Correlation between plasma miR-122 expression and liver injury induced by hepatectomy

Introduction

Hepatocellular carcinoma (HCC) is the most common malignant tumour of the digestive system; ¹ hepatectomy is one of the most effective therapies for HCC. ² To reduce bleeding during hepatectomy, portal triad clamping techniques are usually adopted but can inevitably cause liver injury. ³ It is therefore necessary to find a serological marker that helps evaluate the extent of any liver injury induced by hepatectomy.

MicroRNAs (miRNAs) are a class of post-transcriptional regulators comprised of short (∼22 nucleotide) RNA sequences that bind to complementary sequences in the 3′ UTR of multiple target mRNAs, usually resulting in their silencing. ⁴ MiRNAs have been used as a new class of biomarkers,^5,6 and the fast-growing number of known miRNAs has allowed researchers to link certain groups of miRNAs to specific cellular activities. The evaluation of biomarker sets results in more specific and meaningful information than the identification of a single miRNA as a biomarker, thereby enabling researchers to diagnose diseases, monitor their course and tailor therapies accordingly.^7,8 MiRNA 122 (miR-122) was one of the first examples of a tissue-specific miRNA; it is highly expressed in liver, where it constitutes 70% of the total miRNA pool. ⁹ A plethora of studies using in vivo gene silencing, in vitro experimentation and transcriptome profiling demonstrate how miR-122 regulates the networks of genes that control lipid metabolism, ¹⁰ cell differentiation, ¹¹ hepatic circadian regulation, ¹² hepatitis C virus replication ¹³ and systemic iron homeostasis. ¹⁴ Plasma miR-122 expression has been used to evaluate drug-induced liver injury and liver infection in patients with chronic hepatitis B infection,^15,16 and miR-122 expression could be a more accurate indicator of liver injury than alanine aminotransferase (ALT). ¹⁷

The present study investigated whether miR-122 expression in the plasma of patients with HCC could be a novel serological marker for the evaluation of liver injury induced by hepatectomy.

Patients and methods

Quantitative RT and PCR

TaqMan® MicroRNA Assays (ABI PRISM, Carlsbad, CA, USA) were used to evaluate mature miR-122 expression levels. For the reverse transcription (RT) reactions, 10 ng of total RNA was used in each reaction and mixed with the RT primer as previously described. ¹⁹ RT reactions were performed at 16℃ for 30 min, 42℃ for 30 min, 85℃ for 5  min and then maintained at 4℃. Following the RT reactions, 1.5 µl of cDNA was used for a polymerase chain reaction (PCR) using 2 µl of the TaqMan primers. The quantitative RT–PCR cycling programme involved preliminary denaturation at 94℃ for 2 min followed by 1 cycle of denaturation at 94℃ for 20 s, 60℃ for 30 s and 35 cycles of annealing at 72℃ for 30 s, followed by a final elongation step at 72℃ for 10 min, in an ABI 7500 real-time PCR system (ABI PRISM). The real-time PCR results were analysed and expressed as the relative miRNA level using U6 snRNA for normalization purposes. The RT and PCR primers for miR-122 were purchased from ABI PRISM. Furthermore, U6 snRNA was used as the internal control, and the miRNA-122 expression was obtained using the 2^{−ΔΔ C T} assay, as previously described. ²⁰

Laboratory measurements

For the miR-122 evaluation, laboratory measurements were conducted on the blood samples that were collected from patients preoperatively, and from controls. Total bilirubin in the blood samples was measured by an enzymatic assay using an ADVIA 1650 analyser (Bayer Diagnostics, Tarrytown, NY, USA). For determination of ALT activity, the reference method defined by the International Federation of Clinical Chemistry with pyridoxal phosphate was used and calibrated, as previously described.^21,22

Statistical analyses

Data were expressed as mean ± SD (all SDs were <5%) of three experiments. All statistical analyses were performed using the SPSS® statistical package, version 18.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Statistical comparisons between the groups were undertaken using Student’s t-test; Pearson’s correlation coefficient was used to calculate the correlation between groups. A P-value <0.05 was considered to be statistically significant.

Results

Eighty patients with HCC and 80 control subjects were included in the study. The mean (±SD) age of patients was 51.2 ± 13.7 years and 68 (85.0%) were male. At baseline, expression of miR-122 and ALT in patients with HCC was significantly higher than in the control group (P < 0.05) (Table 1). The correlation coefficient (R) of miR-122 and ALT in patients with HCC was 0.842 (P < 0.01)l the R for miR-122 and ALT in the control group was 0.949 (P < 0.01).

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Table 1.

Plasma levels of microRNA-122 (miR-122) and alanine transaminase (ALT) in patients with hepatocellular carcinoma (HCC) and healthy age-matched controls at baseline (preoperation for patients with HCC).

Group	n	miR-122, 2−ΔΔ C T value	ALT, U/l
Patients  with HCC	80	13.9 ± 4.1 *	48.9 ± 8.7 *
Controls	80	5.7 ± 2.8	15.4 ± 3.4

Expression of ALT and bilirubin increased in patients with HCC on day 1 postoperation, when the highest miR-122 expression was also observed. These three indices returned to preoperative levels ∼7 days postoperation (Table 2). The R for miR-122 and ALT in patients with HCC on days 1 and 7 postoperation were 0.793 (P < 0.001) and 0.869 (P < 0.001), respectively. The R for miR-122 and bilirubin in patients with HCC before surgery and on days 1 and 7 postoperation were 0.742 (P < 0.01), 0.873 (P < 0.001) and 0.903 (P < 0.05), respectively.

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Table 2.

Plasma levels of microRNA-122 (miR-122), bilirubin and alanine transaminase(ALT) in patients with hepatocellular carcinoma (n = 80), preoperatively and on days 1and 7 following hepatectomy.

Timepoint	miR-122, 2−ΔΔ C T value	ALT, U/l	Bilirubin, µmol/l
Preoperative	13.9 ± 4.1	48.9 ± 8.7	13.9 ± 5.2
Day 1 postoperation	67.5 ± 23.8 *	428.3 ± 129.4 *	28.5 ± 14.3 *
Day 7 postoperation	17.2 ± 8.3	98.5 ± 24.1	16.2 ± 7.4

In the subgroup of patients with HCC who received a block of the first hepatic portal during hepatectomy, plasma miR-122 expression on day 1 postoperation was significantly increased, compared with in patients who did not have this block (P < 0.02). Patients with excised tumours >5 cm in diameter also had significantly increased plasma miR-122 expression, compared with those with smaller excised tumours (P < 0.003) on Day 1 postoperation. However, plasma miR-122 expression was not related to clinicopathological characteristics including age, sex or level of intraoperative haemorrhage (Table 3).

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Table 3.

Comparison of plasma miR-122 expression in subgroups of patients with hepatocellular carcinoma (HCC) who exhibited different clinicopathological features; comparison undertaken on 1 day following hepatectomy.

Parameter	n	miR-122, 2−△△CT value	t value	Statistical significance a
Age, years
<50	49	63.4 ± 18.2
≥50	31	72.1 ± 19.5	−0.843	NS
Sex
Male	56	62.6 ± 21.4
Female	24	72.9 ± 24.5	−1.322	NS
Excised tumour size, cm
<5	34	45.8 ± 13.6
≥5	46	89.7 ± 21.5	−8.734	0.003
Block of the first hepatic portal
Yes	58	80.8 ± 23.2
No	22	54.7 ± 28.4	−2.741	0.021
Intraoperative haemorrhage, ml
<400	64	59.4 ± 22.7
≥400	16	76.1 ± 28.6	−1.942	NS

Discussion

The MiRNAs are extensively involved in multiple pathological and physiological processes,^23,24 and their up-regulation and down-regulation influence the progression of disease development. Four miRNAs were found to be significantly up-regulated and three miRNAs were significantly down-regulated in tissue samples from patients with early recurrent HCC.^25,26 MiR-122 is highly expressed in liver, and accounts for >70% of total miRNAs. Additionally, miR-122 expression in liver is at least 1 000 times greater than in other tissues: miR-122 expression in other tissues is so low that it cannot be detected. MiR-122 is characterized by high tissue specificity, and it is an important regulatory factor in the liver. ²⁷

At present, ALT is used as a serological marker that correlates with clinical liver injury. However, numerous extrahepatic conditions can increase ALT levels (including burns, myositis, hypothyroidism and myopathy). Hence, increases in ALT levels are not specific to liver injury and it is therefore not a suitable marker of such injury. ²⁸ It has been reported that miRNA in tumour cells is released into blood using a specific mechanism. ²⁹ Previously, it was believed that RNA was not stable in peripheral blood and that RNA was easily degraded by RNase, ³⁰ therefore, it was difficult to detect the RNA in blood. ³¹ However, miRNA has now been found to maintain its stability well in peripheral blood^32–34 and hence, peripheral miRNA has been used for the diagnosis and prognosis of tumours. ³⁵ MiRNA is specifically expressed in liver cells; therefore, cellular miR-122 may be released into the blood after liver injury. Additionally, peripheral miR-122 expression may reflect the degree of liver injury. Zhang et al. ¹⁷ showed that ALT and miR-122 levels in the plasma of patients with chronic hepatitis B were higher than such levels in healthy volunteers. Hence, they deemed that miR-122 was a useful biomarker for the evaluation of hepatitis B virus-induced liver injury. However, the mechanism involved in the transportation and release of miR-122 is still not clear. In the present study, we found that preoperative miR-122 and ALT levels in the plasma of patients with HCC before were significantly higher than those in healthy controls, and that miR-122 expression was positively correlated with ALT levels.

Data from the present study are consistent with the results of Zhang et al., ¹⁷ and this may be because HCC is commonly accompanied by chronic hepatitis B-induced liver injury. Additionally, miR-122 in the cytoplasm of HCC cells may be released into the blood, and a synergistic action may occur. ³⁶ Hence, detection of plasma miR-122 expression preoperatively could be used to complement the ALT assay for the evaluation of liver injury. Furthermore, the present study showed that ALT and bilirubin expression increased in patients with HCC on day 1 postoperation, when the highest miR-122 expression was also observed. These three indices returned to normal levels 7 days postoperation. The change in plasma miR-122 was similar to ALT and bilirubin. In addition, miR-122 expression was positively correlated with ALT and bilirubin expression (R > 0.7). Hence, these data primarily showed that plasma miR-122 may be a novel biomarker for liver injury induced by hepatectomy.

The present study also analysed the relationship between plasma miR-122 and clinicopathological indices of patients 1 day postoperation, and suggested that plasma miR-122 expression was not associated with age, sex or extent of intraoperative haemorrhage. However, its expression was associated with first hepatic portal control and excised tumour size: receiving first hepatic portal control and having a large excised tumour (>5 cm) induced an increase in plasma miR-122 expression. This may be due to liver cells being injured due to hypoxia after occlusion of hepatic blood flow, and a large excised tumour size means that a large liver section had to be removed, causing more extensive liver injury than would be seen with smaller excisions. However, a larger sample quantity than that studied in the present analysis is needed to further study the mechanisms involved.

The approach presented in our paper has been already proposed for monitoring drug- and viral-induced liver damage, but it should be considered with some caution. Specifically, the 2^{−ΔΔ C T} assay method is widely used to compare control and treated samples, but it is reliable only if the housekeeping RNA levels (in this case U6) are constant and are not modified by the treatment. Future research could involve a standard-curve method, in addition to the 2^{−ΔΔ C T} method, although this is a more technically challenging approach.

The present study findings indicate the potential value of miR-122 expression in patients with HCC, but it should be noted that this test method is currently more expensive compared with other techniques. It is feasible, therefore, that where only standard serological testing is available, severity of liver damage and post-surgery prognosis could be indicated by raised ALT and bilirubin levels, particularly in those with occluded hepatic blood flow and larger excised tumours. However, miR-122 expression is of value because of its good repeatability, sensitivity and specificity.

To conclude, the present study primarily suggested that plasma miR-122 expression in patients with HCC was related to ALT and bilirubin levels, and that hepatic blood flow occlusion and large excised tumour size increased miR-122 expression. MiR-122 may be used as a novel serological marker to evaluate liver injury induced by hepatectomy.