Received: 24 May 2018 Accepted: 18 September 2018 DOI: 10.1002/cbf.3360 RESEARCH ARTICLE Tumorigenic PVT‐1 gene locus is governed by miR‐2909 RNomics Sameena Wani | Deepak Kaul Molecular Biology Unit, Experimental Medicine and Biotechnology Department, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India Correspondence Deepak Kaul, Molecular Biology Unit, Experimental Medicine and Biotechnology Department, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India. Email: dkaul_24@hotmail.com Funding information Indian Council of Medical Research, ICMR, New Delhi, Government of India, Grant/ Award Number: 3/1/3/SRF‐2014/HRD 1 | Genomic regulation and functional significance of PVT‐1 gene locus, in the MYC‐driven cancers, has remained enigmatic ever since its discovery. With the present study, an attempt is made to establish that cellular AATF genome encoded miR‐2909 RNomics pathway involving crucial genes coding for KLF4, Deptor, mTORC1, STAT3, and p53 has the inherent capacity to ensure sustained co‐amplification of PVT‐1 gene locus together with c‐Myc gene. Based upon these results, we propose that miR‐2909 RNomics pathway may play a crucial role in the regulation of tumorigenic PVT‐1 gene locus. KEY W ORDS c‐Myc, Deptor, KLF4, miR‐2909 RNomics, p53, PVT‐1gene locus I N T RO D U CT I O N of rapamycin complex 1 (mTORC1)‐dependent increased expression of STAT3 as well as phosphorylation of the tumour‐suppressor There exists a general recognition of the fact that co‐amplification of eukaryotic translation initiation factor 4E (eIF4G) binding protein genes coding for PVT‐1 and c‐Myc, within human genomic 8q24 1 (4EBP1) during Myc‐driven tumorigenesis.9 Interestingly, AATF region, has been frequently observed to occur in several solid protein has been found to have the capacity to induce the expression tumours.1,2 Ever since the discovery of PVT‐1 non‐protein coding of genes coding for p53, c‐Myc, Deptor, and REDD‐1.5 The Deptor gene locus that also yields a cluster of six microRNAs, this gene gene product is known to inhibit mTORC1 and thereby promote cellular remained enigmatic because of its ability to provide site for tumori- autophagy, whereas REDD‐1 gene‐product induced mTORC2 to genic and retroviral insertions.2 The complexity of this gene locus does ensure cellular survival.5 Further, AATF protein has the ability to inhibit not end here since PVT‐1 is required for high c‐Myc protein levels in the p53‐dependent transcriptional expression of pro‐apoptotic genes the cancer cells.3 Paradoxically, the tumour suppressor protein p53 such as, Puma, Bax, and Bak.10 Keeping in view all the above‐mentioned has been shown to regulate PVT‐1 gene and one of the six encoded findings, the present study attempts to resolve as to how tumorigenic microRNAs (miR‐1204) in a selective fashion.4 This miR‐1204 has PVT‐1 gene locus is governed by miR‐2909 RNomics involving effector been found to increase cellular p53 levels.4 A new dimension was crucial genes such as, KLF4, AATF, c‐Myc, P53, Deptor, mTORC1, added to the cancer biology by the findings that revealed as to how STAT3, and their interplay in terms of mutual regulation. an interplay between cellular AATF genome encoded protein coding transcript and regulatory non‐coding microRNA designated miR‐ 2909 has the ability to govern many master‐genes (especially KLF4, c‐Myc, p53, p21, mTORC1) of crucial importance to the regulation of 2 MATERIALS AND METHODS | cellular processes, such as cell cycle progression, checkpoint control, 2.1 and autophagy/apoptosis.5-8 A surprising coupled with crucial func- Three cellular models with following traits were employed in the pres- tional link was found to exist between c‐Myc and mammalian target ent study: Abbreviations: PVT‐1, Plasmacytoma variant translocation; AATF, Apoptosis antagonizing transcription factor; KLF4, Krupple like factor 4; DEPTOR, DEP domain‐containing MTOR‐interacting protein; mTORC1, Mammalian target of rapamycin complex 1; STAT 3, Signal transducer and activator of transcription 3; REDD1, Regulated in development and DNA damage responses 1 Cell Biochem Funct. 2018;1–5. | Archetype cellular models employed 1. HeLa cervical cancer cells that exhibit no intrinsic miR‐2909 expression. 2. Prostate cancer cells (PC3) that exhibit high intrinsic miR‐2909 expression. wileyonlinelibrary.com/journal/cbf © 2018 John Wiley & Sons, Ltd. 1 WANI AND KAUL 2 3. Human normal peripheral blood mononuclear cells (PBMCs) pro- Needless to mention here that control vector without gene insert was used to transfect the control PBMCs as reported earlier.6-8 grammed to express high levels of KLF4 gene. The human PBMCs were obtained from normal healthy volunteers, who were fasting for 12 hours and had abstained from any medication for 2 weeks before blood donation, by employing standard method reported earlier.6-8 All the cellular models, employed in the 2.3 | Gene expression analysis The isolated RNA from each culture well was subjected to expression present study, were maintained in vitro culture by exposing these cells analysis of genes coding for miR‐2909, KLF4, c‐Myc, AATF, STAT3, to RPMI‐1640 medium enriched with 10% FCS + antibiotics (50 PVT‐1, Deptor, mTORC1, and PVT‐1 encoded microRNAs using gene unitsmL−1 penicillin and 50 μg mL−1 streptomycin) at 37°C in 5% specific primers and Real‐Time PCR method as described earlier.6-8 CO2 atmosphere. U6 and GAPDH were used as invariant controls for the expression of microRNAs and other genes, respectively.11 The isolated proteins from each culture well were subjected to SDS‐PAGE followed by 2.2 | Western immuno‐detection using specific antibodies against KLF4, Cellular transfection experiments c‐Myc, STAT3, p53, and β‐actin (used as an invariant control) and Lentiviral miR‐2909 expression vector (with and without miR‐2909 employing standard methodology described earlier.6-8 6-8 insert coupled with GFP tag) was designed as reported earlier. These vectors were transfected into HeLa cells using ESCORT transfection reagent (Sigma) to ectopically express miR‐2909 within HeLa cells. Control‐vector transfected HeLa cells were used as control cells. 2.4 | Statistical analysis miRCURY LNA AntagomiR‐2909 or its scrambled‐sequence variant The statistical analysis was performed by SPSS windows version 19. was transfected in PC3 cells by employing the above‐mentioned Data were presented as mean ± S.D. Statistical comparisons were transfection reagent and methodology reported earlier.6-8 PMXs‐ made using Student t‐test, Mann Whitney U test, or ANOVA followed hKLF4 obtained from “Addgene plasmid 17967” was transfected in by appropriate post hoc test. Differences were considered significant human normal PBMCs to over‐express KLF gene in these cells. at P < 0.001. FIGURE 1 Ectopic miR‐2909 expression and its effector genes: Ectopic miR‐2909 expression in HeLa cells (A) and its ability to regulate genes coding for KLF4, c‐Myc, AATF, STAT3, mTORC1, Deptor, and PVT‐1 (B,D) together with PVT‐1 gene encoded microRNAs (C). **P < 0.001. Each bar represents mean of experiments done in triplicate WANI AND KAUL 3 | RESULTS 3 coding for AATF and c‐Myc.12Consequently, it became pertinent to explore as to how the downregulation of miR‐2909 within PC3 cells 3.1 | Ectopic cellular miR‐2909 expression regulates PVT‐1 gene locus (having high intrinsic miR‐2909 expression) affects these effector genes involved in the regulation of PVT‐1 gene locus. The ectopic miR‐2909 expression within HeLa cells resulted in significant phenomenon was accompanied by the significant upregulation of genes 3.2 | Cellular miR‐2909 gene downregulation affects PVT‐1 gene locus increase in miR‐2909 expression within these cells (Figure 1A), and this coding for PVT‐1, c‐Myc, AATF, STAT3, mTORC1 coupled with the AntagomiR‐2909 transfected PC3 cells (Figure 2A) exhibited down- downregulation of genes coding for KLF and Deptor (Figure 1B,D). Fur- regulation of PVT‐1 gene along with its encoded microRNAs as well ther, in order to explore whether or not miR‐2909 induced PVT‐1 locus as genes coding for c‐Myc and mTORC1 (Figure 2B‐D). This phenom- also involves the expression of microRNAs encoded by the PVT‐1 gene, enon was accompanied by upregulation of genes coding for KLF4 and attempt was made to examine the effect of miR‐2909 on the expression Deptor (Figure 2C & 2B). Although in our earlier studies, we have of these microRNAs. The results of such a study indeed, revealed that established the ability of miR‐2909 to downregulate KLF4 gene [8] ectopic expression of miR‐2909 within HeLa cells had the capacity to which is in agreement with the results reported here (Figures 1 and induce the expression of microRNAs encoded across the PVT‐1 genome 2), it became pertinent to explore the regulatory role of KLF4 gene (Figure 1C). These results indicated that miR‐2909 expression is accom- on the genes that are known to regulate PVT‐1 gene locus. panied by the upregulation of PVT‐1 gene locus (comprising of PVT‐1 encoded microRNAs as well as c‐Myc) within HeLa cells. Although sevAATF, STAT3, and mTORC1 on c‐Myc gene expression as well as mutual 3.3 | Ectopic cellular KLF4 expression influences genes involved in the regulation of PVT‐1 gene locus regulation of c‐Myc and PVT‐1 genes,3 we have recently established that Ectopic expression of KLF4 within human PBMCs (Figure 3A) resulted in there exists a regulatory relationship between miR‐2909 and genes the significant increase in the expression of genes coding for p53 and eral findings have established the regulatory role of genes coding for FIGURE 2 Cellular miR‐2909 downregulation and its effector genes: AntagomiR‐2909 programmed PC3 cells A, and their expression of genes coding for KLF4, c‐Myc, PVT‐1, Deptor, mTORC1 B, C, as well as PVT‐1 gene encoded microRNAs D. ** P < 0.001. Each bar represents mean of experiments done in triplicate WANI AND KAUL 4 FIGURE 3 EctopicKLF4 expression and its effector genes: Ectopic KLF4 expression in normal human PBMCs A, and KLF4‐dependent regulation of genes coding for p53, c‐Myc, PVT‐1, Deptor, AATF, mTORC1 A, B. ** P < 0.001. Each bar represents mean of experiments done in triplicate FIGURE 4 miR‐2909 RNomics and regulation of PVT‐1 gene locus: Bioinformatic microRNA target analysis exhibiting miR‐2909 target in the 5′‐ UTR region of Deptor mRNA A. Translational expression of Deptor gene within cells transfected with either control vector or miR‐ 2909 expression vector or KLF4 expression vector B. The miR‐2909 RNomics pathway exhibiting the interplay of genes that govern the tumorigenic PVT‐1 gene locus C. ** P < 0.001 indicates level of significance Deptor without any appreciable effect upon either genes coding for PVT‐ which has been found to increase p53 levels,4 and induction of p53 1 or c‐Myc (Figure 3). This phenomenon was accompanied by significant expression is known to inhibit c‐Myc expression13 leading to downregu- downregulation of genes coding for AATF and mTORC1 (Figure 3B). lation of PVT‐1 gene.3 It is in this context, the results reported here assume importance because KLF4 was found to induce p53 expression at both transcriptional and translational levels (Figure 3) as well as Deptor 4 | DISCUSSION gene expression (Figure 3B). Our earlier studies have established mutual regulatory interplay between genes coding for miR‐2909, AATF, c‐Myc, The oncogenic interplay of genes, encoding PVT‐1 (that includes lncRNA and p535-8 as well as the ability of miR‐2909 to suppress KLF4 and its microRNAs) and c‐Myc within human genomic 8q 24 locus, has expression5-8,14 which is also in conformity with the results reported here 1-3 Paradoxically, cellular expres- (Figures 1 and 2). From our findings reported here, it is apparent that sion of PVT‐1 gene is always accompanied by its encoded miR‐1204 miR‐2909 ensures high expression of PVT‐1 and c‐Myc genes through been observed in multiple cancer types. WANI AND KAUL 5 downregulation of KLF4 gene responsible for p53 expression (Figure 3) ORCID as well as repression of mTORC1 gene (Figure 3). It is pertinent to note Deepak Kaul http://orcid.org/0000-0002-6869-5094 here that mTORC1 is known to induce c‐Myc expression through STAT3.15 Based upon these findings as well as those reported here, it is not unlikely that miR‐2909 can initiate sustained expression of PVT‐ 1 gene locus (that includes c‐Myc) through the downregulation of KLF4 gene thereby ensuring reduced expression of genes coding for p53 and Deptor (Figure 4). This phenomenon can ensure sustained mTORC1‐ induced c‐Myc expression through the induction of STAT315 and consequently sustained PVT‐1 expression (Figure 4C). However, miR‐2909 can also induce c‐Myc through AATF and thereby ensure higher expression of PVT‐1 gene either through the induction of c‐Myc or p53 (Figure 4C). Our earlier studies have revealed that c‐Myc can influence p53 protein stability through the induction of Bmi‐1.14 Hence, it is possible that miR‐2909 directed downregulation of Deptor gene, either through KLF4 or direct target of its 5′UTR (Figure 4A,B), may ensure mTORC1‐ dependent upregulation of both c‐Myc and PVT‐1 within 8q 24 genomic locus. Downregulation of miR‐2909 within PC3 cells resulted in the upregulation of KLF4 gene responsible for the downregulation of both genes coding for c‐Myc and PVT‐1 (Figure 2). Paradoxically, the ectopic expression of KLF4 in human PBMCs did not exhibit any appreciable effect upon the c‐Myc or PVT‐1 genes (Figure 3B). This paradox can be resolved if one considers the recent findings regarding modulation of p53 target selectivity by KLF4 through protein‐protein interaction.16 Although KLF4 induced downregulation of AATF coupled with significant upregulation of p53 and Deptor genes should have contributed to the downregulation of PVT‐1 and c‐Myc gens (Figures 2–4), it is not unlikely that the binding affinity of KLF4 with p53 at the protein level may be responsible for their inability to affect expression of either c‐Myc or PVT‐1 within normal human PBMCs (Figures 3B and 4B). There exists a general recognition of the fact that most cancer types are associated with PVT‐1 locus dependence with increased supernumerary copies of c‐Myc gene. Targeting Myc directly with therapeutic interventions has proved challenging; thus, regulation of high c‐Myc gene expression through PVT‐1 bears considerable implication for the treatment of Myc‐driven cancers. 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