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SIRT3 acts as a novel biomarker for the diagnosis of lung cancer

Abstract Lung cancer (LC) is a prevalent malignancy worldwide with increased morbidity and mortality. Mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase sirtuin-3 (SIRT3) has been reported to be involved in tumorigenesis. In this retrospective study, we measured the expression and diagnostic value of SIRT3 in LC patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure serum SIRT3 mRNA level in 150 LC patients and 52 healthy volunteers. SIRT3 protein level was detected using western blot for 84 pairs of LC and adjacent normal tissues. The association of SIRT3 mRNA level with clinical parameters of LC patients was estimated via chi-square test. Receiver operating characteristic curve (ROC) was plotted to evaluate the diagnostic performance of serum SIRT3 in LC patients. SIRT3 mRNA and protein levels were significantly decreased in LC tissues and serum samples, compared with corresponding controls (P < .05). Moreover, the expression of SIRT3 mRNA was negatively associated with tumor size (P = .002), tumor node metastasis stage (P < .001), and metastasis (P < .001). ROC curve demonstrated that serum SIRT3 could distinguish LC patients from healthy individuals, with an area under the curve of 0.918. The optimal cutoff value was 3.12, reaching a sensitivity of 86.4%, and a specificity of 94%. SIRT3 expression is significantly down-regulated in LC serum and tissues. SIRT3 may be employed as a promising biomarker in the early diagnosis of LC.


Introduction
Lung cancer (LC) imposes a great threat to human health because of its high morbidity and mortality. [1] According to incomplete statistics, LC-related deaths account for nearly 29% of total cancer-related deaths annually. [2][3][4] The 5-year survival rate of LC patients is less than 15%. [5,6] Early diagnosis is key for the survival of LC patients. Unfortunately, symptoms in early LC are mild, even without any discomfort. Most of the patients are diagnosed at advanced stages, missing the optimal opportunity for surgical treatment. [6,7] Thus, it is urgent to explore effective diagnostic biomarkers for LC.
Mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase sirtuin-3 (SIRT3) [8] is a well-known tumor suppressor and participates in multiple metabolic processes through deacetylating downstream protein substrates. [9] The dysregulation of SIRT3 has been reported in multiple diseases, such as cancers, cardiovascular diseases, and diabetes. [10] SIRT3 is a member of the sirtuin family (SIRT1-7) whose members function as metabolic sensors to connect their enzyme activities with cell energy and redox state through NAD + cofactor. [11,12] Sirtuin family plays important roles in a variety of cellular processes, such as oxidative stress, genomic stability, apoptosis, and protein and fatty acid metabolism. [13,14] Human SIRT3 gene locates on chromosome 11p15.5, and encodes a 44KD-protein. [15,16] SIRT3 has been reported to exert inhibitory effects against many diseases, but its diagnostic value in LC is rarely reported.
In the present study, we aimed to detect the expression and diagnostic value of serum SIRT3 in LC patients.

Study design and setting
The present study was a retrospective investigation. A total of 150 LC patients and 52 healthy individuals were enrolled in the study from May, 2015 to May, 2017. Blood samples were collected from all participants, while LC and adjacent normal tissues were randomly obtained from 84 LC patients.

Participants
The patients in the current study must meet the following criteria: 1) with an age of ≥18 years; 2) pathologically diagnosed as primary LC without other malignancies; 3) experiencing no antitumor treatments, either chemotherapy, surgery, radiotherapy, or targeted treatments; and 4) with available blood specimens and clinical records. With any one of the following conditions, cases would be excluded: 1) suffering recurrent LC; 2) having benign lung diseases; and 3) with severe heart, liver and/or kidney diseases, inflammation diseases, history of asthma, and recent respiratory infections.
According to the tumor node metastasis (TNM) cancer staging system (2009) set by the Union of International Cancer Control, tumors were classified. Of all LC patients, 84 experienced surgery operation and their diseased tissues and adjacent normal tissues (4 cm away from cancer lesions) were obtained. In addition, 52 healthy volunteers were recruited as healthy controls. The healthy controls did not show any abnormalities in physical examinations and had no malignancy history or lung diseases. Moreover, the healthy controls were matched with LC patients in age and gender. Blood specimens (5 mL venous blood) were collected from LC patients on the day of diagnosis before accepting any surgery or therapy. Basic data of the patients were recorded, including age, sex, tumor size, diseased region, pathologic types, TNM stage, and metastasis status.

Serum preparation and the extraction of RNA
Serum specimen was separated from whole blood via centrifugation. Neither clinical nor pathological data were disclosed during detection. Total RNAs were isolated from serum and tissue samples using TRIzol (Life Technologies, Carlsbad, CA, USA). Only RNA samples with an OD A260/A280 ratio close to 2.0 were used in subsequent analysis.

Quantitative real-time polymerase chain reaction
This process proceeded in a blinded way. Reverse transcription was conducted with 100 ng RNA using PrimeScript RT (Takara, Dalian, China), according to the manufacturer's instructions. Then, quantitative real-time polymerase chain reaction (qRT-PCR) reaction was completed adopting Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems, Foster City, California, USA). GAPDH was employed as the internal reference. The relative expression of SIRT3 was normalized to GAPDH and calculated using 2 ÀDDCt method. Each sample was checked in triplicate.

Western blot
Total proteins were extracted from tissue samples using the One Step Animal Tissue Active Protein Extraction Kit (Sangon, Shanghai). Then, the proteins were lysed and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto PVC membrane. Next, the membranes were blocked, applying TBST with 5% nonfat milk at 4°C for 1 night. The primary antibody anti-SIRT3 was diluted with block solution to proper concentration and then incubated at 4°C overnight. Then, a secondary antibody was added and incubated for 2 h at 4°C. ECL Western blotting kit (Millipore, Boston, MA, USA) was used to visualize the target band of SIRT3. Each test had 3 repeats.

Ethics approval
This study was approved by the Ethics Committee of The First Affiliated Hospital of Jiaxing University (Ethical reference number: 2017KY0402) in October, 2017. All the patients or their families signed written informed consent before sample collection.

Statistical analysis
All statistical analyses were performed using Origin Pro 9.0 software. Statistical power was calculated using G * Power software, and statistical power was fine when obtained values ≥0.8. Continuous data were summarized as mean ± SD. Differences in SIRT3 mRNA and protein levels between the 2 groups were analyzed using Student t test. Chi-square test was adopted to estimate the relationship between serum SIRT3 expression and clinical characteristics of LC cases. To determine the diagnostic value of serum SIRT3 in LC, the receiver operating characteristic (ROC) curve was plotted, and relevant results were estimated using the area under the curve (AUC) combining with sensitivity and specificity. P values <.05 indicated the statistical significance of the results.

The expression level of SIRT3
In our study, serum samples were collected from 150 LC patients and 52 healthy controls. The paired LC and adjacent normal tissues were collected from 84 LC patients receiving surgical operations. qRT-PCR was used to estimate SIRT3 mRNA expression in serum and tissue specimens. Meanwhile, the protein expression of SIRT3 was detected via western blot. As shown in Figure 1A, serum level of SIRT3 was significantly lower in LC patients than in healthy controls (P < .01). The expression level of SIRT3 was significantly higher in LC patients at stage I-II than in stage III-IV LC patients (P < .01, Fig. 1B). In metastatic patients, SIRT3 expression level was significantly decreased, compared with non-metastatic patients (P < .01, Fig. 1C). Similarly, the expression level of SIRT3 mRNA was also obviously lower in LC tissues than in adjacent normal tissues (2.054 ± 0.937 vs 4.077 ± 0.952, P < .05, Fig. 1D). Western blot analysis demonstrated that the expression of SIRT3 protein was weak in LC tissues, compared with adjacent normal tissues (Fig. 1E). Based on the expression patterns of SIRT3 mRNA in LC and healthy groups, the statistical power of study subjects was 1.00, revealing high credibility of our analysis.

Relationship between SIRT3 and clinicopathological characteristics in LC
Clinicopathological characteristics of LC patients were shown in Table 1. Chi-square test showed that SIRT3 mRNA level was negatively associated with tumor size (P = .001), TNM stage (P < .001), and metastasis in LC patients (P < .001). However, there was no significant association between SIRT3 expression  Table 1).

Diagnostic value of SIRT3 in LC
ROC curve showed that SIRT3 expression could distinguish LC patients from healthy controls with a sensitivity and specificity of 86.4% and 94%, respectively. Meanwhile, the AUC was 0.918 ( Figure 2). The cutoff value for serum SIRT3 mRNA diagnosing LC was 3.12.

Discussion
Early diagnosis and timely treatment are very important for the prognosis of LC. [17,18] Currently, the diagnosis of LC mainly depends on histology and iconography, resulting in its delay. [19] Sensitive and efficient biomarkers are in urgent need to facilitate the early diagnosis of LC. In the current study, we estimated the diagnostic significance of serum SIRT3 in LC. The results suggested that the down-regulation of SIRT3 showed a negative association with tumor size, TNM stage, and metastasis. Serum SIRT3 level might be a potential biomarker for the non-invasive detection of LC. SIRT3, a member of the sirtuin family, plays a critical role in cancers. [20,21] Accumulating evidence have demonstrated that SIRT3 plays a regulatory role in mitochondria, including metabolism, ATP production, responses to oxidative stress, and apoptosis. [22][23][24] Previous studies have reported that the Table 1 The expression of SIRT3 and clinicopathological features in LC patients.  www.md-journal.com expression of SIRT3 was decreased in human lung adenocarcinoma tissues, and that SIRT3 exhibited an anti-tumor effect on lung adenocarcinoma cells. [25] However, the potential role of SIRT3 in LC diagnosis is rarely known. In our study, we found that the expression level of SIRT3 was significantly lower in LC patients than in healthy volunteers. The results were in line with previous studies. For example, Zhang et al claimed that the expression of SIRT3 was significantly lower in hepatocellular carcinoma tissues than in normal liver tissues and cirrhotic tissues. [26] Finley et al found SIRT3 expression was reduced in human breast cancer and its overexpression repressed glycolysis and proliferation in breast cancer cells. [27] In the study of Xiao et al, the expression of SIRT3 showed a down-regulated trend in lung adenocarcinoma cells compared with normal cells. [25] But some contrary results were also reported. The study performed by Xiong et al showed that SIRT3 protein and mRNA levels were significantly increased in non-small cell lung cancer. [28] Yang et al found that the expression of SIRT3 in non-small cell lung cancer tissues was obviously higher than that in normal tissues. [29] The controversy may be attributed to different pathological types. Across pathological types of cancers, SIRT3 might trigger different key proteins and signaling pathways, thus initiating oncogenic or anti-tumor actions. [28] In addition, different causes for cancers might also contribute to the discrepancy. Reportedly, SIRT3 might act as an oncogene through enhancing reactive oxygen species resistance of cancer cells. However, under stress conditions, SIRT3 could induce cell apoptosis, thus achieving anti-tumor action. [30] SIRT3 plays dual roles in tumorigenesis.
In addition, we evaluated the relationship between SIRT3 expression and clinicopathological features of LC patients. The results showed that SIRT3 mRNA expression was significantly correlated with tumor size, TNM stage, and metastasis in LC patients, which suggested that SIRT3 might be involved in LC progression. Moreover, we also investigated the diagnostic value of SIRT3 in LC. ROC curve demonstrated that the expression of SIRT3 could distinguish LC patients from healthy individuals. SIRT3 might be an indicator in the early diagnosis of LC. The present research showed the detailed expression levels of SIRT3 mRNA in both tissues and serum for patients at different grades, as well as the protein level in tissues. Besides, the ROC curve presented a high diagnostic value of SIRT3 in LC patients.
However, several shortcomings in the current study should be stated. Firstly, the retrospective design might cause bias in sample collection. Secondly, all individuals in the control group were healthy persons, and the expression of SIRT3 in cases with benign lung diseases or other malignancies remained unclear. Whether the expression of SIRT3 was specific to LC was not determined. In other words, whether serum SIRT3 could distinguish LC patients from other cancer cases or benign lung disease sufferers was not decided. Thirdly, the protein level of SIRT3 was only detected adopting western blot analysis, and immunohistochemistry was necessary to verify obtained results. Besides, due to lacking follow-up information, the clinical value of SIRT3 for prognosis evaluation among LC patients was not explored. Therefore, further investigations are required to verify and improve our findings.
In conclusion, SIRT3 acts as a tumor suppressor in LC and maybe an auxiliary biomarker in early diagnosis of LC.