Emerging role of Telomeric repeat-containing RNA TERRA in
hepatocellular carcinoma
M. Manganelli
1, I. Grossi
1, J. Corsi
2, K. Jurikovà
2, E. Cusanelli
2, V. D’Agostino
2, S. Molfino
3, G. Baiocchi
3, N.Portolani
3,
A. Salvi
1, G. De Petro
11Department of Molecular and Translational Medicine, Division of Biology and Genetics, University of Brescia, Brescia, Italy. m.manganelli@unibs.it 2Department of Cellular, Computational and Integrative Biology, CIBIO, University of Trento, Trento, Italy.
3Department of Clinical and Experimental Sciences, Surgical Clinic, University of Brescia, Brescia, Italy.
BACKGROUND
METHODS
- Hepatocellular carcinoma (HCC) is the most frequent primary
tumor of the liver and the 3rd cause of cancer-related deaths1.
- The identification of candidate molecular targets and biomarkers in HCC clinical practice are needed.
- The signatures of aberrant long non-coding RNAs (lncRNAs) expression in HCC tissues, their extracellular release and stability had led to their exploration as diagnostic and
prognostic tools as well as potential therapeutic targets2.
- Telomeric-Repeat Containing RNA (TERRA) consists of 100nt-9Kb subtelomeric-derived transcripts able to base-pair with
TERC RNA, acting as telomerase allosteric inhibitor3,4. Little is
known on the role of lncRNA TERRA in HCC.
CONCLUSION
REFERENCES
1Bray F. et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers
in 185 countries. CA Cancer J Clin. 2018;68(6):394-424.
2EASL Clinical Practice Guidelines: Management of hepatocellular carcinoma. J Hepatol. 2018;69(1):182-236.
3Azzalin C.M. et al. Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends.
Science. 2007;318(5851):798-801.
4Schoeftner S. et al. Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA
polymerase II. Nat Cell Biol. 2008;10(2):228-236.
5Notarangelo M. et al. Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse
extracellular vesicles from blood. EBioMedicine. 2019;43:114-126.
- By nickel-based isolation method (NBI) we isolated from the conditioned medium (CM) of HA22T/VGH cells the extracellular
vesicles (EVs), subsequently analyzed by qNANO instrument5.
- By qPCR we measured TERRA expression in tumor and peritumoral (PT) tissues of HCC patients (N=25), as well as in plasma and in HCC cells. HCC patients did not receive any treatment before surgical resection.
RESULTS
G L O B A L T E R R A R Q m e a n PT (N =2 5) HC C ( N= 25 ) 0 5 0 1 0 0 1 5 0 2 0 0 *Table 1. Diagnostic performance of TERRA in HCC tissues.
TARGET Sensitivity Specificity AUC SE 95% CI p-value
TERRA1 80% 64% 0,706 0,075 0,560 to 0,826 0.0064 ** TERRA2 84% 52% 0,698 0,074 0.551 to 0.819 0.008 ** TERRA3 84% 52% 0,712 0,072 0.567 to 0.831 0.0036 ** TERRA1+TERRA2 52% 80% 0,686 0,075 0.540 to 0.810 0.0166 * TERRA1+TERRA3 52% 80% 0,71 0,072 0.565 to 0.830 0.0233 * TERRA2+TERRA3 44% 80% 0,712 0,072 0.567 to 0.831 0.0149 * TERRA1+TERRA2+TERRA3 44% 80% 0,709 0,073 0.563 to 0.829 0.0382 *
Figure 3. Expression of TERRA in solid biopsies. A) TERRA relative quantification mean comparison between PT and
HCC. Wilcoxon paired two-tailed t-test. B) ROC curve analysis of TERRA. Sensitivity: percent of correctly classified HCC patients. Specificity: percent of correctly classified non-HCC. AUC: Area Under the ROC Curve. CI: Confidence Interval. *p<0.05, **p<0.01
- Global TERRA expression was significantly downregulated in HCC vs PT tissues (p=0.025) and ROC analysis revealed a significant ability to distinguish HCC from PT (p=0.03).
- Extracellular TERRA transcripts were significantly higher in plasma of HCC patients compared with healthy subjects and logistic regression model strongly evidenced the potential diagnostic ability of circulating TERRA (AUC=0.76; 95% CI= 0.624-0.873; p=0.0004). T E R R A 1 C t m e a n C ( N= 25 ) HC C ( N= 25 ) 0 1 0 2 0 3 0 4 0 ** T E R R A 2 C t m e a n C ( N= 20 ) HC C ( N= 21 ) 1 5 2 0 2 5 3 0 3 5 T E R R A 3 C t m e a n C ( N= 25 ) HC C ( N= 24 ) 1 5 2 0 2 5 3 0 3 5 *
Table 2. Diagnostic performance of circulating TERRA in plasma.
TARGET Sensitivity Specificity AUC SE 95% CI p-value
TERRA1 60% 100% 0.747 0.079 0.604 to 0.859 0,001 ** TERRA2 52% 92% 0.622 0.084 0.474 to 0.756 0,146 TERRA3 56% 88% 0.686 0.08 0.540 to 0.810 0,02 * TERRA1+TERRA2 64% 100% 0.742 0.081 0.599 to 0.856 0,002 ** TERRA1+TERRA3 64% 92% 0.763 0.074 0.622 to 0,872 0,0004 *** TERRA2+TERRA3 52% 96% 0.648 0.083 0.500 to 0.778 0,07 TERRA1+TERRA2+TERRA3 60% 100% 0.765 0.074 0.624 to 0.873 0,0004 ***
Figure 4. Plasma levels of TERRA in healthy subject (C) and HCC patients. A), B), C) TERRA levels in terms of Ct in
plasma of healthy (C) and HCC individuals. Parametric unpaired two-tailed t-test. D) ROC curve analysis of TERRA to discriminate HCC from healthy individuals. Sensitivity: percent of correctly classified HCC patients. Specificity: percent of correctly classified non-HCC. AUC: Area Under the ROC Curve. CI: Confidence Interval. *p<0.05, **p<0.01, ***p<0.001. IN T R A C E L L U L A R G L O B A L E S P R E S S IO N O F T E R R A R Q m e a n HA 22 T/V GH + O .1% DM SO HA 22 T/V GH + 1 5M so rafe nib 0 2 4 6 8 1 0 ** E X T R A C E L L U L A R G L O B A L L E V E L S O F T E R R A IN E V s R Q m e a n BU FF ER HA 22 T/V GH + O .1% D MS O HA 22 T/V GH + 1 5M so rafe nib 0 1 2 3 4 5 5 0 1 0 0 1 5 0 2 0 0 *
- HA22T/VGH cells expressed TERRA, but most of the transcripts are released into the CM, also encapsulated in EVs. Treatment of HCC cells with the multi-kinase inhibitor (KI) sorafenib significantly increased TERRA expression (p=0.001) and decreased (p=0.01) its release in EVs.
Figure 6. Intracellular and extracellular expression of TERRA. The expression levels of TERRA were
measured in HA22T/VGH untreated and treated with 15µM sorafenib and the corresponding EVs in their conditioned medium (CM). Gene expression level in EVs was normalized to the number of particles released. Histograms represent the mean values of 2 experiments, bars are ± SEM. Unpaired two tailed t-test; *p<0.05, **p<0.01.
- Our results provide evidence on TERRA dysregulation in tissues and liquid biopsy of HCC patients, focusing on a novel potential non-invasive biomarker of diagnosis and downstream target of the KI.
- TERRA detected in the EVs of HCC cells open a new field of cancer research to comprehend its role at the extracellular level.
Table 3. Q-NANO analysis of HA22T/VGH secretome
Sample Label Mean Dia (nm) Mode Dia (nm) Raw Conc (ml) Particle Count
HA22T/VGH O.1% DMSO 185 132 1.63 * 10^8 67
HA22T/VGH + 15µM SOR 206 171 2.09 * 10^8 88
Figure 5. Size of EVs particles. The graph shows the
distribution of EVs as a function of their dimension. Dia: diameter; Conc: concentration.
E V s D I S T R I B U T I O N I N T H E S E C R E T O M E O F H A 2 2 T / V G H P a r t i c l e D i a m e t e r ( n m ) C o n c e n tr a ti o n ( p a rt ic le s /m l) 50 100 150 200 250 300 350 0 2 . 01 07 4 . 01 07 6 . 01 07 8 . 01 07 1 . 01 08 H A 2 2 T / V G H + 0 . 1 % D M S O : 1 . 6 3 x 1 08 H A 2 2 T / V G H + 1 5M s o r a f e n ib : 2 . 0 8 x 1 08 Figure 1 Figure 2 A B A B C D A B
10P
UNTREATED HCC CELL LINE
HCC CELL LINE TREATED WITH 15µM SORAFENIB
RNA EXTRACTION AND qPCR ANALYSIS FOR THE TARGET GENE
RNA EXTRACTION AND qPCR ANALYSIS FOR THE TARGET GENE
ADDITION OF FUNCTIONALIZED BEADS CONDITIONED MEDIUM (CM) CONDITIONED MEDIUM (CM) ADDITION OF FUNCTIONALIZED BEADS EVs ISOLATION EVs ISOLATION EXTRACELLULAR VESCICLES (EVs) EXTRACELLULAR VESCICLES (EVs)
LIVER CANCER PERITUMORAL TISSUE (PT)
PLASMA
qPCR ANALYSIS FOR THE TARGET GENE
qPCR ANALYSIS FOR THE TARGET GENE
RNA EXTRACTION WITH TRIZOL RNA EXTRACTION WITH COLUMNS HCC PATIENT RNA RNA
