CHD5 may be a promising novel bio-marker for the diagnosis of lung cancer patients

Chromodomain helicase DNA binding protein 5 (CHD5) is a new tumor suppressor gene in various types of cancer. And it is still not clear about the role of CHD5 in lung cancer. In this study, we aim to assess diagnostic value of CHD5 in patients with lung cancer. CHD5 expression in 108 lung cancer serum samples and 65 healthy controls were determined by quantitative Real-Time PCR (qRT-PCR). A receiver operating characteristic (ROC) curve was established to analyze the effect of CHD5 in the diagnosis of lung cancer. with of ROC analysis showed that the area under ROC curve was 0.855 with a sensitivity of 87.1% and a specicity of 81.3%.


Results
The expression of CHD5 was signi cantly decreased in lung cancer samples compared with the healthy controls (P < 0.0001). Advanced TNM stage (P = 0.020), gender (P = 0.001) and smoking history (P = 0.000) were associated with the decreased CHD5 expression. Besides, the results of ROC analysis showed that the area under ROC curve (AUC) was 0.855 with a sensitivity of 87.1% and a speci city of 81.3%.

Conclusions
In conclusion, this study suggested that CHD5 expression is down-regulated in lung cancer. Furthermore, CHD5 expression could be a potential diagnosis bio-marker in lung cancer patients.

Background
Lung cancer, a serious health-care problem, is one of the deadliest types of cancers in the world [1].
Although the molecular network of lung carcinogenesis at the levels of gene and protein has been studied in recent decades, the high mortality of lung cancer is not markedly changed. One of signi cant factors is diagnosis at late stage, since no obvious symptom was observed at early stage. Besides, due to the metastasis character the tumor often rapidly spreads to distant organs [2]. Although the recent advancements in imaging modalities have improved the detection and evaluation of lung cancer, these non-invasive methods do not provide de nitive con rmation of the disease. There is several limitations by using CT, positron emission tomography and X-ray for early diagnosis [3][4][5]. Therefore, it is urgent and necessary to nd novel non-invasive bio-markers for early diagnosis of lung cancer.
The chromodomain helicase DNA binding domain 5 (CHD5) gene, a member of Chromodomain (CHD) gene family, is known as a part of the lp36 chromosome [6]. The normal mechanism of cancer prevention will be cut off if the CHD5 not work. Like a circuit breaker, CHD5 regulates the tumor suppressor ability of our cells in our body, When it is disconnected, the tumor happens. As a nuclear protein, CHD5 may be di cult to target. However, CHD5 is usually silenced by promoter methylation, which is easy to be modi ed. The suppression of clonigenicity and tumorigenicity, as well as correlation with risk factors and outcome, suggested that CHD5 is a tumor suppressor gene [7][8][9][10]. CHD5 has been found in the breast, colon, ovarian and glioma tumors, revealed that CHD5 acted important roles in tumorigenesis and progression of several cancers [11][12][13]. Moreover, in a recent study, CHD5 is identi ed as a novel tumor suppressor gene in lung cancer [14], but the diagnostic role of CHD5 in lung cancer patients is still unclear.
In the current study, we sought to examine the expression level of CHD5 in lung cancer serum samples compared with normal controls. Besides the association between CHD5 expression and clinical features was analyzed. Furthermore, receiver operating characteristics (ROC) curves was generated for the diagnostic value of CHD5 in lung cancer patients.

Patients and specimens
In the current study, a total of 173 participants were recruited from The First A liated Hospital of Xinxiang Medical University, which included 108 lung cancer patients and 65 healthy controls. The inclusion criteria of lung cancer patients was no previous history of cancer-related diseases and did not receive radiotherapy or chemotherapy prior to research. The healthy control individuals were without tumor-associated lesions before the study. Total of 5ml peripheral blood samples from lung cancer patients and healthy controls were obtained and centrifuged and then stored at -80℃ for further experiments. This study was approved by the Ethical Committee of the hospital and all enrolled patients provided written informed consents prior to sample collection. The clinicopatholgical features for these patients were listed in Table 1. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from the serum samples using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions and the purity of RNAs was measured using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). After puri cation cDNA was synthesized using the First-Strand cDNA Synthesis Kit (ReverTra Ace-a, FSK-100, Toyobo, Osaka, Japan) according to the manufacture's protocol. The real-time PCR was conducted using the 7300 Real-Time PCR System (Applied Biosystems, USA). GAPDH was used as the internal control and each sample was examined in triplicate.

Statistical analysis
In the study, SPSS 21.0 (SPSS Inc., Chicago, IL, USA) software was used to perform all the statistical analyses and GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) software was used to generate all the graphs. The Student's t test was used to assess the difference of CHD5 expression level in lung cancer samples and healthy control samples. The relationship between CHD5 expression and clinicopathological characteristics of lung cancer patients was assessed by Chi-square test. The ROC curve was established to explore the diagnostic value of CHD5 in lung cancer patients. P value less than 0.05 was considered statistically signi cant.

Results
The expression of CHD5 was reduced in lung cancer patients The qRT-PCR was performed to evaluate the expression of CHD5 in lung cancer samples and healthy controls. The results demonstrated that the expression of CHD5 in lung cancer patients signi cantly reduced compared with that of the healthy controls (P < 0.05, Figure 1).

Association of CHD5 expression with clinicopathological features of lung cancer patients
To verify the relationship between CHD5 expression and development of lung cancer, we estimated the association of CHD5 expression with clinicopathological data of lung cancer patients using Chi-square test. The results in Table 1 revealed that the expression level of CHD5 was associated with advanced TNM stage (P=0.020), gender (P=0.001) and smoking history (P=0.000), but there was no signi cant differences with the age (P=0.332) and tumor size (P=0.316) ( Table 1).

Diagnostic accuracy of CHD5 in patients with lung cancer
In order to examine the diagnostic value of CHD5 in lung cancer patients ROC analysis was applied. The ROC curve analysis revealed that the area under the curve (AUC) was 0.855, with the sensitivity of 87.1% and speci city of 81.3%. The optimal cut-off point was 3.880. All these results above suggested that the CHD5 expression was closely correlated with the diagnosis of lung cancer ( Figure 2).

Discussion
As a major global health problem, lung cancer is a leading cause of cancer-related death worldwide, accounting for more than 1.4 million deaths per year [15]. Non-small-cell lung cancer (NSCLC) and smallcell lung cancer (SCLC) are two main histological subtypes of lung cancer [16]. One of the primary risk factors for lung cancer is smoking tobacco [17]. In spite of signi cant improvements in different kinds of therapeutic methods, the 5-year overall survival rate was low [18]. The 30% of patients were usually recommended adjuvant chemotherapy, who experienced relapse after surgery with stage I lung cancer [19]. Recent advances in the eld of lung cancer had showed that several biomarkers acted as noninvasive and cost-effective tools for early-stage prognosis of lung cancer [20]. Although the diagnosis of lung cancer is gradually improving, the detection of tumors at an early stage is still very poor.
Chromodomain (CHD) protein family play important functions in chromatin organization, regulation of transcription and cancer prevention [21]. As one of nine members of the CHD family, CHD5 has been con rmed as the tumor suppressor in various malignant tumors. For example, Ma et al. reported that the expression of CHD5 was signi cantly suppressed in breast cancer tissues and downregulation of CHD5 may contribute to the development and progression of breast cancer [22]. Liu et al. showed that CHD5 mRNA was positively correlated with protein expression and CHD5 gene methylation is associated with clinical and pathological features of colorectal cancer patients [23]. Moreover, in the study of Du et al., they found that CHD5 functioned as a novel tumor suppressor gene for renal cell carcinoma [24]. The tumor suppressor role of CHD5 is also found in other cancers including gastric cancer and neuroblastomas [25,26]. However, only few literatures have investigated the CHD5 gene in lung cancer. Zhao et al. have reported that CHD5 is down-regulated in lung cancer cell lines and primary tumor tissues, and it is a potential tumor suppressor gene in lung cancer [14]. The study of Baykara O et al. showed CHD5 is a potential tumor suppressor in non small cell lung cancer [27]. However, little is known about the diagnostic value of CHD5 in lung cancer patients.
In the current study, the expression of CHD5 in lung cance was decreased compared with the healthy controls. Besides, the Chi-square test was performed to examine the relationship between CHD5 expression level and clinicopathological features of lung cancer patients. The expression of CHD5 in lung cancer is signi cantly associated with TNM stage, gender and smoking history. The results of ROC analysis showed that the value of AUC was high as well as with high sensitivity and speci city. These results suggested CHD5 was a useful diagnostic biomarker for lung cancer patients.
Promoter hypermethylation is one mechanism for the transcriptional inactivation of many tumor suppressor genes. Methylation of the CHD5 gene promoter had been identi ed in several types of cancers [23,24,28]. Previous studies also found that down-regulation of CHD5 may be due to different epigenetic silencing mechanism possibly by miRNAs. Recently, Naraparaju et al. identi ed three different miRNA target regions in the 3'-UTR region of the CHD5 gene using two different prediction algorithms and they also con rmed seven miRNAs that signi cantly downregulated CHD5 expression in Neuroblastoma [29].
The above outcome provides insights for our future studies on the molecular mechanism of CHD5 in lung cancer.

Conclusions
In summary, the expression levels of CHD5 are down-regulated in lung cancer compare with the healthy controls. And CHD5 might serve as a novel biomarkers for early detection of lung cancer. Although all the data suggesting CHD5 may play a broader role in lung cancer, further studies are required to improve the diagnostic ability in lung cancer patients. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests. The relative expression of CHD5 in lung cancer samples and healthy controls. The expression of CHD5 with lung cancer was signi cantly lower than that in healthy controls (P<0.0001).