The diagnostic performance of serum LIM homeobox transcription factor 1 alpha in patients with gastric cancer.

The study was conducted to investigate the diagnostic performance of serum LIM homeobox transcription factor 1 alpha (LMX1A) in patients with gastric cancer (GC).The serum level of LMX1A in GC, benign, and healthy groups was measured using quantitative real time PCR (qRT-PCR) and compared with the student t test. The associations of serum LMX1A levels with clinical parameters were analyzed with chi-square test. The diagnostic value of serum LMX1A in GC was evaluated by receiver operating characteristic (ROC) curve.The level of serum LMX1A in GC group (1.309 ± 0.553) was significantly lower than that in the benign group (2.174 ± 0.676) and healthy group (2.598 ± 0.826) (P < .01 for both). The decreased level of LMX1A was associated with large tumor size (P = .009), positive lymph node metastasis (P = .027), and advanced TNM stages (P = .002). Receiver operating characteristic (ROC) analysis demonstrated that serum LMX1A could discriminate GC patients from the healthy individuals, with the area under the curve (AUC) of 0.889 (95% confidence interval [CI] = 0.838-0.938) combining with the sensitivity and specificity of 82.68% and 82.61%. Additionally, serum LMX1A also exhibited high accuracy in discriminating between GC patients and benign gastric disease cases (AUC = 0.842, 95% CI = 0.782-0.901), with the sensitivity of 81.89% and specificity of 72.41%.Serum LMX1A may be an effective biomarker for early detection of GC.

Introduction

Gastric cancer (GC), originating from gastric mucosal epithelium, is one of the most prevalent malignancies all over the world, especially in the East Asia countries.[ GC represents a primary reason for cancer-related deaths worldwide, posing a severe threat to human health.[ There are various available therapeutic strategies for GC patients, including surgery, chemotherapy, radiotherapy, and other treatments. However, the prognosis of the patients remains unsatisfactory, with high mortality.[ Delay in early diagnosis may be responsible for the dismal outcomes.[ Unfortunately, many patients do not present specific symptoms until advanced stage, consequently limited therapeutic effects, and dismal survival.[ Currently, the early screening of GC mainly dependents on endoscopy and biopsy, but their application value is limited by the high cost and invasive operations.[ Additionally, the commonly used serum biomarkers, such as carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), and carbohydrate antigen 72-4 (CA72-4), exhibit unsatisfactory sensitivity and specificity for GC diagnosis.[ Therefore, it is in urgent need to discover novel and noninvasive biomarkers for early diagnosis of GC to improve the prognosis.

LIM homeobox transcription factor 1 alpha (LMX1A) is mapped to human chromosome 1q24.1, and consists of 11 exons with the length of about 151 kb.[ As a member of the LIM homeobox-containing family, LMX1A contains a homeodomain and 2 LIM domains.[LMX1A plays important roles in various biological progresses, such as cell proliferation, apoptosis, differentiation, and neurogenesis.[ Recently, growing evidences have reported that LMX1A may be involved in tumorigenesis. Dysregulation of LMX1A was observed in diverse types of cancer, such as cervical cancer and ovarian cancer.[ It was reported that LMX1A was down-regulated in malignancy, and played inhibitory roles in multisteps of tumorigenesis. Thus, we speculated that LMX1A might be a candidate biomarker for early detection of GC.

In this study, we aimed to investigate the clinical significance of serum LMX1A in GC. The expression pattern of LMX1A in GC was detected, as well as its association with clinical parameters of the patients. In addition, the diagnostic performance of serum LMX1A for GC was evaluated in the present study.

Materials and methods

Patients and specimens

A total of 127 patients, who were pathologically diagnosed with GC by 2 independent pathologists in The First Affiliated Hospital of Jinzhou Medical University were recruited in the study. All the patients were aged from 25 to 79 years old, including 62 men and 65 women. Other clinical features were listed in Table 1. In addition, 58 patients with benign gastric diseases and 69 healthy blood donors in the same hospital were enrolled in our study. All patients, both the GC and benign cases, had not received chemotherapy, radiotherapy, or other treatments before blood collection. Five milliliter peripheral blood was collected from each participant. Then the serum specimens were prepared from the peripheral blood through centrifugation. This study was supported by the Ethics Committee of The First Affiliated Hospital of Jinzhou Medical University. Written informed consent was obtained from each participant.

Table 1

Relationship between serum LMX1A expression and clinical characteristics in GC.

RNA extraction and quantitative real time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from serum samples of GC patients, benign gastric disease patients, and healthy controls using Trizol reagent (Invitrogen) following the manufacture's instructions. Then the first strand of cDNA was constructed through the reverse transcription using PrimerScript RT reagent kit (Takara, Dalian, China), and the extracted RNA samples served as temple. Finally, the real-time PCR was performed to determine the relative expression of serum LMX1A mRNA. The reaction was performed with SYBR-Green PCR Master Mix (Applied Biosystems, Foster City, CA) on ABI Prism 7900 Sequence Detection System (Applied Biosystems). GAPDH was adopted as the internal reference. The primer sequences were as follows: GAPDH forward, 5′-ATGGGGAAGGTGAAGGTCGG-3′; reverse, 5′-GACGGTGCCATGGAATTTGC-3′. LMX1A forward, 5′-CCCTCAGTAACCTGGGTGATTGT-3′; reverse, 5′-TCTTCCCTGGCCTCCCTGTCCTA-3′. Each sample was repeated in 3 times, and the relative expression of LMX1A was calculated by 2−ΔΔCt method.

Statistical analysis

All data were analyzed by SPSS version 18.0 (SPSS Inc., Chicago, IL) and GraphPad Prism 5.0 software (GraphPad, San Diego, CA). The expression value of serum LMX1A was presented as mean ± standard deviation (SD), and compared by student t test. Chi-square test was conducted to analyze the relationships between LMX1A expression and clinical features of patents. Receiver operating characteristic (ROC) curve was used to assess the diagnostic value of serum LMX1A in GC with the area under the curve (AUC). P value <.05 was considered significant.

Results

Down-regulation of serum LMX1A mRNA in GC

To investigate the level of serum LMX1A mRNA in GC patients, begin gastric disease patients and healthy controls, qRT-PCR was conducted. As shown in Fig. 1, compared with the healthy individuals, LMX1A mRNA level was down-regulated in GC group (P < .0001). Moreover, the serum level of LMX1A was significantly different between GC patients and benign gastric diseases cases (P < .01). GC patients showed decreased serum levels of LMX1A mRNA (Fig. 1).

Figure 1

The expression of serum LMX1A mRNA in GC patients, benign gastric diseases patients, and healthy controls. Serum levels of LMX1A were lower in GC cases than that in the healthy individuals. Furthermore, compared with benign gastric disease patients, GC cases showed down-regulated serum LMX1A level. ∗∗: indicated P < .01; ∗∗∗: suggested P < .001. GC = gastric cancer, LMX1A = LIM homeobox transcription factor 1 alpha.

Relationship of serum LMX1A expression and clinical characteristics of patients

To explore the association of serum LMX1A level with clinical parameters, patients were divided into low LMX1A group (n = 64) and high LMX1A group (n = 63) based on the median LMX1A expression. Chi-square test revealed that serum LMX1A expression exhibited negative association with tumor size (P = .009), lymph node metastasis (P = .027), and TNM stage (P = .002). However, there was no significant association between serum LMX1A expression and age, sex, alcohol abuse, or smoking status (P > .05 for all) (Table 1).

Diagnostic significance of serum LMX1A in GC

To evaluate the diagnostic performance of serum LMX1A in GC, ROC curves were constructed. As shown in Fig. 2, with the benign gastric disease population as reference, the AUC value was 0.842 (95% CI = 0.782–0.901), indicating serum LMX1A could discriminate GC patients from those with benign gastric disease. The optimal cutoff point was 1.725, with the sensitivity and specificity of 81.89% and 72.41%, respectively. Additionally, with the healthy individuals as reference, the AUC value of the curve was 0.889, suggesting serum LMX1A was a candidate for selecting GC patients from healthy population. The optimal cutoff point was 1.755, with the specificity of 82.61% and sensitivity of 82.68% (Fig. 3).

Figure 2

ROC curve was constructed based on serum LMX1A levels in GC patients and benign gastric disease cases. The curve demonstrated that serum LMX1A could discriminate between GC and benign gastric disease with the AUC value of 0.842 (95% CI = 0.782–0.901), combining with the sensitivity and specificity of 81.89% and 72.41%, respectively. The optimal cutoff point was 1.725. AUC = area under the curve, GC = gastric cancer, LMX1A = LIM homeobox transcription factor 1 alpha, ROC = receiver operating characteristic.

Figure 3

The accuracy of serum LMX1A in discriminating between GC cases and healthy individuals. The AUC value of the curve was 0.889, suggesting serum LMX1A might be a candidate biomarker for screening of GC. The optimal cutoff point was 1.755, with the specificity of 82.61% and sensitivity of 82.68%. AUC = area under the curve, GC = gastric cancer, LMX1A = LIM homeobox transcription factor 1 alpha.

Discussion

GC is one of the most common malignant tumors in our countries. Several risk factors may contribute to its occurrence, such as chronic gastric diseases, dysplasia of gastric mucosa epithelial, and Helicobacter pylori (H pylori) infection.[ Radical surgery is the only curative method for GC patients.[ However, the majority of GC patents are at advanced stages when initially diagnosed, missing the operation opportunity. Tumor stage at diagnosis is the key factor for outcomes of patients with GC. Until now, the commonly used detection tools showed unsatisfactory performance for early diagnosis of GC. Therefore, it is necessary to find novel diagnostic biomarkers with high sensitivity and specificity for GC.

The development and progression of GC is a complex process which is implicated with the activation of oncogenes and inactivation of tumor suppressor genes.[ The molecular biomarkers which may provide new insights into the mechanisms of GC are considered as novel and promising candidate biomarkers for early diagnosis, progression prediction, and targeted therapy in management of malignancy. In the previous studies, various genetic biomarkers were confirmed for GC. He et al,[ reported that the expression of Tspan 5 was significantly down-regulated in GC, moreover, its expression patterns showed inverse association with clinical characteristics that might be a potential independent biomarker for prognosis of the patients. The research carried out by Kong et al[ demonstrated that long non-coding RNA PVT1 as a tumor oncogene was involved in malignant progression of GC, and its over-expression predicted poor outcomes of the patients. The altered genes during tumorigenesis can provide accurate information for tumor development and progression which may be candidate biomarkers for human cancers.

LMX1A, a tumor suppressor, is a newly discovered transcription factor, which is implicated in cell proliferation, differentiation, apoptosis, embryonic development, and organ formation. The abnormal behavior of LMX1A has been found in GC with down-regulated expression, indicating LMX1A might be related with the development and progression of GC.[ Thereby, in the present study, we explored the significance of serum LMX1A in the diagnosis of GC. We found that serum LMX1A mRNA level was significantly decreased in GC patients compared with that in healthy controls and benign gastric diseases. Furthermore, down-regulation of LMX1A showed close relation with larger tumor size, positive lymph node metastasis, and advanced TNM stage. All the data indicated that LMX1A played inhibitory roles in aggressive development and progression of GC, revealing its function as a tumor suppressor in the disease.

Given its function in tumorigenesis, we hypothesized that serum LMX1A might serve as a biomarker in GC diagnosis. ROC analysis showed that serum LMX1A could discriminate GC patients from healthy controls and individuals with benign gastric diseases with high accuracy. Serum LMX1A was an efficient biomarker for early detection of GC. Although we had confirmed the low expression of serum LMX1A in GC and identified its diagnostic role in this disease, the accurate inhibitory mechanism of LMX1A in GC was not known yet. The inactivation of LMX1A in GC might be mediated hypermethylation.[ In the next study, the mechanisms of anti-tumor action of LMX1A in GC needed to be identified. Besides, our results might be limited by the relatively small sample size in the current study. Thus, further researches were still required to investigate the application value of serum LMX1A for GC diagnosis in clinical setting.

In conclusion, serum LMX1A is significantly decreased in GC patients compared with patients with benign gastric diseases and healthy controls. Moreover, its down-regulation is obviously correlated with large tumor size, positive lymph node metastasis, and advanced TNM stage. Serum LMX1A may be a potential biomarker for GC diagnosis.

Author contributions

Conceptualization: Dinuo Li.

Data curation: Dinuo Li.

Formal analysis: Dinuo Li, Chen Li.

Funding acquisition: Dinuo Li, Chen Li.

Investigation: Dinuo Li, Chen Li.

Methodology: Dinuo Li, Chen Li.

Project administration: Dinuo Li, Chen Li.

Resources: Dinuo Li, Chen Li.

Software: Dinuo Li, Chen Li.

Supervision: Dinuo Li, Chen Li.

Validation: Dinuo Li, Chen Li.

Visualization: Dinuo Li, Chen Li.

Writing – original draft: Dinuo Li, Chen Li.

Writing – review & editing: Dinuo Li, Chen Li.