It is well established that hypoxia contributes to tumor progression in a HIF-2α-dependent manner in renal cell carcinoma (RCC), yet the role of LncRNAs involved in hypoxia-mediated RCC progression remains unclear. Here we demonstrate that LncRNA-SARCC is differentially regulated by hypoxia in a VHL-dependent manner both in tissue culture and in human RCC clinical samples. LncRNA-SARCC can suppress hypoxic cell cycle progression in the VHL-mutant RCC cells while derepress it in the VHL-restored RCC cells. Mechanism dissection reveals that LncRNA-SARCC can post-transcriptionally regulate androgen receptor (AR) by physically binding and destabilizing AR protein to suppress AR/HIF-2α/C-MYC signals. In return, HIF-2α can transcriptionally regulate the LncRNA-SARCC expression via binding to hypoxia responsive elements (HREs) on the promoter of LncRNA-SARCC. The negative feedback modulation between LncRNA-SARCC/AR complex and HIF-2α signaling may then lead to differentially modulate RCC progression in a VHL-dependent manner. Together, these results may provide us a new therapeutic approach via targeting this newly identified signal from LncRNA-SARCC to AR-mediated HIF-2α/C-MYC signals against RCC progression.
Human samples—surgical specimens from human ccRCC tissues were obtained from 16 patients in the Department of Urology, Shanghai Tenth People’s Hospital, Tongji Medical School (Shanghai, China), freshly frozen in liquid nitrogen and stored at −80 °C until use. OCT-embedded blocks were sectioned until cut planes were >70% tumor. Sections were collected for DNA, RNA, and protein extraction. Samples were cataloged, clinical information on cases was obtained through chart review, and patient identifiers were removed before analysis. Informed consent was obtained from patients and the study was approved by the Institutional Review Board of Tongji Medical College. Immunohistochemistry—immunohistochemical staining was performed as previously described , with antibodies specific for HIF-1α, HIF-2α, C-MYC, Ki-67, AR (Abcam Inc., Cambridge, MA, USA; 1:200 dilution) and CAIX (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200 dilution). The reactivity degree was assessed by at least two pathologists without knowledge of the clinicopathological features of tumors. The degree of positivity was initially classified according to the percentage of positive tumor cells as the following: (−) >5% cells positive, (1+) 6–25% cells positive, (2+) 26–50% cells positive, and (3+) >50% cells positive. For AR, HIF-1α, HIF-2α, C-MYC, Ki-67 and CAIX, samples were scored as negative/weak, intermediate, or strong. Only cells with clear tumor cell morphology were scored. Cell culture and transfection—the human VHL-mut RCC cell lines SW839 (AR positive), OSRC-2 (AR positive), A498 (AR negative), 769-P (AR negative), 786-O (AR negative), and the human VHL-wt RCC cell lines Caki-1 (AR positive), Caki-2 (AR positive), and the immortalized proximal tubule epithelial cell line from normal adult human kidney HK-2 and 293T (AR positive) were originally purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and preserved in our lab. All RCC cells were cultured in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) in the humidified 5% CO2 environment at 37 °C. SW839-VHL cell lines were stably transfected with VHL-wt cDNA into VHL-mut SW839 cells (ATCC). To generate LncRNA-SARCC overexpressing or LncRNA-SARCC knocked-down stable clones of SW839, SW839-VHL and OSRC-2 cells were transfected with lentiviral vectors, pWPI-LncRNA-SARCC vs. pWPI-Vec or pLKO1-sh-LncRNA-SARCC vs. pLKO1-shRNA-control, with the PAX2 packaging plasmid, and PMD2G envelope plasmid, then transfected into 293T cells for 48 hours to obtain the lentivirus soup followed by cryopreservation in −80 °C for later use. The cells were transfected using the lipofectamine 3000 (Invitrogen) reverse transfection protocol, according to the manufacturer’s instructions. Hypoxia—hypoxia (0.5% O2, 5% CO2, 94.5% N2) was achieved using an In Vivo2 hypoxic workstation (Ruskinn Technologies) or in a positive pressure chamber receiving gas from a custom-mixed tank (Airgas). Oxyrase® (Oxyrase) was used as a hypoxia mimetic at a final concentration of 100 mM while CoCl2 was used as a hypoxia mimetic at a final concentration of 200 µM. RNA immunoprecipitation (RIP)—native RIP was performed as described previously . Briefly, SW839, SW839-VHL and OSRC-2 cells were lysed in RIPA lysis buffer (20 mM Tris-HCl/pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin) supplemented with RNase inhibitor, protease inhibitor cocktail. RNase-free DNase (NEB) (400 U) was then added to the lysates and incubated on-ice for 30 minutes. The cell lysates were diluted in the RIPA buffer and 50 µL of the supernatant saved as input for PCR analysis. A total of 500 µL of the supernatant was incubated with 4 µg of AR antibody overnight (normal rabbit IgG as control). Protein A/G beads were pre-blocked by 15 mg/mL BSA in PBS. Then pre-blocked beads were added to the antibody-lysate mixture and incubated for another 2 hours. The RNA/antibody complex was washed four times by RIPA buffer supplemented with RNase inhibitor, protease inhibitor cocktail. The RNA was extracted using Trizol (Invitrogen) according to the manufacturer’s protocol and subjected to RT-qPCR analysis. For UV cross-linking and RIP, cells were first subjected to formaldehyde cross-linking and then native RIP protocol was conducted. RNA-pull down assay—LncRNA-SARCC, LncRNA-SARCC (1.6 kb), LncRNA-SARCC (1.2 kb) or LncRNA-SARCC (0.8 kb) were in vitro transcribed respectively from the PCR generated T7 promoter driven DNA fragments and biotin-labeled with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). One milligram of whole-cell lysates from SW839 cells were incubated with 3 µg of purified biotinylated transcripts for 1 hour at 25 °C and complexes were isolated with streptavidin agarose beads (Invitrogen). The AR protein present in the pull-down material was detected by standard immunoblot analysis. Chromatin immunoprecipitation assay (ChIP)—cells were cross-linked with 4% formaldehyde for 10 minutes followed by cell collection and sonication with a predetermined power to yield genomic DNA fragments of 300–1,000 bp long. Lysates were precleared sequentially with normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and protein A-agarose. Anti-AR antibody (2.0 µg) was added to the cell lysates and incubated at 4 °C overnight. For the negative control, IgG was used in the reaction. Cell-cycle analysis—cells were plated at a density such that they would be 50% confluent on the day of analysis. Treatment (hypoxia) was then initiated over the next several days, so that all cells were in culture for the same amount of time and at similar confluency when harvested. BrdU analysis was performed following the standard protocol (Becton Dickinson) after a 20 min pulse with 10 mM BrdU. Cells were stained with Alexa 488 anti-BrdU (Invitrogen) and 0.1 M propidium iodide and analyzed in an LSR FACS machine (Becton Dickinson). For proliferation analysis with Hoechst staining, 104 cells were plated on 6-cm2 plates, with staining and counting done according to the manufacturer’s instructions (Invitrogen). Luciferase assay—cells were plated in 24-well plates and transfected with pGL3 reporter constructs using lipofectamine (Invitrogen) according to the manufacturer’s instruction. After transfection, DMEM media was added into the culture with dihydrotestosterone (DHT) with ethanol as vehicle control. pRL-TK was used as internal control. Luciferase activity was measured by Dual-Luciferase Assay (Promega) according to the manufacturer’s manual. Subcutaneous and renal capsule implantation—SW839 cells expressing pLKO1-sh-LncRNA-SARCC vs. pLKO1-shRNA-control (2×106) and SW839-VHL cells expressing pWPI-LncRNA-SARCC vs. pWPI-mock (2×106), were subcutaneously injected into one flank of 6-week-old male athymic nude mice (NCI) (n=8 mice per group). After 8–9 weeks, mice were sacrificed, and tumors were excised and weighed. Similarly, cells were injected into left renal capsule of 6-week-old male athymic nude mice (NCI) (n=8 mice per group). After 7–8 weeks, mice were sacrificed, and tumors were excised and weighed. Studies on animals were conducted with approval from the Animal Research Ethics Committee of the University of Rochester Medical Center. Statistical analysis—unless otherwise stated, all data were shown as mean ± SD. The SPSS 12.0 statistical software (SPSS Inc., Chicago, IL, USA) was applied for statistical analysis. The χ2 analysis and Fisher exact probability analysis were applied for comparison among the expression of AR, HIF-1α, HIF-2α, C-MYC and Ki-67 and individual clinicopathological features. Difference of tumor cells was determined by t test or analysis of variance.
(I) Hypoxia differentially regulates RCC cell proliferation in a VHL-dependent manner; (II) LncRNA-SARCC is differentially modulated by hypoxia in a VHL-dependent manner and is physically associated with AR; (III) LncRNA-SARCC interacts with AR and decreases AR protein stability; (IV) LncRNA-SARCC suppressed AR which alters HIF-2α/C-MYC signals under hypoxia; (V) mechanism dissection how AR modulates HIF-2α expression at the transcriptional level under hypoxia; (VI) mechanism dissection how HIF-2α modulates LncRNA-SARCC expression at the transcriptional level under hypoxia; (VII) LncRNA-SARCC differentially modulates RCC proliferation under hypoxia in the RCC in vivo mouse model.
LncRNA-SARCC is differentially regulated by hypoxia in a VHL-dependent manner both in tissue culture and in human RCC clinical samples. LncRNA-SARCC can suppress hypoxic cell cycle progression in the VHL-mutant RCC cells while derepress it in the VHL-restored RCC cells.