Circulating ncRNAs as promising non-invasive molecular biomarkers of HCC

Circulating ncRNAs as promising non-invasive molecular

biomarkers of HCC

Ilaria Grossi

1

, Michele Manganelli

1

, Teresa Faranda

1

, Paola Guerriero

2

, Massimo Negrini

2

,

Gianluca Baiocchi

3

, Nazario

Portolani

3

, Michele Ghidini

4

, Chiara Senti

4

, Rodolfo Passalacqua

4

, Giuseppina De Petro

1

, Alessandro Salvi

1

1

_{Department of Molecular and Translational Medicine, Division of Biology and Genetics, University of Brescia, Brescia, Italy; alessandro.salvi@unibs.it}

2

_{Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy.}

3

_{Department of Clinical and Experimental Sciences, Surgical Clinic, University of Brescia, Brescia, Italy.}

4

_{Department of Oncology, Azienda Socio Sanitaria Territoriale of Cremona, Cremona, Italy.}

INTRODUCTION.

Human hepatocellular carcinoma (HCC) is the most frequent primary tumor of the liver and it is the third cause of cancer-related deaths. The prognosis of HCC is poor

and thus the identification of novel molecular biomarkers for the early diagnosis in at-high risk patients is needed. Circulating ncRNAs (including lncRNAs and miRNAs more and less than

200 nt long respectively) have been detected in different human body fluids, including serum, plasma and urine. In order to identify promising circulating diagnostic molecular biomarkers, we

measured the levels of selected ncRNAs in plasma from HCC patients. In particular, we evaluated the levels of plasma circulating miR-23b-3p and miR-126-3p that we previously found

significantly down-regulated in primary HCCs and the levels of plasma circulating lncRNA GAS5 since it resulted up-regulated following sorafenib treatment of a panel of cancer cells (HCC,

breast and renal cancers).

miR-23b-3p levels are down-regulated in

plasma of HCC patients

CONCLUSIONS. The levels of plasmatic circulating miR-23b-3p and miR-126-3p were significantly lower and higher respectively in HCC patients respect to healthy subjects, while the levels of lncRNA GAS5 were significantly lower in HCC patients respect to healthy subjects. Sorafenib treatment promoted variations in plasmatic levels of miR-23b-3p and miR-126-3p during the treatment of patients with advanced HCC.

Our results contribute to identify potential novel non-invasive biomarkers of diagnosis and response to therapy in liquid biopsies of HCC patients. The levels of plasma circulating miR-23b-3p measured by

digital-droplet PCR (ddPCR) were significantly lower in HCC patients (n=25) respect to healthy subjects (n=37)

The ROC analysis displayed a discrete capability of miR-23b-3p to discriminate HCC from control individuals (AUC=0.67; P-value=0.019).

The levels of circulating miR-126-3p were significantly higher in HCC patients compared to controls (upper graph). The plotting was made on the average raw cycle thresholds

(C_t) since there are no established endogenous miRNAs

acting as normalizers for plasma miRs. The average Ct values of the spike-in cel-miR-39 (lower graph) were the same in the 2 groups demonstrating a good and a constant performance of RT and qPCR reactions.

The ROC curve analysis supported the diagnostic potential of plasmatic miR-126-3p in distinguishing HCC patients from control subjects (AUC= 0.78; P-value= 0.0007).

The ROC curve analysis evidenced the diagnostic potential of GAS5 in distinguishing HCC patients from healthy subjects (AUC= 0.72; P-value= 0.007; right).

Flowcharts of patient history and their treatment. The objective was to measure the expression variation of miR-23b-3p (by ddPCR) and miR-126-3p (by qPCR) in liquid biopsies (plasma) of HCC patients during the course of treatment with Sorafenib, an oral multikinase inhibitor used to treat the unresectable HCC. Currently, six patients with advanced HCC were analyzed.

In the majority of patients, the expression of miR-126-3p is superimposable to the expression of miR-23b-3p. These preliminary data would suggest these two miRNAs as possible candidates as circulating biomarkers for the prediction of resistance to Sorafenib therapy to be monitored in liquid biopsy during the therapeutic treatment.

miR-126-3p levels are up-regulated in

plasma of HCC patients

Variation of miR-23b-3p and miR-126-3p expression

evaluated by ddPCR and qPCR in plasma of HCC patients

during the treatment with sorafenib

lncRNA GAS5 levels are down-regulated in plasma

of HCC patients

In order to assess whether GAS5 can be considered a circulating diagnostic molecular biomarker for HCC, we determined its levels in the plasma by qPCR. The expression levels of GAS5 were significantly lower in HCC patients compared to controls. MEAN=27.67 MEAN=26.60 MEAN=25.61 MEAN=25.49 R O C c u r v e : R O C o f m iR - 2 3 b - 3 p 1 0 0 % - S p e c if ic i t y % S e n s it iv it y % 0 5 0 1 0 0 1 5 0 0 5 0 1 0 0 1 5 0 S e n s itiv ity % Id e n t ity % AUC = 0.68 95%CI = 0.54 – 0.82 P value = 0.019