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Abstract

OBJECTIVE:

The study aims to evaluate the predictive value of microRNA-143 (miR-143) for the prognosis of patients with hepatocellular carcinoma (HCC).

METHODS:

Between October 2010 and October 2012, 131 HCC patients were selected as a case group; meanwhile, 122 healthy controls were enrolled as a control group. The miR-143 expression in serum was detected by quantitative real-time polymerase chain reaction (qRT-PCR). These HCC patients were divided into the high miR-143 expression group and the low miR-143 expression group based on the threshold of receiver operating characteristic (ROC) curve. Kaplan-Meier method was applied to analyze the prognosis of HCC patients.

RESULTS:

MiR-143 exhibited decreased expression in the case group significantly compared to the control group. The areas under the ROC curve (AUC), sensitivity value and specificity value of the miR-143 expression for the diagnosis of HCC were 0.831, 80.30% and 82.40%, respectively. The miR-143 expression was negatively correlated with vascular invasion, TNM staging, tumor recurrence, metastasis and survival of HCC patients.

CONCLUSIONS:

Our study provides evidence that miR-143 may be negatively correlated with the prognosis of HCC and provides a promising strategy for HCC treatment and prognosis improvement.

Keywords

MicroRNA-143 hepatocellular carcinoma prognosis predictive value

1. Introduction

Hepatocellular carcinoma (HCC) is a common and deadly malignancy. In 2008, there were an estimated 748,300 new cases and 695,900 deaths of HCC worldwide [1]. HCC has already been the second main reason of cancer death in China and its mortality rate accounts for 19.33% of all cancer deaths [2]. The main causes for HCC include the Hepatitis B virus (HBV) infection, Hepatitis C virus (HCV) infection and aflatoxin [3]. In recent years, some non-viral risk factors, including obesity, diet and alcohol drinking, may also lead to HCC [4, 5]. Currently, the main therapeutic strategies for this disease include systemic chemotherapy, hepatic arterial infusion chemotherapy and molecular targeted therapy of sorafenib [6, 7]. In recent years, studies have shown that microRNA (miR) played a critical role in modulating the development of tumor, making miR therapy a research hotspot [8, 9].

MiR is a class of small noncoding RNAs with a length of 21 $\sim$ 25 nucleotides (nt), which plays an critical role in regulating the progression of cancer. They could bind to the complementary sequences of the 3’ untranslated region (3’ UTR) in target gene and regulate gene expression, thus repressing the protein translation and down-regulating protein expression [10, 11]. MiR-143 is located in the chromosome 5 with a mature length of 22 nt [9]. A previous study has revealed that miR-143 exhibited reduced level in many cancers, which may prove that high miR-143 expression could inhibit cell proliferation and induce tumor cell apoptosis in the development of cancer [12]. MiR-143 could inhibit the translation of multiple proteins including the complement regulatory protein CD46 and promote the necrosis and apoptosis of tumor cells [13]. Besides, a research proved that miR-143 suppressed the migration and invasion of prostate cancer cell by acting on the Matrix Metallo Preteinases-13 (MMP-13) [14]. However, the concrete mechanism of miR-143 on the development and progression of HCC is still unclear. Our study aims to investigate the predictive value of miR-143 for the prognosis of HCC patients with the expectation to identify potential targets for effective treatment.

2. Materials and methods

2.1 Ethical statement

The study was conducted in accordance with approval from the Ethics Committee of our hospital. Written informed consent was collected from each of the eligible patient prior to the experiment.

2.2 Study subjects

Between October 2010 and October 2012, 131 HCC patients who received radical resection in our hospital were selected in this study. The staging system established by Korean Liver Cancer Study Group (KLCSG) and National Cancer Institute (NCC) [15] was adopted for evaluation of postoperative pathological staging. Inclusion criteria: (1) Patients who received radical resection in our hospital and were diagnosed as HCC by histopathological examination after surgery; (2) Patients who did not receive any anti-tumor treatment prior to the operation; (3) Complete medical data of all the cases were recorded; (4) No major organ diseases or dysfunctions were observed and the examinations of blood routine, urine routine, stool routine, liver function, kidney function, cardiac function were normal. Exclusion criteria: (1) Patients with secondary HCC; (2) Patients with other malignant tumors; (3) Patients who have already received concurrent chemotherapy and radiotherapy before the radical resection; (4) Patients who received molecular targeted therapy recently. Healthy controls ( $n=$ 122) at the same time were selected as the control group.

Table 1
Comparisons of baseline characteristics between the case group and the control group

Characteristic Case Control t/ $\chi^{2}$ $P$

( $n=$ 131) ( $n=$ 122)

Mean age (years) 50.49 $\pm$ 11.04 49.35 $\pm$ 11.81 0.794 0.428

Gender (men/women) 88/43 79/43 0.165 0.685

Smoking history (yes/no) 50/81 43/79 0.232 0.630

Drinking history(yes/no) 40/91 25/97 3.337 0.068

Characteristic	Case	Control	t/ $\chi^{2}$	$P$
	( $n=$ 131)	( $n=$ 122)
Mean age (years)	50.49 $\pm$ 11.04	49.35 $\pm$ 11.81	0.794	0.428
Gender (men/women)	88/43	79/43	0.165	0.685
Smoking history (yes/no)	50/81	43/79	0.232	0.630
Drinking history(yes/no)	40/91	25/97	3.337	0.068

2.3 Sample collection and preservation

Venous blood of the two groups was collected and then placed at 37 ${}^{\circ}$ C for blood coagulation. Then the samples were kept at 4 ${}^{\circ}$ C overnight until the serum was separated out. Then samples were centrifuged at 3000 rpm for 10 min. The supernatant was harvested in new EP tubes, then sub-packaged and stored in the ultra-low temperature freezer for use. The exposure time of the samples should be shortened during the whole process in case of the RNA degradation.

2.4 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from 200 $\mu$ L of the serum sample using miRNeasy Mini Kit (Qiagen Company, Hilden, Germany). A volume of 5 $\mu$ L RNA was diluted 20 times with ultra-pure water (Tiangen Biotech Co., Ltd, Beijing, China) without RNAse. The optical density (OD) at 260 nm and 280 nm was determined by ultraviolet spectrophotometry to analyze the quantity and quality of the RNA sample. The qualified sample with the OD260/OD280 ratio of 1.7 $\sim$ 2.1 was preserved for further study. The PCR-Cycler was used for the cDNA synthesis. The qRT-PCR was performed using the ABI 7500 System (Applied Biosystems, Shanghai, China). The 20 $\mu$ L PCR reaction mixture included 10 $\mu$ L of 2 $\times$ real time PCR buffer (3 mmol/L Mg ${}^{2+}$ , 0.2 mmol/ L dNTP and SYBR Green I, all purchased from Invitrogen), 0.32 $\mu$ L of forward and reverse primers(5 mmol/ $\mu$ L), 0.2 $\mu$ L of Taq DNA polymerase and 2 $\mu$ L cDNA sample. The PCR cycling conditions were as follows: 95 ${}^{\circ}$ C for 3 min followed by 30 cycles of 94 ${}^{\circ}$ C for 30 s, 55 ${}^{\circ}$ C for 30 s and 72 ${}^{\circ}$ C for 30 s. Each sample was repeated three times. The results were analyzed by Opticon Monitor 3 software (Bio-Rad Laboratories, Inc. CA, USA). The lowest point of the logarithm curve was selected as the threshold and the respective threshold cycle (Ct) values were obtained. The data was analyzed using the 2 ${}^{-\Delta\Delta Ct}$ method. The 2 ${}^{-\Delta\Delta Ct}$ was defined as the fold-expression change of the experimental group and control group with the following formula: $\Delta\Delta$ CT $=$ $\Delta$ Ct (experimental group) $-$ $\Delta$ Ct (control group) and the $\Delta$ Ct $=$ Ct miRNA – Ct (GAPDH). The experiment was replicated three times.

2.5 Follow-up

Patients were routinely followed up by telephone, outpatient follow-up and referring to medical records since the cases were confirmed. The deadline of the follow-up was October 30, 2015. After surgery, regular imaging examination and $\alpha$ -fetoprotein (AFP) determination were performed to monitor the recurrence and metastasis of the tumor. The patients were diagnosed according to clear tumor images. The follow-up was conducted for 3 $\sim$ 36 months with 5 cases lost to follow up and the follow-up rate was 96.18%. No Progression Free Survival (PFS) was defined as the period from the beginning time of treatment to the recurrence or death.

2.6 Statistical analysis

All the data were analyzed using SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA). The measurement data were expressed as $\bar{x}$ $\pm$ s, and the comparisons were conducted using $t$ test. The comparisons of the enumeration data were performed using the $\chi^{2}$ test. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficiency of miR-143 in HCC. Kaplan-Meier method was used to analyze the survival and prognostic condition of patients with HCC. $P<$ 0.05 was considered as statistically significant.

3. Results

3.1 Comparisons of baseline characteristics between the case and control groups

The case group included 131 HCC patients (88 males and 43 females; age range: 26 $\sim$ 74 years; mean age: 50.49 $\pm$ 11.04 years) and the control group included 122 healthy subjects (79 males and 43 females; age range: 23 $\sim$ 74 years; mean age: 49.35 $\pm$ 11.81 years). No significant differences were observed between the two groups in age distribution, sex, smoking history and drinking history ( $P>$ 0.05) (Table 1).

3.2 Comparison of miR-143 expression between the case and control groups

The relative expressions of miR-143 in the case and control groups were 0.50 $\pm$ 0.10 and 0.65 $\pm$ 0.12, respectively. MiR-143 exhibited significantly decreased expression in the case group than in the control group ( $P<$ 0.05) (Fig. 1).

Figure 1.

Comparison of the relative microRNA-143 expression between the case group and the control group. Note: ${}^{*}$ , compared with the control group, $P<$ 0.05.

3.3 Predictive value of miR-143 for the diagnosis of HCC

The areas under the ROC curve (AUC), sensitivity and specificity of miR-143 expression for the diagnosis of HCC were 0.831, 80.30% and 82.40%, respectively (Fig. 2). The threshold of the ROC curve was 0.58.

Figure 2.

ROC curve of microRNA-143 for the diagnosis of hepatocellular carcinoma. Note: ROC, receiver operating characteristic.

3.4 Correlations between miR-143 expression and clinicopathological features of HCC patients

These HCC patients were divided into the high miR-143 expression group and the low miR-143 expression group according to the threshold of ROC curve. The clinicopathological features showed that the miR-143 expression was not significantly correlated with sex, age, AFP level, hepatitis B surface antigen (HBsAg), cirrhosis, the size and number of tumors, tumor capsule and tumor differentiation (all $P>$ 0.05), but was significantly correlated with vascular invasion and tumor-node-metastasis (TNM) staging (both $P<$ 0. 05) (Table 2).

Table 2
Correlations between microRNA-143 expression and clinicopathological features of patients with hepatocellular carcinoma

Feature microRNA-143 $P$

Low expression High expression

( $n=$ 108) ( $n=$ 23)

Gender 0.231

Female 33 10

Male 75 13

Age (years) 0.958

$\leqslant$ 51 57 12

$>$ 51 51 11

Preoperative AFP 0.850

$\leqslant$ 20 54 12

$>$ 20 54 11

HBsAg 0.699

Negative 61 14

Positive 47 9

Cirrhosis 0.313

No 50 8

Yes 58 15

ALT (U/L) 0.626

$\leqslant$ 75 53 10

$>$ 75 55 13

Tumor size (cm) 0.958

$\leqslant$ 5 51 11

$>$ 5 57 12

Tumor number 0.913

Single 55 12

Multiple 53 11

Tumor capsule 0.913

No 55 12

Completed 53 11

Vascular invasion 0.007

No 26 12

Yes 82 11

TNM staging 0.001

I 26 13

II 26 7

III 56 3

Differentiation degree 0.117

High 57 8

Low 51 15

Feature	microRNA-143	$P$
	Low expression	High expression
	( $n=$ 108)	( $n=$ 23)
Gender			0.231
Female	33	10
Male	75	13
Age (years)			0.958
$\leqslant$ 51	57	12
$>$ 51	51	11
Preoperative AFP			0.850
$\leqslant$ 20	54	12
$>$ 20	54	11
HBsAg			0.699
Negative	61	14
Positive	47	9
Cirrhosis			0.313
No	50	8
Yes	58	15
ALT (U/L)			0.626
$\leqslant$ 75	53	10
$>$ 75	55	13
Tumor size (cm)			0.958
$\leqslant$ 5	51	11
$>$ 5	57	12
Tumor number			0.913
Single	55	12
Multiple	53	11
Tumor capsule			0.913
No	55	12
Completed	53	11
Vascular invasion			0.007
No	26	12
Yes	82	11
TNM staging			0.001
I	26	13
II	26	7
III	56	3
Differentiation degree			0.117
High	57	8
Low	51	15

Note: AFP, $\alpha$ -fetoprotein; HBsAg, hepatitis B surface antigen; ALT, glutamate pyruvic transaminase; TNM, Tumor Node Metastasis; LC, liver cancer.

3.5 Correlation between miR-143 expression and the prognosis of HCC

As shown Table 3, the rate of high miR-143 expression in HCC patients with recurrence/metastasis was significantly lower than those without recurrence/metastasis (8.97% vs. 30.19%, $P=$ 0. 0038). The Fig. 3 revealed that the mean survival time of 131 HCC patients WAS 25.86 $\pm$ 0.93 months. HCC patients in the low miR-143 expression group had lower mean survival time than those in the high miR-143 expression group (24.15 $\pm$ 1.02 months vs. 33.82 $\pm$ 1.28 months, $P<$ 0.05).

Table 3
Correlation of microRNA-143 expression with the recurrence/ metastasis of patients with hepatocellular carcinoma

Group n microRNA-143

Low expression High expression

With recurrence/metastasis 78 71 (91.02) 7 (8.97) ${}^{*}$

Without recurrence/metastasis 53 37 (69.81) 16 (30.19)

Group	n	microRNA-143
With recurrence/metastasis	78	71 (91.02)	7 (8.97) ${}^{*}$
Without recurrence/metastasis	53	37 (69.81)	16 (30.19)

Note: ${}^{*}$ , compared with patients without recurrence/metastasis, $P<$ 0.05.

Figure 3.

Correlations between microRNA-143 expression and the prognosis of patients with hepatocellular carcinoma.

4. Discussion

HCC is a common malignancy with high mortality rate and nearly 85% of the cases occur in developing countries, which is a serious threat to the health worldwide [16]. Although the survival rate has already increased with the advances in medical technology, the 5-year overall survival rate still remains poor [17]. Moreover, the metastasis and recurrence rate is still high even after radical resection [18]. Therefore, more and more studies are carried out to investigate the causes of HCC and provide new materials for HCC treatment.

Our study found that miR-143 exhibited reduced levels in HCC patients. MiR-143 is one of the well-studied miRNAs and has been found to be downregulated significantly in several cancers such as prostate cancer [14] and colorectal cancer [9], proving that miR-143 acts as a tumor suppressor in the tumor progression. In addition, a study revealed that the target genes and mechanisms of miR-143 in many cancers were different [12]. MiR-143 may target Toll-like receptor 2 (TLR2) and regulate its expression [19]. TLR2 is one of the pattern-recognition receptors which promote tumor growth and immune escape once being activated by damage-associated molecular patterns (DAMP) released from the injured or tumor tissues [19, 20]. Nuclear factor kappa B (NF- $\kappa$ B), a downstream factor in the signaling pathway of TLR, is activated by the TLR2 activation and then results in the activation of the adaptive immune response [21]. It has been proved that the NF- $\kappa$ B could promote the metastasis and migration of the breast cancer cells as a carcinogen [22]. The mechanism research about the tumor inhibition of miR-143 in HCC proved that when miR-143 exhibited decreased level, the inhibition effect on TLR2 and NF- $\kappa$ B reduced significantly, leading to the occurrence and development of HCC [23].

Our research also revealed that the recurrence or metastasis rate of the patients with higher miR-143 expression was significantly lower than that of the group with lower miR-143 expression, indicating that the prognosis in the higher miR-143 expression group was more favorable. Metastasis is a complex process, involving in multiple factors such as cancerous cells, tumor microenvironment and host immune status [24]. Previous studies have also proved that miR-143 suppressed cancer cells proliferation, invasion, and migration by targeting KRAS, Bcl-2 and then promoted the apoptosis of cancer cells [25, 26]. A previous study proposed that the high miR-143 expression may inhibit the HCC proliferation by down regulating the expression of TLR2 and inhibiting the activity of NF- $\kappa$ B [19]. Moreover, a research also proved that the activation of TLR2/phosphoinositide kinase-3 (PI3k)/NF- $\kappa$ B signaling pathway could produce inflammatory cytokines and chemotactic effects and involve in tumor metastasis [27]. Therefore, patients with higher miR-143 expression could obtain better prognosis.

In summary, our study suggest that miR-143 may be negatively correlated with the prognosis of HCC. Therefore, increasing miR-143 expression may be an effective approach for suppressing the HCC incidence and metastasis as well as improving the prognosis. However, the sample size is limited and further studies with large sample size are still required for a more comprehensive understanding of the clinical potential about our research.

Footnotes

Acknowledgments

We would like to acknowledge the helpful comments on this paper received from our reviewers.

Conflict of interest

None.

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Predictive value of microRNA-143 in evaluating the prognosis of patients with hepatocellular carcinoma

Abstract

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Ethical statement

2.2 Study subjects

2.4 Quantitative real-time polymerase chain reaction (qRT-PCR)

2.5 Follow-up

2.6 Statistical analysis

3. Results

3.1 Comparisons of baseline characteristics between the case and control groups

3.2 Comparison of miR-143 expression between the case and control groups

Table 3 Correlation of microRNA-143 expression with the recurrence/ metastasis of patients with hepatocellular carcinoma Group n microRNA-143 Low expression High expression With recurrence/metastasis 78 71 (91.02) 7 (8.97) * Without recurrence/metastasis 53 37 (69.81) 16 (30.19)

Footnotes

Acknowledgments

Conflict of interest

References

Table 3
Correlation of microRNA-143 expression with the recurrence/ metastasis of patients with hepatocellular carcinoma

Group n microRNA-143

Low expression High expression

With recurrence/metastasis 78 71 (91.02) 7 (8.97) ${}^{*}$

Without recurrence/metastasis 53 37 (69.81) 16 (30.19)