LHX3 is an advanced-stage prognostic biomarker and metastatic oncogene in hepatocellular carcinoma

Abstract

BACKGROUND:

Hepatocellular carcinoma (HCC) is a common cancer and exhibits high morbidity and mortality in the world. We recently identified LHX3 as a preferentially expressed gene with a possible involvement in HCC.

OBJECTIVE:

To determine the expression, clinical relevance, prognostic significance and functions of LHX3 in HCC.

MATERIALS AND METHODS:

LHX3 expression was assessed in 190 cancerous and 40 adjacent non-cancerous tissues by PCR, western blot and immunohistochemistry. Associations between LHX3 expression and clinicopathological characteristics of patients were investigated. Correlations between LHX3 expression and overall survival of patients were analyzed by Kaplan-Meier and Cox-regression methods. Functional roles of LHX3 were evaluated by transwell assays.

RESULTS:

LHX3 expression is significantly increased in carcinoma tissues, and associated with clinical stage and metastasis of patients. LHX3 expression is much higher in the advanced-stage patients than the early-stage patients, and is sharply increased in metastasic patients. High LHX3 expression is associated with unfavorable overall survival, and is an independent prognostic factor of patients. Moreover, LHX3 is an unfavorable and independent prognostic factor unique to advanced-stage patients. Knockdown expression of LHX3 obviously inhibits tumor cell migration and invasion.

CONCLUSION:

LHX3 is an advanced-stage prognostic biomarker, and acts as a new potential metastatic oncogene in HCC.

Keywords

LHX3 prognostic biomarker metastasis oncogene hepatocellular carcinoma

1. Introduction

Hepatocellular carcinoma (HCC) is one of the most common cancers with high morbidity and mortality worldwide [1, 2, 3]. Although the most effective treatment is surgery for HCC patients at early stage, the patients at advanced HCC is un-resectable. Moreover, there is still deficiency of effective methods for patients diagnosed at the advanced stages [4]. In general, despite advances in treatment strategies, the rate of 5-year survival remains very poor due to limit treatments and lack of effective prognostic markers [5, 6]. Therefore, identification of new targets for treatment and practical prognostic markers for prognostic evaluation are very important in HCC.

LIM homeobox (LHX) genes are important LIM-homeodomain factors. Previous studies have revealed that LHX members play key roles in development of various tissues [7, 8, 9, 10, 11, 12]. Researches have identified a few LHX genes involved in various type cancers, such as Lhx2, Lhx4 and Lhx1 [13, 14, 15, 16, 17, 18]. LHX3, as an important LHX member, is critical in the development of spinal cord motor neurons and pituitary [19, 20, 21, 22]. Recently, a study has revealed a clear correlation between LHX3 methylation and breast cancer [23]. However, the clinical, prognostic significance and potential role of LHX3 in cancer, especially in HCC, remain unknown.

In our present study, we identified LHX3 as a new differentially expression gene in tumor and normal liver tissues. The clinical, prognostic significance and potential roles on metastasis of LHX3 were explored in human HCC. Our study indicates that LHX3 is an important advanced-stage prognostic factor and functions as a potential metastatic promoter in HCC.

2. Materials and methods

2.1 Patient samples and cell lines

Total 190 HCC patients undergone surgical resection between 2004 and 2010 were obtained from the Southwest Hospital in Chongqing, China. The clinico-pathologic information includes gender, age, tumor size, histological grade, lymph node status, clinical stage, radiotherapy, tumor location and overall survival (OS). Patients were followed up for a maximum period of 80 months, with a median of 32.9 months. OS was defined as the period from surgery to death or the last observation. The clinico-pathologic information also includes AFP level and the expressions of Ki67, P53, C-ERBB-2, CK19, HEP1, CK8 and CD34. The level or expressions of these markers were measured by the Hospital. When the tumor cell staining of cancer samples was completely no positive signal, it was identified as negative. The HCC cell lines (Huh7 and 7721) were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China), and cultured in RPMI-1640 (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (HyClone). This study was approved by the ethics committee of Southwest Hospital Affiliated to the Third Military Medical University. All experiments were carried out in accordance with approved guidelines of Third Military Medical University. Informed consent was signed by all the patients.

2.2 Total RNA isolation

Total RNA was extracted from frozen HCC or normal liver tissues. About 3.5 $\mu$ g of RNA was treated with DNase I in order to eliminate the genomic DNA, and was then reverse-transcribed to cDNA and stored at $-$ 20 ${{}^{\circ}}$ C.

2.3 RT-PCR

Series of PCR with different cycles were performed and the appropriate cycles were chosen. Human $\beta$ -actin was used as an endogenous control. The primers of LHX3 and $\beta$ -actin for RT-PCR are: LHX3-F, 5’-AA GTGTCTCAAGTGCAGCGA-3’ and LHX3-R, 5’-GT GGTACACGAAGTCCTGGG-3’; $\beta$ -actin-F, 5’-TTCT ACAATGA GCTGCGTGTG-3’ and $\beta$ -actin-R, 5’-GG GGTGTTGAAGGTCTCAAA-3’.

2.4 RT-qPCR

RT-qPCR was performed using GoTaq ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ qPCR Master Mix (Promega, USA) by Real-Time Detection system (iQ5, Bio-Rad). The relative expression was calculated by equation 2 ${}^{-\Delta\Delta\text{CT}}$ . The primers of LHX3 and $\beta$ -actin for RT-qPCR are: LHX3-F1, 5’-ACGGA CCCAGTTCTGACCTA-3’ and LHX3-R1, 5’-TGGT CTACCTCAT CCAGCCA-3’; $\beta$ -actin-F2, 5’-TG ACG TGGACATCCGCAAAG-3’ and $\beta$ -actin-R2, 5’-CTG GAAGGTGGACAGCGAG G-3’.

2.5 Tissue microarray

To construct the tissue microarray (TMA), two cores were taken from cancerous and matched adjacent noncancerous liver tissues within a distance of 35 mm. The adjacent noncancerous tissues were stained with hematoxylin & eosin and determined by comparing with normal tissues. The core size of TMA is 1.5 mm. The TMA was constructed as previously described [24].

Figure 1.

LHX3 expression is sharply increased in HCC. (A) RT-PCR and qRT-PCR analysis of LHX3 mRNA expression in normal and cancerous tissues. N represents normal tissue; M represents marker; T represent cancerous tissue. The $\beta$ -actin was used as internal control. ${}^{**}$ , $p<$ 0.01. Error bar indicates SEM. (B) LHX3 mRNA expression was analyzed in normal and tumor tissues from the TCGA database (https://genome-cancer.ucsc.edu, the accession date is 2017.2.22). The expression in TCGA was performed by RNAseq (liver hepatocellular carcinoma (LIHC) gene expression by RNAseq, IlluminaHiSeq percentile; $n=$ 423). (C) Immunohisto-chemical analysis of LHX3 protein expression in 190 cancerous and 40 adjacent noncancerous samples. LHX3 expression was higher in cancerous tissues than adjacent noncancerous tissues. LHX3 was expressed in both the cytoplasm and nuclei of tumor cells. Scale bars represent 50 $\mu$ m.

2.6 Immunohisto-chemical (IHC) analysis

IHC was used to determine the expression of LHX3 in HCC tissues with LHX3 antibody (1:150, Abcam) as previously described [25]. The staining percentage of tumor cells was quantified into 5 grades: $<$ 5% positive cells for 0 score; 5% to 25% for 1 score; 26% to 50% for 2 score; 51% to 75% for 3 score and $\geqslant$ 76% for 4 score. The staining intensity of tumor cells was graded as negative (0 score), weak (1 score), moderate (2 score) and strong (3 score). The expression levels of LHX3 were defined by product of percentage and intensity for staining with a score ranged from 0 (the minimum score) to 12 (the maximum score) [26, 27]. All the stained tissues were independently reviewed by two blinded pathologists.

2.7 LHX3 siRNA

The LHX3 siRNA used in the present study was purchased from Santa Cruz Biotechnology (sc-38712).

2.8 Western blotting analysis

Western blotting (WB) was performed as previous study [28]. Briefly, the protein was run on 10% SDS-PAGE, transferred onto PVDF membrane (Millipore, USA), and incubated with LHX3 antibody (1:1500, Abcam) or $\beta$ -actin antibody (1:2000, Sigma). The protein on the membrane was detected using chemiluminescence (Pierce, USA).

2.9 Migration and invasion assays

Transwell assays were performed to determine migration or invasion activities of cancer cells in 24-well plates without or with matrigel (8 $\mu$ m, Corning) [29]. The transfected cells were suspended in the up well of chamber with serum-free medium, and the low well contained fully 10% FBS medium. The cells migrated to low chamber after 16 h for migration and 18 h for invasion were stained with crystal violet and counted.

Figure 2.

The expression of LHX3 is increased in the HCC patients at advanced stage or with metastasis. (A) LHX3 protein expression is shown in the patients at different clinical stages. Scale bars represent 50 $\mu$ m. (B) LHX3 protein expression is increased in the HCC patients at advanced stage. I $+$ II represents the patients at clinical stage I and II. III $+$ IV represents the patients at clinical stage III and IV. (C) LHX3 expression is increased in the HCC patients with metastasis. M0 represents the patients with non-metastasis. M1 represents the patients with metastasis.

2.10 Analysis of available datasets

To get the expression of LHX3 in cancer and normal samples in an independent cohort, we used the UCSC Cancer Genomics Browser (https://genome-cancer.soe.ucsc.edu/) to analyze the RNAseq expression in HCC cohort (IlluminaHiSeq; $n=$ 423). The TCGA data in the UCSC Cancer Browser was chose. The UCSC Cancer Browser allows researchers to explore cancer genomics data and its clinical information. Data can be viewed by gene-set of interest. It is also possible to quickly perform and easily view statistical analysis on subsets of the data.

2.11 Statistical analysis

SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses. The difference in categorical variables was analyzed by Chi-square or Linear-by-Linear test. Kaplan-Meier method and log-rank test were used to evaluate the OS. Cox regression was performed for multivariate analysis of prognostic predictors. The high or low expression was categorized by the median. $P<$ 0.05 was taken as statistically significant.

3. Results

3.1 Increased LHX3 expression is observed in HCC tissues

To further investigate the expression pattern of LHX3 in HCC, LHX3 expression level was checked in normal and tumor tissues by RT-PCR and RT-qPCR. LHX3 expression was increased in tumor tissues compared to normal liver tissues (Fig. 1A). The mean level of LHX3 expression was 4.511 $\pm$ 0.4351 in tumor tissues higher than 2.609 $\pm$ 0.4252 in normal tissues, which was further confirmed in an independent cohort of HCC patients using The Cancer Genome Atlas (TCGA) database (Fig. 1A and B). To validate the altered LHX3 expression at protein level, we performed an immunohisto-chemistry (IHC) for LHX3 on a tissue microarray (TMA) containing 190 carcinoma and 40 corresponding para-carcinoma tissues. The positive staining of tumor cells was quantified and the mean protein expression of LHX3 was 8.279 $\pm$ 0.2025 in cancer tissues also sharply higher than 5.100 $\pm$ 0.5838 in para-carcinoma tissues ( $p<$ 0.0001, Fig. 1C). These results indicate that LHX3 expression is significantly elevated in HCC.

3.2 LHX3 expression is associated with clinical stage and metastasis of HCC patients

Based on quantified expression, LHX3 expression was classified into high (greater than and equal to the mean expression, $\geqslant$ 8.279) and low (less than the mean expression, $<$ 8.279) groups. After investigating correlations between LHX3 expression and clinico-pathologic features, LHX3 expression was highly associated with clinical stage ( $n=$ 186, $p=$ 0.009) and metastasis ( $n=$ 184, $p=$ 0.000) of HCC patients (Table 1). Whereas, LHX3 expression was not associated with age ( $p=$ 0.150), histological grade ( $p=$ 0.171), gender ( $p=$ 0.840) and tumor size ( $p=$ 0.273, Table 1). To further confirm the association, the changes of LHX3 expression were analyzed in the early-stage (I $+$ II) patients ( $n=$ 68) or advanced-stage (III $+$ IV) patients ( $n=$ 118) and the patients with ( $n=$ 166) or without ( $n=$ 18) metastasis. LHX3 expression was much higher in the advanced-stage patients than the early-stage patients ( $p=$ 0.0019, Fig. 2A and B), and notly LHX3 expression is sharply increased in the patients with metastasis ( $p=$ 0.0027, Fig. 2B). These data show that LHX3 is closely associated with clinical stage and metastasis of HCC patients.

Table 1
Correlations of LHX3 expression with clinicopathologic features in human HCC patients ( $n=$ 190)

		LHX3 expression
Clinical feature	Total	High	Low	Chi-	$P$ value
		( $n=$ 61)	( $n=$ 129)	square
Age (years)
$<$ 51	86	23	63	2.072	0.150
$\geqslant$ 51	104	38	66
Gender
Female	10	4	6	0.041	0.840 ${}^{\text{a}}$
Male	180	57	123
Metastasis
M0	166	44	122	15.873	0.000
M1	18	13	5
Histological grade
1 $+$ 2	134	39	95	1.878	0.171
3	56	22	34
Tumor size
$<$ 7	92	31	61	1.200	0.273
$\geqslant$ 7	70	18	52
Clinical stage
I $+$ II	68	18	50	6.757	0.009
III	94	26	68
IV	24	15	9

${}^{\text{a}}$ Continuity correction.

Figure 3.

High LHX3 expression is associated with poor OS of HCC patients. (A) High and low expression level of LHX3 as seen in representative HCC tissues. Low represents staining weak (the expression score $\leqslant$ 8). High represents staining strong (the expression score $>$ 8). (B) Kaplan-Meier and Cox regression survival analysis of LHX3 expression in 190 HCC patients. LHX3 is associated with poor OS of the patients, and is an independent prognostic factor in HCC. HR represents hazard ratio.

3.3 LHX3 is associated with HCV and CD34 status of HCC patients

We then analyzed the correlations of LHX3 expression with other maker expression or virus infection in HCC patients. LHX3 expression is clearly associated with hepatitis C virus (HCV, $p=$ 0.002) and cluster of differentiation-34 (CD34, $p=$ 0.043) expression status of HCC patients (Table 2). However, it was not associated with makers HEP1 ( $p=$ 0.077), C-ERBB-2 ( $p=$ 0.655), KI67 ( $p=$ 0.499), P53 ( $p=$ 0.413), HBCAG ( $p=$ 0.709), AFP ( $p=$ 0.319), CK8 ( $p=$ 0.877), CK19 ( $p=$ 0.843) and HBSAG ( $p=$ 0.648, Table 2). These results suggest that LHX3 expression is associated with HCV infection and CD34 expression status of HCC patients.

Table 2
Correlations of LHX3 expression with other maker expression in human patients ( $n=$ 144)

		LHX3 expression
Clinica	Total	High	Low	Chi-	$P$ value
feature		( $n=$ 42)	( $n=$ 102)	square
HBCAG
Negative	94	24	70	0.139	0.709
Positive	40	9	31
HCV
Negative	100	18	82	9.324	0.002
Positive	34	15	19
KI67
Negative	34	12	22	0.457	0.499
Positive	103	30	73
P53
Negative	48	9	39	0.669	0.413
Positive	80	20	60
C-ERBB-2
Negative	24	8	16	0.199	0.655
Positive	22	6	16
AFP
Negative	26	4	22	0.994	0.319
Positive	106	26	80
CK19
Negative	60	14	46	0.039	0.843 ${}^{\text{a}}$
Positive	22	4	18
HEP1
Negative	18	7	11	3.315	0.077 ${}^{\text{a}}$
Positive	78	13	65
CD34
Negative	16	0	16	4.106	0.043 ${}^{\text{a}}$
Positive	74	20	54
CK8
Negative	10	2	8	0.024	0.877 ${}^{\text{a}}$
Positive	64	18	46
HBSAG
Negative	58	14	44	0.208	0.648
Positive	76	21	55

${}^{\text{a}}$ Continuity correction.

3.4 High LHX3 expression is associated with poor OS of HCC patients

LHX3 expression was then analyzed with respect to survival data of the HCC patients. Kaplan-Meier analysis revealed a poor OS in the patients with high LHX3 expression compared to the patients with low LHX3 expression ( $p=$ 0.000, Fig. 3A and B). To correct for the bias, LHX3 expression as well as other parameters (gender, age, tumor size, histological grade and clinical stage) were analyzed by multivariate Cox-regression analysis. LHX3 was an independent prognostic factor (HR $=$ 1.964, $p=$ 0.004) for OS of the HCC patients (Fig. 3B, Table 3). These results show that LHX3 is an unfavorable and independent prognostic factor of HCC patients.

Table 3
Multivariate analysis of different prognostic factors in human HCC patients ( $n=$ 190)

Expression	Variable	HR	95% CI	$P$ value
level
LHX3 protein	Age	0.980	0.955–1.006	0.137
expression	Gender	0.286	0.065–1.251	0.096
	Histological grade	0.986	0.675–1.442	0.944
	Metastasis	1.618	0.672–3.892	0.283
	Clinical stage	1.164	0.795–1.706	0.434
	Tumor size	1.020	0.980–1.061	0.339
	LHX3 expression	1.964	1.244–3.100	0.004

Abbreviations: HR, hazard ratio; CI, confidence interval.

3.5 LHX3 is an advanced-stage prognostic factor of the HCC patients

The correlations between LHX3 expression and survival of the patients at early stages (I $+$ II) or advanced stages (III $+$ IV) were then evaluated. Survival analyses by Kaplan-Meier and Cox-regression indicated that high LHX3 expression was strikingly associated with the unfavorable OS ( $p=$ 0.000) and was an independent prognostic factor of HCC patients at advanced stages (HR $=$ 2.285, $p=$ 0.008), but not associated with OS of the patients at early stages ( $p=$ 0.167) (Fig. 4, Table 4). These data show that LHX3 is an advanced-stage prognostic biomarker of HCC patients.

Figure 4.

LHX3 is an advanced stage prognostic factor of the HCC patients. Kaplan-Meier and Cox regression survival analysis of LHX3 expression in 68 early stage (stage I $+$ II) or 118 advanced stage (stage III $+$ IV) HCC patients. LHX3 is associated with poor OS of the patients, and is an independent prognostic factor in advanced stage HCC. HR represents hazard ratio.

3.6 LHX3 stimulates cancer cell migration and invasion

LHX3 expression is associated with clinical stages and metastasis of HCC patients, indicating a role in tumor metastasis. To investigate the effects of LHX3 on HCC cell migration and invasion, we transfected LHX3 siRNA into HCC Huh7 cells with high LHX3 expression and performed the transwell assays with or without matrigel (Fig. 5A and B). The data showed that a significant decrease of cancer cell migration and invasion was found in LHX3 siRNA cells compared with negative cells (Fig. 5C). These data indicate that LHX3 strongly stimulates cancer cell migration and invasion.

Table 4
Multivariate analysis of different prognostic factors in advanced HCC patients ( $n=$ 118)

Expression	Variable	HR	95% CI	$P$ value
level
LHX3 protein	Age	0.977	0.939–1.016	0.236
expression	Gender	0.409	0.088–1.905	0.255
	Histological grade	1.081	0.625–1.870	0.781
	Metastasis	1.898	0.939–3.836	0.074
	Tumor size	1.029	0.987–1.073	0.172
	LHX3 expression	2.285	1.244–4.195	0.008

Abbreviations: HR, hazard ratio; CI, confidence interval.

Figure 5.

LHX3 knockdown inhibits cancer cell migration and invasion. (A) RT-PCR analysis of LHX3 mRNA expression in HCC cell lines and tissues. N represents normal tissue; T represents cancerous tissue. The $\beta$ -actin was used as internal control. (B) RT-PCR and WB analyses of LHX3 expression in LHX3 siRNA or Negative transfected Huh7 cells. LHX3 siRNA was purchased from Santa Cruz (sc-38712). $\beta$ -actin was used as internal control. (C) The cancer cell migration and invasion were measured by transwell assays without or with matrigel after transfection in Huh7 cells. The quantified cells were determined by the average count of at least five random microscopic fields. ${}^{**}$ , $p<$ 0.01. Error bars indicate s.d. ( $n=$ 5).

4. Discussion

In our present study, the clinical relevance and functional roles of LHX3 for the first time were investigated in HCC. LHX3 expression is generally increased in HCC tissues, and is associated with clinical stages and metastasis. LHX3 expression is an unfavorable and independent prognostic factor of patients in HCC. Moreover, LHX3 expression is an advanced-stage prognostic factor of HCC patients. Functionally, LHX3 strongly promotes HCC cell migration and invasion.

Previous studies have demonstrated that LHX genes are involved in tumorigenesis of different cancers [13, 14, 15, 16, 17, 18]. However, little is known about their clinical and prognostic relevance in these cancers. In this study, LHX3 expression is found to be closely associated with clinical stages and metastasis of HCC patients. To evaluate the prognostic significance of LHX3 expression on HCC patients, Survival analyses were performed by Kaplan-Meier and Cox-regression. The patients with low LHX3 expression have correspondingly prolonged OS compared to the patients with high LHX3 expression. Furthermore, LHX3 expression is an independent prognostic factor for OS in HCC patients. Noticeably, LHX3 is an advanced-stage prognostic factor in HCC patients. These data demonstrates that LHX3 expression is an important prognostic biomarker in HCC patients at advanced stages. It is worth noting that the number of cases in some tables is not consistent in the text, which is due to missing the clinico-pathologic features of some patients. In addition, this study here mainly focuses on the clinical and prognostic relevance of LHX3. So, we included the clinico-pathologic features, and excluded the factors of AFP level and the expressions of Ki67, P53, C-ERBB-2, CK19, HEP1, CK8, and CD34 in multivariate analysis.

Previous evidences have revealed that LHX3 plays key roles in the development of spinal cord motor neurons and pituitary [19, 20, 21, 22]. Recently, it is reported that the DNA methylation of LHX3 is closely related to breast cancer [23]. However, there is few functional study of LHX3 on tumorigenesis. In the present study, LHX3 expression is significantly elevated in cancer tissues. LHX3 expression is much higher in the advanced-stage patients than in the early-stage patients. Moreover, LHX3 expression is sharply increased in the patients with metastasis. These data suggest that LHX3 may be involved in HCC metastasis. So, gain/loss-of-function assays were investigated to determine the roles of LHX3 on tumor metastasis. LHX3 indeed significantly promotes cancer cell migration and invasion, revealing that LHX3 functions as a metastatic promoter in HCC. However, the effects of LHX3 on cancer cell proliferation, apoptosis, and cycle still remain unclear, and further studies are needed to solve this problem in HCC, as well as confirm its function in vivo.

It seems that LHX3 is expressed in the non-neoplastic stromal cells between HCC cells of stage III $+$ IV patients. It also seems that LHX3 is expressed in nuclei of HCC cells from stage I $+$ II patients, whereas it is expressed mainly in cytoplasm and weakly in nuclei of HCC cells from stage III $+$ IV patients. These issues are very interesting, and further studies are needed to explore these questions.

In conclusion, LHX3 expression is a potential advanced-stage biomarker for prognosis evaluation of patient, and LHX3 acts as a candidate metastatic promoter in HCC. However, further studies are needed be performed to explore the molecular mechanism of LHX3 in HCC.

Footnotes

Acknowledgments

This work was supported by the Natural Science Foundation of Chongqing (cstc2018jcyjAX0473), and the Natural Science Foundation of Chongqing (cstc 2015jcyjBX0060). The authors thank all the patients in this study.

Conflict of interest

None declared.

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LHX3 is an advanced-stage prognostic biomarker and metastatic oncogene in hepatocellular carcinoma

Abstract

BACKGROUND:

OBJECTIVE:

MATERIALS AND METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Patient samples and cell lines

2.2 Total RNA isolation

2.3 RT-PCR

2.4 RT-qPCR

2.5 Tissue microarray

2.7 LHX3 siRNA

2.8 Western blotting analysis

2.9 Migration and invasion assays

2.11 Statistical analysis

3. Results

3.1 Increased LHX3 expression is observed in HCC tissues

3.2 LHX3 expression is associated with clinical stage and metastasis of HCC patients

Table 1 Correlations of LHX3 expression with clinicopathologic features in human HCC patients ( n = 190)

Table 2 Correlations of LHX3 expression with other maker expression in human patients ( n = 144)

Table 3 Multivariate analysis of different prognostic factors in human HCC patients ( n = 190)

Table 4 Multivariate analysis of different prognostic factors in advanced HCC patients ( n = 118)

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Correlations of LHX3 expression with clinicopathologic features in human HCC patients ( $n=$ 190)

Table 2
Correlations of LHX3 expression with other maker expression in human patients ( $n=$ 144)

Table 3
Multivariate analysis of different prognostic factors in human HCC patients ( $n=$ 190)

Table 4
Multivariate analysis of different prognostic factors in advanced HCC patients ( $n=$ 118)