Identification of genes involved in the regulation of TERT in hepatocellular carcinoma - PubMed (original) (raw)

. 2019 Feb;110(2):550-560.

doi: 10.1111/cas.13884. Epub 2019 Jan 4.

Affiliations

Identification of genes involved in the regulation of TERT in hepatocellular carcinoma

Masataka Amisaki et al. Cancer Sci. 2019 Feb.

Abstract

Telomerase reverse transcriptase (TERT) promotes immortalization by protecting telomeres in cancer cells. Mutation of the TERT promoter is one of the most common genetic alterations in hepatocellular carcinoma (HCC), indicating that TERT upregulation is a critical event in hepatocarcinogenesis. Regulators of TERT transcription are, therefore, predicted to be plausible targets for HCC treatment. We undertook a genome-wide shRNA library screen and identified C15orf55 and C7orf43 as regulators of TERT expression in HepG2 cells. Promoter assays showed that C15orf55- and C7orf43-responsive sites exist between base pairs -58 and +36 and -169 and -59 in the TERT promoter, respectively. C15orf55 upregulates TERT expression by binding to two GC motifs in the SP1 binding site of the TERT promoter. C7orf43 upregulates TERT expression through Yes-associated protein 1. The expression levels of C15orf55 and C7orf43 also correlated with that of TERT, and were significantly increased in both HCC tissues and their adjacent non-tumor tissues, compared to normal liver tissues from non-HCC patients. Analysis of 377 HCC patients in The Cancer Genome Atlas dataset showed that overall survival of patients with low levels of C15orf55 and C7orf43 expression in tumor tissues was better compared with patients with high levels of C15orf55 and/or high C7orf43 expression. These results indicate that C15orf55 and C7orf43 are involved in the incidence and progression of HCC by upregulating TERT. In conclusion, we identified C15orf55 and C7orf43 as positive regulators of TERT expression in HCC tissues. These genes are promising targets for HCC treatment.

Keywords: C15orf55; C7orf43; hepatocellular carcinoma; telomerase reverse transcriptase; transcription.

© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Lentiviral sh

RNA

screening identified C15orf55 and C7orf43 as activators of TERT expression. A, Outline of the sh

RNA

screening strategy. B, Efficiency of sh

RNA

gene knockdown (

KD

). All genes except SLC22A16 were significantly knocked down by sh

RNA

s. C, Downregulation of TERT

mRNA

levels by sh

RNA KD

. Two sh

RNA

s, to C15orf55 and C7orf43, produced significant suppression of TERT expression. D, Upregulation of TERT

mRNA

levels by transient overexpression of C15orf55 and C7orf43. Results expressed as the mean ±

SD

, n = 3. *P < .05

Figure 2

Figure 2

HepG2 cells with stable C15orf55 (

OE

‐C15orf55) or C7orf43 (

OE

‐C7orf43) expression have activated TERT and telomerase expression. A, Western blot analysis for

OE

‐C15orf55 and

OE

‐C7orf43. B, Quantitative telomerase repeat amplification protocol assay. Results are expressed as the mean ±

SD

, n = 3. *P < .05

Figure 3

Figure 3

Overexpression of C15orf55 or C7orf43 promotes cell proliferation, whereas knockdown (

KD

) of C15orf55 or C7orf43 inhibits colony formation ability. A, Cell proliferation plots for cells stably expressing each gene. n = 4. B, Colony formation assay using sh

RNA

lentivirus. Number of colonies 20 days after induction of sh

RNA

s using lentivirus; n = 3. C, Typical colonies in the colony formation assay. Scale bar = 10 mm. Results are expressed as the mean ±

SD

. *P < .05

Figure 4

Figure 4

C15orf55 and C7orf43 interact with different sites in the TERT promoter. A,B, Luciferase reporter assays to determine the response sites of each gene. Expression vectors were cotransfected into HepG2 cells with a luciferase reporter plasmid containing the designated length of TERT promoter. Data are normalized with the control in each assay. C, Luciferase reporter assay to show a synergic effect of C15orf55 and C7orf43. Half the amount of each expression vector used in the reporter assays shown in A and B were cotransfected. Average values are shown above each bar charts. Results are expressed as the mean ±

SD

; n = 3. *P < .05. RLU, relative light unit

Figure 5

Figure 5

C15orf55 and C7orf43 activate the TERT promoter through

SP

1 and Yes‐associated protein 1 (

YAP

1), respectively. A, Reporter assay using luciferase driven by

GC

motifs of the TERT promoter region. B, Recruitment of

SP

1 to the

GC

motifs in the TERT promoter. Chromatin immunoprecipitation was carried out using an anti‐

SP

1 antibody. Quantitative

PCR

was carried out with primers for two

GC

motifs in the TERT promoter. Promoter enrichment is shown relative to input. C, Reporter assay to detect C7orf43 response site. Fragments #3 and #6 are shown to have C7orf43 responsiveness. D, Western blot analysis for

YAP

1 and

YAP

1‐related protein. E, Intensity of nuclear

YAP

1 by immunofluorescence analysis of

OE

‐Empty and

OE

‐7orf43 cells. F, Schematic diagram depicting TERT activation by C15orf55 and C7orf43. Results are expressed as the mean ±

SD

; n = 3. *P < .05

Figure 6

Figure 6

C15orf55 and C7orf43 are upregulated in both tumor and nontumor tissues from hepatocellular carcinoma (

HCC

) patients. A, B, Comparison between normal liver from benign liver disease patients and tumor‐adjacent tissue (nontumor) or tumor from

HCC

patients. C,D, Relationship between gene expression and Child‐Pugh score, a clinical surrogate marker for the degree of cirrhosis and hepatitis. Relative gene expression levels of C15orf55 and C7orf43 to ACTB were normalized against that of normal liver

Figure 7

Figure 7

Expression of C15orf55 and C7orf43 correlates with TERT expression and patient survival. A‐D, Gene expression and TERT expression in nontumor (A,C) and tumor (B,D) in patients with hepatocellular carcinoma. E,F, Survival curves of hepatocellular carcinoma patients from The Cancer Genome Atlas database. Overall survival rates (A), and recurrence‐free survival rates (B) are shown for patients with low C15orf55 and C7orf43 expression (C15low and C7low), and high C15orf55 and/or high C7orf43 expression (C15high and/or C7high). Relative gene expression levels of TERT to ACTB were normalized against that in the tumor

References

    1. Donati B, Valenti L. Telomeres, NAFLD and chronic liver disease. Int J Mol Sci. 2016;17:383. - PMC - PubMed
    1. Griffith JDCL, Rosenfield S, Stansel RM, Bianchi A, Moss H, de Lange T. Mammalian telomeres end in a large duplex loop. Cell. 1999;97:503‐514. - PubMed
    1. Deng Y, Chan SS, Chang S. Telomere dysfunction and tumour suppression: the senescence connection. Nat Rev Cancer. 2008;8:450‐458. - PMC - PubMed
    1. Calado RT, Young NS. Telomere diseases. N Engl J Med. 2009;361:2353‐2365. - PMC - PubMed
    1. Shay JW, Wright WE. The reactivation of telomerase activity in cancer progression. Trends Genet. 1996;12:129‐131. - PubMed

MeSH terms

Substances

LinkOut - more resources