The transcript NR 134251.1 of lncRNA APTR with an opposite function to all transcripts inhibits proliferation and induces apoptosis by regulating proliferation and apoptosis-related genes

Abstract

Arsenic (As) exposure has been a global public health concern for hundreds of millions worldwide. LncRNA APTR (Alu-mediated p21 transcriptional regulator) plays an essential role in tumor growth and development. However, its function in arsenic-induced toxicological responses is still unknown. In this study, we found that the expressions of all transcripts and the transcript NR 134251.1 of APTR were increased in a dose-dependent manner in 16HBE cells treated with sodium arsenite (NaAsO₂). Silencing the transcript NR 134251.1 of APTR inhibited cell proliferation and induced apoptosis. However, silencing all transcripts of APTR had the opposite function to the transcript NR 134251.1. Then we examined the protein level of the proliferation and apoptosis-related genes after silencing the transcript NR 134251.1 of APTR. The results showed that silencing the transcript NR 134251.1 of APTR up-regulated the expression of transcription factor E2F1 and regulated its downstream genes involved in proliferation and apoptosis, including p53, phospho-p53-S392, phospho-p53-T55, p21, Cyclin D1, PUMA, Fas, Bim, BIK, Caspase-3, Caspase-7, and Cyt-c. In conclusion, arsenic induced APTR expression and the transcript NR 134251.1 of APTR have an opposite function to all transcripts, providing a theoretical basis for the prevention and treatment of arsenic exposure.

Keywords

Arsenic APTR Transcript Proliferation Apoptosis E2F1

Introduction

Arsenic (As) is a toxic metalloid widely present in the environment. Arsenic can exist in plants, air, water, rocks, animals, and soil.¹ Arsenic can live in natural environments in oxidized, organic, and inorganic forms.² There is growing evidence that organic arsenic is less harmful than inorganic arsenic.³ Chronic exposure to inorganic arsenic adversely affects human health. For example, groundwater in vast parts of Bangladesh and West Bengal was contaminated with inorganic arsenic. India is one of the deadiest incidents of mass poisoning in the past.⁴ Water contamination with arsenic has become an ongoing public health problem affecting hundreds of millions worldwide. Therefore, inorganic arsenic in drinking water is classified as a human class 1 carcinogen by the International Agency for Research on Cancer (IARC).⁵ Many studies have shown that arsenic and its compounds have critical medicinal applications. Arsenic trioxide, for example, is the standard treatment for acute promyelocytic leukemia, for which it is still the drug of choice.⁶ Therefore, the role of arsenic in the development of human cancer remains an issue worthy of continued exploration.

Long non-coding RNA (lncRNA) is the RNA transcripts of over 200 proteins in length that do not encode protein nucleotides.⁷ They are emerging regulators of gene expression and various physiological and pathological processes.⁸ LncRNAs are also involved in the arsenic-induced apoptosis and carcinogenesis processes. In the study of Jiang et al.,⁹ they showed that the sodium arsenite significantly inhibited the expression of lncRNA DICER1-AS1 in a dose-dependent manner and inhibited cell proliferation by regulating the cell cycle pathway. In another independent study, Luo et al.¹⁰ also demonstrated arsenite could induce the overexpression of lncRNA MALAT1 and participate in the malignant transformation and carcinogenesis of cells.

Alu-mediated p21 transcriptional regulator (APTR) is a newly discovered lncRNA. APTR was found to be involved in the transcriptional regulation of Cyclin-dependent kinase inhibitor 1A (CDKN1A/p21) by Nedishi et al. in 2014. APTR has four transcripts: NR 038361.1, NR 134251.1, NR 134253.1, and NR 134254.1. For the transcript of NR_038361.1, its length is 1978 bp. This variant (1) represents the shortest transcript. The length of the transcript of NR 134251.1 is 2016 bp. This variant (2) uses an alternate splice site in the 5′ region and contains two alternate 3′ exons, compared to variant 1. The length of the transcript of NR_134253.1 is 2156 bp. This variant (3) uses an alternate 3′-terminal exon, compared to variant 1. The transcript length of NR_134254.1 is 2029 bp. This variant (4) lacks an internal exon and uses an alternate 3′-terminal exon, compared to variant 1. Several studies have shown that APTR is highly present in osteosarcoma, glioma, and other cancers and promotes tumors. For example, APTR can recruit PRC2 to the p21 promoter of malignant glioma cells to inhibit p21 expression and accelerate tumor progression.¹¹ Silencing APTR in osteosarcoma cells inhibits cell proliferation, invasion, and migration.¹² APTR promotes the proliferation of leiomyoma cells by targeting the Era Wnt pathway.¹³ However, the expression of APTR is down-regulated in thyroid cancer, and the expression of APTR is negatively correlated with TNM stage and distant metastasis.¹⁴ These studies have demonstrated that APTR has different effects on different cancer cells, which may be related to the transcripts of APTR. However, the function and mechanism of APTR in arsenic-induced toxicological responses remain unclear. In this study, we detected the expression levels of all transcripts and the transcript NR 134251.1 of APTR in arsenite-treated 16HBE cells. Moreover, our research group selected the transcriptional regulator E2F1 and its downstream genes p53, phospho-p53-S392, phospho-p53-T55, p21, Cyclin D1, PUMA, Fas, Bim, BIK, Caspase-3, Caspase-7, and Cyt-c to determine the potential function and mechanism of the transcript NR 134251.1 of APTR in 16HBE cells, providing a theoretical basis for the prevention and treatment of arsenic exposure.

Material and method

Cell culture and treatment

Human bronchial epithelial cell lines 16HBE were purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences. The cells were cultured in MEM medium supplemented with 10% FBS (QuaCell Biotechnology, Co. Ltd, China) and 1% penicillin/streptomycin (Beijing Solarbio Science and Technology Co., Ltd, China) at 37°C and 5% CO₂ incubator.

16HBE cells were seeded into 6-well plates at a density of 1.5 × 10⁵ cells/well in the log growth phase. After incubating for 20 h, MEM medium (10% FBS and 1% penicillin/streptomycin) containing NaAsO₂ (Xiya Chemical Industry Co. Ltd, China) with different concentrations (0, 1.5, 3, 4.5 μmol/L) was added. The cells were continued to culture in a 6-well plate for 48 h.

Cell transfection

Small interfering RNA (siRNA) with APTR (siRNA1, siRNA2, and siRNA3) and its control (si-NC) were synthesized and purchased from Shanghai GenePharma Co., Ltd (China). siRNA-1 interfered with all transcripts of APTR, and siRNA-2 and siRNA-3 interfered with the transcript NR 134251.1. The sequences were all listed in Table 1.

Table 1.

The sequences of siRNAs used for transfection.

Gene	Sequences (5′ → 3′)	Interfered with transcripts
NC	Sense 5′ - UUCUCCGAACGUGUCACGU-3′
siRNA-1	Sense 5′ -CCAGGUACUGCCUUCUAAC-3′	All transcripts (NR 038361.1, NR 134251.1, NR 134253.1, NR 134254.1)
siRNA-2	Sense 5′ -CCAUGAUCCGGUAUCACCA-3′	Transcript NR 134251.1
siRNA-3	Sense 5′ -GUAGCACCAUCACUGCUCA-3′	Transcript NR 134251.1

16HBE cells were seeded into 6-well plates at a density of 1.0 × 10⁵ cells/well in the log growth phase. After 18 h, the 16HBE cells were transfected using the RFect siRNA/miRNA transfection reagent (40 μmol/L, Changzhou Bio-generation Biotechnology Co., China). After incubating for 72 h, transfection efficiency was measured by detecting the expression level of APTR with qRT-PCR.

cDNA preparation and quantitative real-time PCR analysis

The total RNA of 16HBE cells was extracted using RNAiso plus reagent (TaKaRa, Beijing, China) and by using HiFiScript cDNA synthesis kit reverse transcription into cDNA (CoWinBiotech, Beijing, China). The qRT PCR was performed under the LightCycler® 96 Real-Time PCR System (Roche Molecular Systems, California) using SYBR Green PCR Master Mix (Kewan Bioscience, China). β-actin was used as an internal reference. We designed two pairs of primers based on APTR transcripts, APTR (all transcripts) could amplify four transcripts, and APTR (transcript NR 134251.1) could amplify only transcript NR 134251.1. The sequences of primers are all listed in Table 2. The reaction conditions were denaturation for 8 min at 95°C, followed by 50 cycles of 95°C for 10 s, annealing for 10 s at 60°C, and a final extension for 10 s at 72°C. Ct values were analyzed using the 2^−ΔΔCT method.

Table 2.

The sequences of primer used for qRT-PCR.

Gene	Primer sequences(5′→ 3′)	Amplified transcripts
APTR (all transcripts)	Forward 5′- TGTCAGATAGGACCAACTTGC-3′	All transcripts (NR 038361.1, NR 134251.1, NR 134253.1, NR 134254.1)
APTR (all transcripts)	Reverse 5′ -GCTACAGTGAGCAGTGATGG-3′
APTR (transcript NR 134251.1)	Forward 5′- TGTGGGTACAAAAGGAGAGTAACAT-3′	Transcript NR 134251.1
APTR (transcript NR 134251.1)	Reverse 5′ -GTAGATCTGGAGCTGCAACTACAG-3′	Transcript NR 134251.1
β-actin	Forward 5′- CCTGTACGCCAACACAGTGC-3′
β-actin	Reverse 5′ -ATACTCCTGCTTGCTGATCC-3′

Cell viability assay

Cell viability was determined using the Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). 16HBE cells were seeded and cultured in 96-well culture plates at a concentration of 8.0 × 10³ cells/well for sodium arsenite treatment. After 22 h, sodium arsenite was added in different concentrations of 0, 1.5, 3, and 4.5 μmol/L medium for 48 h. 16HBE cells were seeded into 6-well plates at a density of 4.5 × 10³ cells/well in the log growth phase for transfection. After transfection, 200 μL MEM complete medium cultured cell for 72 h. After treatment, add 10 μL CCK-8 reagents to each well, incubate at 37°C according to the instructions, and measure the absorbance at 450 nm with a microplate reader (Bio-Rad, Hercules, CA, USA).

Mitochondrial membrane potential

Mitochondrial membrane potential was detected by JC-1 staining (US Everbright® Inc., USA). For the transfected cells, 16HBE cells were seeded into 6-well plates at a density of 4.5 × 10³ cells/well in the log growth phase. After transfection, 200 μL MEM complete medium cultured cell for 72 h. After the treatments, cells were collected and stained with 1× JC-1 solution in MEM medium for 15 min at 37°C. The fluorescence value was measured using a microplate reader (Bio-Rad, Hercules, CA, USA).

EdU (5-ethynyl-2′-deoxyuridine) stains

Cell proliferation was detected using Beyoclick™ EdU Cell Proliferation Kit and alexafluor555 (C10310-3, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. 16HBE cells were added reaction mixture and left in the dark for 30 min. After that, samples were stained with Hoechst 33342 for 15 min (Salic and Mitchison, 2008). 16HBE cells were visualized under a fluorescence microscope, and the number of EdU-labeled cells and Hoechst33342-stained cells were counted using the ImageJ software. Photographs were taken three times at random.

Hoechst 33342 and propidium iodide double staining method

Cells were washed twice with PBS, then 550 μL of Hoechst 33342 (HO) and propidium iodide (PI) (Beyotime, China) mixed solution was added to each well, placed at room temperature for 15 min, and were observed and photographed with a fluorescence microscope. They could stain by HO for apoptotic cells, showing bright blue fluorescence. They could be marred by HO/PI for necrotic cells, showing bright blue and red fluorescence. Normal cells cannot be impaired by HO/PI, revealing a dark blue fluorescence. Photographs were taken three times at random. They used the ImageJ software to calculate the number of Hoechst 33342(HO) cells and propidium iodide (PI) cells.

Western blot analysis

Cells (1.0 × 10⁵) were collected and lysed in RIPA (Thermo Fisher Scientific inc. USA) buffer. Total protein was determined using a protein detection kit (BCA; China Bi Biotechnology Co., Ltd). Complete proteins were separated by SDS-PAGE, transferred to PVDF membranes in Tris-glycine methanol buffer, and incubated with specific antibodies (E2F1, p21, p53, Phospho-p53-S392, Phospho-p53-T55, Caspase-3, Caspase-7, Bim, BIK, MDM2, PUMA, Fas, Cytochrome C (Cyt-c), Cyclin D1, p65, Phospho-AKT, β-actin). The primary antibodies were incubated overnight at 4°C, whereas the horseradish peroxidase-conjugated secondary antibodies were incubated for 2 h at room temperature. The bands were visualized using BeyoECL Plus chromogenic substrate (Beyotime Biotechnology Co., China), and the intensity was quantified using Gel-Pro Analyzer software (Media Cybernetics). The band intensity of western blotting was quantified by ImageJ software. Data were normalized β-actin as the loading control.

Statistical analysis

All data were analyzed using SPSS 20.0 software, GraphPad Prism 5.0, and ImageJ software and expressed as means ± standard error. The Student’s t-test performed two-group comparisons. Pearson chi-square was used to assess the efficiency of cell proliferation and apoptosis between the si-APTR groups and the NC group. p < 0.05 indicated statistically significant differences. For all data, *p < 0.05, **p < 0.01.

Results

Sodium arsenite up-regulated the expression levels of all transcripts and the transcript NR 134251.1 of alu-mediated p21 transcriptional regulator in 16HBE cells in a dose-dependent manner

To investigate whether sodium arsenite could affect the expression levels of all transcripts and the transcript NR 134251.1 of APTR in 16HBE cells, after NaAsO₂ treatment, the cell proliferation capacity was measured by the CCK-8 assay. The results showed that NaAsO₂ significantly hindered cell viability (p < 0.05) (Figure 1(a)). Therefore, we detected the APTR levels in 16HBE cells treated with 0, 1.5, 3, and 4.5 μmol/L concentrations of NaAsO₂. As shown in Figure 1(b) and (c), sodium arsenite significantly increased the expression levels of all transcripts and the transcript NR 134251.1 of APTR in a dose-dependent manner in 16HBE cells (p < 0.05).

Figure 1.

Effect of sodium arsenite on APTR RNA expression in 16HBE cells. (a) Cell viability after 48 h of treatment with different concentrations of sodium arsenite (0, 1.5, 3, 4.5 μM). (b) qRT-PCR examined the expression of all transcripts of APTR after 48 h of sodium arsenite treatment. (c) The expression of the APTR transcript, NR134251.1, was determined after 48 h of sodium arsenite treatment by qRT-PCR. Data are expressed as mean ± SE *p < 0.05, **p < 0.01, compared with the control group.

Detection of alu-mediated p21 transcriptional regulator silencing efficiency in 16HBE cells

To test the silencing efficiency of the APTR, we transfected siRNA-1/siRNA-2/siRNA-3 and si-NC into 16HBE cells. The transfection efficiency of APTR was assessed by fluorescence microscopy (Figure 2(a)). The qRT-PCR showed that the expression of all transcripts of APTR was significantly reduced after transfection of siRNA-1, and the expression of the transcript NR 134251.1 was significantly reduced after transfection of siRNA-2 or siRNA-3. These results indicate that all transcripts and the transcript NR 134251.1 of APTR were knocked down successfully (p < 0.05) (Figure 2(b) and (c)).

Figure 2.

Detection of efficiency after APTR knockdown in 16HBE cells. (a) 16HBE cells were transfected with FAM-siRNA. Transfection efficiency was determined by fluorescence microscopy. (b) After the 16HBE cells were transfected with NC and siAPTR-1 for 72 h, the RNA levels of all transcripts of APTR were measured with qRT-PCR. (c) 16HBE cells were transiently transfected with NC, siAPTR-2 and siAPTR-3 for 72 h and the RNA level of the transcript NR 134251.1 of APTR was evaluated by qRT-PCR. Data are expressed as mean ± SE *p < 0.05, **p < 0.01, compared with the NC group.

Silencing of all transcripts of alu-mediated p21 transcriptional regulator promoted proliferation, while silencing of the transcript NR 134251.1 of alu-mediated p21 transcriptional regulator had an opposite function

To investigate the functions of APTR in cell proliferation, the CCK-8 and EdU were used to detect cell viability and proliferation, respectively. The CCK-8 assay revealed that after silencing all transcripts of the APTR, the cell viability growth increased by 3.270%, there was no statistically significant, however silencing the transcript NR 134251.1 of APTR significantly inhibited proliferation of 16HBE cells with growth inhibition of 23.18% and 18.83%, respectively (p < 0.05) (Figure 3(a)). The EdU results showed that the addition of 16HBE cells increased after silencing all transcripts of APTR and decreased after silencing the transcript NR 134251.1 of APTR (Figure 3(b)). Quantitative analysis of the ImageJ software showed that the number of proliferation cells accounts for 21.2%, 27.5%, 1.08%, and 10.81% in the si-NC, siRNA-1, siRNA-2, and siRNA-3 groups, respectively (p < 0.05) (Figure 3(c)). These results suggested that silencing of all transcripts of APTR promoted the proliferation of 16HBE cells, and silencing the transcript NR 134251.1 of APTR suppressed the proliferation of 16HBE cells.

Figure 3.

CCK-8 and EdU in 16HBE cells were analyzed after silencing all transcripts and the transcript NR 134251.1 of APTR for 72 h. (a) After silencing all transcripts and the transcript NR 134251.1 of APTR, CCK-8 detection was performed in 16HBE cells. Data are expressed as mean ± SE *p < 0.05, **p < 0.01, compared with the NC group. (b) After transient transfection of 16HBE cells with NC, siAPTR-1, siAPTR-2, and siAPTR-3 for 72 h. The images were detected by a fluorescence microscopy, and the nuclei of the EdU-positive cells were stained in red. Blue nuclei indicate Hoechst33342. (c) ImageJ software counted the number of cells with red and blue fluorescence in (b) (percentage, with Pearson chi-square).

Silencing of all transcripts of alu-mediated p21 transcriptional regulator suppressed cell apoptosis, while silencing of the transcript NR 134251.1 of alu-mediated p21 transcriptional regulator had an opposite function

To investigate the functions of APTR in cell apoptosis, JC-1 was used to detect the mitochondrial membrane potential. JC-1 results showed that after silencing all transcripts of the APTR, the red-green fluorescence ratio increased by 20.60%. However, silencing the transcript NR 134251.1 of APTR, the red-green fluorescence ratio decreased by 52.10% and 16.59%, respectively (p < 0.05) (Figure 4(a)). To detect the apoptosis and necrosis of 16HBE cells after APTR silencing, the HO-PI cell apoptosis assay determined apoptosis after siRNA 72 h transfection. The results showed that apoptosis and necrosis were decreased after the silence of all transcripts of APTR. In contrast, the apoptosis and necrosis of 16HBE cells were increased after silencing the transcript NR 134251.1 of APTR (Figure 4(b)). Furthermore, quantitative analysis of the ImageJ software showed that the number of apoptotic and necrotic cells was 0.840%, 0.443%, 3.38%, and 0.985% in the si-NC, siRNA-1, siRNA-2, and siRNA-3 groups, respectively (p < 0.05) (Figure 4(c)). These results suggested that silencing of all transcripts of APTR suppressed the apoptosis of 16HBE cells, and silencing the transcript NR 134251.1 of APTR promoted the apoptosis of 16HBE cells.

Figure 4.

JC-1 and HO-PI in 16HBE cells were analyzed after silencing all transcripts and the transcript NR 134251.1 of APTR for 72 h. (a) The JC-1 assay was used to examine the mitochondrial membrane potential in 16HBE cells after transfection with APTR-siRNA. Data are expressed as mean ± SE *p < 0.05, **p < 0.01, compared with the NC group. (b) 16HBE cells were transiently transfected with NC, siAPTR-1, siAPTR-2, and siAPTR-3 for 72 h. The levels of apoptosis and necrosis were determined by double staining with Hoechst33342 and PI. Apoptotic cells appear in bright blue within the nucleus and necrotic cells in red. (c) The apoptosis frequency was calculated by ImageJ software for the number of cells with weak red and robust blue fluorescence in (b) (percentage, with Pearson chi-square). *p < 0.05, **p < 0.01, compared with the NC group.

Change of proliferation and apoptosis-related genes after knockdown of the transcript NR 134251.1 of alu-mediated p21 transcriptional regulator

To explore the mechanism of the transcript NR 134251.1 of APTR in cell apoptosis and proliferation, protein levels of proliferation and apoptosis-related genes were measured by western blotting. The results are shown in Figure 5 (p < 0.05). Knockdown of APTR inhibited expression levels of Cyclin D1, and induced expression levels of E2F1, MDM2, p53, phospho-p53-S392, phospho-p53-T55, p21, Caspase-3, Caspase-7, Bim, BIK, PUMA, Fas, and Cyt-c in 16HBE cell lines. However, there were no changes in p65 and phospho-AKT expression.

Figure 5.

Changes in protein expression levels of apoptosis and proliferation-related genes after silencing the transcript NR 134251.1 of APTR in 16HBE cells. (a and c) Protein levels of E2F1, MDM2, p53, phospho-p53-S392 and phospho-p53-T55, p21, Cyclin D1, p65, phospho-AKT were detected by western blotting. (b and d) Protein levels of PUMA, Fas, Bim, BIK, Caspase-3, Caspase-7, and Cyt-c were detected by western blotting.*p < 0.05, compared with the NC group.

Discussion

Arsenic is one of the most serious environmental pollutants. People may be exposed to arsenic through food, air pollution, drinking water, etc. Therefore, human exposure to arsenic is a significant public health concern.¹ Numerous studies have linked the expression of human genes to arsenic exposure. For example, arsenic exposure affects the expression of lncRNA MEG3.¹⁵ Many studies have shown that arsenic can lead to apoptosis in several ways. For example, arsenic can lead to apoptosis by reducing the mitochondrial membrane potential, enhancing oxidative stress and caspase activation, etc.⁴ However, the mechanisms of lncRNAs regulation involved are poorly understood. In this study, we examined the role of lncRNA APTR in sodium arsenite in promoting apoptosis. APTR has different functions in cells and may be related to the transcripts. Studies to date have shown that the BCL-x gene, a member of the BCL-2 family, enables different transcripts from the same gene to have the same or different expression patterns and functions through alternative splicing.¹⁶ Therefore, we selected all transcripts and the transcript NR 134251.1 of APTR in this study to research its function and mechanism. 16HBE cells were treated with different concentrations of sodium arsenite for 48 h, and the expression of all transcripts and the transcript NR 134251.1 of lncRNA APTR showed a dose-dependent promotion. Arsenic can contribute to apoptosis through multiple signaling pathways. For example, the ATF3-mediated expression enhancement of DR5 expression and the inhibition of BCL-xL expression could thereby promote arsenic-induced apoptosis.¹⁷ In BEAS-2B cells, As₂O₃-induced apoptosis by the upregulation of BCL-2, phosphorylation, and nuclear localization.¹⁸ However, the function of all transcripts and the transcript NR 134251.1 of APTR in cell proliferation and apoptosis remains unclear. Therefore, we evaluated the proliferation and apoptosis of 16HBE cells by knockdown of APTR in 16HBE cells. The results showed that silencing all transcripts of APTR could promote 16HBE cell proliferation and inhibit apoptosis while silencing the transcript NR 134251.1 of APTR inhibited 16HBE cell proliferation and promoted apoptosis. These results suggested that all transcripts of APTR played a dominant role in arsenic-induced apoptosis and proliferation inhibition.

To further study the mechanism of the transcript NR 134251.1 of APTR, the western blot was used to examine the expression of genes involved in proliferation and apoptosis after silencing the transcript NR 134251.1 of APTR in 16HBE cells. The results showed that the expression level of E2F1 was increased. E2F1 is a critical transcription factor in cells. It is the first protein of the E2Fs to be cloned and plays a crucial role in cell proliferation, differentiation, and apoptosis.¹⁹ E2F1 is involved in the transition of the cell cycle from the G1 to the S phase.²⁰ However, under conditions of DNA damage, E2F1 promotes cell apoptosis mainly through the p53 pathway.^21,22 E2F1 activates the transcription of p14^ARF, which prevents the binding of MDM2 to p53 and prevents the degradation of p53.²² However, in the absence of p14^ARF, E2F1 directly acts on p53 and results in the phosphorylation of p53.²³ Several studies have shown that the direct role of the P53/P21/Cyclin D1 pathway is to arrest the cell cycle in the G0/G1 phase.^24,25 Peng et al.¹⁹ showed that E2F1 regulates Cyclin D1 expression because the Cyclin D1 promoter contains an E2F1 consensus site, and E2F1 can inhibit Cyclin D1 gene expression . Our results also showed that silencing the transcript NR 134251.1 of APTR up-regulated the expressions of p53, phospho-p53-S392, phospho-p53-T55, MDM2, p21, and down-regulated Cyclin D1 expression. Studies have shown that E2F1 directly activates the pro-apoptotic protein PUMA, Fas, Bim, and BIK through transcriptional mechanisms.^26,27 Meanwhile, E2F1 induces Apaf-1 expression, Caspase-3, and Caspase-7 activation, and Cyt-c release, then leading to cell apoptosis.²⁸ Our results also showed that silencing the transcript NR134251.1 of APTR elevated the expression of PUMA, Fas, Bim, BIK, Caspase-3, Caspase-7, and Cyt-c. These results suggested that the transcript NR134251.1 of APTR silencing induced cell apoptosis by regulating E2F1 and its downstream genes involved in proliferation and apoptosis.

Conclusion

In summary, our results demonstrated that arsenic up-regulated the expression of all transcripts and the transcript NR 134251.1 of lncRNA APTR expression in 16HBE cells. Silencing all transcripts of APTR promoted 16HBE cell proliferation and inhibited apoptosis, and silencing the transcript NR 134251.1 of APTR inhibited 16HBE cell proliferation and promoted apoptosis. The transcript NR134251.1 of APTR silencing could increase the expression level of the transcription factor E2F1 and regulate its downstream genes involved in proliferation and apoptosis, including p53, phospho-p53-S392, phospho-p53-T55, p21, Cyclin D1, PUMA, Fas, Bim, BIK, Caspase-3, Caspase-7, and Cyt-c. We will further investigate the function and mechanisms of other transcripts of APTR in the effects of arsenic exposure.

Footnotes

Author contributions

Jinyi Yu analysed and interpreted the present study experiment and data and was a major contributor in writing the manuscript. Shuting Li analysed data. Simin Shen, Qian Zhou, Jinyao Yin, Ruihuan Z hao, and Jingwen Tan prepared the instrument for the experiment. Chenglan Jiang and Yuefeng He had contributed in the conceptualization and designing of this article. All authors read and approved the final manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by the National Natural Science Foundation of China (Grant No. 82160607), the Innovative Research Team of Yunnan Province (Grant No. 202005AE16002), College Students Innovative Pilot Projects (2021JXD002), Yunnan Applied Basic Research Projects-Union Foundation, Yunnan Provincial Science and Technology Department and Kunming Medical University, China (Grant Nos. 202001AY070001-134 and 202001AY070001-203).

ORCID iDs

Chenglan Jiang

Yuefeng He

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