Role of MYC-regulated long noncoding RNAs in cell cycle regulation and tumorigenesis

Abstract

Background: The functions of long noncoding RNAs (lncRNAs) have been identified in several cancers, but the roles of lncRNAs in colorectal cancer (CRC) are less well understood. The transcription factor MYC is known to regulate lncRNAs and has been implicated in cancer cell proliferation and tumorigenesis.

Methods: CRC cells and tissues were profiled to identify lncRNAs differentially expressed in CRC, from which we further selected MYC-regulated lncRNAs. We used luciferase promoter assay, ChIP, RNA pull-down assay, deletion mapping assay, LC-MS/MS and RNA immunoprecipitation to determine the mechanisms of MYC regulation of lncRNAs. Moreover, soft agar assay and in vivo xenograft experiments (four athymic nude mice per group) provided evidence of MYC-regulated lncRNAs in cancer cell transformation and tumorigenesis. The Kaplan-Meier method was used for survival analyses. All statistical tests were two-sided.

Results: We identified lncRNAs differentially expressed in CRC (P < .05, greater than two-fold) and verified four lncRNAs upregulated and two downregulated in CRC cells and tissues. We further identified MYC-regulated lncRNAs, named MYCLos. The MYC-regulated MYCLos may function in cell proliferation and cell cycle by regulating MYC target genes such as CDKN1A (p21) and CDKN2B (p15), suggesting new regulatory mechanisms of MYC-repressed target genes through lncRNAs. RNA binding proteins including HuR and hnRNPK are involved in the function of MYCLos by interacting with MYCLo-1 and MYCLo-2, respectively. Knockdown experiments also showed that MYCLo-2, differentially expressed not only in CRC but also in prostate cancer, has a role in cancer transformation and tumorigenesis.

Conclusions: Our results provide novel regulatory mechanisms in MYC function through lncRNAs and new potential lncRNA targets of CRC.

Figure 1.

Identification of long noncoding RNAs (lncRNAs) differentially expressed in colorectal cancer (CRC). A and B) Heatmaps of protein-coding transcripts (A) and lncRNAs (B) dysregulated in CRC (HCT116, RKO, SW620, HT29, and CRC tissues), compared with normal colon (CCD-18Co, CCD-33Co, CCD112CoN, CCD-841-CoN and colon normal adjacent tissues [NATs]). C) Volcano map indicating up- or downregulation of CRC-associated lncRNAs (CCAT1, CCAT3~8). D-G) Verification of induced expression of CCAT3 (D), CCAT4 (E), CCAT5 (F), and CCAT6 (G) in CRC cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). Data are means ± SD of three independent experiments and each measured in triplicate. H and I) Verification of repressed expression of CCAT7 (H) and CCAT8 (I) in CRC cell lines by using qRT-PCR. Data are means ± SD of three independent experiments and each measured in triplicate. J-O) Differential expression of CCAT3 (J), CCAT4 (K), CCAT5 (L), CCAT6 (M), CCAT7 (N), and CCAT8 (O) in 52 matched CRC and NAT tissues. Shown are box plots with lower and upper bounds of the boxes representing the 25th and 75th quartiles, respectively; whiskers demarcate values within the 10^th to 90th percentiles, and solid circles indicate values less than the 10th percentile and greater than the 90th percentile.

Figure 2.

Identification of colorectal cancer [CRC]–associated lncRNAs directly regulated by MYC. A) Comparison of mRNA levels of MYC in normal colon–derived (CCD-18Co, CCD-33Co, CCD112CoN, CCD-841-CoN, and colon normal adjacent tissue [NAT] samples) and CRC cells/tissues (HCT116, RKO, SW620, HT29, and CRC tissue samples). Data are means ± SD of three independent experiments and each measured in triplicate. B and C) Heatmaps of lncRNAs dysregulated by MYC knockdown in HCT116 (B) and RKO (C). D) Venn diagrams showing the number of upregulated long noncoding RNAs (lncRNAs) in a MYC-dependent manner. Hypergeometric probability of three lncRNAs commonly found in all criteria is P(X=3) = 0.01. E-G) Quantitative real-time polymerase chain reaction (qRT-PCR) results showing MYC-mediated positive regulation of MYCLo-1 (E), -2 (F), and -3 (G) in various types of cancer cells. Data are means ± SD of three independent experiments and each measured in triplicate (**P = .0062; †P = .001; ‡P = .0079; ¶P = .004; *P < .001). Statistical significance determined by unpaired two-sided Student’s t test. H-K) Expressional associations between MYC (H) and MYCLos (MYCLo-1 [I], -2 [J], and -3 [K]) in 50 colorectal tissue samples. The number of samples in each group is eight (Group A), 17 (Group B), 15 (Group C), and 10 (Group D). The P values between Group A and Group D are indicated. Shown are box plots with lower and upper bounds of the boxes represent the 25th and 75th quartiles, respectively; whiskers demarcate values within the 10th to 90th percentiles, and solid circles indicate values less than the 10th percentile and greater than the 90th percentile. Statistical significance was determined by the unpaired two-sided Student’s t test. L-N) Luciferase assays showing MYC-dependent regulation of 5’ promoter activities of MYCLo-1 (L), -2 (M), and -3 (N). The promoter region of each MYCLo for luciferase assay is indicated with a red box in (O-Q), respectively. Data are means ± SD of three independent experiments and each measured in triplicate (**P = .01; †P = .03; *P = .004). Statistical significance was determined by the unpaired two-sided Student’s t test. O-Q) The database of open chromatin TFBS by ChIP-seq from ENCODE/Open Chrom (UT Austin) shows that MYC binds to 5’ promoter regions of MYCLo-1 (O), -2 (P), and -3 (Q) in various types of cells such as GM12878, K562, HeLa, HepG2, and HUVEC. MYC-binding regions are indicated with red boxes.

Figure 3.

Effect of MYCLos knockdown on cell proliferation and cell cycle progression. A and B) Cell proliferation assays. HCT116 (n = 5, A) or PC3 (n = 4, B) cells were treated with siRNAs (final concentration: 50nM), as indicated and subjected to proliferation assay every 24 hours (†P = .013; ‡P = .0048; ¶P = .009; *P < .001). Error bars represent standard deviation. C) Cell proliferation assay. CCD-18Co cells were transfected with pcDNA3.3 plasmids (EV) expressing MYCLo-1, -2, or -3 and subjected to a cell proliferation assay every 24 hours (n = 3) (*P = .001; †P = .0045; ‡P = .0025). Error bars represent standard deviation. D) Proportion of cells in each cell cycle phase determined by flow cytometry analysis. HCT116 cells were treated with siRNAs (50nM) as indicated and analyzed 48 hours after transfection. Data are mean ± SD of three independent experiments and each measured in triplicate (**P = .047; †P = .0085; ‡P = .00662; *P < .001). E) Heatmap representing cell cycle regulator genes dysregulated by MYCLo-1. The genes that are commonly regulated by MYC are indicated. Expression values displayed in gradient of red and blue are Log2-transformed fold change. The list of the dysregulated genes is available in Supplementary Table 4 (available online). F) Heatmap representing cell cycle regulator genes dysregulated by MYCLo-2. The genes that are commonly regulated by MYC are indicated. Expression values displayed in gradient of red and blue are Log2-transformed fold change. The list of the dysregulated genes is available in Supplementary Table 4 (available online). G) Quantitative real-time polymerase chain reaction (qRT-PCR) results showing MYC-independent regulation of CDKN1A (left) and CDKN2B (middle) expression by MYCLo-1 knockdown (right). HCT116 cells were treated with 50nM siRNAs targeting MYC or MYCLo-1 for 72 hours. Data are mean ± SD of three independent experiments, measured in triplicate (††P = .003; §P = .0017; ∥P = .013; †P = .013; ‡P = .001; #P = .002; *P < .001). H) qRT-PCR results showing MYC-independent regulation of CDKN2B (left), CDKN1A, YWHAB, CFL1, and SFN (middle) expression by MYCLo-2 knockdown (right). HCT116 cells were treated with 50nM siRNAs targeting MYC or MYCLo-2 for 72 hours. Data are mean ± SD of three independent experiments and each measured in triplicate (**P = .03; ††P = .002; ∥P = .003; §P = .01; †P = .001; ‡P = .005; #P = .016; ¶P = .004; *P < .001). Statistical significance was determined by the unpaired two-sided Student’s t test.

Figure 4.

Interactions between MYCLo-1/2 and RNP binding proteins (HuR and hnRNPK) and their involvement in the regulation of 5’ promoter activities of CDKN1A and CDKN2B. A and B) Schematics (top) of pGL4 luciferase vectors inserted with distal 5’ promoter regions (blue bars) of CDKN1A (A) and CDKN2B (B). Red bars depict proximal promoter regions where MYC binds (MB). Luciferase reporter assays (bottom) representing transcription activity of the distal 5’ promoter regions of CDKN1A (A) and CDKN2B (B) in HCT116 cells transfected with the pGL4 luciferase vectors and siRNAs as indicated. Data are mean ± SD of three independent experiments and each measured in triplicate (**P = .024; *P < .001). C and D) Luciferase reporter assays to identify specific 5’ promoter regions of CDKN1A (C) and CDKN2B (D), where MYCLo-1 and -2 regulate their promoter activities, respectively. Data are mean ± SD of three independent experiments and each measured in triplicate (**P = .027; †P = .014; *P < .001). E and F) RNA pull-down assays showing the interactions between MYCLo-1 and HuR (E), and MYCLo-2 and hnRNPK (F). Total protein in SDS-PAGE gel was analyzed by liquid chromatography–tandem mass spectrometry. Western blot data are representative of two independent experiments. G and H) RIP assays and quantitative real-time polymerase chain reaction detection demonstrating the specific interactions between HuR and MYCLo-1 (G), and hnRNPK and MYCLo-2 (H). Data are mean ± SD of three independent experiments and each measured in triplicate (*P < .001). I and J) Deletion mapping analyses showing the importance of central region of MYCLo-1 (I, 401~690bp) and of marginal regions of MYCLo-2 (J, 71~170bp and 608~658bp) in the interactions with HuR and hnRNPK, respectively. Data are representative of three independent experiments. K and L) ChIP assays showing that HuR binds to the specific 5’ promoter region of CDKN1A, where MYCLo-1 regulates its transcription activity (K) and that hnRNPK binds to the specific 5’ promoter region of CDKN2B, where MYCLo-2 regulates its transcription activity (L). Data are representative of three independent experiments.

Figure 5.

Function of MYCLo-2 in cancer cell transformation and tumorigenesis. A) Comparison of expression level of MYCLo-2 in each pair of CRC tissues (red) and their matched normal adjacent tissues (NATs) (black). B) Quantitative real-time polymerase chain reaction (qRT-PCR) results showing significant overexpression of MYCLo-2 in PC-derived cell lines, compared with normal prostate-derived cell lines. Data are mean ± SD of three independent experiments and each measured in triplicate. C) Box and whisker plot (left) showing the significant overexpression of MYCLo-2 in PC, compared with their matched NAT (25 pairs of primary tissues). Comparison of expression level (right) of MYCLo-2 in each pair of PC tissues (red) and their matched NATs (black). D) Soft agar colony formation assays indicating that knockdown of MYCLo-2 causes a decreased number of cells (left) and colonies (right) in CRC-derived (HCT116) and PC-derived (PC3) cells. Data are mean ± SD of three independent experiments, each measured in triplicate (#P = .0066; §P = .00315; *P = .01185; ∥P = .00151; **P = .003322; †P = .001821; ¶P = .0107; ‡P = .02474). E) Kaplan-Meier plot of tumor-free survival analysis in athymic nude mice xenografted with siRNA-treated HCT116 (left) or PC3 (right) cells as indicated. The Kaplan-Meier method was used to estimate survival curves, and the log-rank test was used to test for differences between curves using SPSS Statistical Software (SPSS Inc., Chicago, IL).