MicroRNA‑9‑5p downregulates Klf4 and influences the progression of hepatocellular carcinoma via the AKT signaling pathway

Dong,Xiao; Wang,Fan; Xue,Ying; Lin,Zhipeng; Song,Weifeng; Yang,Ning; Li,Qi

doi:10.3892/ijmm.2019.4062

MicroRNA‑9‑5p downregulates Klf4 and influences the progression of hepatocellular carcinoma via the AKT signaling pathway

Authors:
- Xiao Dong
- Fan Wang
- Ying Xue
- Zhipeng Lin
- Weifeng Song
- Ning Yang
- Qi Li
View Affiliations

Affiliations: Department of Oncology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, P.R. China, The 5th Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai 200082, P.R. China
Published online on: January 14, 2019 https://doi.org/10.3892/ijmm.2019.4062
Pages: 1417-1429
Copyright: © Dong et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Krüppel‑like factor 4 (Klf4) is a transcriptional factor involved in the progression of hepatocellular carcinoma (HCC). However, the underlying regulatory mechanisms associated with the Klf4 gene as a tumor suppressor in HCC remain unclear. microRNAs (miRNAs or miRs) are a series of small non‑coding RNAs that serve a vital role in regulating gene expression via their influence on protein translation and the associated degradation of mRNA. The mRNA expression levels of the miRNA, miR‑9‑5p, and Klf4 were measured using reverse transcription‑quantitative polymerase chain reaction. The protein expression levels of Klf4, protein kinase B (AKT), phosphorylated (p‑)AKT, mechanistic target of rapamycin (mTOR), p‑mTOR, B cell lymphoma‑2 (Bcl‑2) and Bcl‑2‑associated X protein (Bax) were determined by western blot analysis. Dual luciferase reporter assay was used to confirm a direct interaction between miR‑9‑5p and the 3'‑untranslated region (3'‑UTR) sequence of Klf4. Cell counting kit‑8 assay, wound healing assay, Transwell migration assay and flow cytometric analysis were performed to evaluate the proliferative, migratory and apoptotic capabilities of the HCC cells. In the present study, miR‑9‑5p was revealed to be overexpressed in HCC as a novel upstream gene of Klf4. miR‑9‑5p expression was inversely associated with Klf4 expression in clinical samples. Additionally, Kaplan‑Meier analysis revealed a markedly poor prognosis of HCC in the miR‑9‑5p high‑expression group. Bioinformatics analysis revealed that miR‑9‑5p bound directly to the 3'‑UTR of Klf4, which reduced the expression levels of Klf4. The miR‑9‑5p/Klf4 axis promoted HCC proliferation and migration, and inhibited HCC apoptosis. Furthermore, miR‑9‑5p upregulated the Bcl‑2/Bax protein ratio and activated AKT/mTOR signaling. Taken together, these data demonstrated that the miR‑9‑5p/Klf4 axis was able to promote HCC progression, which may occur via regulation of the AKT signaling pathway, highlighting a potential novel target in HCC treatment.

View Figures

View References

1	Siegel RL, Miller KD and Jemal A: Cancer statistics, 2018. CA Cancer J Clin. 68:7–30. 2018. View Article : Google Scholar : PubMed/NCBI
2	Guo H, Wu T, Lu Q, Li M, Guo JY, Shen Y, Wu Z, Nan KJ, Lv Y and Zhang XF: Surgical resection improves long-term survival of patients with hepatocellular carcinoma across different Barcelona clinic liver cancer stages. Cancer Manag Res. 10:361–369. 2018. View Article : Google Scholar : PubMed/NCBI
3	Li T, Xie J, Shen C, Cheng D, Shi Y, Wu Z, Deng X, Chen H, Shen B, Peng C, et al: Upregulation of long noncoding RNA ZEB1-AS1 promotes tumor metastasis and predicts poor prognosis in hepatocellular carcinoma. Oncogene. 35:1575–1584. 2016. View Article : Google Scholar
4	Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, et al: Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 359:378–390. 2008. View Article : Google Scholar : PubMed/NCBI
5	Kudo M: Systemic therapy for hepatocellular carcinoma: Latest advances. Cancers (Basel). 10:pii: E412. 2018. View Article : Google Scholar :
6	Augello G, Emma MR, Cusimano A, Azzolina A, Mongiovi S, Puleio R, Cassata G, Gulino A, Belmonte B, Gramignoli R, et al: Targeting HSP90 with the small molecule inhibitor AUY922 (luminespib) as a treatment strategy against hepatocellular carcinoma. Int J Cancer. Nov 29–2018.Epub ahead of print. View Article : Google Scholar : PubMed/NCBI
7	Harris WP, Wong KM, Saha S, Dika IE and Abou-Alfa GK: Biomarker-driven and molecular targeted therapies for hepato-biliary cancers. Semin Oncol. 45:116–123. 2018. View Article : Google Scholar : PubMed/NCBI
8	Zhang Q, Wei L, Yang H, Yang W, Yang Q, Zhang Z, Wu K and Wu J and Wu J: Bromodomain containing protein represses the Ras/Raf/MEK/ERK pathway to attenuate human hepatoma cell proliferation during HCV infection. Cancer Lett. 371:107–116. 2016. View Article : Google Scholar
9	Chai S, Ng KY, Tong M, Lau EY, Lee TK, Chan KW, Yuan YF, Cheung TT, Cheung ST, Wang XQ, et al: Octamer 4/microRNA-1246 signaling axis drives Wnt/β-catenin activation in liver cancer stem cells. Hepatology. 64:2062–2076. 2016. View Article : Google Scholar : PubMed/NCBI
10	Ou DL, Lee BS, Lin LI, Liou JY, Liao SC, Hsu C and Cheng AL: Vertical blockade of the IGFR- PI3K/Akt/mTOR pathway for the treatment of hepatocellular carcinoma: The role of survivin. Mol Cancer. 13:22014. View Article : Google Scholar : PubMed/NCBI
11	Vivanco I and CL S: The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer. 7:489–501. 2002. View Article : Google Scholar
12	Presnell JS, Schnitzler CE and Browne WE: KLF/SP Transcription factor family evolution: Expansion, diversification, and innovation in eukaryotes. Genome Biol Evol. 7:2289–2309. 2015. View Article : Google Scholar : PubMed/NCBI
13	Hu JH, Navas P, Cao H, Stamatoyannopoulos G and Song CZ: Systematic RNAi studies on the role of Sp/KLF factors in globin gene expression and erythroid differentiation. J Mol Biol. 366:1064–1073. 2007. View Article : Google Scholar : PubMed/NCBI
14	Carrano AC, Dillin A and Hunter T: A Krüppel-like factor downstream of the E3 ligase WWP-1 mediates dietary-restriction-induced longevity in Caenorhabditis elegans. Nat Commun. 5:37722014. View Article : Google Scholar
15	Kim CK, He P, Bialkowska AB and Yang VW: SP and KLF transcription factors in digestive physiology and diseases. Gastroenterology. 152:1845–1875. 2017. View Article : Google Scholar : PubMed/NCBI
16	Chen HY, Lin YM, Chung HC, Lang YD, Lin CJ, Huang J, Wang WC, Lin FM, Chen Z, Huang HD, et al: miR-103/107 promote metastasis of colorectal cancer by targeting the metastasis suppressors DAPK and KLF4. Cancer Res. 72:3631–3641. 2012. View Article : Google Scholar : PubMed/NCBI
17	Malak PN, Dannenmann B, Hirth A, Rothfuss OC and Schulze-Osthoff K: Novel AKT phosphorylation sites identified in the pluripotency factors OCT4, SOX2 and KLF4. Cell Cycle. 14:3748–3754. 2015. View Article : Google Scholar : PubMed/NCBI
18	Hsu YY, Liu CM, Tsai HH, Jong YJ, Chen IJ and Lo YC: KMUP-1 attenuates serum deprivation-induced neurotoxicity in SH-SY5Y cells: Roles of PKG, PI3K/Akt and Bcl-2/Bax pathways. Toxicology. 268:46–54. 2010. View Article : Google Scholar
19	Muir KR, Lima MJ, Docherty HM, McGowan NW, Forbes S, Heremans Y, Forbes SJ, Heimberg H, Casey J and Docherty K: Krüppel-Like factor 4 overexpression initiates a mesenchymal-to-epithelial transition and redifferentiation of human pancreatic cells following expansion in long term adherent culture. PLoS One. 10:e01403522015. View Article : Google Scholar
20	Sun H, Peng Z, Tang H, Xie D, Jia Z, Zhong L, Zhao S, Ma Z, Gao Y, Zeng L, et al: Loss of KLF4 and consequential downregulation of Smad7 exacerbate oncogenic TGF-β signaling in and promote progression of hepatocellular carcinoma. Oncogene. 36:2957–2968. 2017. View Article : Google Scholar : PubMed/NCBI
21	Chang YL, Zhou PJ, Wei L, Li W, Ji Z, Fang YX and Gao WQ: MicroRNA-7 inhibits the stemness of prostate cancer stem-like cells and tumorigenesis by repressing KLF4/PI3K/Akt/p21 pathway. Oncotarget. 6:24017–24031. 2015. View Article : Google Scholar : PubMed/NCBI
22	Li Z, Zhao J, Li Q, Yang W, Song Q, Li W and Liu J: KLF4 promotes hydrogen-peroxide-induced apoptosis of chronic myeloid leukemia cells involving the bcl-2/bax pathway. Cell Stress Chaperones. 15:905–912. 2010. View Article : Google Scholar : PubMed/NCBI
23	Li Q, Gao Y, Jia Z, Mishra L, Guo K, Li Z, Le X, Wei D, Huang S and Xie K: Dysregulated Krüppel-like factor 4 and vitamin D receptor signaling contribute to progression of hepatocellular carcinoma. Gastroenterology. 143:799–810.e2. 2012. View Article : Google Scholar
24	Bartel DP: MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 116:281–297. 2004. View Article : Google Scholar : PubMed/NCBI
25	Wang LQ, Zhang Y, Yan H, Liu KJ and Zhang S: MicroRNA-373 functions as an oncogene and targets YOD1 gene in cervical cancer. Biochem Biophys Res Commun. 459:515–520. 2015. View Article : Google Scholar : PubMed/NCBI
26	Yuan J, Ji H, Xiao F, Lin Z, Zhao X, Wang Z, Zhao J and Lu J: MicroRNA-340 inhibits the proliferation and invasion of hepa-tocellular carcinoma cells by targeting JAK1. Biochem Biophys Res Commun. 483:578–584. 2017. View Article : Google Scholar
27	Yao S, Tian C, Ding Y, Ye Q, Gao Y, Yang N and Li Q: Down-regulation of Krüppel-like factor-4 by microRNA-135a-5p promotes proliferation and metastasis in hepatocellular carcinoma by transforming growth factor-β1. Oncotarget. 7:42566–42578. 2016. View Article : Google Scholar : PubMed/NCBI
28	Mirihana Arachchilage G, Kharel P, Reid J and Basu S: Targeting of G-quadruplex harboring Pre-miRNA 92b by LNA rescues PTEN expression in NSCL cancer cells. ACS Chem Biol. 13:909–914. 2018. View Article : Google Scholar : PubMed/NCBI
29	Varnholt H, Drebber U, Schulze F, Wedemeyer I, Schirmacher P, Dienes HP and Odenthal M: MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma. Hepatology. 47:1223–1232. 2008. View Article : Google Scholar : PubMed/NCBI
30	Amin MB, Greene FL, Edge SB, Compton CC, Gershenwald JE, Brookland RK, Meyer L, Gress DM, Byrd DR and Winchester DP: The eighth edition AJCC cancer staging manual: Continuing to build a bridge from a population-based to a more 'personalized' approach to cancer staging. CA Cancer J Clin. 67:93–99. 2017. View Article : Google Scholar : PubMed/NCBI
31	Kamarajah SK, Frankel TL, Sonnenday C, Cho CS and Nathan H: Critical evaluation of the American joint commission on cancer (AJCC) 8th edition staging system for patients with hepatocellular carcinoma (HCC): A surveillance, epidemiology, end results (SEER) analysis. J Surg Oncol. 117:644–650. 2018. View Article : Google Scholar
32	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar
33	Zawada AM, Rogacev KS, Muller S, Rotter B, Winter P, Fliser D and Heine GH: Massive analysis of cDNA Ends (MACE) and miRNA expression profiling identifies proatherogenic pathways in chronic kidney disease. Epigenetics. 9:161–172. 2014. View Article : Google Scholar :
34	Wang GC, He QY, Tong DK, Wang CF, Liu K, Ding C, Ji F and Zhang H: MiR-367 negatively regulates apoptosis induced by adriamycin in osteosarcoma cells by targeting KLF4. J Bone Oncol. 5:51–56. 2016. View Article : Google Scholar : PubMed/NCBI
35	Guo Y, An R, Zhao R, Sun Y, Liu M and Tian L: miR-375 exhibits a more effective tumor-suppressor function in laryngeal squamous carcinoma cells by regulating KLF4 expression compared with simple co-transfection of miR-375 and miR-206. Oncol Rep. 36:952–960. 2016. View Article : Google Scholar : PubMed/NCBI
36	Braconi C and Patel T: Non-coding RNAs as therapeutic targets in hepatocellular cancer. Curr Cancer Drug Targets. 12:1073–1080. 2012.PubMed/NCBI
37	Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN, Harborth J, Heyes JA, Jeffs LB, John M, et al: RNAi-mediated gene silencing in non-human primates. Nature. 441:111–114. 2006. View Article : Google Scholar : PubMed/NCBI
38	Klingenberg M, Matsuda A, Diederichs S and Patel T: Non-coding RNA in hepatocellular carcinoma: Mechanisms, biomarkers and therapeutic targets. J Hepatol. 67:603–618. 2017. View Article : Google Scholar : PubMed/NCBI
39	Hayes CN and Chayama K: MicroRNAs as biomarkers for liver disease and hepatocellular carcinoma. Int J Mol Sci. 17:2802016. View Article : Google Scholar : PubMed/NCBI
40	He S, Zhang DC and Wei C: MicroRNAs as biomarkers for hepa-tocellular carcinoma diagnosis and prognosis. Clin Res Hepatol Gastroenterol. 39:426–434. 2015. View Article : Google Scholar : PubMed/NCBI
41	Yang N, Ekanem NR, Sakyi CA and Ray SD: Hepatocellular carcinoma and microRNA: New perspectives on therapeutics and diagnostics. Adv Drug Deliv Rev. 81:62–74. 2015. View Article : Google Scholar
42	Fang F, Chang RM, Yu L, Lei X, Xiao S, Yang H and Yang LY: MicroRNA-188-5p suppresses tumor cell proliferation and metastasis by directly targeting FGF5 in hepatocellular carcinoma. J Hepatol. 63:874–885. 2015. View Article : Google Scholar : PubMed/NCBI
43	Ma S, Tang KH, Chan YP, Lee TK, Kwan PS, Castilho A, Ng I, Man K, Wong N, To KF, et al: miR-130b promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1. Cell Stem Cell. 7:694–707. 2010. View Article : Google Scholar : PubMed/NCBI
44	Missiaglia E, Shepherd CJ, Aladowicz E, Olmos D, Selfe J, Pierron G, Delattre O, Walters Z and Shipley J: MicroRNA and gene co-expression networks characterize biological and clinical behavior of rhabdomyosarcomas. Cancer Lett. 385:251–260. 2017. View Article : Google Scholar :
45	Guo LM, Pu Y, Han Z, Liu T, Li TX, Liu M, Li X and Tang H: MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-kappaB1. FEBS J. 276:5537–5546. 2009. View Article : Google Scholar : PubMed/NCBI
46	Han D, Li J, Wang H, Su X, Hou J, Gu Y, Qian C, Lin Y, Liu X, Huang M, et al: Circular RNA circMTO1 acts as the sponge of microRNA-9 to suppress hepatocellular carcinoma progression. Hepatology. 66:1151–1164. 2017. View Article : Google Scholar : PubMed/NCBI
47	Tang W, Zhu Y, Gao J, Fu J, Liu C, Liu Y, Song C, Zhu S, Leng Y, Wang G, et al: MicroRNA-29a promotes colorectal cancer metastasis by regulating matrix metalloproteinase 2 and E-cadherin via KLF4. Br J Cancer. 110:450–458. 2014. View Article : Google Scholar :
48	Calvisi DF, Wang C, Ho C, Ladu S, Lee SA, Mattu S, Destefanis G, Delogu S, Zimmermann A, Ericsson J, et al: Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma. Gastroenterology. 140:1071–1083. 2011. View Article : Google Scholar :
49	Fang Y, Xue JL, Shen Q, Chen J and Tian L: MicroRNA-7 inhibits tumor growth and metastasis by targeting the phos-phoinositide 3-kinase/Akt pathway in hepatocellular carcinoma. Hepatology. 55:1852–1862. 2012. View Article : Google Scholar : PubMed/NCBI
50	Hata AN, Engelman JA and Faber AC: The BCL2 family: Key mediators of the apoptotic response to targeted anticancer therapeutics. Cancer Discov. 5:475–487. 2015. View Article : Google Scholar : PubMed/NCBI