A testis-specific lncRNA functions as a post-transcriptional regulator of MDM2 and stimulates apoptosis of testicular germ cell tumor cells

A testis-specific lncRNA functions as a post-transcriptional regulator of MDM2 and stimulates apoptosis of testicular germ cell tumor cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A testis-specific lncRNA functions as a post-transcriptional regulator of MDM2 and stimulates apoptosis of testicular germ cell tumor cells Saya Ito, Akihisa Ueno, Takashi Ueda, Ryota Ogura, Satoshi Sako, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4234181/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Aug, 2024 Read the published version in Cell Death Discovery → Version 1 posted You are reading this latest preprint version Abstract Germ cells preferentially induce apoptosis in response to DNA damage to avoid genomic mutations. Apoptosis of germ cells is closely related to cancer development and chemotherapy resistance; however, its regulatory mechanism is unclear. Here, we suggest that testis-specific lncRNA LINC03074 is involved in male germ cell apoptosis by regulating the expression of the proto-oncogene MDM2 . LINC03074 is highly expressed in the sperm of healthy adult testes and cancer cells of testes with testicular germ cell tumors (TGCTs). LINC03074 binds to MDM2 mRNA via an Alu element, thereby reducing MDM2 protein levels. LINC03074 stimulates STAU1-mediated nuclear export of MDM2 mRNA by increasing STAU1 binding to MDM2 mRNA in the cell nucleus, thereby promoting PKR-mediated translational repression in the cytoplasm. The induction of apoptosis in TGCT cells and their responsiveness to the anticancer drug cisplatin is enhanced by LINC03074 . Notably, LINC03074 increased E2F1 expression without increasing p53, the primary target of MDM2, and upregulated the apoptotic gene p73 , the target gene of E2F1. LINC03074 -mediated regulation of apoptosis contributes to the responsiveness of TGCTs to anticancer drug-induced DNA damage. Biological sciences/Molecular biology/Non-coding RNAs Biological sciences/Cancer/Testicular cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Testicular germ cell tumors (TGCTs) are generally highly sensitive to platinum-based chemotherapy, such as cisplatin, but some are chemotherapy-resistant [ 1 ]. Mouse double minute 2 (MDM2) is amplified in various human malignancies, including TGCTs, and MDM2 overexpression is associated with chemotherapy resistance [ 2 , 3 ]. MDM2 is a major negative regulator of p53, promoting ubiquitin-dependent proteasomal degradation of p53 as an E3 ubiquitin ligase and repressing p53 transcriptional activation [ 4 , 5 ]. The tumor suppressor p53 is commonly mutated in various types of cancers [ 6 , 7 ], whereas it is often overexpressed and rarely mutated in TGCTs [ 3 ]. Accordingly, MDM2 overexpression in TGCTs is predicted to affect the activity of wild-type p53; however, the molecular mechanisms underlying the sensitivity or resistance of TGCTs to chemotherapy remain unclear. MDM2 expression and activity are regulated at multiple levels, from transcription to numerous post-translational modifications, in addition to genomic alterations, such as copy number variations, mutations, and polymorphisms [ 8 ]. Post-transcriptional regulation of MDM2 at the mRNA level has been widely reported to modulate transcript stability via miRNAs [ 9 ]. Human MDM2 has a very long 3′ UTR (~ 5.7 kb) that retains a multitude of potential miRNA targets [ 10 ]. Notably, MDM2 contains multiple transposable elements containing Alu in the 3′ UTR [ 11 ]. Alu elements are abundant retrotransposon elements that spread throughout the human genome and occupy a significant portion of the 3′ UTR [ 12 ]. The inverted repeat Alu element (IR Alus ) in the 3′ UTR forms a double-stranded RNA (dsRNA) structure, which serves as a target for dsRNA-binding factors such as Staufen1 (STAU1) and adenosine deaminase acting on RNA (ADAR1) [ 13 , 14 ]. STAU1 binds to IR Alu in the 3′ UTR to undergo various RNA metabolic processes, such as RNA synthesis, folding, modification, processing, translation, and decay [ 13 ]. ADAR1, an adenosine-to-inosine (A-to-I) RNA-editing enzyme, competes with STAU1 for the occupancy of target RNAs, thereby inhibiting STAU1-mediated nuclear retention or decay of RNAs [ 15 , 16 ]. In MDM2 gene expression, STAU1 and ADAR1 appear to mediate post-transcriptional regulation via binding to the IR Alus of the MDM2 3′ UTR, a mechanism independent of alterations in mRNA stability and miRNA targeting [ 16 ]. Here, we show the role of LINC03074 , a testis-specific lncRNA with an Alu element, in TGCT cells. LINC03074 binds to MDM2 mRNA via Alu , thereby stimulating STAU1-mediated nuclear export of MDM2 mRNA. Consequently, MDM2 is reduced by PKR-mediated translational repression, which in turn promotes the apoptosis of TGCT cells. Our findings provide molecular mechanistic insights into the drug responsiveness of TGCTs by demonstrating a regulatory mechanism of apoptosis in TGCT cells. Results LINC03074 shows different expression patterns between cancerous and normal sperm To elucidate the characteristics of TGCTs, we compared the gene expression profiles of cancer tissues from seminoma patients with those of matched normal adjacent tissues [ 17 ]. A total of 565 genes, among the 50,599 genes tested, exhibited a more than 2-fold increase in RNA expression in seminoma tissues compared to normal adjacent tissues, including 18 genes encoding lncRNAs (data not shown). In contrast, 431 genes exhibited a > 2-fold decrease in expression in cancer tissues, including 59 genes encoding lncRNAs (data not shown). To identify the lncRNA that determines the characteristics of testicular tumor cells, we focused on LINC03074 ( LOC100505685 ), which showed marked differences in expression between cancerous and normal tissues. According to the database, LINC03074 is expressed specifically in the testes of humans (Fig. 1 A). Moreover, a recent study identified LINC03074 as a testis-specific lncRNA [ 18 ]. The expression pattern of LINC03074 , which was significantly higher in normal tissues than in cancerous tissues of the testes of patients with seminoma, was confirmed via relative quantitative analysis using RT-qPCR ( P = 0.00178, Fig. 1 B). We performed ISH with an LINC03074 detection probe in the testes of healthy adults and patients with seminomas. The results revealed that LINC03074 was localized to the nucleus and cytoplasm of normal spermatids, whereas it was mainly localized to the nucleus of seminoma cells (Fig. 1 C). We further quantified the expression of LINC03074 in four types of cultured cells derived from seminoma and non-seminoma tissues and found that its expression was significantly higher in TCam-2 seminoma cells (Fig. S1 ) [ 35 ]. These results suggest that LINC03074 functions in both testis-derived seminoma cells and normal cells, although it is differentially expressed in cancer and normal cells. LINC03074 interacts with MDM2 mRNA via Alu element To elucidate the function of LINC03074 in testicular cells, we searched databases to identify the elements with which this lncRNA could interact. Using lncRRIsearch, MDM2 mRNA was identified as a candidate interacting factor for LINC03074 [ 19 ]. LINC03074 contained one Alu element, while MDM2 mRNA contained five Alu elements and a pair of inverted- Alu s in the 3′ UTR (Fig. 2 A). There were multiple candidate sequences for the interaction between LINC03074 and MDM2 mRNA within each Alu element (Fig. 2 A). The sequences of LINC03074 and the sense strand of MDM2 mRNA were nearly complementary, as shown in an example of a candidate interaction region (DG = -60.91 kcal/mol, lower part of Fig. 2 A). We decided to use TCam-2 cells as a model for seminoma cells in the following experiments, considering that LINC03074 is likely to function in seminomas according to its expression pattern (Fig. 1 C and Fig. S1 ). To determine whether LINC03074 binds to MDM2 mRNA, we performed CHART assays using TCam-2 cells. CHART enables the identification of associated targets of lncRNAs by enriching lncRNAs with their targets using affinity-tagged oligonucleotides (C-oligo) to capture endogenous lncRNAs in cross-linked cell extracts [ 20 ]. LINC03074 was enriched in TCam-2 cell extracts by CHART using a C-oligo for LINC03074 (Fig. 2 B). We then tested whether mRNAs of MDM2 , 18S-rRNA and GAPDH were enriched using LINC03074 CHART, and found that MDM2 mRNA was associated with LINC03074 (Fig. 2 B). Approximate estimates using qPCR showed that the molecular ratio of LINC03074 to MDM2 mRNA in TCam-2 cells was approximately 1:166 (Fig. S2 ). To confirm whether LINC03074 and MDM2 mRNA interact via their respective Alu elements, we generated Alu element-deficient LINC03074 and MDM2 expression constructs (Fig. 2 C). We performed an RNA pull-down assay with the 3′ UTR region of biotin-labeled MDM2 mRNA using total RNA extracted from HEK293 cells transiently expressing LINC03074 . We found that LINC03074 (FL) binds to the MDM2 3′ UTR (FL) (Fig. 2 D). In comparison, LINC03074 (FL) binds to the MDM2 3′ UTR (D5′-Alu) was attenuated (Fig. 2 D). In addition, LINC03074 (DAlu) showed significantly weaker binding to MDM2 than LINC03074 (FL) (Fig. 2 D). In contrast, no binding was detected between 18S-rRNA and MDM2 3′ UTR (either FL or D5′-Alu) (Fig. 2 D). These results indicated that LINC03074 binds to the Alu elements of MDM2 mRNA via its own Alu element. LINC03074 inhibits MDM2 gene expression by binding to MDM2 mRNA We speculated that LINC03074 may affect MDM2 gene expression by interacting with MDM2 mRNA. Three different siRNAs were used to knockdown LINC03074 , all of which decreased MDM2 mRNA levels and increased MDM2 protein levels (Fig. 3 A, B). The increase in MDM2 protein by LINC03074 knockdown was rescued by the transient expression of full-length LINC03074 (FL), but not by the DAlu mutant (DAlu) (Fig. 3 B). Next, we verified that LINC03074 -induced alterations in MDM2 protein levels were caused by the binding of LINC03074 to MDM2 mRNA. Flag-tag fusion MDM2 protein expression plasmids were generated by inserting 3′ UTR sequences (FL or D5′-Alu) downstream of the CDS of MDM2 (Fig. 3 C). These plasmids were transfected together with LINC03074 (FL or DAlu) expression plasmids into HEK293 cells, and the protein levels of FLAG-MDM2 were quantified by immunoblotting using an anti-FLAG antibody. In the absence of the 3′ UTR, FLAG-MDM2 levels remained unchanged regardless of the presence of LINC03074 (Fig. 3 D). However, in the presence of the 3′ UTR (FL), FLAG-MDM2 levels were significantly reduced by LINC03074 (FL), but not by LINC03074 (DAlu) (Fig. 3 D). In the case of Alu elements in the 3′ UTR were absent (D5′-Alu), FLAG-MDM2 levels were only slightly reduced by LINC03074 (Fig. 3 D). These findings suggest that LINC03074 binding to MDM2 mRNA via Alu elements may influence the post-transcriptional or translational processes of MDM2 gene expression. LINC03074 enhances STAU1-mediated nuclear export and PKR-induced translational repression of MDM2 mRNA It has been reported that the inverted repeat Alu elements (IR Alus ) in the 3′ UTR of mRNA serve as a binding site for ADAR1, a dsRNA-specific enzyme that performs A-to-I RNA editing [ 16 ]. The mRNA with 3′ UTR IR Alus edited by ADAR1 is retained in the nucleus through interaction with paraspeckle, which is formed by the nuclear lncRNA, NEAT1 , and its binding partner, NONO [ 21 ]. Alternatively, IR Alus in the 3′ UTR of mRNA binds to the dsRNA-binding protein STAU1, which is involved in various RNA metabolic regulations [ 13 ]. STAU1 binds to IR Alu to facilitate the export of IR Alu mRNA from the nucleus to the cytoplasm, while competitive inhibition of NONO binding to IR Alu prevents NONO-mediated mRNA retention in the nucleus [ 22 ]. We examined the binding of MDM2 mRNA to STAU1, ADAR1, or NONO in the nuclei of TCam-2 cells and the effect of LINC03074 on their binding. RIP assays using nuclear extracts of TCam-2 cells showed that MDM2 mRNA binds to STAU1 and ADAR1, but not NONO (Fig. 4 A). Knockdown of LINC03074 suppressed the binding of MDM2 mRNA to STAU1 while increasing its binding to ADAR1 (Fig. 4 A). In contrast, LINC03074 bound only to STAU1 but not to ADAR1 and NONO (Fig. 4 A). Considering that STAU1 is responsible for RNA shuttling, we investigated whether LINC03074 affected the nuclear export of MDM2 mRNA. MDM2 mRNA was increased in the nucleus and decreased in the cytoplasm following LINC03074 and STAU1 knockdown (Fig. 4 B). These results suggested that LINC03074 promotes the recruitment of STAU1 to MDM2 mRNA in the nucleus and facilitates STAU1-mediated nuclear export. Taken together, the downregulation of LINC03074 increased intracellular MDM2 protein levels, despite decreasing MDM2 mRNA levels in the cytoplasm (Fig. 3 B and 4 B). We speculated that LINC03074 -mediated enhancement of STAU1 and MDM2 mRNA interactions in the nucleus leads to the translational repression of MDM2 . STAU1 binding to IR Alu mRNA promotes nuclear export and translation [ 22 ]. However, STAU1-mediated mRNA nuclear export is promoted when the paraspeckle component is downregulated, whereas protein kinase R (PKR)-mediated translational repression in the cytoplasm is promoted [ 22 ]. PKR is activated by binding to virus-derived dsRNA and phosphorylates eukaryotic translation initiation factor 2A (eIF2a), resulting in translational inhibition [ 23 , 24 ]. To determine whether PKR can bind to MDM2 mRNA in TGCT cells, we performed RIP assays with cytoplasmic extracts from TCam-2 cells using a PKR antibody. PKR was found to bind to MDM2 mRNA in the cytoplasm, and this interaction was reduced by LINC03074 and STAU1 knockdown (Fig. 4 C). Furthermore, STAU1 and MDM2 mRNA binding in the cytoplasm was attenuated by LINC03074 and STAU1 knockdown (Fig. 4 C). Finally, we determined whether MDM2 was translationally repressed by PKR activation. MDM2 protein levels were increased by PKR inhibitor treatment of TCam-2 cells (Fig. 4 D). The increase in MDM2 protein expression induced by PKR inhibitors was not detected with LINC03074 and STAU1 knockdown (Fig. 4 D). These results indicated that LINC03074 , similar to paraspeckle components, modulates the nuclear export of STAU1-bound MDM2 mRNA, thereby facilitating PKR-mediated translational repression. LINC03074 enhances cisplatin-induced apoptosis and cell growth inhibition To assess whether LINC03074 affects the proliferation of TGCT cells, CCK8 analysis was performed using cisplatin, a platinum chemotherapeutic agent that induces DNA damage in cancer cells by inhibiting DNA repair [ 25 ]. The growth of TCam-2 cells was inhibited by cisplatin treatment in a concentration-dependent manner (Fig. 5 A). LINC03074 knockdown enhanced the growth of TCam-2 cells and attenuated cisplatin-induced inhibition of cell growth (Fig. 5 A). In contrast, LINC03074 knockdown had no effect on the growth of non-seminoma-derived NEC8 cells expressing low LINC03074 levels (Fig. S3). First, the effect of LINC03074 on the cell cycle was assessed; however, no cell cycle abnormalities due to LINC03074 knockdown or cisplatin treatment were observed under the conditions examined (Fig. S4). Next, the effect of LINC03074 on apoptosis was examined using FACS analysis. LINC03074 knockdown reduced the frequency of spontaneous and cisplatin-induced apoptosis (Fig. 5 B and S5). These results indicate that LINC03074 inhibits the proliferation and promotes the apoptosis of seminoma cells. Furthermore, the responsiveness of seminoma cells to cisplatin-induced DNA damage was enhanced by LINC03074 . LINC03074 increases E2F1 levels and upregulates p73 gene expression MDM2 is a major negative regulator of p53; MDM2 acts as an E3 ubiquitin ligase that recognizes p53 and acts as a transcriptional repressor of p53 [ 4 , 5 ]. To test whether LINC03074 affected p53 protein levels and function, we performed immunoblotting using anti-p53 and anti-phosphorylated p53 (Ser15) antibodies. p53 is activated by phosphorylation in response to DNA damage, and its Ser15 residue is the major phosphorylation site [ 26 ]. Immunoblotting confirmed that cisplatin-induced DNA damage markedly increased the p53 protein levels and promoted p53 phosphorylation (Fig. 6 A). Interestingly, LINC03074 knockdown did not affect the p53 or phosphorylated p53 levels (Fig. 6 A). MDM2 has been reported to interact with various proteins other than p53, and the apoptosis-related transcription factor E2F1 is one of its target proteins [ 27 , 28 ]. Immunoblotting with an anti-E2F1 antibody showed that E2F1 levels were increased by cisplatin treatment and decreased by LINC0374 knockdown, in the presence or absence of cisplatin (Fig. 6 A). E2F1 induces apoptosis through several mechanisms, including activation of p53-dependent and -independent pathways and inhibition of survival signaling [ 29 ]. To elucidate the mechanism by which LINC03074 mediates apoptosis, we examined the effects of LINC03074 knockdown on E2F1 target gene expression. Among the apoptotic genes targeted by E2F1, p73 , and BIM are transcriptionally regulated by E2F1, whereas PUMA and NOXA are regulated by E2F1 and p53 [ 29 ]. Of the four apoptosis-related genes subjected to mRNA quantification, only p73 exhibited decreased mRNA levels following LINC03074 knockdown with cisplatin (Fig. 6 B). In addition, p73 mRNA levels increased in response to cisplatin treatment, regardless of LINC03074 knockdown (Fig. 6 B). BIM , PUMA , and NOXA mRNAs showed a tendency to increase with LINC03074 knockdown with cisplatin, but no cisplatin addition-dependent increase was observed without LINC03074 knockdown (Fig. 6 B). Our results indicate that cisplatin-induced apoptosis of seminoma cells is associated with the increased expression of p73 . LINC03074 contributes to the upregulation of p73 by increasing E2F1 expression, which may indirectly affect the expression of other apoptotic genes. Discussion MDM2 expression levels are associated with chemotherapy resistance in human malignancies and are regulated at multiple levels [ 8 ]. In this study, we suggest that MDM2 levels are regulated by the testis-specific lncRNA LINC03074 during post-transcription. The LINC03074-MDM2-mediated apoptosis regulatory pathway provides new insights into the mechanisms of the DNA damage response in TGCT cells. A total of 17 pairs of interaction sequences were predicted within the Alu element of LINC03074 with the five Alu elements in the 3′ UTR of MDM2 mRNA (data from lncRRIsearch; shown in Fig. 2 A). Our results suggest the following: (1) LINC03074 binds complementarily to either Alu element of the MDM2 3′ UTR via its own Alu element; (2) LINC03074 modulates the binding of STAU1 and ADAR1 to the IR Alus of the MDM2 3′ UTR; and (3) LINC03074 interacts with STAU1 but not ADAR1 in the nucleus. These results suggest that LINC03074 functions as an RNA chaperone for MDM2 mRNA and that LINC03074 -induced conformational changes convert the dsRNA-binding factor that binds to the 3′ UTR of MDM2 . It is speculated that ADAR1 and STAU1 competitively bind to MDM2 mRNA because the interaction of ADAR1 with MDM2 mRNA has been reported to suppress STAU1-MDM2 mRNA binding [ 16 ]. Therefore, we concluded that LINC03074 binds to MDM2 mRNA to promote STAU1 recruitment, thereby indirectly suppressing ADAR1-MDM2 mRNA binding. The intracellular molecular ratio of LINC03074 to MDM2 mRNA in the TCam-2 cells was approximately 1:166 (Fig. S2 ). As an example of how a small amount of lncRNA can act on a large number of target molecules, previous reports have shown that SLERT is recycled to induce conformational changes in DDX21, which has approximately 1000 times more molecules than SLERT [ 30 ]. Further detailed molecular analysis is needed to determine the mechanism by which LINC03074 exerts its action and modifies more targets than its stoichiometry. In addition, the significance of the differences in LINC03074 levels between carcinoma and normal testis tissues needs to be investigated. PKR is activated by binding to virus-derived dsRNA and phosphorylates eukaryotic translation initiation factor 2A (eIF2A), resulting in translational repression [ 24 ]. It has been reported that downregulation of paraspeckle components, such as NEAT1 and NONO, increases cytoplasmic 3′ UTR IR Alus mRNAs, which increases phosphorylation of PKR and eIF2A, resulting in intracellular translational repression [ 22 ]. In this study, we demonstrated that downregulation of LINC03074 suppresses STAU1-mediated nuclear export of 3′ UTR IR Alus mRNA, resulting in attenuated PKR-mediated translational repression (Fig. 4 ). Taken together, LINC03074 had an opposite effect to that of paraspeckle components on the post-transcriptional regulation of IR Alus mRNA, but no interaction between LINC03074 and NONO was detected (Fig. 4 A). Additional examination of the molecular mechanism of LINC03074- mediated translational repression, involving its association with paraspeckles, is expected to provide new insights into the post-transcriptional regulation of the 3′ UTR IR Alus mRNAs. The relationship between MDM2 and p53 expression in TGCTs remains unclear, although MDM2 overexpression plays an important role in suppressing p53 activity in numerous tumors that retain wild-type p53 [ 31 , 32 ]. Several reports have suggested that p53 degradation by MDM2 is ineffective in TGCTs. Some studies have shown a positive correlation between MDM2 and wild-type p53 expression levels in TGCTs, while others have shown no correlation [ 3 , 33 ]. We found that attenuation of MDM2 levels by LINC03074 led to an increase in E2F1 but not p53 (Fig. 6 A). E2F1 is negatively and positively regulated by MDM2 in a p53-independent manner via both direct and indirect mechanisms [ 27 , 28 ]. Furthermore, E2F1 plays an important role in regulating cell proliferation and differentiation by significantly influencing cell cycle progression and survival through extensive crosstalk with p53 [ 34 ]. In this study, we demonstrated that the p73 , a target gene of E2F1 related to apoptosis, is involved in the responsiveness of seminoma cells to cisplatin, and its expression is regulated by LINC03074 (Fig. 6 B). Considering that the MDM2-E2F1-p73 pathway is predicted to play an important role in chemotherapy resistance of TGCTs, further insights into this pathway may lead to the development of new therapies for TGCTs. Materials and Methods Human tumor samples Samples of histologically normal testicular lesions and cancerous lesions were obtained from the surgical specimens of patients who underwent radical orchiectomy at the Kyoto Prefectural University of Medicine. The use of surgical and autopsy specimens for molecular analysis was approved by the Institutional Ethics Committee of the hospital. RNA immunoprecipitation TCam-2 cells were transfected with siLINC03074 and incubated for 72 h. Nuclear and cytoplasmic extracts were prepared as described previously (described in detail in the next section) [ 16 ]. To detect the interaction between RNA and protein, cellular lysates were incubated with anti-ADAR1 (15.8.6; Santa Cruz Biotechnology), anti-NONO (11058-1-AP; Proteintech), anti-STAU1 (C-4; Santa Cruz Biotechnology), anti-PKR (18244-1-AP; Proteintech) or anti-IgG (I5006; Sigma) antibodies at 4°C for 18 h and then mixed with Dynabeads Protein G (Thermo Fisher) at 4°C for 1 h. Immunoprecipitated RNAs were isolated using ISOGEN (NIPPON GENE), and quantified via RT-qPCR, as described above. Nuclear and Cytoplasmic fractionation Nuclear and cytoplasmic fractions were obtained as previously reported, with some modifications [ 16 ]. TCam-2 cells were lysed with the nuclear fractionation buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.2% NP-40, 3 mM MgCl 2 , 100 U/ml RNase Inhibitor at 4°C for 10 min and centrifuged at 13000 rpm at 4°C for 10 min. The supernatant was used as the cytoplasmic fraction. The pellet was washed with the nuclear fractionation buffer and centrifuged at 13000 rpm at 4°C for 10 min. The pellet was used as the nuclear fraction. The respective markers of the nuclear and cytoplasmic fractions, 5S-rRNA and GAPDH, respectively, were used as controls. Statistical analysis Statistical analyses were performed using t -tests or ANOVA, as appropriate. Statistical significance was set at P < 0.05. Each experiment was repeated at least three times. Information on statistical measures is provided in the legend of each figure. Declarations Competing Interests The authors declare no conflicts of interest. Author Contributions SI wrote the main manuscript text, made substantial contributions to the conception, design, acquisition of data, and analysis, and approved the final version to be submitted. AU, RO, TS, and YG contributed to the acquisition of clinical samples. TU and OU contributed to the acquisition of clinical samples and polished the manuscript. All authors have reviewed and approved the manuscript. Acknowledgment We thank Dr. Riko Kitazawa for providing us with the TCam-2 cell line. The authors would like to thank Dr. Hideo Nakagawa for assisting in the preparation of research samples from human clinical specimens. We would like to thank Editage ( www.editage.jp ) for the English language editing. This work was supported in part by the Japan Society for the Promotion of Science (JSPS) through Grants-in-Aid for Scientific Research fund (grant numbers 16K15695 and 19K09698). References Singh R, Fazal Z, Freemantle SJ, Spinella MJ. Mechanisms of cisplatin sensitivity and resistance in testicular germ cell tumors. Cancer Drug Resist. 2019; 2: 580–594. Epub 20190919. doi: 10.20517/cdr.2019.19, PubMed PMID: 31538140, PubMed Central PMCID: PMC6752046. 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Mizuno Y, Gotoh A, Kamidono S, Kitazawa S. [Establishment and characterization of a new human testicular germ cell tumor cell line (TCam-2)]. Nihon Hinyokika Gakkai Zasshi. 1993; 84: 1211–1218. doi: 10.5980/jpnjurol1989.84.1211, PubMed PMID: 8394948. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files ItoetalSupportingInformation.docx Cite Share Download PDF Status: Published Journal Publication published 03 Aug, 2024 Read the published version in Cell Death Discovery → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4234181","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":289244272,"identity":"5d36b199-707b-4f95-b688-85fb38f9a59b","order_by":0,"name":"Saya Ito","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBACNigtJwGmDmBIYNEClTKWYGAmUgtMKnEGmhbcgE+++fCHn2126TPb+w8+YDhzJ7FB7ADjhx8MfHm4HcaWJtnblpw7m+cwswHDjWeJDdIJzJI9DGzFuLXwmDHwtjHnzpNIZpNg+HA4cf/tBAZpoERiA24txh//ttWny8G0gGz5TUCLgTRv2+EEabCWG2AtbARsSUuTljl33HBmz2Fjg4Qzz4wbpBPbLHsMcPsFGGCHP74pq5aXON748MGHY3dkG6STD9/4UXEMZ4iBASMs3hLAEcMIdJLBsQS8Whj+wFkHYIwaAlpGwSgYBaNgBAEAXClRjdNWcZAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3077-8975","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Saya","middleName":"","lastName":"Ito","suffix":""},{"id":289244273,"identity":"25ff7063-3242-4bde-abb2-c01a56d11f58","order_by":1,"name":"Akihisa Ueno","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Akihisa","middleName":"","lastName":"Ueno","suffix":""},{"id":289244274,"identity":"30f5870a-3d58-4ac0-96aa-3b422c44ea11","order_by":2,"name":"Takashi Ueda","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Ueda","suffix":""},{"id":289244275,"identity":"5f9289cb-fc63-43f6-9e6b-ec368be36bde","order_by":3,"name":"Ryota Ogura","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ryota","middleName":"","lastName":"Ogura","suffix":""},{"id":289244276,"identity":"70d0366c-1e34-4eb0-8f4b-6d993334fb73","order_by":4,"name":"Satoshi Sako","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Sako","suffix":""},{"id":289244277,"identity":"6618f8a6-90a7-4a60-abb8-d19e4c5c401c","order_by":5,"name":"Yusuke Gabata","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Gabata","suffix":""},{"id":289244278,"identity":"55ae6e17-52d7-445a-9ad5-5b22acd5c14c","order_by":6,"name":"Osamu Ukimura","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Osamu","middleName":"","lastName":"Ukimura","suffix":""}],"badges":[],"createdAt":"2024-04-08 05:55:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4234181/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4234181/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-024-02119-8","type":"published","date":"2024-08-03T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57082287,"identity":"6b7a1752-0497-42b1-91ef-184b0b0432f2","added_by":"auto","created_at":"2024-05-24 10:51:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1000174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLINC03074\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in testis. A\u003c/strong\u003e Expression of \u003cem\u003eLINC03074\u003c/em\u003e in major human tissues. The data resulted from an RNA-Seq CAGE analysis of human tissues of the RIKEN FANTOM5 project. \u003cstrong\u003eB\u003c/strong\u003e \u003cem\u003eLINC03074\u003c/em\u003e levels in normal and cancer regions of testis tissues from patients with seminoma. Paired normal and cancer tissue samples were obtained from ten different testicles (paired dots are connected by gray lines). Relative expression levels of \u003cem\u003eLINC03074\u003c/em\u003e to \u003cem\u003eGAPDH\u003c/em\u003e were measured using RT-qPCR. *\u003cem\u003eP \u003c/em\u003e= 0.00178. \u003cstrong\u003eC\u003c/strong\u003e \u003cem\u003eIn situ\u003c/em\u003e hybridization with detection probes for antisense or sense strands of \u003cem\u003eLINC03074\u003c/em\u003eon testes of healthy adults and of patients with seminoma. The right small panels show enlarged views of the corresponding left panels. The sense strand of \u003cem\u003eLINC03074\u003c/em\u003e was localized to the nucleus and cytoplasm in normal sperm cells and mainly to the nucleus in seminoma cells.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4234181/v1/b82e615e9e99f84a84253c55.png"},{"id":57081072,"identity":"c361c0ea-308a-4449-88e8-c7fea8b7000c","added_by":"auto","created_at":"2024-05-24 10:35:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":390601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLINC03074\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMDM2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mRNA. A\u003c/strong\u003e Schematic drawing of predicted interaction regions between human \u003cem\u003eLINC03074\u003c/em\u003e and \u003cem\u003eMDM2 mRNA\u003c/em\u003e. Interaction regions predicted by the lncRRIsearch database (gray dot lines), \u003cem\u003eAlu \u003c/em\u003eelements (orange arrows), and inverted \u003cem\u003eAlu\u003c/em\u003e pairs (yellow arrows) are shown (upper panel). An example of the predicted base pairs for the regions shown in red and blue in the upper panel is shown (DG = -60.91 kcal/mol, bottom panel). Sx, Sx1, FLAM_C, Sz and Y, \u003cem\u003eAlu\u003c/em\u003e subfamilies; 5¢ UTR, 5¢ untranslated region; CDS, coding sequence; 3¢ UTR, 3¢ untranslated region. \u003cstrong\u003eB\u003c/strong\u003eEnrichment of RNAs by \u003cem\u003eLINC03074\u003c/em\u003e CHART as measured using RT-qPCR. Each enrichment value is shown as a percentage of the measurement for each mock (without C-oligo). Error bars represent + SEM for three qPCR experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eC\u003c/strong\u003e Schematic representation of full length (FL) and \u003cem\u003eAlu\u003c/em\u003e element-deficient (DAlu) \u003cem\u003eLINC03074\u003c/em\u003eand biotin-tagged \u003cem\u003eMDM2\u003c/em\u003e 3¢ UTR (FL and D5¢-Alu). \u003cstrong\u003eD\u003c/strong\u003eRNA pull-down assays using the biotin-tagged (bio)-\u003cem\u003eMDM2 \u003c/em\u003e3¢ UTR. \u003cem\u003eIn vitro,\u003c/em\u003e transcribed bio-\u003cem\u003eMDM2\u003c/em\u003e was incubated with total RNA extracted from HEK293 cells overexpressing \u003cem\u003eLINC03074\u003c/em\u003e. RNAs associated with bio-\u003cem\u003eMDM2 \u003c/em\u003e3¢ UTRs were detected via RT-qPCR. Error bars represent + SEM for three qPCR experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4234181/v1/c458e2468c68426f04d70692.png"},{"id":57081071,"identity":"242f592c-dc22-4079-8282-364424a67399","added_by":"auto","created_at":"2024-05-24 10:35:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":573873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduction of MDM2 protein levels by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLINC03074. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA\u003c/strong\u003e \u003cem\u003eMDM2\u003c/em\u003eand \u003cem\u003eLINC03074\u003c/em\u003e RNA levels in \u003cem\u003eLINC03074\u003c/em\u003e-knockdown TCam-2 cells measured via RT-qPCR. TCam-2 cells were transfected with siRNAs for \u003cem\u003eLINC03074\u003c/em\u003e (\u003cem\u003esiLINC03074\u003c/em\u003e) for 72 h. Data represent the average of three independent measurements normalized to \u003cem\u003eGAPDH\u003c/em\u003e mRNA expression. \u003cstrong\u003eB\u003c/strong\u003e MDM2 protein levels in \u003cem\u003eLINC03074\u003c/em\u003e knockdown cells transiently expressing the \u003cem\u003eLINC03074\u003c/em\u003e mutant. Western blotting was performed with anti-MDM2 antibody using TCam-2 cells transfected with \u003cem\u003esiLINC03074\u003c/em\u003e for 48 h followed by \u003cem\u003eLINC03074 \u003c/em\u003eexpression plasmids for 24 h. Band intensity was quantified by Image Lab 6.1. The measurements were normalized to the \u003cem\u003esiControl\u003c/em\u003eprotein levels that are indicated at the bottom of each band. \u003cstrong\u003eC\u003c/strong\u003e Schematic representation of the constructs containing FLAG tag-fused \u003cem\u003eMDM2\u003c/em\u003eCDS alone or in combination with 3¢ UTR. \u003cstrong\u003eD\u003c/strong\u003eWestern blot analysis using an anti-FLAG antibody against HEK293 cells transfected with FLAG-tagged MDM2 and \u003cem\u003eLINC03074\u003c/em\u003eexpression plasmids (left panels). The relative intensity to the band of the control in each first left lane is shown in the bar graph (right panel).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4234181/v1/1cdefdfc070372ff5d3b7903.png"},{"id":57081073,"identity":"d8843ba4-e9f1-4d0c-92ca-c2878dcf4e7e","added_by":"auto","created_at":"2024-05-24 10:35:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":378690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDown-regulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLINC03074\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e suppresses STAU1-mediated mRNA nuclear export and PKR-induced translational repression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMDM2.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e A \u003c/strong\u003eEffect of \u003cem\u003eLINC03074 \u003c/em\u003eon the binding of dsRNA binding proteins and \u003cem\u003eMDM2\u003c/em\u003e mRNA in TCam-2 cell nuclei. RIP assay was performed with each dsRNA binding protein antibody using TCam-2 cell nuclear extracts with \u003cem\u003eLINC03074\u003c/em\u003e knockdown. The level of RNA binding with each dsRNA binding protein was measured via RT-qPCR and is shown as a relative value to the IgG binding level. Error bars represent + SEM for three qPCR experiments. An asterisk above each bar indicates statistical significance for IgG values. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eB\u003c/strong\u003e Effect of \u003cem\u003eLINC03074\u003c/em\u003e on \u003cem\u003eMDM2\u003c/em\u003e mRNA levels in the nucleus and cytoplasm of TCam-2 cells. The relative expression of \u003cem\u003eMDM2\u003c/em\u003e to \u003cem\u003e5S-rRNA\u003c/em\u003e (nucleus) or \u003cem\u003eGAPDH\u003c/em\u003e (cytoplasm) was measured using RT-qPCR. Error bars represent + SEM for three qPCR experiments. An asterisk above each bar indicates statistical significance for \u003cem\u003esiControl\u003c/em\u003e values. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003cstrong\u003e C\u003c/strong\u003e RIP assay using anti-PKR and anti-STAU1 antibodies with cytoplasmic extracts from TCam-2 with knockdown of \u003cem\u003eLINC03074\u003c/em\u003e or STAU1. Error bars represent + SEM for three qPCR experiments. An asterisk above each bar indicates statistical significance for IgG values. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eD\u003c/strong\u003e Western blotting of \u003cem\u003eLINC03074\u003c/em\u003e or STAU1 knocked-down TCam-2 cells treated with PKR inhibitor. TCam-2 cells were transfected with \u003cem\u003esiLINC03074\u003c/em\u003e and siSTAU1 for 24 h, followed by treatment with 1mM PKR inhibitor for 24 h. Band intensity was quantified by Image Lab 6.1. The measurements were normalized to control (\u003cem\u003esiControl\u003c/em\u003e without PKR inhibitor) protein levels that are indicated at the bottom of each band.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4234181/v1/86f386511e6eef8b45df395d.png"},{"id":57081074,"identity":"aec83865-346a-4cf5-9be5-96f4bd04423a","added_by":"auto","created_at":"2024-05-24 10:35:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":263708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLINC03074\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e enhances cisplatin-induced apoptosis.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Cell growth assay using TCam-2 cells transfected with \u003cem\u003esiLINC03074\u003c/em\u003e and treated with different concentrations of cisplatin\u003cem\u003e.\u003c/em\u003e Absorbance at 450 nm (OD\u003csub\u003e450\u003c/sub\u003e) was used to estimate cell concentration. Data represent the means ± SEM (n = 3). \u003cstrong\u003eB\u003c/strong\u003e Apoptosis assay using \u003cem\u003eLINC03074-\u003c/em\u003eknockdown TCam-2 cells treated with cisplatin. TCam-2 cells were transfected with \u003cem\u003esiLINC03074\u003c/em\u003e and treated with 20 mM cisplatin for 48 h. Apoptotic cells were identified by the increase in the fluorescence intensity of FITC-labeled Annexin-V using flow cytometry. Percentage of TCam-2 cells (either with or without \u003cem\u003esiLINC03074\u003c/em\u003e) in apoptosis in the presence or absence of cisplatin (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cem\u003esiControl\u003c/em\u003e, TCam-2 cells transfected with a negative control siRNA.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4234181/v1/fb82ea5b53afecfb86dfbf0e.png"},{"id":57081075,"identity":"cbe6f3e7-c774-4217-998a-8e28e4459dc2","added_by":"auto","created_at":"2024-05-24 10:35:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":572744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLINC03074\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e activates E2F1 and p73 pathways.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003eWestern blotting using TCam-2 cells transfected with \u003cem\u003esiLINC03074\u003c/em\u003e for 72 h. Cisplatin was added to the culture medium at a concentration of 10 mM for 72 h before cell extraction. Band intensity was quantified using Image Lab 6.1, and all measurements were normalized to the protein levels of the \u003cem\u003esiControl\u003c/em\u003ewithout cisplatin (indicated at the bottom of each band). \u003cstrong\u003eB\u003c/strong\u003e Relative expression levels of apoptotic genes in \u003cem\u003eLINC03074\u003c/em\u003e-knockdown cells were measured using RT-qPCR. TCam-2 cells were transfected with \u003cem\u003esiLINC03074\u003c/em\u003e and treated with cisplatin for 48 h (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eC\u003c/strong\u003e Predicted schematic of \u003cem\u003eLINC03074\u003c/em\u003e-mediated apoptosis-promoting mechanism.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4234181/v1/f59c2074be41497583fc9fea.png"},{"id":61711845,"identity":"d0574d70-abb1-4430-aa65-e0f6fa817270","added_by":"auto","created_at":"2024-08-04 07:05:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3988158,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4234181/v1/fb791d65-51d7-4638-8e78-0d94d0b08814.pdf"},{"id":57081747,"identity":"9b9734c8-34a0-42a6-b9a1-7e120ece4bff","added_by":"auto","created_at":"2024-05-24 10:43:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":142791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"ItoetalSupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4234181/v1/f170a17ce1e6161f9dafd847.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"A testis-specific lncRNA functions as a post-transcriptional regulator of MDM2 and stimulates apoptosis of testicular germ cell tumor cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTesticular germ cell tumors (TGCTs) are generally highly sensitive to platinum-based chemotherapy, such as cisplatin, but some are chemotherapy-resistant [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mouse double minute 2 (MDM2) is amplified in various human malignancies, including TGCTs, and MDM2 overexpression is associated with chemotherapy resistance [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. MDM2 is a major negative regulator of p53, promoting ubiquitin-dependent proteasomal degradation of p53 as an E3 ubiquitin ligase and repressing p53 transcriptional activation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The tumor suppressor p53 is commonly mutated in various types of cancers [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], whereas it is often overexpressed and rarely mutated in TGCTs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Accordingly, MDM2 overexpression in TGCTs is predicted to affect the activity of wild-type p53; however, the molecular mechanisms underlying the sensitivity or resistance of TGCTs to chemotherapy remain unclear.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMDM2\u003c/em\u003e expression and activity are regulated at multiple levels, from transcription to numerous post-translational modifications, in addition to genomic alterations, such as copy number variations, mutations, and polymorphisms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Post-transcriptional regulation of \u003cem\u003eMDM2\u003c/em\u003e at the mRNA level has been widely reported to modulate transcript stability via miRNAs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Human \u003cem\u003eMDM2\u003c/em\u003e has a very long 3\u0026prime; UTR (~\u0026thinsp;5.7 kb) that retains a multitude of potential miRNA targets [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Notably, \u003cem\u003eMDM2\u003c/em\u003e contains multiple transposable elements containing \u003cem\u003eAlu\u003c/em\u003e in the 3\u0026prime; UTR [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eAlu\u003c/em\u003e elements are abundant retrotransposon elements that spread throughout the human genome and occupy a significant portion of the 3\u0026prime; UTR [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The inverted repeat \u003cem\u003eAlu\u003c/em\u003e element (IR\u003cem\u003eAlus\u003c/em\u003e) in the 3\u0026prime; UTR forms a double-stranded RNA (dsRNA) structure, which serves as a target for dsRNA-binding factors such as Staufen1 (STAU1) and adenosine deaminase acting on RNA (ADAR1) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. STAU1 binds to IR\u003cem\u003eAlu\u003c/em\u003e in the 3\u0026prime; UTR to undergo various RNA metabolic processes, such as RNA synthesis, folding, modification, processing, translation, and decay [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. ADAR1, an adenosine-to-inosine (A-to-I) RNA-editing enzyme, competes with STAU1 for the occupancy of target RNAs, thereby inhibiting STAU1-mediated nuclear retention or decay of RNAs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In \u003cem\u003eMDM2\u003c/em\u003e gene expression, STAU1 and ADAR1 appear to mediate post-transcriptional regulation via binding to the IR\u003cem\u003eAlus\u003c/em\u003e of the \u003cem\u003eMDM2\u003c/em\u003e 3\u0026prime; UTR, a mechanism independent of alterations in mRNA stability and miRNA targeting [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we show the role of \u003cem\u003eLINC03074\u003c/em\u003e, a testis-specific lncRNA with an \u003cem\u003eAlu\u003c/em\u003e element, in TGCT cells. \u003cem\u003eLINC03074\u003c/em\u003e binds to \u003cem\u003eMDM2\u003c/em\u003e mRNA via \u003cem\u003eAlu\u003c/em\u003e, thereby stimulating STAU1-mediated nuclear export of \u003cem\u003eMDM2\u003c/em\u003e mRNA. Consequently, MDM2 is reduced by PKR-mediated translational repression, which in turn promotes the apoptosis of TGCT cells. Our findings provide molecular mechanistic insights into the drug responsiveness of TGCTs by demonstrating a regulatory mechanism of apoptosis in TGCT cells.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eLINC03074\u003c/b\u003e \u003cb\u003eshows different expression patterns between cancerous and normal sperm\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the characteristics of TGCTs, we compared the gene expression profiles of cancer tissues from seminoma patients with those of matched normal adjacent tissues [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A total of 565 genes, among the 50,599 genes tested, exhibited a more than 2-fold increase in RNA expression in seminoma tissues compared to normal adjacent tissues, including 18 genes encoding lncRNAs (data not shown). In contrast, 431 genes exhibited a\u0026thinsp;\u0026gt;\u0026thinsp;2-fold decrease in expression in cancer tissues, including 59 genes encoding lncRNAs (data not shown). To identify the lncRNA that determines the characteristics of testicular tumor cells, we focused on \u003cem\u003eLINC03074\u003c/em\u003e (\u003cem\u003eLOC100505685\u003c/em\u003e), which showed marked differences in expression between cancerous and normal tissues. According to the database, \u003cem\u003eLINC03074\u003c/em\u003e is expressed specifically in the testes of humans (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Moreover, a recent study identified \u003cem\u003eLINC03074\u003c/em\u003e as a testis-specific lncRNA [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The expression pattern of \u003cem\u003eLINC03074\u003c/em\u003e, which was significantly higher in normal tissues than in cancerous tissues of the testes of patients with seminoma, was confirmed via relative quantitative analysis using RT-qPCR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00178, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). We performed ISH with an \u003cem\u003eLINC03074\u003c/em\u003e detection probe in the testes of healthy adults and patients with seminomas. The results revealed that \u003cem\u003eLINC03074\u003c/em\u003e was localized to the nucleus and cytoplasm of normal spermatids, whereas it was mainly localized to the nucleus of seminoma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We further quantified the expression of \u003cem\u003eLINC03074\u003c/em\u003e in four types of cultured cells derived from seminoma and non-seminoma tissues and found that its expression was significantly higher in TCam-2 seminoma cells (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These results suggest that \u003cem\u003eLINC03074\u003c/em\u003e functions in both testis-derived seminoma cells and normal cells, although it is differentially expressed in cancer and normal cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLINC03074\u003c/b\u003e \u003cb\u003einteracts with\u003c/b\u003e \u003cb\u003eMDM2\u003c/b\u003e \u003cb\u003emRNA via\u003c/b\u003e \u003cb\u003eAlu\u003c/b\u003e \u003cb\u003eelement\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the function of \u003cem\u003eLINC03074\u003c/em\u003e in testicular cells, we searched databases to identify the elements with which this lncRNA could interact. Using lncRRIsearch, \u003cem\u003eMDM2\u003c/em\u003e mRNA was identified as a candidate interacting factor for \u003cem\u003eLINC03074\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eLINC03074\u003c/em\u003e contained one \u003cem\u003eAlu\u003c/em\u003e element, while \u003cem\u003eMDM2\u003c/em\u003e mRNA contained five \u003cem\u003eAlu\u003c/em\u003e elements and a pair of inverted-\u003cem\u003eAlu\u003c/em\u003es in the 3\u0026prime; UTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). There were multiple candidate sequences for the interaction between \u003cem\u003eLINC03074\u003c/em\u003e and \u003cem\u003eMDM2\u003c/em\u003e mRNA within each \u003cem\u003eAlu\u003c/em\u003e element (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The sequences of \u003cem\u003eLINC03074\u003c/em\u003e and the sense strand of \u003cem\u003eMDM2\u003c/em\u003e mRNA were nearly complementary, as shown in an example of a candidate interaction region (DG = -60.91 kcal/mol, lower part of Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We decided to use TCam-2 cells as a model for seminoma cells in the following experiments, considering that \u003cem\u003eLINC03074\u003c/em\u003e is likely to function in seminomas according to its expression pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To determine whether \u003cem\u003eLINC03074\u003c/em\u003e binds to \u003cem\u003eMDM2\u003c/em\u003e mRNA, we performed CHART assays using TCam-2 cells. CHART enables the identification of associated targets of lncRNAs by enriching lncRNAs with their targets using affinity-tagged oligonucleotides (C-oligo) to capture endogenous lncRNAs in cross-linked cell extracts [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. \u003cem\u003eLINC03074\u003c/em\u003e was enriched in TCam-2 cell extracts by CHART using a C-oligo for \u003cem\u003eLINC03074\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We then tested whether mRNAs of \u003cem\u003eMDM2\u003c/em\u003e, \u003cem\u003e18S-rRNA\u003c/em\u003e and \u003cem\u003eGAPDH\u003c/em\u003e were enriched using \u003cem\u003eLINC03074\u003c/em\u003e CHART, and found that \u003cem\u003eMDM2\u003c/em\u003e mRNA was associated with \u003cem\u003eLINC03074\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Approximate estimates using qPCR showed that the molecular ratio of \u003cem\u003eLINC03074\u003c/em\u003e to \u003cem\u003eMDM2\u003c/em\u003e mRNA in TCam-2 cells was approximately 1:166 (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). To confirm whether \u003cem\u003eLINC03074\u003c/em\u003e and \u003cem\u003eMDM2\u003c/em\u003e mRNA interact via their respective \u003cem\u003eAlu\u003c/em\u003e elements, we generated \u003cem\u003eAlu\u003c/em\u003e element-deficient \u003cem\u003eLINC03074\u003c/em\u003e and \u003cem\u003eMDM2\u003c/em\u003e expression constructs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). We performed an RNA pull-down assay with the 3\u0026prime; UTR region of biotin-labeled \u003cem\u003eMDM2\u003c/em\u003e mRNA using total RNA extracted from HEK293 cells transiently expressing \u003cem\u003eLINC03074\u003c/em\u003e. We found that \u003cem\u003eLINC03074\u003c/em\u003e (FL) binds to the \u003cem\u003eMDM2\u003c/em\u003e 3\u0026prime; UTR (FL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In comparison, \u003cem\u003eLINC03074\u003c/em\u003e (FL) binds to the \u003cem\u003eMDM2\u003c/em\u003e 3\u0026prime; UTR (D5\u0026prime;-Alu) was attenuated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In addition, \u003cem\u003eLINC03074\u003c/em\u003e (DAlu) showed significantly weaker binding to \u003cem\u003eMDM2\u003c/em\u003e than \u003cem\u003eLINC03074\u003c/em\u003e (FL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In contrast, no binding was detected between \u003cem\u003e18S-rRNA\u003c/em\u003e and \u003cem\u003eMDM2\u003c/em\u003e 3\u0026prime; UTR (either FL or D5\u0026prime;-Alu) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These results indicated that \u003cem\u003eLINC03074\u003c/em\u003e binds to the \u003cem\u003eAlu\u003c/em\u003e elements of \u003cem\u003eMDM2\u003c/em\u003e mRNA via its own \u003cem\u003eAlu\u003c/em\u003e element.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLINC03074\u003c/b\u003e \u003cb\u003einhibits\u003c/b\u003e \u003cb\u003eMDM2\u003c/b\u003e \u003cb\u003egene expression by binding to\u003c/b\u003e \u003cb\u003eMDM2\u003c/b\u003e \u003cb\u003emRNA\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe speculated that \u003cem\u003eLINC03074\u003c/em\u003e may affect \u003cem\u003eMDM2\u003c/em\u003e gene expression by interacting with \u003cem\u003eMDM2\u003c/em\u003e mRNA. Three different siRNAs were used to knockdown \u003cem\u003eLINC03074\u003c/em\u003e, all of which decreased \u003cem\u003eMDM2\u003c/em\u003e mRNA levels and increased MDM2 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). The increase in MDM2 protein by \u003cem\u003eLINC03074\u003c/em\u003e knockdown was rescued by the transient expression of full-length \u003cem\u003eLINC03074\u003c/em\u003e (FL), but not by the DAlu mutant (DAlu) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Next, we verified that \u003cem\u003eLINC03074\u003c/em\u003e-induced alterations in MDM2 protein levels were caused by the binding of \u003cem\u003eLINC03074\u003c/em\u003e to \u003cem\u003eMDM2\u003c/em\u003e mRNA. Flag-tag fusion MDM2 protein expression plasmids were generated by inserting 3\u0026prime; UTR sequences (FL or D5\u0026prime;-Alu) downstream of the CDS of \u003cem\u003eMDM2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These plasmids were transfected together with \u003cem\u003eLINC03074\u003c/em\u003e (FL or DAlu) expression plasmids into HEK293 cells, and the protein levels of FLAG-MDM2 were quantified by immunoblotting using an anti-FLAG antibody. In the absence of the 3\u0026prime; UTR, FLAG-MDM2 levels remained unchanged regardless of the presence of \u003cem\u003eLINC03074\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, in the presence of the 3\u0026prime; UTR (FL), FLAG-MDM2 levels were significantly reduced by \u003cem\u003eLINC03074\u003c/em\u003e (FL), but not by \u003cem\u003eLINC03074\u003c/em\u003e (DAlu) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In the case of \u003cem\u003eAlu\u003c/em\u003e elements in the 3\u0026prime; UTR were absent (D5\u0026prime;-Alu), FLAG-MDM2 levels were only slightly reduced by \u003cem\u003eLINC03074\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These findings suggest that \u003cem\u003eLINC03074\u003c/em\u003e binding to \u003cem\u003eMDM2\u003c/em\u003e mRNA via \u003cem\u003eAlu\u003c/em\u003e elements may influence the post-transcriptional or translational processes of \u003cem\u003eMDM2\u003c/em\u003e gene expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLINC03074\u003c/b\u003e \u003cb\u003eenhances STAU1-mediated nuclear export and PKR-induced translational repression of\u003c/b\u003e \u003cb\u003eMDM2\u003c/b\u003e \u003cb\u003emRNA\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIt has been reported that the inverted repeat \u003cem\u003eAlu\u003c/em\u003e elements (IR\u003cem\u003eAlus\u003c/em\u003e) in the 3\u0026prime; UTR of mRNA serve as a binding site for ADAR1, a dsRNA-specific enzyme that performs A-to-I RNA editing [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The mRNA with 3\u0026prime; UTR IR\u003cem\u003eAlus\u003c/em\u003e edited by ADAR1 is retained in the nucleus through interaction with paraspeckle, which is formed by the nuclear lncRNA, \u003cem\u003eNEAT1\u003c/em\u003e, and its binding partner, NONO [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Alternatively, IR\u003cem\u003eAlus\u003c/em\u003e in the 3\u0026prime; UTR of mRNA binds to the dsRNA-binding protein STAU1, which is involved in various RNA metabolic regulations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. STAU1 binds to IR\u003cem\u003eAlu\u003c/em\u003e to facilitate the export of IR\u003cem\u003eAlu\u003c/em\u003e mRNA from the nucleus to the cytoplasm, while competitive inhibition of NONO binding to IR\u003cem\u003eAlu\u003c/em\u003e prevents NONO-mediated mRNA retention in the nucleus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We examined the binding of \u003cem\u003eMDM2\u003c/em\u003e mRNA to STAU1, ADAR1, or NONO in the nuclei of TCam-2 cells and the effect of \u003cem\u003eLINC03074\u003c/em\u003e on their binding. RIP assays using nuclear extracts of TCam-2 cells showed that \u003cem\u003eMDM2\u003c/em\u003e mRNA binds to STAU1 and ADAR1, but not NONO (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Knockdown of \u003cem\u003eLINC03074\u003c/em\u003e suppressed the binding of \u003cem\u003eMDM2\u003c/em\u003e mRNA to STAU1 while increasing its binding to ADAR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, \u003cem\u003eLINC03074\u003c/em\u003e bound only to STAU1 but not to ADAR1 and NONO (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Considering that STAU1 is responsible for RNA shuttling, we investigated whether \u003cem\u003eLINC03074\u003c/em\u003e affected the nuclear export of \u003cem\u003eMDM2\u003c/em\u003e mRNA. \u003cem\u003eMDM2\u003c/em\u003e mRNA was increased in the nucleus and decreased in the cytoplasm following \u003cem\u003eLINC03074\u003c/em\u003e and STAU1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results suggested that \u003cem\u003eLINC03074\u003c/em\u003e promotes the recruitment of STAU1 to \u003cem\u003eMDM2\u003c/em\u003e mRNA in the nucleus and facilitates STAU1-mediated nuclear export.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, the downregulation of \u003cem\u003eLINC03074\u003c/em\u003e increased intracellular MDM2 protein levels, despite decreasing \u003cem\u003eMDM2\u003c/em\u003e mRNA levels in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). We speculated that \u003cem\u003eLINC03074\u003c/em\u003e-mediated enhancement of STAU1 and \u003cem\u003eMDM2\u003c/em\u003e mRNA interactions in the nucleus leads to the translational repression of \u003cem\u003eMDM2\u003c/em\u003e. STAU1 binding to IR\u003cem\u003eAlu\u003c/em\u003e mRNA promotes nuclear export and translation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, STAU1-mediated mRNA nuclear export is promoted when the paraspeckle component is downregulated, whereas protein kinase R (PKR)-mediated translational repression in the cytoplasm is promoted [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. PKR is activated by binding to virus-derived dsRNA and phosphorylates eukaryotic translation initiation factor 2A (eIF2a), resulting in translational inhibition [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To determine whether PKR can bind to \u003cem\u003eMDM2\u003c/em\u003e mRNA in TGCT cells, we performed RIP assays with cytoplasmic extracts from TCam-2 cells using a PKR antibody. PKR was found to bind to \u003cem\u003eMDM2\u003c/em\u003e mRNA in the cytoplasm, and this interaction was reduced by \u003cem\u003eLINC03074\u003c/em\u003e and STAU1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, STAU1 and \u003cem\u003eMDM2\u003c/em\u003e mRNA binding in the cytoplasm was attenuated by \u003cem\u003eLINC03074\u003c/em\u003e and STAU1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Finally, we determined whether \u003cem\u003eMDM2\u003c/em\u003e was translationally repressed by PKR activation. MDM2 protein levels were increased by PKR inhibitor treatment of TCam-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The increase in MDM2 protein expression induced by PKR inhibitors was not detected with \u003cem\u003eLINC03074\u003c/em\u003e and STAU1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results indicated that \u003cem\u003eLINC03074\u003c/em\u003e, similar to paraspeckle components, modulates the nuclear export of STAU1-bound \u003cem\u003eMDM2\u003c/em\u003e mRNA, thereby facilitating PKR-mediated translational repression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLINC03074\u003c/b\u003e \u003cb\u003eenhances cisplatin-induced apoptosis and cell growth inhibition\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess whether \u003cem\u003eLINC03074\u003c/em\u003e affects the proliferation of TGCT cells, CCK8 analysis was performed using cisplatin, a platinum chemotherapeutic agent that induces DNA damage in cancer cells by inhibiting DNA repair [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The growth of TCam-2 cells was inhibited by cisplatin treatment in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). \u003cem\u003eLINC03074\u003c/em\u003e knockdown enhanced the growth of TCam-2 cells and attenuated cisplatin-induced inhibition of cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, \u003cem\u003eLINC03074\u003c/em\u003e knockdown had no effect on the growth of non-seminoma-derived NEC8 cells expressing low \u003cem\u003eLINC03074\u003c/em\u003e levels (Fig. S3). First, the effect of \u003cem\u003eLINC03074\u003c/em\u003e on the cell cycle was assessed; however, no cell cycle abnormalities due to \u003cem\u003eLINC03074\u003c/em\u003e knockdown or cisplatin treatment were observed under the conditions examined (Fig. S4). Next, the effect of \u003cem\u003eLINC03074\u003c/em\u003e on apoptosis was examined using FACS analysis. \u003cem\u003eLINC03074\u003c/em\u003e knockdown reduced the frequency of spontaneous and cisplatin-induced apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and S5). These results indicate that \u003cem\u003eLINC03074\u003c/em\u003e inhibits the proliferation and promotes the apoptosis of seminoma cells. Furthermore, the responsiveness of seminoma cells to cisplatin-induced DNA damage was enhanced by \u003cem\u003eLINC03074\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLINC03074\u003c/b\u003e \u003cb\u003eincreases E2F1 levels and upregulates\u003c/b\u003e \u003cb\u003ep73\u003c/b\u003e \u003cb\u003egene expression\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMDM2 is a major negative regulator of p53; MDM2 acts as an E3 ubiquitin ligase that recognizes p53 and acts as a transcriptional repressor of \u003cem\u003ep53\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To test whether \u003cem\u003eLINC03074\u003c/em\u003e affected p53 protein levels and function, we performed immunoblotting using anti-p53 and anti-phosphorylated p53 (Ser15) antibodies. p53 is activated by phosphorylation in response to DNA damage, and its Ser15 residue is the major phosphorylation site [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Immunoblotting confirmed that cisplatin-induced DNA damage markedly increased the p53 protein levels and promoted p53 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Interestingly, \u003cem\u003eLINC03074\u003c/em\u003e knockdown did not affect the p53 or phosphorylated p53 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). MDM2 has been reported to interact with various proteins other than p53, and the apoptosis-related transcription factor E2F1 is one of its target proteins [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Immunoblotting with an anti-E2F1 antibody showed that E2F1 levels were increased by cisplatin treatment and decreased by \u003cem\u003eLINC0374\u003c/em\u003e knockdown, in the presence or absence of cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). E2F1 induces apoptosis through several mechanisms, including activation of p53-dependent and -independent pathways and inhibition of survival signaling [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To elucidate the mechanism by which \u003cem\u003eLINC03074\u003c/em\u003e mediates apoptosis, we examined the effects of \u003cem\u003eLINC03074\u003c/em\u003e knockdown on E2F1 target gene expression. Among the apoptotic genes targeted by E2F1, \u003cem\u003ep73\u003c/em\u003e, and \u003cem\u003eBIM\u003c/em\u003e are transcriptionally regulated by E2F1, whereas \u003cem\u003ePUMA\u003c/em\u003e and \u003cem\u003eNOXA\u003c/em\u003e are regulated by E2F1 and p53 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Of the four apoptosis-related genes subjected to mRNA quantification, only \u003cem\u003ep73\u003c/em\u003e exhibited decreased mRNA levels following \u003cem\u003eLINC03074\u003c/em\u003e knockdown with cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In addition, \u003cem\u003ep73\u003c/em\u003e mRNA levels increased in response to cisplatin treatment, regardless of \u003cem\u003eLINC03074\u003c/em\u003e knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). \u003cem\u003eBIM\u003c/em\u003e, \u003cem\u003ePUMA\u003c/em\u003e, and \u003cem\u003eNOXA\u003c/em\u003e mRNAs showed a tendency to increase with \u003cem\u003eLINC03074\u003c/em\u003e knockdown with cisplatin, but no cisplatin addition-dependent increase was observed without \u003cem\u003eLINC03074\u003c/em\u003e knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Our results indicate that cisplatin-induced apoptosis of seminoma cells is associated with the increased expression of \u003cem\u003ep73\u003c/em\u003e. \u003cem\u003eLINC03074\u003c/em\u003e contributes to the upregulation of \u003cem\u003ep73\u003c/em\u003e by increasing E2F1 expression, which may indirectly affect the expression of other apoptotic genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMDM2 expression levels are associated with chemotherapy resistance in human malignancies and are regulated at multiple levels [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In this study, we suggest that MDM2 levels are regulated by the testis-specific lncRNA \u003cem\u003eLINC03074\u003c/em\u003e during post-transcription. The LINC03074-MDM2-mediated apoptosis regulatory pathway provides new insights into the mechanisms of the DNA damage response in TGCT cells.\u003c/p\u003e \u003cp\u003eA total of 17 pairs of interaction sequences were predicted within the \u003cem\u003eAlu\u003c/em\u003e element of \u003cem\u003eLINC03074\u003c/em\u003e with the five \u003cem\u003eAlu\u003c/em\u003e elements in the 3\u0026prime; UTR of \u003cem\u003eMDM2\u003c/em\u003e mRNA (data from lncRRIsearch; shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Our results suggest the following: (1) \u003cem\u003eLINC03074\u003c/em\u003e binds complementarily to either \u003cem\u003eAlu\u003c/em\u003e element of the \u003cem\u003eMDM2\u003c/em\u003e 3\u0026prime; UTR via its own \u003cem\u003eAlu\u003c/em\u003e element; (2) \u003cem\u003eLINC03074\u003c/em\u003e modulates the binding of STAU1 and ADAR1 to the IR\u003cem\u003eAlus\u003c/em\u003e of the \u003cem\u003eMDM2\u003c/em\u003e 3\u0026prime; UTR; and (3) \u003cem\u003eLINC03074\u003c/em\u003e interacts with STAU1 but not ADAR1 in the nucleus. These results suggest that \u003cem\u003eLINC03074\u003c/em\u003e functions as an RNA chaperone for \u003cem\u003eMDM2\u003c/em\u003e mRNA and that \u003cem\u003eLINC03074\u003c/em\u003e-induced conformational changes convert the dsRNA-binding factor that binds to the 3\u0026prime; UTR of \u003cem\u003eMDM2\u003c/em\u003e. It is speculated that ADAR1 and STAU1 competitively bind to \u003cem\u003eMDM2\u003c/em\u003e mRNA because the interaction of ADAR1 with \u003cem\u003eMDM2\u003c/em\u003e mRNA has been reported to suppress STAU1-MDM2 mRNA binding [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, we concluded that \u003cem\u003eLINC03074\u003c/em\u003e binds to \u003cem\u003eMDM2\u003c/em\u003e mRNA to promote STAU1 recruitment, thereby indirectly suppressing ADAR1-MDM2 mRNA binding. The intracellular molecular ratio of \u003cem\u003eLINC03074\u003c/em\u003e to \u003cem\u003eMDM2\u003c/em\u003e mRNA in the TCam-2 cells was approximately 1:166 (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). As an example of how a small amount of lncRNA can act on a large number of target molecules, previous reports have shown that \u003cem\u003eSLERT\u003c/em\u003e is recycled to induce conformational changes in DDX21, which has approximately 1000 times more molecules than \u003cem\u003eSLERT\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Further detailed molecular analysis is needed to determine the mechanism by which \u003cem\u003eLINC03074\u003c/em\u003e exerts its action and modifies more targets than its stoichiometry. In addition, the significance of the differences in \u003cem\u003eLINC03074\u003c/em\u003e levels between carcinoma and normal testis tissues needs to be investigated.\u003c/p\u003e \u003cp\u003ePKR is activated by binding to virus-derived dsRNA and phosphorylates eukaryotic translation initiation factor 2A (eIF2A), resulting in translational repression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It has been reported that downregulation of paraspeckle components, such as \u003cem\u003eNEAT1\u003c/em\u003e and NONO, increases cytoplasmic 3\u0026prime; UTR IR\u003cem\u003eAlus\u003c/em\u003e mRNAs, which increases phosphorylation of PKR and eIF2A, resulting in intracellular translational repression [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this study, we demonstrated that downregulation of \u003cem\u003eLINC03074\u003c/em\u003e suppresses STAU1-mediated nuclear export of 3\u0026prime; UTR IR\u003cem\u003eAlus\u003c/em\u003e mRNA, resulting in attenuated PKR-mediated translational repression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Taken together, \u003cem\u003eLINC03074\u003c/em\u003e had an opposite effect to that of paraspeckle components on the post-transcriptional regulation of IR\u003cem\u003eAlus\u003c/em\u003e mRNA, but no interaction between \u003cem\u003eLINC03074\u003c/em\u003e and NONO was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additional examination of the molecular mechanism of \u003cem\u003eLINC03074-\u003c/em\u003emediated translational repression, involving its association with paraspeckles, is expected to provide new insights into the post-transcriptional regulation of the 3\u0026prime; UTR IR\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eAlus\u003c/span\u003e mRNAs.\u003c/p\u003e \u003cp\u003eThe relationship between MDM2 and p53 expression in TGCTs remains unclear, although MDM2 overexpression plays an important role in suppressing p53 activity in numerous tumors that retain wild-type p53 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Several reports have suggested that p53 degradation by MDM2 is ineffective in TGCTs. Some studies have shown a positive correlation between MDM2 and wild-type p53 expression levels in TGCTs, while others have shown no correlation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. We found that attenuation of MDM2 levels by \u003cem\u003eLINC03074\u003c/em\u003e led to an increase in E2F1 but not p53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). E2F1 is negatively and positively regulated by MDM2 in a p53-independent manner via both direct and indirect mechanisms [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, E2F1 plays an important role in regulating cell proliferation and differentiation by significantly influencing cell cycle progression and survival through extensive crosstalk with p53 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, we demonstrated that the \u003cem\u003ep73\u003c/em\u003e, a target gene of E2F1 related to apoptosis, is involved in the responsiveness of seminoma cells to cisplatin, and its expression is regulated by \u003cem\u003eLINC03074\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Considering that the MDM2-E2F1-p73 pathway is predicted to play an important role in chemotherapy resistance of TGCTs, further insights into this pathway may lead to the development of new therapies for TGCTs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHuman tumor samples\u003c/h2\u003e \u003cp\u003eSamples of histologically normal testicular lesions and cancerous lesions were obtained from the surgical specimens of patients who underwent radical orchiectomy at the Kyoto Prefectural University of Medicine. The use of surgical and autopsy specimens for molecular analysis was approved by the Institutional Ethics Committee of the hospital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation\u003c/h2\u003e \u003cp\u003eTCam-2 cells were transfected with \u003cem\u003esiLINC03074\u003c/em\u003e and incubated for 72 h. Nuclear and cytoplasmic extracts were prepared as described previously (described in detail in the next section) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To detect the interaction between RNA and protein, cellular lysates were incubated with anti-ADAR1 (15.8.6; Santa Cruz Biotechnology), anti-NONO (11058-1-AP; Proteintech), anti-STAU1 (C-4; Santa Cruz Biotechnology), anti-PKR (18244-1-AP; Proteintech) or anti-IgG (I5006; Sigma) antibodies at 4\u0026deg;C for 18 h and then mixed with Dynabeads Protein G (Thermo Fisher) at 4\u0026deg;C for 1 h. Immunoprecipitated RNAs were isolated using ISOGEN (NIPPON GENE), and quantified via RT-qPCR, as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eNuclear and Cytoplasmic fractionation\u003c/h2\u003e \u003cp\u003eNuclear and cytoplasmic fractions were obtained as previously reported, with some modifications [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. TCam-2 cells were lysed with the nuclear fractionation buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.2% NP-40, 3 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 100 U/ml RNase Inhibitor at 4\u0026deg;C for 10 min and centrifuged at 13000 rpm at 4\u0026deg;C for 10 min. The supernatant was used as the cytoplasmic fraction. The pellet was washed with the nuclear fractionation buffer and centrifuged at 13000 rpm at 4\u0026deg;C for 10 min. The pellet was used as the nuclear fraction. The respective markers of the nuclear and cytoplasmic fractions, 5S-rRNA and GAPDH, respectively, were used as controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using \u003cem\u003et\u003c/em\u003e-tests or ANOVA, as appropriate. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Each experiment was repeated at least three times. Information on statistical measures is provided in the legend of each figure.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eSI wrote the main manuscript text, made substantial contributions to the conception, design, acquisition of data, and analysis, and approved the final version to be submitted. AU, RO, TS, and YG contributed to the acquisition of clinical samples. TU and OU contributed to the acquisition of clinical samples and polished the manuscript. All authors have reviewed and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eWe thank Dr. Riko Kitazawa for providing us with the TCam-2 cell line. The authors would like to thank Dr. Hideo Nakagawa for assisting in the preparation of research samples from human clinical specimens. We would like to thank Editage (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.editage.jp\" target=\"_blank\"\u003ewww.editage.jp\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.editage.jp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the English language editing. This work was supported in part by the Japan Society for the Promotion of Science (JSPS) through Grants-in-Aid for Scientific Research fund (grant numbers 16K15695 and 19K09698).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSingh R, Fazal Z, Freemantle SJ, Spinella MJ. Mechanisms of cisplatin sensitivity and resistance in testicular germ cell tumors. Cancer Drug Resist. 2019; 2: 580\u0026ndash;594. 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Apoptosis of germ cells is closely related to cancer development and chemotherapy resistance; however, its regulatory mechanism is unclear. Here, we suggest that testis-specific lncRNA \u003cem\u003eLINC03074\u003c/em\u003e is involved in male germ cell apoptosis by regulating the expression of the proto-oncogene \u003cem\u003eMDM2\u003c/em\u003e. \u003cem\u003eLINC03074\u003c/em\u003e is highly expressed in the sperm of healthy adult testes and cancer cells of testes with testicular germ cell tumors (TGCTs). \u003cem\u003eLINC03074\u003c/em\u003e binds to \u003cem\u003eMDM2\u003c/em\u003e mRNA via an \u003cem\u003eAlu\u003c/em\u003e element, thereby reducing MDM2 protein levels. \u003cem\u003eLINC03074\u003c/em\u003e stimulates STAU1-mediated nuclear export of \u003cem\u003eMDM2\u003c/em\u003e mRNA by increasing STAU1 binding to \u003cem\u003eMDM2\u003c/em\u003e mRNA in the cell nucleus, thereby promoting PKR-mediated translational repression in the cytoplasm. The induction of apoptosis in TGCT cells and their responsiveness to the anticancer drug cisplatin is enhanced by \u003cem\u003eLINC03074\u003c/em\u003e. Notably, \u003cem\u003eLINC03074\u003c/em\u003e increased E2F1 expression without increasing p53, the primary target of MDM2, and upregulated the apoptotic gene \u003cem\u003ep73\u003c/em\u003e, the target gene of E2F1. \u003cem\u003eLINC03074\u003c/em\u003e-mediated regulation of apoptosis contributes to the responsiveness of TGCTs to anticancer drug-induced DNA damage.\u003c/p\u003e","manuscriptTitle":"A testis-specific lncRNA functions as a post-transcriptional regulator of MDM2 and stimulates apoptosis of testicular germ cell tumor cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-24 10:34:57","doi":"10.21203/rs.3.rs-4234181/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5baff64a-95db-43eb-9d8c-6b0dc19a47bc","owner":[],"postedDate":"May 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30456278,"name":"Biological sciences/Molecular biology/Non-coding RNAs"},{"id":30456279,"name":"Biological sciences/Cancer/Testicular cancer"}],"tags":[],"updatedAt":"2024-08-04T07:05:38+00:00","versionOfRecord":{"articleIdentity":"rs-4234181","link":"https://doi.org/10.1038/s41420-024-02119-8","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2024-08-03 04:00:00","publishedOnDateReadable":"August 3rd, 2024"},"versionCreatedAt":"2024-05-24 10:34:57","video":"","vorDoi":"10.1038/s41420-024-02119-8","vorDoiUrl":"https://doi.org/10.1038/s41420-024-02119-8","workflowStages":[]},"version":"v1","identity":"rs-4234181","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4234181","identity":"rs-4234181","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}