Yonsei Med J.  2018 Sep;59(7):816-826. 10.3349/ymj.2018.59.7.816.

XIST Induced by JPX Suppresses Hepatocellular Carcinoma by Sponging miR-155-5p

• Lin XQ
• Huang ZM
• Chen X
• Wu F
• Wu W

• KMID: 2428907
• DOI: http://doi.org/10.3349/ymj.2018.59.7.816

Abstract

PURPOSE
The influence of X-inactive specific transcript (XIST) and X-chromosome inactivation associated long non-coding RNAs (lncRNAs) just proximal to XIST (JPX) on hepatocellular carcinoma (HCC) remains controversial in light of previous reports, which the present study aimed to verify.
MATERIALS AND METHODS
The DIANA lncRNA-microRNA (miRNA) interaction database was used to explore miRNA interactions with JPX or XIST. JPX, XIST, and miR-155-5p expression levels in paired HCC specimens and adjacent normal tissue were analyzed by RT-qPCR. Interaction between XIST and miR-155-5p was verified by dual luciferase reporter assay. Expression levels of miR-155-5p and its known target genes, SOX6 and PTEN, were verified by RT-qPCR and Western blot in HepG2 cells with or without XIST knock-in. The potential suppressive role of XIST and JPX on HCC was verified by cell functional assays and tumor formation assay using a xenograft model.
RESULTS
JPX and XIST expression was significantly decreased in HCC pathologic specimens, compared to adjacent tissue, which correlated with HCC progression and increased miR-155-5p expression. Dual luciferase reporter assay revealed XIST as a direct target of miR-155-5p. XIST knock-in significantly reduced miR-155-5p expression level and increased that of SOX6 and PTEN, while significantly inhibiting HepG2 cell growth in vitro, which was partially reversed by miR-155-5p mimic transfection. JPX knock-in significantly increased XIST expression and inhibited HepG2 cell growth in vitro or tumor formation in vivo in a XIST dependent manner.
CONCLUSION
JPX and XIST play a suppressive role in HCC. JPX increases expression levels of XIST in HCC cells, which suppresses HCC development by sponging the cancer promoting miR-155-5p.

Keyword

JPX; XIST; lncRNA; hepatocellular carcinoma; miR-155-5p; X-chromosome inactivation

MeSH Terms

Blotting, Western
Carcinoma, Hepatocellular*
Hep G2 Cells
Heterografts
In Vitro Techniques
Luciferases
MicroRNAs
RNA, Long Noncoding
Transfection
Luciferases
MicroRNAs
RNA, Long Noncoding

Figure

• 

  Fig. 1 XIST and JPX expression levels inversely correlated with HCC development and miR-155-5p expression level. (A and B) Expression levels of XIST and JPX significantly decreased in 20 HCC tissue specimens compared to adjacent counterparts and further decreased in advanced staged HCC tissue specimens, compared to early staged ones, while increases in miR-155-5p expression are significantly correlated with HCC oncogenesis and progression. Data were normalized to the mean of adjacent tissue and are presented as fold changes. (C) Representative images of JPX and XIST hybridization histochemistry in situ (magnification, ×200). Long non-coding RNAs in the frozen section were hybridized with biotin-labeled riboprobes in a complementary base pairing manner (pale brown). Cell nuclei were counterstained with hematoxylin (cyan). (D and E) Correlation analysis showing that expression levels of JPX and XIST are inversely correlated with those of miR-155-5p in all 20 pairs of HCC and adjacent tissue. Relative expression in (A) and (B) was used for correlation analysis in (D) and (E). *p<0.05, †p<0.01, ‡p<0.001. XIST, X-inactive specific transcript; JPX, just proximal to XIST; HCC, hepatocellular carcinoma.

• 

  Fig. 2 XIST is a direct target of miR-155-5p. (A) Demonstration of XIST cDNA inserted into the 3′UTR of firefly luciferase gene with putative binding sites of miR-155-5p. (B) Dual luciferase reporter assay results suggesting that miR-155-5p directly targets XIST. (C and D) XIST expression was significantly increased by knocking in and decreased by miR-155-5p mimic transfection in wild type (knock-in control) and XIST knock-in HepG2 cells. (E and F) Western blot results showing that protein expression levels of previously identified miR-155-5p targeting genes PTEN and SOX6 were significantly decreased by miR-155-5p mimic transfection and partially rescued by XIST knock-in in HepG2 cells. Semi-quantification of protein analysis was performed by comparing the gray scale of PTEN or SOX6 protein bands to that of β-actin. (G) Representative results of Western blots evaluating PTEN and SOX6 expression in HepG2 cells with different miR-155-5p and XIST expression statuses. Data are presented as fold changes compared to the mean of a control group (presented as the first bar on the left of each graph). *p<0.05, †p< 0.01, ‡p<0.001. XIST, X-inactive specific transcript; JPX, just proximal to XIST; KI NC, knock-in control; XIST KI, XIST knock-in.

• 

  Fig. 3 XIST suppresses cell growth while inducing apoptosis in HepG2 cells by antagonizing the cancer promoting effect of miR-155-5p. Cells were treated with cisplatin at indicated concentrations for 24 h before analysis. (A) Cell proliferation assay showing that increase in cell proliferation rate induced by miR-155-5p mimic transfection can be repressed by XIST knock-in. (B–D) XIST knock-in significantly reduced cell viability while increasing apoptosis and cisplatin susceptibility of HepG2 cells with or without miR-155-5p mimic transfection. (C) is a representative result of flow cytometry counting for the percentage of Annexin V positive cells in each group. (E–G) miR-155-5p mimic transfection decreased, while XIST knock-in increased, apoptosis-related protein parameters in HepG2 cells with or without cisplatin treatment. (G) is a representative result of Bim, Bax, and Bcl-2 protein expression levels in different groups. Semi-quantitative analysis of Western blot results was performed as described in Fig. 2. Western blot data were presented as fold-changes compared to that of a control group. *p<0.05, †p< 0.01, ‡p<0.001, §p<0.0001. XIST, X-inactive specific transcript; JPX, just proximal to XIST; KI NC, knock-in control; XIST KI, XIST knock-in.

• 

  Fig. 4 JPX suppresses HepG2 cell growth while inducing apoptosis in HepG2 cells via XIST. (A and B) JPX expression levels were significantly increased by knocking in. XIST expression levels were significantly increased by JPX knock-in. Meanwhile, miR-155-5p mimic transfection did not alter JPX expression levels. (C and D) The anti-proliferation and apoptosis-inducing mechanism of JPX in HepG2 cells requires the integrity of XIST. (E–G) Results of Western blot analysis on apoptosis-related protein parameters correlated with cell apoptosis in graph (D). Semi-quantitative analysis of Western blot results was performed as described in Fig. 2. Western blot data are presented as fold-changes compared to that of controls. *p<0.05, †p< 0.01, ‡p<0.001. XIST, X-inactive specific transcript; JPX, just proximal to XIST; JPX KI, JPX knock-in; KI NC, knock-in control; sh-XIST, XIST knockdown.

• 

  Fig. 5 Tumor formation in nude mice reveals the HCC suppressive role of XIST and JPX. Twelve nude mice were randomly assigned to each group for cell injection on the left side of the back. (A) Tumor mass was weighed at 1, 2, 3, and 4 week(s) post-injection. (B) is a representative result of tumor formation at week 4. Statistical analysis was performed on tumor weight at week 4. *p<0.01. HCC, hepatocellular carcinoma; XIST, X-inactive specific transcript; JPX, just proximal to XIST.

Reference

1. Tay Y, Rinn J, Pandolfi PP. The multilayered complexity of ceRNA crosstalk and competition. Nature. 2014; 505:344–352.

2. Karreth FA, Pandolfi PP. ceRNA cross-talk in cancer: when ce-bling rivalries go awry. Cancer Discov. 2013; 3:1113–1121.

3. Yuan SX, Wang J, Yang F, Tao QF, Zhang J, Wang LL, et al. Long noncoding RNA DANCR increases stemness features of hepatocellular carcinoma by derepression of CTNNB1. Hepatology. 2016; 63:499–511.

4. Fu WM, Zhu X, Wang WM, Lu YF, Hu BG, Wang H, et al. Hotair mediates hepatocarcinogenesis through suppressing miRNA-218 expression and activating P14 and P16 signaling. J Hepatol. 2015; 63:886–895.

5. Cui M, Xiao Z, Wang Y, Zheng M, Song T, Cai X, et al. Long non-coding RNA HULC modulates abnormal lipid metabolism in hepatoma cells through an miR-9-mediated RXRA signaling pathway. Cancer Res. 2015; 75:846–857.

6. Ma X, Yuan T, Yang C, Wang Z, Zang Y, Wu L, et al. X-inactive-specific transcript of peripheral blood cells is regulated by exosomal Jpx and acts as a biomarker for female patients with hepatocellular carcinoma. Ther Adv Med Oncol. 2017; 9:665–677.

7. Ma W, Wang H, Jing W, Zhou F, Chang L, Hong Z, et al. Downregulation of long non-coding RNAs JPX and XIST is associated with the prognosis of hepatocellular carcinoma. Clin Res Hepatol Gastroenterol. 2017; 41:163–170.

8. Mo Y, Lu Y, Wang P, Huang S, He L, Li D, et al. Long non-coding RNA XIST promotes cell growth by regulating miR-139-5p/PDK1/AKT axis in hepatocellular carcinoma. Tumour Biol. 2017; 39:1010428317690999.

9. Li C, Wan L, Liu Z, Xu G, Wang S, Su Z, et al. Long non-coding RNA XIST promotes TGF-β-induced epithelial-mesenchymal transition by regulating miR-367/141-ZEB2 axis in non-small-cell lung cancer. Cancer Lett. 2018; 418:185–195.

10. Zhang Q, Chen B, Liu P, Yang J. XIST promotes gastric cancer (GC) progression through TGF-β1 via targeting miR-185. J Cell Biochem. 2018; 119:2787–2796.

11. Du Y, Weng XD, Wang L, Liu XH, Zhu HC, Guo J, et al. LncRNA XIST acts as a tumor suppressor in prostate cancer through sponging miR-23a to modulate RKIP expression. Oncotarget. 2017; 8:94358–94370.

12. Xiao C, Sharp JA, Kawahara M, Davalos AR, Difilippantonio MJ, Hu Y, et al. The XIST noncoding RNA functions independently of BRCA1 in X inactivation. Cell. 2007; 128:977–989.

13. Liu X, Cui L, Hua D. Long non-coding RNA XIST regulates miR-137-EZH2 axis to promote tumor metastasis in colorectal cancer. Oncol Res. 2018; 03. 01. [Epub]. DOI: 10.3727/096504018X15195193936573.

14. Huang YS, Chang CC, Lee SS, Jou YS, Shih HM. Xist reduction in breast cancer upregulates AKT phosphorylation via HDAC3-mediated repression of PHLPP1 expression. Oncotarget. 2016; 7:43256–43266.

15. Yan XL, Jia YL, Chen L, Zeng Q, Zhou JN, Fu CJ, et al. Hepatocellular carcinoma-associated mesenchymal stem cells promote hepatocarcinoma progression: role of the S100A4-miR155-SOCS1-MMP9 axis. Hepatology. 2013; 57:2274–2286.

16. Wang B, Majumder S, Nuovo G, Kutay H, Volinia S, Patel T, et al. Role of microRNA-155 at early stages of hepatocarcinogenesis induced by choline-deficient and amino acid-defined diet in C57BL/6 mice. Hepatology. 2009; 50:1152–1161.

17. Chen G, Wang D, Zhao X, Cao J, Zhao Y, Wang F, et al. miR-155-5p modulates malignant behaviors of hepatocellular carcinoma by directly targeting CTHRC1 and indirectly regulating GSK-3β-involved Wnt/β-catenin signaling. Cancer Cell Int. 2017; 17:118.

18. Fu X, Wen H, Jing L, Yang Y, Wang W, Liang X, et al. MicroRNA-155-5p promotes hepatocellular carcinoma progression by suppressing PTEN through the PI3K/Akt pathway. Cancer Sci. 2017; 108:620–631.

19. Wong CM, Tsang FH, Ng IO. Non-coding RNAs in hepatocellular carcinoma: molecular functions and pathological implications. Nat Rev Gastroenterol Hepatol. 2018; 15:137–151.

20. Pintacuda G, Young AN, Cerase A. Function by structure: spotlights on Xist long non-coding RNA. Front Mol Biosci. 2017; 4:90.

21. Robert Finestra T, Gribnau J. X chromosome inactivation: silencing, topology and reactivation. Curr Opin Cell Biol. 2017; 46:54–61.

22. Xing Y, Wen X, Ding X, Fan J, Chai P, Jia R, et al. CANT1 lncRNA triggers efficient therapeutic efficacy by correcting aberrant lncing cascade in malignant uveal melanoma. Mol Ther. 2017; 25:1209–1221.

23. Yildirim E, Kirby JE, Brown DE, Mercier FE, Sadreyev RI, Scadden DT, et al. Xist RNA is a potent suppressor of hematologic cancer in mice. Cell. 2013; 152:727–742.

24. Jiang H, Zhang H, Hu X, Li W. Knockdown of long non-coding RNA XIST inhibits cell viability and invasion by regulating miR-137/PXN axis in non-small cell lung cancer. Int J Biol Macromol. 2018; 111:623–631.

25. Zhang R, Xia T. Long non-coding RNA XIST regulates PDCD4 expression by interacting with miR-21-5p and inhibits osteosarcoma cell growth and metastasis. Int J Oncol. 2017; 51:1460–1470.

26. Lin C, Yang L. Long noncoding RNA in cancer: wiring signaling circuitry. Trends Cell Biol. 2018; 28:287–301.

27. Bhan A, Soleimani M, Mandal SS. Long noncoding RNA and cancer: a new paradigm. Cancer Res. 2017; 77:3965–3981.

28. Chang S, Chen B, Wang X, Wu K, Sun Y. Long non-coding RNA XIST regulates PTEN expression by sponging miR-181a and promotes hepatocellular carcinoma progression. BMC Cancer. 2017; 17:248.

29. Sun S, Del Rosario BC, Szanto A, Ogawa Y, Jeon Y, Lee JT. Jpx RNA activates Xist by evicting CTCF. Cell. 2013; 153:1537–1551.

30. Tian D, Sun S, Lee JT. The long noncoding RNA, Jpx, is a molecular switch for X chromosome inactivation. Cell. 2010; 143:390–403.

31. Romito A, Rougeulle C. Origin and evolution of the long non-coding genes in the X-inactivation center. Biochimie. 2011; 93:1935–1942.

32. Chow JC, Hall LL, Clemson CM, Lawrence JB, Brown CJ. Characterization of expression at the human XIST locus in somatic, embryonal carcinoma, and transgenic cell lines. Genomics. 2003; 82:309–322.

33. Paraskevopoulou MD, Vlachos IS, Karagkouni D, Georgakilas G, Kanellos I, Vergoulis T, et al. DIANA-LncBase v2: indexing microRNA targets on non-coding transcripts. Nucleic Acids Res. 2016; 44:D231–D238.

34. Wang M, Yang F, Qiu R, Zhu M, Zhang H, Xu W, et al. The role of mmu-miR-155-5p-NF-κB signaling in the education of bone marrow-derived mesenchymal stem cells by gastric cancer cells. Cancer Med. 2018; 7:856–868.

35. Zhang Y, Wei C, Guo CC, Bi RX, Xie J, Guan DH, et al. Prognostic value of microRNAs in hepatocellular carcinoma: a meta-analysis. Oncotarget. 2017; 8:107237–107257.

36. Zhang L, Wang W, Li X, He S, Yao J, Wang X, et al. MicroRNA-155 promotes tumor growth of human hepatocellular carcinoma by targeting ARID2. Int J Oncol. 2016; 48:2425–2434.

37. Xie Q, Chen X, Lu F, Zhang T, Hao M, Wang Y, et al. Aberrant expression of microRNA 155 may accelerate cell proliferation by targeting sex-determining region Y box 6 in hepatocellular carcinoma. Cancer. 2012; 118:2431–2442.

38. Liu F, Yuan JH, Huang JF, Yang F, Wang TT, Ma JZ, et al. Long non-coding RNA FTX inhibits hepatocellular carcinoma proliferation and metastasis by binding MCM2 and miR-374a. Oncogene. 2016; 35:5422–5434.

39. Zhuang LK, Yang YT, Ma X, Han B, Wang ZS, Zhao QY, et al. MicroRNA-92b promotes hepatocellular carcinoma progression by targeting Smad7 and is mediated by long non-coding RNA XIST. Cell Death Dis. 2016; 7:e2203.

40. Kristiansen M, Knudsen GP, Maguire P, Margolin S, Pedersen J, Lindblom A, et al. High incidence of skewed X chromosome inactivation in young patients with familial non-BRCA1/BRCA2 breast cancer. J Med Genet. 2005; 42:877–880.

41. Manoukian S, Verderio P, Tabano S, Colapietro P, Pizzamiglio S, Grati FR, et al. X chromosome inactivation pattern in BRCA gene mutation carriers. Eur J Cancer. 2013; 49:1136–1141.