.

Cell counting kit (CCK-8) assay. The effects of circ006029 on MIN6 cell proliferation were determined by Cell Counting Kit-8 (CCK-8) (Abiowell, China) in accordance with the manufacturer’s protocol. MIN6 cells (1.7 × 10³) were seeded into 96-well plates. After transfection of circ006029 or NC siRNA, the cells were cultured for an additional 12 h, 24 h, and 48 h, respectively. The CCK-8 reagent was added to each well and the plate was incubated at 37°C for 2 h. After incubation, the absorbance at 562 nm was measured by using Varioskan LUX (ThermoScientific, USA).

Subcellular Hierarchical Analysis. Subcellular separation of MIN6 cells was conducted by using Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Belmont, CA, USA) prior to reverse transcription process, following the manufacturer’s instruction. β-actin and U6 were considered as internal reference of cytoplasmic and nuclear, respectively.

CircRNA stability determination. The total RNA of MIN6 cells was extracted using TRIzol, and then digested with RNase R using kit obtained from Yeasen Biotechnology (China), at a concentration of 1 U per 3 ug of RNA at 37℃ for 5 min, followed by an enzyme deactivation step at 70℃ for 10 min. Besides, MIN6 cells were incubated with 5 ug/ml of Actinomycin D (Aladdin, China) for 2, 4, 6, 12 h to inhibit RNA transcription. Then, qRT-PCR was performed as described in RNA isolation and Quantitative Real-Time PCR section to validate the characteristic of circ006029.

Flow Cytometry. Apoptosis analysis of MIN6 cells was performed by flow cytometry using Annexin V-FITC /PI Apoptosis Assay Kit (Biosharp, China) according to the manufacturer’s instruction. Briefly, MIN6 cells were transfected with circ006029 or NC siRNA. MIN6 cells in the culture medium and the adherent cells were both harvested. After being gently washed and resuspended with phosphate buffer solution (PBS, BasalMedia, China), cells were stained with an appropriate amount of Annexin V and PI in the dark. The measurement of apoptosis was performed on CytoFLEX SRT (Beckman Coulter, USA), and the percentage of cell apoptosis was analyzed by Flowjo.

The Luciferase Reporter Assay. MIN6 cells were transfected with circ006029 or NC siRNA along with NF-κB activity luciferase reporter plasmids using lipofectamine 3000. After 48 h, cells were lysed in Passive Lysis Buffer and underwent further handling. By using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instruction, the luciferase activity was measured by luminometry, and relative light units were normalized by Renilla luciferase.

Immunofluorescence. MIN6 cells were seeded into 12-well plate with microscope cover glass one day prior to transfection. Cells were co-transfected with circ006029 or NC siRNA and the mCherry-GFP-LC3b plasmid. Forty-eight hours post-transfection, the cells were treated with or without 100 nM Rapamycin (Abiowell, China) for 10 h. Then, the cells were fixed with 4% paraformaldehyde and the LC3b fluorescence was examined with a fluorescence microscope (KEYENCE BZ-X800, China) under a 60x oil immersion objective.

Immunoblot Analysis. Immunoblot was conducted to measure corresponding protein expression. The MIN6 cells were washed gently with PBS and lysed with NP-40 (Beyotime Biotechnology, Shanghai, China) on ice for 20 min. All samples were centrifuged at 4℃ for 20 min at 12000xg. The supernatant was collected to the new Eppendorf tube, mixed well with 5× loading buffer (NCM Biotech, China), and then boiled at 97℃ for 5 min. Lysates were electrophoresed by SDS-PAGE and transferred to PVDF membrane. After 5% bovine serum albumin (BSA, Biosharp, China) blocking and TBS-T washing at room temperature, membranes were then incubated with primary antibodies and species-specific secondary antibodies respectively. GAPDH and β-tubulin were employed as internal control.

Differentially Expressed mRNA Sequencing and Clustering Analysis. The MIN6 cells were transfected with circ006029 or NC siRNA. After 48 h, the total RNA was extracted from the cells by using TRIzol reagents (Thermo Fisher Scientific). RNA integrity was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and RNA quantity and quality were determined by NanoDrop 2000. The mRNA sequencing was performed on a llumina Novaseq 6000 platform. P value < 0.05 and fold change > 2 or fold change < 0.5 was set as the threshold for significantly differential expression gene (DEGs). The normalized expression level of each group was further analyzed with hierarchical clustering (HCL), and data was presented by using TreeView software (v. 1.5). The color blue represents low expression, while red represents high expression. In addition, the differentially expressed mRNAs from sequencing data were applied for GO and KEGG analysis.

Statistical Analysis. GraphPad Prism 9.0 software was used for data analysis. All data was expressed as mean ± standard deviation (mean ± SD). T-test was conducted to compare differences among groups. Difference with p < 0.05 was considered statistically significant.

Acknowledgments

Not applicable.

Author contributions

All authors contributed to the study. Kunlin Huang and Jiaxing Feng contributed to experiment accomplishment. Zhen Wang and Ying Zheng contributed to the conception and design. Zhiguang Zhou was responsible for the technical guide. The first draft of the manuscript was written by Kunlin Huang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Additional Information

Competing interests

The authors declare no conflict of interest.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Funding

This work was supported by the Science and Technology Innovation Program of Hunan Province (2022RC1010, 2023SK2027), Hunan Provincial Natural Science Foundation of China (2022JJ30851, 2023JJ70061).

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No competing interests reported.

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Inhibition of circular RNA 006029 alleviates pancreatic β-cell injury through the AKT/mTOR signaling pathway

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Version 1

Abstract

Figures

Introduction

Results

circ006029 is increased in cytokine-induced injured MIN6 cells

Knockdown of circ006029 facilitates proliferation and alleviates injury of MIN6 pancreatic β-cells

Knockdown of circ006029 increases MIN6 cell autophagy through the AKT/mTOR signaling pathway

Knockdown of circ006029 increases autophagy and concomitantly attenuates apoptosis of MIN6 cells

Circ006029 may be involved in the inflammatory response of β-cells

Changed Expression Profile of mRNAs in circ006029-depleted MIN6 cells

Discussion

Methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1