Abstract
Cancer-testis (CT) genes participate in the initiation and progression of cancer, but the role of CT-associated long non-coding RNAs (CT-lncRNAs) in hepatocellular carcinoma (HCC) is still elusive. Here, we discovered a conserved CT-lncRNA, named lnc-CTHCC, which was highly expressed in the testes and HCC. A lnc-CTHCC-knockout (KO) mouse model further confirmed that the global loss of lnc-CTHCC inhibited the occurrence and development of HCC. In vitro and in vivo assays also showed that lnc-CTHCC promoted HCC growth and metastasis. Mechanistically, lnc-CTHCC bound to heterogeneous nuclear ribonucleoprotein K (hnRNP K), which was recruited to the YAP1 promoter for its activation. Additionally, the N6-methyladenosine (m6A) modification was mediated by N6-adenosine-methyltransferase 70-kDa subunit (METTL3) and recognized by insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)/IGF2BP3, which maintained lnc-CTHCC stability and increased its expression in HCC. Together, our results show that lnc-CTHCC directly binds to hnRNP K and promotes hepatocellular carcinogenesis and progression by activating YAP1 transcription, suggesting that lnc-CTHCC is a potential biomarker and therapeutic target of HCC.
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Data availability
RNA-sequencing data have been deposited in GEO under accession codes GSE160912 and GSE160913. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. GTEx is available at https://gtexportal.org/home/, and TCGA is available at http://cancergenome.nih.gov/. Source data are provided with this paper.
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Acknowledgements
This work was supported by grants from the National Key Research and Development Program of China (2016YFC0905900 to B.S.), Funds for International Cooperation and Exchange of the National Natural Science Foundation of China (82120108012 to B.S.), State Key Program of the National Natural Science Foundation (81930086 to B.S.; 31530047 to Z.H.), National Natural Science Foundation (82073114 and 81773383 to S.W.; 81902836 to Y.G.) and Innovative Research Groups of the National Natural Science Foundation (81521004 to B.S.). B.S. is a Distinguished Professor Yangtze River Scholar.
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A.X. performed the experiments and wrote the paper. A.X., Q.W., W.Y., Y.G., L.Z. and Z.W. analyzed the data. Y.G., W.Y. and Z.H. generated the lnc-CTHCC-KO mouse model using the CRISPR–Cas9 system. W.Y. and Y.G. performed some data analysis from TCGA and GTEx databases. J.X., C.C., Y.G., D.W., Q.H., W.Y., F.W., C.X., Y.Z., G.B., X.T. and S.L. provided the samples. A.X., B.S., S.W. and Z.H. designed the study and revised the paper.
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Nature Cancer thanks Sven Diederichs and Lars Zender for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 The characteristics of cancer-testis-associated lncRNA (lnc-CTHCC).
(a) Expression in TPM (Transcripts Per Kilobase Million) of lnc-CTHCC among the GTEx normal tissue RNA-seq database (n = 17382 samples). The lower bound of the box is 25% quantile of the data, the upper bound of the box is 75% quantile of the data and the centre bound of the box is 50% quantile of the data. The IQR (Inter-Quartile Range) is the distance between the lower and upper bound of the box. The lower whisker extends from the lower bound to the lowest value within 1.5×IQR and the upper whisker extends from the upper bound to the highest value within 1.5×IQR. (b) Expression in FPKM (Fragments Per Kilobase Million) of lncCTHCC among the various tumors of TCGA (n = 9669 samples). The box plots are defined in the same terms as (A). (c-d) Representative images of PCR products from the human (C) and the mouse (D) 5’ RACE and 3’ RACE and sequencing of RACE products. The results are representative of three independent experiments. (e) The expression of the long versus short lnc-CTHCC transcripts in HCC tissues (n = 18 tumors, P = 0.0144). Two-tailed paired t-test was performed. (f) The aligned sequences of human and mouse lnc-CTHCC transcripts. (g-i) The protein-coding potential of lnc-CTHCC using the Coding Potential Assessment Tool (http://lilab.research.bcm.edu/cpat/), Coding Potential Calculator (http://cpc.cbi.pku.edu.cn/) and ORF finder software from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/orffinder/). GAPDH and β-actin served as the positive controls of coding genes, and PVT1 and HOTAIR served as the positive control of non-coding genes. (j) The secondary structure of lnc-CTHCC by the Vienna RNA Web Services (http://rna.tbi.univie.ac.at/). (k) Univariate Cox regression analysis in the HCC cohort (n = 64 patients). All the bars corresponded to 95% confidence intervals. (l) Multivariate Cox regression analysis from TCGA with complete clinical data (n = 240 patients). All the bars corresponded to 95% confidence intervals. *P < 0.05.
Extended Data Fig. 2 The expression and prognoses of lnc-CTHCC in cholangiocarcinoma and other types of cancer from TCGA.
(a) The RNA levels of lnc-CTHCC in cholangiocarcinoma and paired non-tumor tissues (n = 18 patients). Data are shown as mean ± SEM. Two-tailed paired t-test was performed. (b) The RNA levels of lnc-CTHCC in tumors from HCC (n = 64 patients) and cholangiocarcinoma (n = 18 patients, P = 0.0353, two-tailed unpaired t-test). Data are shown as mean ± SEM. (c-l) Kaplan–Meier survival curves of overall survival among other types of cancer using TCGA database. Log-rank test was performed. CHOL: cholangiocarcinoma (n = 36 patients), LUAD: lung adenocarcinoma (n = 471 patients), THYM: thymoma (n = 118 patients), BRCA: breast invasive carcinoma (n = 1061 patients), OV: ovarian serous cystadenocarcinoma (n = 422 patients), COAD: colon adenocarcinoma (n = 265 patients), ESCA: esophageal carcinoma (n = 178 patients), KIRC: kidney renal clear cell carcinoma (n = 508 patients), PAAD: pancreatic adenocarcinoma (n = 178 patients), STAD: stomach adenocarcinoma (n = 379 patients). *P < 0.05.
Extended Data Fig. 3 The effect of lnc-CTHCC KO in mice HCC and the localization of lnc-CTHCC in single cells from mouse liver cancer.
(a) The expression of adjacent genes, such as MAPK3, GDPD3, INO80E, SEZ6L2, and MVP, in the livers of WT and KO mice (n = 3 mice per group). Data are shown as mean ± SEM. Two-tailed unpaired t-test was performed. (b) Schematic representation of the DEN-induced HCC model. Two-week-old mice were intraperitoneally injected with DEN (25 mg/kg) and then fed a normal diet for 34 weeks (n = 6 mice per group). (c) Representative images of gross morphology from the livers of WT and KO mice. The images are representative of n = 6 animals. (d) The ratio of liver to body weight (P = 0.0003), the number of tumors (P = 0.0001) and the largest tumor diameter (P = 0.0022) from WT and KO mice. (e) ALT and AST levels in the serums of WT and KO mice. P values are as follows: P = 0.0003 (ALT); P = 0.0171 (AST). (f-g) HE, Ki67, TUNEL, and CD31 staining of the livers from WT and KO mice (scale bars = 50 μm). P values are as follows: P < 0.0001 (Ki67); P < 0.0001 (TUNEL); P < 0.0001 (CD31). For D-G, data are shown as mean ± SEM (n = 6 mice per group). Two-tailed unpaired t-test was performed. (h) FISH of lnc-CTHCC in the H22 cell line (scale bars = 5 μm). The results are representative of three independent experiments. (i-j) Liver cancer tissues from WT mice were dissociated into single cells for IF staining and FISH (scale bars = 5 μm). The markers of macrophages, epithelial cells, endothelial cells, and immune cells were F4/80, KRT, CD31, and CD45, respectively. The results are representative of three biologically independent samples. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Fig. 4 Overexpression of lnc-CTHCC promotes cell growth in HCC.
(a) The knockdown and overexpression efficiencies at the RNA levels in different HCC cells. P values are as follows: P < 0.0001 (Hep3B-sh1); P = 0.0001 (Hep3B-sh2); P = 0.0002(Hep3B-sh3); P < 0.0001 (MHCC97L-sh1); P = 0.0001 (MHCC97L-sh2); P = 0.0006(MHCC97L-sh3); P = 0.0006 (SMMC7721); P = 0.0003 (MHCC97H); P = 0.0004 (L02). (b) CCK8 assays in different HCC cells with the knockdown or overexpression of lnc-CTHCC. For Hep3B cells, P values are as follows: P = 0.0057 (48 h); P = 0.0029 (72 h); P = 0.0037 (96 h). For MHCC97L cells, P values are as follows: P = 0.0022 (48 h); P = 0.0011(72 h); P = 0.0003 (96 h). For SMMC7721 cells, P values are as follows: P = 0.0013 (72 h); P = 0.0002 (96 h); For MHCC97H cells, P values are as follows: P = 0.0011 (48 h); P = 0.0021 (72 h); P = 0.0011 (96 h). (c) Colony formation assays in different HCC cells with the knockdown or overexpression of lnc-CTHCC. P values are as follows: P = 0.0006 (Hep3B); P = 0.0013 (MHCC97L); P = 0.0008 (SMMC7721); P = 0.0026 (MHCC97H). (d) EdU assays in SMMC7721 and MHCC97H cells with lncCTHCC overexpression (scale bars = 50 μm). P values are as follows: P = 0.0010(SMMC7721); P < 0.0001 (MHCC97H). (e) Soft agar assays in SMMC7721 cells with lnc-CTHCC overexpression (scale bars = 50 μm, P = 0.0080). (f) Angiogenesis assays in HUVECs cultured with medium from SMMC7721 cells with lnc-CTHCC overexpression or control cells (scale bars = 50 μm). P values are as follows: P = 0.0022 (number of tubes); P = 0.0412 (cell growth). (g) The cytokine array analysis of the conditioned medium from the control groups and lnc-CTHCC-overexpressing SMMC7721 cells. (h) Heat map showing the differentially expressed proangiogenic factors, such as MCP-1, EGF, VEGF, Angiogenin, FGF-6, and MIG, in SMMC7721 cells with lnc-CTHCC overexpression. These experiments were performed twice. For A-F, data are from three biologically independent experiments and shown as mean ± SEM. Two-tailed paired t-test was performed. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Fig. 5 Overexpression of lnc-CTHCC promotes cell metastasis in HCC.
(a) Wound-healing assays in different HCC cells with the knockdown of lnc-CTHCC (scale bars = 50 μm). P values are as follows: P = 0.0002 (Hep3B); P = 0.0007 (MHCC97L). (b) Wound-healing assays in different HCC cells with the overexpression of lnc-CTHCC (scale bars = 50 μm). P values are as follows: P < 0.0001 (SMMC7721); P = 0.0033 (MHCC97H). (c) Representative images of cell migration and invasion assays in different HCC cells with the knockdown of lnc-CTHCC (scale bars = 50 μm). P values are as follows: P = 0.0051 (Hep3B-migration); P = 0.0022 (Hep3B-invasion); P = 0.0013 (MHCC97L-migration); P = 0.0042 (MHCC97L-invasion). (d) Representative images of cell migration and invasion assays in different HCC cells with the overexpression of lnc-CTHCC (scale bars = 50 μm). P values are as follows: P = 0.0012 (SMMC7721-migration); P = 0.0019 (SMMC7721-invasion); P = 0.0011 (MHCC97H-migration); P = 0.0034 (MHCC97H-invasion). (e-g) Overexpression of lnc-CTHCC significantly promoted HCC lung metastasis in nude mice (n = 10 mice per group). (E) Representative bioluminescent images in lung regions (P < 0.0001). (F) Representative images of the metastatic nodes in the lungs (n = 10 mice per group, P = 0.0007). For E-F, data are shown as mean ± SEM and two-tailed unpaired t-test was performed. (G) HE-stained lung sections (scale bars = 50 μm). (h-i) The expressions of EMT-related markers were analyzed by western blots, which indicated that lnc-CTHCC regulated EMT genes specifically via hnRNP K or YAP. The results are representative of three independent experiments. For A-D, data are from three biologically independent experiments and shown as mean ± SEM. Two-tailed paired t-test was performed. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Fig. 6 Lnc-CTHCC has no effect on the expression and localization of hnRNP K.
(a,b) The knockdown efficiencies of hnRNP K at the RNA and protein levels in Hep3B cells. P values are as follows: P = 0.0008 (si-1); P = 0.0004 (si-2); P = 0.0001 (si-3). (c) Overexpression of hnRNP K rescued the colony formation ability of Hep3B cells with lnc-CTHCC knockdown. P values are as follows: P = 0.0004 (sh-NC + NC and sh-lnc-CTHCC + NC); P = 0.0017 (sh-lnc-CTHCC + NC and sh-lnc-CTHCC + hnRNP K). (d) Knockdown of hnRNP K rescued the colony formation ability of SMMC7721 cells with lnc-CTHCC overexpression. P values are as follows: P < 0.0001 (NC + si-NC and lnc-CTHCC + si-NC); P = 0.0006 (lnc-CTHCC + si-NC and lnc-CTHCC + sihnRNP K). (e) Overexpression of hnRNP K rescued the invasion ability of Hep3B cells with lnc-CTHCC knockdown (scale bars = 50 μm). P values are as follows: P = 0.0004 (sh-NC + NC and sh-lncCTHCC + NC); P = 0.0005 (sh-lnc-CTHCC + NC and sh-lnc-CTHCC + hnRNP K). (f) Knockdown of hnRNP K rescued the invasion ability of SMMC7721 cells with lnc-CTHCC overexpression (scale bars = 50 μm). P values are as follows: P < 0.0001 (NC + si-NC and lncCTHCC + si-NC); P = 0.0018 (lnc-CTHCC + si-NC and lnc-CTHCC + si-hnRNP K). (g) The RNA levels of hnRNP K in different HCC cells with knockdown or overexpression of lnc-CTHCC. (h) Western blot assays of hnRNP K in different HCC cells with knockdown or overexpression of lnc-CTHCC. (i) The hnRNP K staining of the xenograft tumors with lnc-CTHCC knockdown or overexpression (scale bars = 50 μm). (j) Cellular immunofluorescence assays of hnRNP K in different HCC cells with knockdown or overexpression of lnc-CTHCC (scale bars = 25 μm). For B and H-J, the results are representative of three independent experiments. For A and C-G, data are from three biologically independent experiments and shown as mean ± SEM. Two-tailed paired t-test was performed. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Fig. 7 Transcriptome sequencing analysis after knockdown or knockout of lnc-CTHCC in HCC.
(a,b) Heat map showing the differentially expressed genes in Hep3B cells with lnc-CTHCC knockdown (A) and in liver cancer tissues from wild-type and knockout mice (B). (c) The RNA levels of YAP in Hep3B cells with lnc-CTHCC knockdown (n = 3 independent experiments, P = 0.0007, two-tailed paired t-test) or in the tumors from WT and KO mice (n = 3 mice per group, P < 0.0001, two-tailed unpaired t-test). Data are shown as mean ± SEM. (d) IHC staining of YAP in the xenograft tumors with lnc-CTHCC overexpression (scale bars = 50 μm). The results are representative of three biologically independent samples. (e) The RNA levels of YAP target genes (FGF2, CTGF, CYR61, CDC20 and SERPINE1) in MHCC97L cells with lnc-CTHCC knockdown. P values are as follows: P = 0.0006 (SERPINE1); P = 0.0048 (CDC20); P = 0.0035 (CTGF); P = 0.0004 (CYR61); P = 0.0004 (FGF2). (f) Luciferase reporter assays for YAP activity in Hep3B cells with sh-lnc-CTHCC and hnRNP K overexpression. P values are as follows: P < 0.0001 (sh-NC + NC and sh-lnc-CTHCC + NC); P = 0.0017 (sh-lnc-CTHCC + NC and sh-lnc-CTHCC + hnRNP K); P = 0.0008 (sh-NC + NC and sh-NC + hnRNP K). (g) The protein levels of YAP in Hep3B cells with sh-lnc-CTHCC and hnRNP K overexpression. The results are representative of three independent experiments. (h) The RNA levels of YAP in HCC and normal tissues from TCGA (P = 0.0001, two-tailed unpaired t-test) or in HCC and paired non-tumor tissues from the HCC cohort (n = 50 patients, P < 0.0001, two-tailed paired t-test). Data are shown as mean ± SEM. (i) Pearson correlation analysis between lnc-CTHCC expression and YAP expression (n = 50 patients, P < 0.0001). (J) Western blot assays of YAP in HCC and paired non-tumor tissues (n = 10 paired samples). (k) IHC staining of YAP from HCC tissues with relatively high or low lnc-CTHCC expression (scale bars = 50 μm). (l) Kaplan-Meier survival curves of OS from TCGA (P = 0.0413, log-rank test). For E-F, data are shown as mean ± SEM of three independent experiments. Two-tailed paired t-test was performed. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Fig. 8 YAP is involved in the biological function of lnc-CTHCC in HCC.
(a) The knockdown efficiencies of YAP at the RNA and protein levels in Hep3B cells. P values are as follows: P = 0.0002 (sh-1); P = 0.0001 (sh-2). (b,c) Knockdown of YAP suppressed HCC cell proliferation and metastasis in vitro. (B) Colony formation assays. P values are as follows: P = 0.0034 (sh-YAP-1); P = 0.0057 (sh-YAP-2). (C) Transwell assays. Scale bars = 50 μm. P values are as follows: P = 0.0044 (migration-sh-YAP-1); P = 0.0046 (migration-sh-YAP2); P = 0.0009(invasion-sh-YAP-1); P = 0.0013 (invasion-shYAP-2). (d-f) Knockdown of YAP suppressed lnc-CTHCC-induced increase in HCC cell proliferation and metastasis in vitro. (D) Colony formation assays. P values are as follows: P = 0.0004 (Ctrl + sh-NC and Ctrl + sh-YAP); P = 0.0065 (lnc-CTHCC + sh-NC and lnc-CTHCC + sh-YAP). (E) EdU assays. Scale bars = 50 μm. P values are as follows: P = 0.0117 (Ctrl + sh-NC and Ctrl + shYAP); P = 0.0102 (lnc-CTHCC + sh-NC and lnc-CTHCC + sh-YAP). (F) Transwell assays. Scale bars = 50 μm. P values are as follows: P = 0.0006 (Ctrl + sh-NC and Ctrl + sh-YAP); P = 0.0031 (lnc-CTHCC + sh-NC and lnc-CTHCC + sh-YAP). (g,h) The tumor growth curves and tumor weight (n = 6 mice per group, P < 0.0001). Data are shown as mean ± SEM. Two-tailed unpaired t-test was performed. (i,j) Overexpression of YAP rescued the proliferation and invasion abilities of Hep3B cells with lnc-CTHCC knockdown (scale bars = 50 μm). (I) P values are as follows: P = 0.0012 (sh-NC + NC and sh-lnc-CTHCC + NC); P = 0.0003 (sh-lnc-CTHCC + NC and sh-lnc-CTHCC + YAP). (J) P values are as follows: P = 0.0006 (sh-NC + NC and sh-lnc-CTHCC + NC); P = 0.0005 (sh-lnc-CTHCC + NC and sh-lnc-CTHCC + YAP). For A-F and I-J, data are from three biologically independent experiments and shown as mean ± SEM. Two-tailed paired t-test was performed. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Fig. 9 METTL3 promotes cell proliferation and metastasis in HCC.
(a-c) The knockdown and overexpression efficiencies of METTL3 at the RNA and protein levels in Hep3B cells. (A) P = 0.0003. (B) P = 0.0013. (C) The results are representative of three independent experiments. (d-f) Knockdown of METTL3 inhibited cell proliferation and metastasis while overexpression of METTL3 promoted cell proliferation and metastasis in Hep3B cells. (D) P values are as follows: P = 0.0009 (sh-METTL3); P = 0.0012 (Lv-METTL3). (E) Scale bars = 50 μm. P values are as follows: P < 0.0001 (sh-METTL3); P = 0.0010 (Lv-METTL3). (F) Scale bars = 50 μm. P values are as follows: P = 0.0020 (sh-METTL3); P = 0.0004 (Lv-METTL3). (g,h) Knockdown of lnc-CTHCC rescued the proliferation and invasion abilities of Hep3B cells with METTL3 overexpression. (G) P values are as follows: P = 0.0003 (NC + sh-NC and METTL3 + sh-NC); P = 0.0009 (METTL3 + sh-NC and METTL3 + sh-lnc-CTHCC). (H) Scale bars = 50 μm. P values are as follows: P = 0.0022(NC + sh-NC and METTL3 + sh-NC); P = 0.0003 (METTL3 + sh-NC and METTL3 + sh-lnc-CTHCC). For A-B and D-H, data are from three biologically independent experiments and shown as mean ± SEM. Two-tailed paired t-test was performed. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Fig. 10 Knockdown of IGF2BP1/3 inhibits cell proliferation and metastasis in HCC.
(a) The RNA levels of IGF2BP1/3 in Hep3B cells with lnc-CTHCC knockdown. (b) Western blot assays of IGF2BP1/3 in different cells with lnc-CTHCC knockdown or overexpression. The results are representative of three independent experiments. (c,d) The knockdown efficiencies of IGF2BP1/3 at the RNA and protein levels in Hep3B cells. P values are as follows: P = 0.0006 (si-IGF2BP1-1); P = 0.0006 (si-IGF2BP1-2); P = 0.0002 (si-IGF2BP3-1); P = 0.0002 (si-IGF2BP3-2). For D, the results are representative of three independent experiments. (e,f) Knockdown of IGF2BP1/3 inhibited cell proliferation and metastasis in Hep3B cells. (e) P values are as follows: P = 0.0016 (si-IGF2BP1-1); P = 0.0027 (si-IGF2BP1-2); P < 0.0001 (si-IGF2BP3-1); P < 0.0001 (si-IGF2BP3-2). (f) Scale bars = 50 μm. P values are as follows: P = 0.0012 (migration-si-IGF2BP1-1); P = 0.0003 (migration-si-IGF2BP1-2); P = 0.0009 (invasionsi-IGF2BP1-1); P < 0.0001 (invasion-si-IGF2BP1-2); P = 0.0006 (migration-si-IGF2BP3-1); P = 0.0009 (migration-si-IGF2BP3-2); P = 0.0008 (invasion-si-IGF2BP3-1); P = 0.0009 (invasion-siIGF2BP3-2). (g) Schematic diagram showing the molecular mechanisms underlying lnc-CTHCC action in hepatocellular carcinoma. For A, C and E-F, data are from three biologically independent experiments and shown as mean ± SEM. Two-tailed paired t-test was performed. **P < 0.01; ***P < 0.001; ****P < 0.0001.
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Xia, A., Yuan, W., Wang, Q. et al. The cancer-testis lncRNA lnc-CTHCC promotes hepatocellular carcinogenesis by binding hnRNP K and activating YAP1 transcription. Nat Cancer 3, 203–218 (2022). https://doi.org/10.1038/s43018-021-00315-4
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DOI: https://doi.org/10.1038/s43018-021-00315-4
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