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Cancer cells have evolved various mechanisms to overcome cellular stress and continue to progress. Protein kinase R (PKR) and its protein activator (PACT) are the initial responders that monitor various stress signals leading to inhibition of cell proliferation and apoptosis. However, the regulation of the PACT-PKR pathway in cancer cells remains largely unknown. Here, we found that long non-coding RNA (lncRNA) aspartyl tRNA synthetase antisense RNA 1 (DARS-AS1) is directly involved in the inhibition of the PACT-PKR pathway and promotes cancer cell proliferation. Using large-scale functional screening of CRISPRi 971 cancer-associated lncRNA, we found that DARS-AS1 was associated with significantly enhanced cancer cell proliferation. Therefore, DARS-AS1 knockout inhibits cell proliferation and promotes cancer cell apoptosis in various cancer cell lines in vitro and significantly reduces tumor growth in vivo. Mechanically, DARS-AS1 binds directly to the PACT activation domain and prevents PACT-PKR interaction, thereby reducing PKR activation, eIF2α phosphorylation, and inhibiting apoptotic cell death. Clinically, DARS-AS1 is widely expressed in multiple cancers, and overexpression of this lncRNA is indicative of a poor prognosis. This study elucidates cancer-specific regulation of the PACT-PKR pathway by DARS-AS1 lncRNA and provides another target for cancer prognosis and treatment.
The ability to adapt to stress is an important characteristic of cancer cell survival and proliferation. The rapid proliferation and metabolic hallmarks of cancer peak in harsh microenvironments—nutrient deprivation, hypoxia, and low pH—that can trigger cell death signaling pathways. Dysregulation of stress-sensitive genes such as p535, heat shock proteins 6, 7, KRAS8, 9, and HIF-110, 11, 12, 13 is frequently observed in cancer, thereby blocking apoptosis and promoting survival.
Protein kinase R (PKR) is an important stress sensor and subunit kinase of eukaryotic initiation factor 2α (eIF2α), a translational regulator that links cellular stress to cell death. PKR was originally identified as an antiviral protein by the detection of a foreign double-stranded RNA (dsRNA). Upon activation, PKR phosphorylates eIF2α to inhibit viral and cellular protein synthesis14,15,16. PACT (PKR activator protein) has been identified as the first PKR activator protein in the absence of dsRNA17,18,19,20,21,22,23. Through direct interaction with PKR, PACT transduces various stresses (serum starvation, peroxide or arsenite treatment) to PKR and downstream signaling pathways. In addition to eIF2α phosphorylation, PACT-mediated PKR activation triggers various events associated with the stress response, including altered redox status via the PI3K/Akt24 pathway, enhanced DNA damage checking via p5325,26 and NF-κB27,28 Regulates transcription, 29. Given their critical role in stress response, proliferation, apoptosis and other key cellular processes, PKR and PACT are promising therapeutic targets for many diseases, especially cancer30,31,32,33. However, despite this pleiotropic functional and biological significance, the regulation of PACT/PKR activity in cancer cells remains elusive.
lncRNAs are transcripts larger than 200 nucleotides with no protein-coding potential. Since cutting-edge whole genome sequencing projects have identified thousands of lncRNAs,35,36 much effort has been made to elucidate their biological functions. A growing body of research has shown that lncRNAs are involved in many biological processes37 including the regulation of X-chromosome inactivation38,39, imprinting40, transcription41,42, translation43 and even cancer growth44,45,46,47. These studies reported that many lncRNAs are involved in the PACT/PKR pathway. One such study showed that lncRNA ASPACT inhibited PACT transcription and increased nuclear retention of PACT mRNA. Other studies have shown that lncRNA nc886 binds to PKR and inhibits its phosphorylation49,50. So far, lncRNA regulating PACT-mediated PKR activation has not been reported.
Aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) has been identified as an oncogenic lncRNA51,52,53,54. Through the regulation of miP-194-5p53, miP-12952 and miP-532-3p51, DARS-AS1 has been shown to promote the growth of clear cell renal cell carcinoma, thyroid carcinoma and non-small cell lung carcinoma, respectively. Tong and colleagues also found that DARS-AS1 promotes myeloma progression by maintaining the stability of the protein 39 (RBM39) RNA-binding motif. However, no studies have been conducted on whether this lncRNA is involved in the regulation of PACT-PKR activation and the stress response of cancer cells.
Here, we performed a large-scale loss-of-function screen using the CRISPRi system and determined that DARS-AS1 lncRNA promotes the proliferation of several types of cancer cells. In addition, we have identified a major mechanism: DARS-AS1 binds directly to PACT, inhibits PACT and PKR binding, prevents phosphorylation of eIF2α, a lower PKR substrate, and ultimately inhibits apoptotic cell death. In conclusion, our work reveals the DARS-AS1 lncRNA as a regulator of the PACT-PKR pathway and a potential target for cancer treatment and prognosis.
Extensive genomic profiling studies have identified hundreds of lncRNAs associated with cancer. However, their function remains largely unknown56. To identify promising lncRNA candidates involved in cancer progression, we performed a loss-of-function screen for reduced proliferation in the SW620 colorectal cancer cell line using the CRISPRi system (Fig. 1a). The unique feature of the SW480 and SW620 colon cancer cell lines is that they are derived from primary and secondary tumors in a single patient. This provides a valuable comparison for studying genetic changes in the progression of advanced colon cancer. Therefore, we analyzed the transcriptomes of colorectal cancer cell lines (SW480 and SW620) using RNA sequencing and collected some potential functional lncRNAs from the published literature. Based on these results, we designed a pooled sgRNA library containing 7355 sgRNA oligos targeting 971 cancer-associated lncRNAs and 500 untargeted sgRNA oligos for a negative control (Supplementary Data 1).
Schematic representation of screening using the CRISPRi system. b sgRNA enrichment after screening. The horizontal dotted line represents log2 (fold change) = ±0.58. The vertical dotted line indicates p value = 0.05. Black dots represent non-target sgRNA (designated as NC). Red dots are sgRNAs targeting DARS-AS1. Blue dots are sgRNAs targeting LINC00205, a previously described oncogenic lncRNA. fold change = (normalized reading, day 17)/(normalized reading, day 0). c DARS-AS1 sgRNA knockdown inhibited cell growth. Error bars represent ± standard deviation of the three experiments. * p ≤ 0.05, ** p ≤ 0.01 two-tailed Student’s t-test. d DARS-AS1 expression in tumors (TCGA dataset). em Expression of DARS-AS1 in paired normal and tumor samples from patients with BLCA, KIRC, PRAD, LUSC, UCEC, LUAD, LIHC, KIRP, and COAD, respectively (TCGA dataset). p-values ​​were obtained using paired two-tailed Student’s t-test.
After constructing the plasmid and packaging the lentivirus, we transduced the dCas9-SW620 colorectal cancer cell line with the above library in four independent infection experiments. The multiplicity of infection (MOI) for these infections was 0.1–0.3, indicating that each cell can only be transfected with one sgRNA. After 18 days of in vitro culture, the enrichment profile of target sgRNAs decreased or increased after screening, while the number of non-targeted control oligonucleotides remained relatively unchanged compared to the pre-screening profile, indicating that our target has a highly screen-specific library. Rice. 1b and supplementary table 1). LINC00205, which was previously reported to promote lung cancer and liver cancer progression58,59,60, was screened out (log2 (foldchange) < −0.58, p value < 0.05), confirming the reliability of this screening (Fig. 1b). LINC00205, which was previously reported to promote lung cancer and liver cancer progression58,59,60, was screened out (log2 (foldchange) < −0.58, p value < 0.05), confirming the reliability of this screening (Fig. 1b). LINC00205, о котором ранее сообщалось, что он способствует прогрессированию рака легких и рака печени58, 59, 60, был исключен (log2 (кратное изменение) <-0,58, значение p <0,05), что подтверждает надежность этого скрининга (рис. 1b). LINC00205, previously reported to promote the progression of lung cancer and liver cancer58,59,60, was excluded (log2 (fold change) <-0.58, p-value <0.05), confirming the robustness of this screening (Fig. .1b). LINC00205 之前被报道可促进肺癌和肝癌进展58,59,60，被筛选掉（log2（倍数变化）< -0.58，p 值< 0.05），证实了该筛选的可靠性（图1b）。 LINC00205 之前被报道可促进肺癌和肝癌进展58,59,60，被筛选掉（log2（倍数变化）< -0.58，p 值< 0.05），证实了该筛选的可靠性（图1b）。 LINC00205, о котором ранее сообщалось, что он способствует прогрессированию рака легких и печени58, 59, 60, был исключен (log2 (кратное изменение) <-0,58, p-значение <0,05), что подтверждает надежность этого скрининга (рис. 1b). LINC00205, previously reported to promote lung and liver cancer progression58,59,60, was excluded (log2 (fold change) <-0.58, p-value <0.05), confirming the robustness of this screening (Fig. .1b).
Among all lncRNAs tested, DARS-AS1 was also screened, with the three cognate sgRNA oligonucleotides significantly reduced after 18 days of culture, suggesting that knockdown of this lncRNA resulted in reduced cancer proliferation (Fig. 1b). This result was further supported by MTS analysis in colorectal cancer cells showing that the growth rate of DARS-AS1 knockdown cells was only halved compared to control cells (Figure 1c) and was consistent with previous reports of several other cancer types. : clear cell kidney cancer, thyroid cancer and non-small cell lung cancer51,52,53,55. However, its function and molecular mechanisms in colorectal cancer remain unexplored. Therefore, we chose this lncRNA for further study.
To study DARS-AS1 expression in patients, we comprehensively analyzed 10,327 tumor samples from the Cancer Genome Atlas (TCGA) project. Our results show that DARS-AS1 is widely expressed and significantly upregulated in healthy cells in a variety of tumors, including colon adenocarcinoma (COAD), renal clear cell carcinoma (KIRC), and renal papillary cell carcinoma (KIRP). . Very few (Fig. 1d and Supplementary Fig. 1a, b). Analysis of paired healthy/tumor samples further confirmed a significantly higher expression of DARS-AS1 in the tumors of bladder urothelial carcinoma (BLCA), kidney renal clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC), uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), liver hepatocellular carcinoma (LIHC), kidney renal papillary cell carcinoma (KIRP), and colon adenocarcinoma (COAD) (p value < 0.05) (Fig. 1e–m). Analysis of paired healthy/tumor samples further confirmed a significantly higher expression of DARS-AS1 in the tumors of bladder urothelial carcinoma (BLCA), kidney renal clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) , uterine corpus endometrial carcinoma (UCEC), lung adenocarcinoma (LUAD), liver hepatocellular carcinoma (LIHC), kidney renal papillary cell carcinoma (KIRP), and colon adenocarcinoma (COAD) (p value < 0.05) (Fig. 1e–m) . Analysis of paired healthy/tumor samples also confirmed significantly higher expression of DARS-AS1 in bladder urothelial carcinoma (BLCA), clear cell renal and renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), lung squamous cell carcinoma (LUSC) tumors. , карцинома эндометрия тела матки (UCEC), аденокарцинома легкого (LUAD), гепатоцеллюлярная карцинома печени (LIHC), папиллярно-клеточная карцинома почки (KIRP) и аденокарцинома толстой кишки (COAD) (значение p <0,05) (рис. 1e–m) . , endometrial carcinoma of the corpus uteri (UCEC), adenocarcinoma of the lung (LUAD), hepatocellular carcinoma of the liver (LIHC), papillary cell carcinoma of the kidney (KIRP), and adenocarcinoma of the colon (COAD) (p value <0.05) (Fig. 1e– m) .配对健康/肿瘤样本的分析进一步证实了DARS-AS1 在膀胱尿路上皮癌(BLCA)、肾肾透明细胞癌(KIRC)、前列腺腺癌(PRAD)、肺鳞状细胞癌(LUSC) 肿瘤中的显着更高表达，子宫体子宫内膜癌（UCEC），肺腺癌（LUAD），肝肝细胞癌（LIHC），肾肾乳头状细胞癌（KIRP）和结肠腺癌（COAD）（p值<0.05）（图1e-m） .配对 健康/肿瘤样本 的 分析 证实 了 dars-os1 在 尿路 上 皮癌 皮癌 皮癌 皮癌 、 肾 肾 细胞癌 细胞癌 细胞癌 细胞癌 前列 腺腺癌 腺腺癌 (prad) 、 细胞癌 细胞癌 (lusc) 肿瘤 的 的 的 的 中 中 中 中 中 中 中 中 中 中 中显着 更 高 表达 ， 内膜 癌 （（ucel） 肺腺癌 （luad） 肝肝 细胞癌 （lihc） 肾 肾 乳头状 细胞癌 （kirp） （coad） （p 值<0.05）（图1e-m） . Analysis of healthy/tumor paired samples further supported the role of DARS-AS1 in bladder urothelial carcinoma (BLCA), clear cell renal cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), and lung squamous cell carcinoma (LUSC) tumors. экспрессия при карциноме тела матки (UCEC), аденокарциноме легкого (LUAD), гепатоцеллюлярной карциноме (LIHC), почечно-почечной папиллярно-клеточной карциноме (KIRP) и аденокарциноме толстой кишки (COAD) (значение p <0,05) (рис. 1e-m). expression in corpus uterine carcinoma (UCEC), lung adenocarcinoma (LUAD), hepatocellular carcinoma (LIHC), renal papillary cell carcinoma (KIRP), and colon adenocarcinoma (COAD) (p value <0.05) (Figure 1e -m). Taken together, these results indicate that DARS-AS1 is widely and highly expressed in a variety of cancers.
Because DARS-AS1 and DARS (the gene encoding the antisense strand) share the same promoter and are located next to each other, we designed shRNA to specifically knockdown DARS-AS1 but not DARS (Supplementary Fig. 2a,b and Supplementary Table 2 ). In addition to SW620, we also used three other cell lines highly expressing DARS-AS1 to study the efficacy and function of shRNA knockdown (Supplementary Table 3). Our results indicated that all three shRNAs developed achieved at least 80% DARS-AS1 knockdown efficiency with little effect on the amount of DARS mRNA (Supplementary Fig. 2c–f). In addition, we found that DARS-AS1 knockdown with these shRNAs significantly inhibited cell growth in colorectal cancer cell lines SW620 (49.7%) and HCT116 (27.7%), breast cancer cell line MBA-MD-231 (53.4%). ) and the HepG2 hepatoma cell line (92.7% reduction), as well as their ability to form unanchored spheres (average reduction of ~50.8%, 44.6%, 40.7% and 75.7% per cell line) (Fig. 2a,b). In SW620, the results of the colony formation assay further confirmed that DARS-AS1 shRNA significantly inhibited cell proliferation with an average decrease of approximately 69.6% (Fig. 2c).
Effect of control shRNA and DARS-AS1 shRNA on cell proliferation (a) and spheroid formation (b) in SW620, HCT116, MBA-MD-231, and HepG2 cells. c Effect of control shRNA and DARS-AS1 shRNA on colony formation in SW620 cells. Cell proliferation (d), spheroid formation (e), and colony formation (f) of SW620 cells overexpressing DARS-AS1. Data shown are the mean ± standard deviation of three experiments. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 by two-tailed Student’s t-test.
To complement loss-of-function studies, we next created SW620 cells overexpressing DARS-AS1 (Supplementary Fig. 2g). DARS-AS1 overexpression significantly increased cell growth (1.8-fold), unanchored spheroid formation (1.4-fold), and colony formation (3.3-fold) in SW620 cells (Fig. 2d–f). We confirmed this result using another DARS-AS1 expressing cell line, A549. This enhanced cell proliferation due to DARS-AS1 overexpression was further observed in A549 cells (Supplementary Fig. 2h, i and Supplementary Table 3). Taken together, these gain and loss studies demonstrate that DARS-AS1 promotes cancer cell proliferation in vitro.
To explore the underlying mechanism by which DARS-AS1 regulates cell proliferation, we performed an RNA pull-down analysis to identify its potential protein-binding partners. RT-qPCR results showed that about 86.2% of DARS-AS1 is located in the cytoplasm of SW620 cells (Supplementary Fig. 3a). The in vitro transcribed biotinylated DARS-AS1 or pseudoRNA was then incubated with SW620 cell lysates followed by SDS-PAGE separation. Subsequent silver staining showed that a distinct band (~38 kDa) was significantly enriched in DARS-AS1 pull samples but not in dummy RNA or beads samples (Fig. 3a). This band was identified as a PKR activating protein (PACT) by mass spectrometry (MS) and further confirmed by immunoblotting in SW620, HCT116, and HepG2 cell lines (Fig. 3a,b). The enrichment of DARS and related PACT proteins – PKR and TRBP – was also investigated using RNA analysis by Western blotting (WB). The results indicated that no direct interaction between DARS-AS1 RNA and these three proteins was found (Supplementary Fig. 3b). The specific interaction between DARS-AS1 and PACT was further confirmed by RNA immunoprecipitation (RIP) analysis, which showed that DARS-AS1 was significantly enriched in anti-PACT antibodies but not other control RNAs (Figure 3c). To determine if DARS-AS1 interacts directly with PACT in the absence of any other cellular components, an in vitro biolayer interferometry (BLI) assay was performed using purified PACT. Biotin-labeled DARS-AS1 or dummy RNA was immobilized on streptavidin (SA) biosensors and then incubated in kinetic buffer containing 1 μM PACT. Notably, PACT bound strongly to DARS-AS1 (KD value ~26.9 nM), but not to mimic RNA (Figure 3d). Taken together, these results demonstrate a direct interaction and high affinity between DARS-AS1 and PACT.
RNA pull analysis identified DARS-AS1 interacting with PACT in SW620 cells. Above, silver staining of related proteins. Lower immunoblots were performed with anti-PACT antibody. b RNA pull-down analysis was performed in HCT116 (top) and HepG2 (bottom) cells. PACT enrichment was detected by immunoblotting. cRNA immunoprecipitation (RIP) assays were performed in SW620 cells using the indicated antibodies. d PACT binding curves to full-length DARS-AS1 or control RNA were obtained using biolayer interferometry (BLI). RNA was immobilized on a streptavidin biosensor. 1 μM PACT was used to measure association. e RNA pull assay was performed using biotinylated full-length DARS-AS1 or truncated (top). Immunoblot showing PACT received (bottom). f Purified flagged PACT was incubated with biotinylated full-length DARS-AS1 or truncated (as in e) for in vitro RIP assay. The extracted RNA was verified by RT-qPCR. g The relative affinity of different RNA fragments for PACT was obtained using biolayer interferometry. For analysis, 100 nM RNA and 1 μM RAST were used. h In vitro RIP assays were performed using purified intact or truncated labeled PACT. The extracted RNA was verified by RT-qPCR. i Growth rate of SW620 cells overexpressing DARS-AS1, PACT, or both. j Overexpression of full-length or truncated DARS-AS1 in SW620 cells had different effects on cell growth. k Apoptosis was detected by immunoblotting with anti-PARP antibody. l Knockout of DARS-AS1 induces apoptosis of SW620 cells as shown by flow cytometry. Data shown are the mean ± standard deviation of three experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, by two-tailed Student’s t test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, by two-tailed Student’s t test. *p ≤ 0,05, **p ≤ 0,01, ***p ≤ 0,001, ****p < 0,0001 по двустороннему критерию Стьюдента. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001 by two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001，通过双尾学生t 检验。 *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001，通过双尾学生t 检验。 *p ≤ 0,05, **p ≤ 0,01, ***p ≤ 0,001, ****p <0,0001 по двустороннему критерию Стьюдента. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001 by two-tailed Student’s t-test.
We then generated three biotinylated DARS-AS1 RNA fragments by in vitro transcription to identify the DARS-AS1 region required for PACT association (Figure 3e). The RNA analysis results showed that each fragment was able to interact with PACT, but the 3′-terminal region (384–768 nucleotides labeled A3) showed more than 1–384 nucleotides labeled A1) (Fig. 3e). Similar results were observed in the in vitro RIP assay using recombinant PACT (Figure 3f). Consistent with these results, experiments to bind immobilized RNA fragments to PACT using BLI also showed that PACT has a higher affinity for A3 (384–768 nt) (KD value of approximately 94.6 nM), while almost no links with other areas. (Fig. 3d).
We also examined the associated binding regions in PACT. PACT contains three functional domains, two of which are conserved double-stranded RNA-binding domains (dsRBD) and a third domain (designated D3) that acts as an activator of protein interactions. To examine the lncRNA binding capacity of each domain, we engineered three mutations that removed each of the three domains and performed an in vitro RIP assay. Our results showed that deletion of the third domain (D3) of PACT significantly reduced its interaction with DARS-AS1 (by 0.11-fold compared with intact PACT) compared to the other two mutations (Fig. 3h), it was shown that the release of D3 interacted with DARS. -AC1. Taken together, these results suggest that interaction between DARS-AS1 and PACT may occur primarily through the 3′ end of DARS-AS1 and the D3 domain of PACT.
We noted that DARS-AS1 had no effect on PACT expression and PACT had no effect on DARS-AS1 (Supplementary Fig. 3c). We then examined the effect of PACT knockdown on cell growth. In contrast to DARS-AS1, relative cells grew 1.5–3 times faster when PACT was knocked down (Supplementary Fig. 3d). The results of the colony formation assay indicated that the cells formed 2-3-fold colonies after shRNA treatment with PACT (Supplementary Fig. 3e). To test whether DARS-AS1 regulates cell proliferation through PACT, we generated SW620 cells overexpressing PACT, DARS-AS1, or both. Overexpression of PACT showed significant inhibition of cell proliferation (Figure 3i). While DARS-AS1 overexpression per se significantly promoted cell proliferation, there was no significant difference in the growth rate of cells overexpressing DARS-AS1 and PACT. These results suggest that PACT may counteract the increased proliferation caused by DARS-AS1 overexpression.
Since different regions of DARS-AS1 have different PACT-binding abilities, we investigated their relative influence on cell proliferation by different overexpression of DARS-AS1 fragments. Compared to the other two fragments, DARS-AS1 was overexpressed at the 3′ end (384–768 nt), the main PACT-related region in DARS-AS1, which had the highest ability to stimulate cell proliferation (Fig. 3j). These results indicate a positive correlation between the binding capacity and biological function of DARS-AS1.
PACT has been reported to be a pro-apoptotic protein19. Therefore, we investigated the effect of DARS-AS1 on apoptosis. As expected, DARS-AS1 knockdown significantly increased PARP cleavage in SW620 cells and increased the proportion of annexin V-positive cells in SW620, HCT116, HepG2, and MBA-MD-231 cell lines (Fig. 3k). 3). 3f–h), indicating that the anti-apoptotic effect of DARS-AS1 in cancer cells is opposite to the apoptosis-inducing function of PACT. Taken together, these results suggest that the mechanism of DARS-AS1 oncogenic function may be through inhibition of PACT function.
Next, we explored the functional implications of the DARS-AS1-PACT association. PACT has been reported to activate PKR through direct interaction, which subsequently enhances eIF2α phosphorylation, causing translational deletion and apoptosis17. First, we examined whether DARS-AS1 affects the cellular localization of PACT and PKR. Confocal fluorescence microscopy showed that PACT and PKR were highly colocalized in SW620 cells with an average Pearson correlation coefficient of 0.72. Meanwhile, DARS-AS1 overexpression significantly reduced PACT and PKR co-localization (mean Pearson correlation coefficient 0.61) (Figure 4a). To investigate whether DARS-AS1 could modulate the PACT-PKR interaction, we performed a co-immunoprecipitation (co-IP) assay with anti-PACT antibody in SW620 cell lysates. PKR was highly enriched in anti-PACT in control cells, while PKR recovery was significantly reduced in lysates from cells overexpressing DARS-AS1 (Fig. 4b). Purified labeled PACT and PKR were used for in vitro protein binding assays. Accordingly, those that provided DARS-AS1 but no control RNA showed suppressed PACT-PKR interaction (Figure 4c). All results showed that DARS-AS1 disrupted PACT and PKR communication.
a Co-localization of PACT and PKR in control cells or cells overexpressing DARS-AS1 was observed using confocal fluorescence microscopy. The nuclei were stained with DAPI. Statistical results were obtained from 16 photographs. b Co-immunoprecipitation (co-IP) using anti-PACT antibody in cell lysates of control SW620 cells or cells overexpressing DARS-AS1. c Labeled PACT, purified PKR and transcribed in vitro with DARS-AS1 or mock RNA were incubated for in vitro protein binding analysis. Anti-flag antibodies were used for immunoprecipitation. d Immunoblots with the indicated antibodies were performed in SW620 and HCT116 cells transfected with control shRNA or DARS-AS1-shRNA followed by serum starvation. e DARS-AS1 expression levels altered cellular sensitivity to thapsigargin. SW620 cells were transfected with DARS-AS1 shRNA, DARS-AS1 overexpression plasmid or control plasmid. Cells were treated with thapsigargin for 48 hours and cell viability was determined using the MTS reagent. f In vitro transcribed DARS-AS1 or dummy RNA and purified PACT were used for in vitro activation assay and immunoblot detection. g Immunoblots using these antibodies were performed on SW620-ctrl cells (left) or cells overexpressing PKR mutants (right). These cells were then transfected with control shRNA or DARS-AS1-shRNA followed by serum starvation. h Flow cytometry showed that inactivation of the mutant PKR compensated for DARS-AS1-induced apoptosis in SW620 cells. i Immunoblots with the indicated antibodies were performed in SW620 (left) or HCT116 (right) cells. Cells transfected with control shRNA or DARS-AS1 shRNA are serum-deprived and supplemented with 100 nM PKR C16 inhibitor or DMSO. Scale bar = 5 µm. Data shown are the mean ± standard deviation of three experiments. * p ≤ 0.05 two-tailed Student’s t-test.
It is generally believed that once PACT interacts with PKR17, PKR phosphorylation at Thr451 can be induced. Our results indicated that the level of PKR phosphorylation was significantly elevated in DARS-AS1 knockdown cells after serum starvation (Fig. 4d and Supplementary Fig. 4a). Accordingly, we found that phosphorylation of eIF2α, the main PKR substrate, was also significantly increased by DARS-AS1 shRNA (Fig. 4d and Supplementary Fig. 4a). Thapsigargin is an ER stressor that causes the ER to release Ca2+. Treatment with thapsigargin has been reported to induce the expression and activation of PACT, which further interacts with and activates PKR, leading to apoptosis by increasing eIF2α phosphorylation 18,61 . Here, we used thapsigargin as a stimulator of the PACT/PKR pathway to investigate whether DARS-AS1 can help cells overcome stress by inhibiting the PACT/PKR pathway. We observed that the level of DARS-AS1 expression positively correlated with cell resistance to thapsigargin. SW620 cells overexpressing DARS-AS1 survived better when treated with thapsigargin, while cells with DARS-AS1 knockdown became more susceptible (Fig. 4e). Consistent with these results, DARS-AS1 overexpression reduced thapsigargin-induced PKR phosphorylation (Supplementary Fig. 4b). In contrast, after thapsigargin treatment, PKR and eIF2α were phosphorylated to a higher extent in DARS-AS1 knockdown cells compared to control cells (Supplementary Fig. 4b). Interestingly, thapsigargin induced DARS-AS1 expression in a dose-dependent manner, which may indicate an anti-stress function of DARS-AS1 (Supplementary Fig. 4c). In addition, we performed in vitro activation assays to confirm these observations. Briefly, PKR was purified from cell lysates using an anti-PKR antibody, then incubated with recombinant PACT and DARS-AS1 transcribed in vitro. After the enzymatic reaction, phospho-PKR was detected using WB. Our results indicated that PKR phosphorylation was significantly inhibited by DARS-AS1, but not by control RNA (Figure 4f). These in vitro and in vivo results suggest that DARS-AS1 inhibits PACT-mediated PKR activation. At the same time, we also observed a decrease in PACT recovery in the presence of DARS-AS1 (Figure 4f). This result is consistent with the results of the in vitro protein binding assay (Figure 4c) and again illustrates the blocking function of DARS-AS1 for PACT-PKR association.
Ser246 and Ser287 in the D3 domain of PACT are required for PKR activation under cellular stress. Substitution of two serine residues for alanine gave mutant PACT (mutD), which activated PKR in the absence of stress, and substitution for alanine (mutA) reversed the protocol. Since we have demonstrated the importance of this domain in direct association with DARS-AS1, we generated these two PACT mutants to test whether these residues could also be involved in interaction with DARS-AS1. Interestingly, both mutants lost the ability to bind to DARS-AS1 (Supplementary Fig. 4d), suggesting that the complete structure of the PACT protein may be required for efficient interaction with DARS-AS1.
Furthermore, our results also suggest that DARS-AS1-shRNA-induced inhibition of cell proliferation can be partially restored by overexpressing a dominant negative PACT mutant (PACTmutA) or a dominant negative PKR mutant (PKRmut) (Supplementary Fig. 4e. e). Overexpression of dominant-negative PKR mutants reduced PKR phosphorylation induced by DARS-AS1 knockdown as well as eIF2α phosphorylation in serum-deprived cells (Fig. 4g). More importantly, the proportion of apoptotic cells induced by DARS-AS1 knockdown was also reduced in cells overexpressing PKRmut (Fig. 4h and Supplementary Fig. 4g). Inhibition of PKR kinase activity also impairs DARS-AS1 function, as results showed that DARS-AS1 knockdown rarely triggered PKR and eIF2α phosphorylation when cells were treated with a PKR-specific C16 inhibitor (Fig. 4i and Supplementary Fig. 4h). ). Taken together, our results suggest that DARS-AS1 promotes cell proliferation, at least in part, by inhibiting PACT-mediated PKR activation.
To further explore the role of DARS-AS1 in tumorigenesis, we performed in vivo experiments using a mouse xenograft model. Results show that knockdown of DARS-AS1 dramatically decreased tumor growth in mice (p value < 0.0001) (Fig. 5a). Results show that knockdown of DARS-AS1 dramatically decreased tumor growth in mice (p value < 0.0001) (Fig. 5a). Результаты показывают, что нокдаун DARS-AS1 резко снижает рост опухоли у мышей (значение p <0,0001) (рис. 5а). The results show that DARS-AS1 knockdown drastically reduces tumor growth in mice (p value < 0.0001) (Figure 5a).结果表明，DARS-AS1 的敲低显着降低了小鼠的肿瘤生长（p 值< 0.0001）（图5a）。结果表明，DARS-AS1 的敲低显着降低了小鼠的肿瘤生长（p值<0.0001）（图5a）。 Результаты показали, что нокдаун DARS-AS1 значительно снижает рост опухоли у мышей (значение р <0,0001) (рис. 5а). The results showed that DARS-AS1 knockdown significantly reduced tumor growth in mice (p value < 0.0001) (Figure 5a). Thus, in the DARS-AS1 knockdown group, there was a significant decrease in mean tumor volume by about 72.9% and mean tumor mass by about 87.8% (Figure 5b-d). These results strongly suggest that DARS-AS1 can significantly promote tumor growth in vivo.
Effects of ad DARS-AS1 knockdown on colorectal oncogenesis in nude mice. Growth curves (a), tumor size (b), weight (c), and tumor images (d) are shown. Error bars represent ±SEM. n = 10. ****p < 0.0001, by two-tailed Student’s t test. n = 10. ****p < 0.0001, by two-tailed Student’s t test. n = 10. ****p < 0,0001 по двустороннему критерию Стьюдента. n = 10. ****p < 0.0001 two-tailed Student’s t-test. n = 10. ****p < 0.0001，通过双尾学生t 检验。 ****p < 0.0001，通过双尾学生t检验。 ****p < 0,0001 по двустороннему критерию Стьюдента. ****p < 0.0001 two-tailed Student’s t-test. e Kaplan-Meier analyzed the correlation between DARS-AS1 expression levels and overall survival in patients with UVM, KICH, KIRP, MESO, GBM, and LGG. High levels of DARS-AS1 expression in patients were in the top 50%; the low level of DARS-AS1 expression in patients was in the bottom 50%. p-values ​​were determined using the log rank test. f Proposed model in which DARS-AS1 regulates the PACT-PKR pathway and tumor growth.
To better understand the clinical impact of DARS-AS1, we examined the correlation between its expression and patient survival. By analyzing the TCGA dataset, we found that higher DARS-AS1 expression was associated with uveal melanoma (UVM), renal chromophobia (KICH), renal papillary cell carcinoma (KIRP), mesothelioma (MESO), multiplex. Lower survival was significantly associated with glioblastoma morphosis (GBM) and patients with low-grade brain glioma (LGG) (Figure 5e). These results suggest that DARS-AS1 may play an important role in clinical tumor progression and may be a potential predictive biomarker in multiple cancers.
In this study, using large-scale CRISPRi functional screening, we determined that DARS-AS1 lncRNA overcomes cancer cell stress by regulating two key stress responders, PACT and PKR. By interacting directly with PACT, DARS-AS1 inhibited PACT-mediated PKR activation, thereby preventing apoptotic cell death and promoting cell proliferation (Fig. 5f). Upregulation of DARS-AS1 has been observed in multiple types of cancer, suggesting that its function of promoting cancer cell survival under stressful conditions may be broadly applicable to multiple types of cancer.
PACT has been identified as a PKR activator protein, and PACT-mediated PKR activation plays an important role in stress responses by regulating transcription, translation, apoptosis, and other important cellular processes62. For decades, attempts have been made to understand the cancer-specific regulation of the PACT-PKR cascade. Here, our study revealed a different mechanism of regulation of PACT-PKR in cancer cells through cellular lncRNA DARS-AS1, which directly binds to PACT, blocks PACT-PKR interaction, inhibits PKR activation and eIF2α phosphorylation, thereby inhibiting stress-induced apoptosis and stimulating eventual cancer proliferation. cells. This discovery sheds light on potential lncRNA targets for cancer prognosis and therapy.
Our data showed that DARS-AS1 knockdown sensitizes cells to serum starvation with a significant increase in phosphorylated PKR and eIF2α. These results suggest that DARS-AS1 promotes cancer cell survival under harsh conditions by inhibiting PACT/PKR activity. Several other non-coding RNAs, such as ASPACT and nc886, are also involved in the PACT/PKR axis by downregulating PACT48 mRNA or regulating autophosphorylation by binding to PKR49,50,64. Among them, DARS-AS1 acts as a disruptor of the PACT-PKR association. This study enriches our understanding of PACT/PKR axis regulation and the role of lncRNAs in stress responses.
PACT contains three separate domains. Each of the first two dsRBDs is sufficient to achieve high affinity binding of PACT to PKR, while the third domain (D3) is required for PKR activation in vitro and in vivo. Our study showed that DARS-AS1 preferentially interacts with the D3 domain (Fig. 3h). Given the large size of the lncRNA (768 nucleotides), DARS-AS1 binding to D3 can physically inhibit the interaction between the PACT domain of dsRBD and PKR, thereby blocking the association of PACT and PKR. PACT point mutations that replaced Ser246 and Ser287 in D3 with alanine or aspartate disrupted its binding affinity for DARS-AS1, pointing to the importance of D3′s overall structural and electrical properties in their association. Further details of this mechanism will be required in the future, using more precise biochemical analysis and high resolution PACT structural analysis.
Previous studies have reported that DARS-AS1 promotes cell proliferation through several mechanisms51,52,53. In one example, investigators observed that DARS-AS1 upregulated its antisense protein-encoding DARS gene by targeting miP-194-5p in kidney cancer cells. However, in the present study, DARS-AS1 knockdown had little effect on DARS transcription in multiple types of cancer, including at least colorectal, breast, and liver cancers. Because lncRNAs exhibit cell- and tissue-specific expression patterns, functional mechanisms may not be conserved across cancer types, which may contribute to this discrepancy between our observations and previous assessments of different cancers. Special studies are needed to elucidate the specific mechanisms of various physiological and pathological processes.
An analysis of clinical data showed that DARS-AS1 expression in tumors is inversely correlated with the survival of cancer patients, which underlines the importance of the DARS-AS1/PACT/PKR axis in cancer prognosis. In conclusion, our study shows that DARS-AS1 is a regulator of the PACT/PKR signaling axis, promotes cancer cell proliferation, and inhibits apoptosis during the stress response, which provides another line of research and is of interest for future research into potential treatments.
Human cell lines SW620, A549, MBA-MD-231, HCT116, HepG2 and HEK293T were obtained from the National Cell Line Resource Infrastructure in China. All cells were maintained in DMEM medium (DMEM, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS (Gemini, Brooklyn, NY) and 1% penicillin-streptomycin (Thermo Fisher Scientific) at 37°C, 5% CO2. incubator.
Anti-PACT, Abcam (ab31967); Anti-PKR, Abcam (ab184257); Anti-PKR (phospho-T451), Abcam (ab81303); Anti-Flag, Abcam (ab125243); Anti-eIF2α, Abcam (A0764)) ; anti-eIF2α (phosphorus S51), Abcam (ab32157); anti-PACT (phosphorus S246), Abgent (AP7744b); anti-β-tubulin, CST (2128); normal mouse IgG, CST (5415S); normal rabbit IgG, CST (2729S). Antibodies were diluted 1:1000 in PBST for Western blotting and 1:100 for IP.
sgRNAs were developed using a publicly available tool called CRISPR-ERA66. We used the default tool parameters for sgRNA development and the algorithm computed sgRNA binding sites in the 3 kb region. centered at TSS. Pools of sgRNA oligonucleotides were synthesized at CustomArray, Inc. (Bothewell, WA) and cloned into humanized pgRNA plasmids (Addgene #44248). A total of 12 µg of pooled humanized pgRNA plasmid, 7.2 µg of psPAX2 (Addgene #12260), and 4.8 µg of pMD2.G (Addgene #12259) were co-transfected into 5 x 106 HEK293T in 10 cm dishes using DNAfect Transfection Reagent cells ( CWBIO, Beijing, China) following the manufacturer’s instructions. Virus-containing supernatants were collected 48 and 72 hours after transfection and filtered through a 0.45 µm filter. For screening, SW620 cells expressing the dCas9/KRAB fusion protein were obtained by virus transduction. Modified SW620 cells were infected with the virus library in four independent infection experiments at an MOI of 0.1-0.3 and were sampled with 2 μg/ml puromycin (Sigma, St. Louis, MO) for 2 days. Thereafter, cells were cultured for 18 days in vitro with a minimum library coverage of 500 cells/sgRNA for screening.
Genomic DNA was extracted according to the instructions of the QIAamp DNA Blood Midi Kit (QIAGEN, Düsseldorf, Germany; 51183). In total, 100 μg of genomic DNA per biological repeat was used to build the library. The sgRNA region was amplified by two rounds of PCR and linked to a barcode.
PCR products were purified using NucleoSpin® gel and PCR purification kit (MACHEREY-NAGEL, Düren, Germany; 740609.250) and quantified using Qubit™ HS double-stranded DNA detection kit (Thermo Fisher Scientific; Q32854).
The MTS assay was used to measure cell proliferation. Cells were seeded in 96-well plates at an initial density of 2000 cells/well. The relative number of cells was measured daily at the indicated time for a total of 4-6 days. For each well, 20 μl of MTS reagent (Promega) was diluted with 100 μl of DMEM, incubated with cells for 4 h at 37°C, and then OD490 was measured.
The capacity for unanchored growth was discovered by analyzing the formation of spheres. Briefly, 2000 cells transfected with shRNA DARS-AS1 or control shRNA were cultured in ultra low attachment microplates (Corning) with medium change every 4 days. The spheroids were counted after 14 days. 500 cells transfected with the DARS-AS1 overexpression plasmid or a control plasmid were used for the enhancement assay, otherwise the method was unchanged.
RNA was transcribed using T7 RNA polymerase and biotin-16-UTP (Roche 1138908910) according to the instructions of Riboprobe® Combination Systems (Promega P1440). The primers used here are listed in Supplementary Table 4.
Protein-coding PACT or PKR regions were cloned into pET15b (Addgene #73619) and transformed into BL21(DE3). The bacteria were incubated overnight in LB supplied with ampicillin and then diluted 100-fold with fresh LB. When the OD600 of the medium reached 0.8, 1 mM IPTG was added to induce protein expression. After incubation overnight with gentle shaking (250 rpm at 20°C), the cell pellet was collected by centrifugation (4000 rpm, 10 min, 4°C). Resuspend the cell pellet in lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 1 mM PMSF) and incubate on ice for 30 min, then sonicate (15 min, 5 s on/off, on ice) and centrifuge (13,000 rpm). , 30 min, 4°С). The supernatant was then loaded onto Ni-NTA resin (QIAGEN) 3 times at 4°C, washed 4 times with wash buffer (50 mM Tris, pH 8.0, 40 mM imidazole, 250 mM NaCl) and eluted 3 times, with a total of 10 ml eluent buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole). Purified protein was determined using WB and the concentration was determined using the Qubit™ protein assay kit (Thermo Fisher Scientific; Q 33212).
RIP assays were performed as previously described, with modifications. Briefly, 1x RIP buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% NP-40, RNasin ribonuclease inhibitor (Promega), PMSF (Beyotime Biotechnology), 1 mM DDM, protease) lyses cytostatic 1 x 107 cocktail (Roche, 1 mM DTT) and centrifuge at 13,000 rpm for 15 min at 4 °C. The supernatant was then incubated with protein A+G magnetic beads (Millipore) conjugated with 5 μg of anti-PACT antibody (Abeam) or IgG (CST). The beads were washed 5 times with 5x RIP buffer, then digested with proteinase K (NEB). RNA was extracted with Trizol and determined by RT-qPCR. Primers are presented in Supplementary Table 5.
The in vitro RIP assay was performed according to a modified standard RIP assay protocol. A total of 5 pmol of in vitro transcribed RNA was diluted 1x with RIP buffer and annealed by incubation at 65°C for 5 minutes followed by slow cooling to room temperature. A total of 5 pmol of intact or mutated flag-labeled PACT proteins were purified from E. coli. Incubate with renatured RNA for 2 hours at 4°C and follow the above procedure for RIP analysis for anti-flag IP.
For RNA extension analysis, 1×107 cells were lysed with 1xRIP buffer. After centrifugation at 13,000 rpm for 15 min at 4°C, the supernatant was pretreated with 30 μl of streptavidin magnetic beads (Beckman) for 2 h at 4°C. The purified lysate was then supplied with yeast tRNA and incubated with 40 pmol of renatured RNA overnight at 4°C, then for another 2 hours and 20 μl of new streptavidin magnetic beads blocked with BSA was added. The washing step consisted of 4 times with 5x RIP buffer and 4 times with 1x RIP buffer. The corresponding proteins were eluted with biotin elution buffer (25 mM Tris-HCl, pH 7.5, 12.5 mM D-biotin, PMSF) and separated on NuPAGE 4-12% Bis-Tris Gel (Invitrogen). After silver staining (Beyotime Biotechnology), certain bands were excised and analyzed by MS.
Co-IP analysis was performed to test the interaction between PACT and PKR. Briefly, supernatant lysates were prepared by incubating 1 x 107 lysed cells in 1 x RIP buffer followed by centrifugation at 13,000 rpm for 15 minutes at 4°C. Lysates were loaded with protein A + G magnetic beads, conjugated with 5 µg of anti-PACT antibody, and gently rotated overnight at 4°C. The beads were washed 3 times with 5×RIP buffer, twice with 1×RIP buffer and eluted with 1×SDS buffer. The recovered protein was analyzed by SDS-PAGE gel and detected by WB.
Two pmol of flagged PACT and 1 pmol of PKR were purified from E. coli. Dilute in 1× RIP buffer and incubate with 10 pmol of renatured RNA for 2 hours at 4 °C. After that, they were incubated with protein A+G magnetic bead-conjugated anti-labeled antibody for an additional two hours. The beads were then washed four times with 1x RIP buffer and eluted with 1x SDS buffer. The resulting PACT and PKR were detected by WB.