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Abstract

OBJECTIVE:

The tripartite motif-containing protein (TRIM) family is involved in important biological processes such as the cell cycle, cell apoptosis, and innate immunity of virus. This study aimed to investigate TRIM66 expression and its predictive role in non-small cell lung cancer (NSCLC) patients.

METHODS:

We detected the expression levels of TRIM66 protein and TRIM66 mRNA in NSCLC tissues, and evaluated the prognostic role of TRIM66 in NSCLC.

RESULTS:

TRIMM66 was highly expressed in NSCLC tissues compared with normal paracancerous tissues ( $P=$ 0.001). The high TRIM66 expression closely associated with lymph node metastasis and TNM stage in NSCLC patients ( $P<$ 0.05). Kaplan-Meier survival model indicated that survival time of NSCLC patients in the high TRIM66 expression group were markedly lower than those in the low expression group ( $P<$ 0.05). Cox regression analysis showed that high expression of TRIM66 is associated with poor prognosis in NSCLC patients.

CONCLUSION:

TRIM66 can be serve as an important molecular marker for predicting the prognosis in NSCLC patients.

Keywords

Non-small cell lung cancer TRIM66 expression prognosis

1. Introduction

Lung cancer is associated with highest morbidity and mortality among all cancers, with approximately 1.2 million annual deaths worldwide [1]. Non-small cell lung cancer (NSCLC) accounts for over 80% of the total lung cancer cases. Most NSCLC patients are diagnosed at an advanced stage, and the postoperative 5-year survival rate for early NSCLC patients is low [2]. Therefore, investigating the pathogenesis of NSCLC and identifying new therapeutic targets for NSCLC are current research focuses.

The tripartite motif-containing protein (TRIM) family is involved in important biological processes such as the cell cycle, cell apoptosis, and innate immunity of virus [3, 4, 5]. In addition, TRIM is associated with the genesis and development of diseases, including tumorigenesis. TRIM family proteins can affect the sensitivity of tumor cells to chemotherapeutics [6]. TRIM protein 66 (TRIM66) is a member of the TRIM family [7], which is not only a participant in regulating protein expression but also in the malignant behavior of tumor cells. To our knowledge, TRIM66 is abnormally expressed in multiple malignant tumor tissues. However, studies on the correlation between TRIM66 expression and NSCLC are lacking. In this study, we determined the expression of TRIM66 in NSCLC tissues to preliminarily probe its association with the genesis, development, and malignancy grade of NSCLC.

2. Materials and methods

2.1 Patients

NSCLC and para-carcinoma tissue specimens were obtained from 130 patients (75 male and 55 female) who had undergone surgical resection in The First Hospital of Lanzhou University from May 2008 to August 2012 and with pathologic diagnosis of NSCLC. Complete clinical information was available for all patients; the average age of patients was 54.2 $\pm$ 8.5 years (range: 38–75 years). Patients were classified according to the tumor node metastasis (TNM) classification system formulated jointly by American Joint Committee on Cancer (AJCC) and Union for International Cancer Control (UICC) after surgery [8]. None of the 130 NSCLC patients had received chemotherapy, radiotherapy, or biotherapy before surgery. All patients or their family members had signed the informed consent form. This study was approved by the ethics committee of The First Hospital of Lanzhou University.

2.2 Immunohistochemistry

The streptavidin-peroxidase (SP) method was adop-ted for immunohistochemical analysis. Paraffin specimens were cut into slices (4 $\mu$ m thickness), which were mounted on slides, baked, deparaffinized, and hydrated following conventional methods; 3% hydrogen peroxide was used to block endogenous peroxidase. All procedures were performed in strict accordance with the instructions of kit. Rabbit anti-TRIM66 antibody (Proteintech; 1:100, ProteinTech Group, Inc. Chicago, USA) was added dropwise; next, after washing thoroughly with phosphate buffer saline (PBS), secondary antibody (ZsBio; Zhongshan Golden Bridge Biotechnology, Beijing, China) was added dropwise. The obtained mixture was incubated at 37 ${}^{\circ}$ C for 10 min and washed three times with PBS. Subsequently, horseradish enzyme-labeled streptavidin was added followed by an incubation at 37 ${}^{\circ}$ C for 10 min and three times of washing with PBS. The color development reagent 3,3’-diaminobenzidine was added; color development was controlled by observation using microscopy. Finally, the reaction was terminated using distilled water.

Single-blind review of sections was adopted in which the pathologists did not know of the corresponding clinical data. The Allred score was used by pathologist during evaluation of immunohistochemical staining [9]. Staining intensity was classified as follows: 0 point, no staining; 1 point, faint yellow staining; 2 points, claybank staining; and 3 points, brown staining. The proportion of nuclear positive cells was classified as 0–100%. In this study, the product of nuclear staining intensity and proportion of nuclear positive cells greater than 0 was considered as indicative of positive TRIM66 expression; otherwise, it was TRIM66 negative. Thus, TRIM66 staining intensities of 0 or 1 point and 2 or 3 points were deemed low and high expression, respectively. Immunohistochemical staining were evaluated with a double-headed microscope (Olympus, Tokyo, Japan).

Figure 1.

Immunohistochemistry staining of TRIMM66 in NSCLC tissues and adjacent normal tissues. (A and B) lung squamous cell carcinoma; (C and D) lung adenocarcinoma; (E and F) normal adjacent lung tissues. (A, C and E) the TRIM66 located in the nuclei of tumor cells; (B, D and F) the TRIM66 located in the cytoplasm of tumor cells. Abbreviations: NSCLC, nonsmall cell lung cancer.

Figure 2.

The TRIM66 mRNA was detected by RT-PCR. Abbreviations: T, tumor tissues; N, normal tissues; RT-PCR, Reverse transcription-polymerase chain reaction.

2.3 Reverse transcription-polymerase chain reaction (RT-PCR)

RNA was extracted in strict accordance with TRIzol (Life Technologies Corporation, Carlsbad, CA) instructions. RT of sample mRNA into cDNA was conducted, followed by PCR amplification. The reaction conditions were as follows: pre-degeneration at 94 ${}^{\circ}$ C for 5 min, degeneration at 94 ${}^{\circ}$ C for 30 s, annealing at 56.2 ${}^{\circ}$ C for 30 s (annealing temperature of internal reference $\beta$ -actin was 65 ${}^{\circ}$ C), and extension at 72 ${}^{\circ}$ C for 45 s for 35 loops, followed by extension at 72 ${}^{\circ}$ C for 2 min. Agarose gel electrophoresis (1.5%–2.0% agarose gels) was performed using a mixture of 6 $\mu$ L of target gene PCR product and 6 $\mu$ L of internal reference PCR product as samples. A gel-imaging system was used to measure the gray intensity of the electrophoretic bands, and the internal reference of each sample was set as 1. The ratio of the gray value of the TRIM66 mRNA band to the internal reference $\beta$ -actin band was treated as the expression quantity of TRIM66 mRNA. Sequences of PCR primer are as follows: forward primer 5 $\prime$ -GCCCTCTGTGCTACTTACTC-3 $\prime$ , reverse primer 5 $\prime$ -GCTGGTTGTGGGTTACTCTC-3 $\prime$ .

2.4 Statistical methods

By using $\chi^{2}$ testing, the relationship between TRIM66 expression and clinical pathological parameters of patients with NSCLC were analyzed. The Kaplan-Meier method and log-rank testing were applied to conduct survival analysis of the 130 patients with NSCLC. Disease-free survival (DFS) and overall survival (OS) were the primary outcomes of this study. The Cox proportional hazard model was used to analyze the joint effects of various variables. SPSS 19.0 statistical software (SPSS Inc., Chicago, USA) was used for statistical analysis. If $P<$ 0.05, the difference was considered statistically significant.

3. Results

3.1 TRIMM66 expression in NSCLC tissues

Immunohistochemical results indicated that TRIM66 was mainly located in the nuclei of tumor cells, but TRIM66 staining could also be found in the cytoplasm of a few tumor cells (Fig. 1). Of the 130 NSCLC tissue samples, 75 showed high TRIM66 expression, which was remarkably higher than that found in para-carcinoma tissues (61.2% vs. 33.1%; Pearson $\chi^{2}=$ 11.508, $P=$ 0.001). To further verify the results as mentioned earlier, 40 NSCLC tissues and adjacent normal tissues were randomly selected, and the expression level of TRIM66 mRNA was detected using RT-PCR. It was discovered that the expression level of TRIM66 mRNA in NSCLC tissues was notably higher than in corresponding normal lung tissues ( $P<$ 0.001, Fig. 2).

3.2 TRIM11 expression and clinicopathological features of NSCLC patients

Based on the immunohistochemical results, 130 cases of NSCLC patients were classified into the low or high TRIM66 expression groups. Simultaneously, the correlation between TRIM66 expression and clinicopathological characteristics of NSCLC patients was analyzed. The high TRIM66 expression closely associated with lymph node metastasis and TNM stage in NSCLC patients ( $P<$ 0.05); however, it was not related to age, sex, smoking history, pathological type, tumor size, and tumor differentiation of NSCLC patients ( $P>$ 0.05). Above results were summarized in Table 1.

Table 1
Correlation of clinicopathologic variables with TRIM66 in NSCLC

Variables	N	TRIM66 expression		$\chi^{2}$	$P$ -value
		Low	High
Age (years)				0.665	0.415
$<$ 60	75	34	41
$\geqslant$ 60	55	21	34
Sex				0.009	0.923
Male	75	32	43
Female	55	23	32
Smoking history				2.224	0.136
Yes	61	30	31
No	69	25	44
Histological type				0.368	0.544
SCC	74	33	41
AD	56	22	34
Differentiation				1.850	0.397
Well	28	9	19
Moderate	63	27	36
Poor	39	19	20
Tumor size (cm)				0.005	0.945
$\leqslant$ 3	61	26	35
$>$ 3	69	29	40
Lymph node metastasis				6.864	0.009
N0	56	31	25
N1 $+$ N2	74	24	50
TNM stage				5.464	0.019
Stage I	40	23	17
Stage II $+$ III	90	32	58

Abbreviations: SCC, squamous cell carcinoma; AD, adenocarcinoma; NSCLC, non-small cell lung cancer; TNM, tumor node metastasis.

Figure 3.

Kaplan-Meier survival curves for disease-free survival of NSCLC patients based on TRIM66 expression.

Figure 4.

Kaplan-Meier survival curves for overall survival of NSCLC patients based on TRIM66 expression.

Figure 5.

Kaplan-Meier survival curves for overall survival of NSCLC patients based on lymph node status.

3.3 Prognostic value of TRIM66 in NSCLC

Table 2
DFS and OS of NSCLC patients according to TRIM66 expression

	N	Survival time (months)		$P$ -value
		Mean $\pm$ SE	95% Cl
DFS				0.001
Low expression	55	47.36 $\pm$ 3.45	40.60–54.13
High expression	75	32.48 $\pm$ 2.65	27.29–37.67
OS				0.003
Low expression	55	53.76 $\pm$ 2.84	48.20–59.32
High expression	75	42.28 $\pm$ 2.52	37.35–47.22

Abbreviations: NSCLC, non-small cell lung cancer; DFS, disease-free survival; OS, overall survival; Cl, confidence interval; SE, standard error.

Table 3

Univariate and multivariate analysis for overall survival

Variables	Univariate analyses		Multivariate analysis
	HR (95% Cl)	$P$ -value	HR (95% Cl)	$P$ -value
Age	1.16 (0.73–1.82)	0.532	1.03 (0.64–1.66)	0.910
Sex	1.10 (0.70–1.73)	0.679	1.24 (0.77–1.99)	0.376
Smoking	1.19 (0.76–1.86)	0.453	1.30 (0.80–2.10)	0.289
Histology	1.27 (0.81–2.00)	0.294	1.28 (0.80–2.06)	0.300
Differentiation	0.82 (0.59–1.14)	0.237	0.91 (0.64–1.29)	0.598
Tumor size	0.88 (0.56–1.38)	0.584	0.78 (0.48–1.25)	0.301
Lymph node metastasis	2.75 (1.69–4.48)	$<$ 0.001	1.74 (1.02–3.33)	0.044
TNM stage	3.06 (1.68–5.56)	$<$ 0.001	2.00 (1.03–4.41)	0.035
TRIM66 expression	2.04 (1.26–3.32)	0.004	1.80 (1.06–3.05)	0.029

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

Long-term follow-up was conducted in all NSCLC patients to probe the relation between TRIM66 expression and prognosis for NSCLC patients. The survival curve of the 130 NSCLC patients was drawn at the end of follow-up. Kaplan-Meier survival model indicated that DFS and OS of NSCLC patients in the high TRIM66 expression group were markedly lower than those in the low expression group ( $P<$ 0.05, Table 2). Please see Figs 3 and 4. Moreover, the prognosis for NSCLC patients with lymph node metastases was worse ( $P<$ 0.05, Fig. 5). The Cox proportional hazard model was utilized to analyze each variable that affected OS of NSCLC patients. The results of the univariate analysis suggested that the prognosis in NSCLC patients was closely associated with TRIM66 expression ( $P=$ 0.004, Table 3). The data revealed that advanced TNM stage, lymphovascular invasion, lymph node etastases, and high TRIM66 expression were associated with poorer prognosis in NSCLC patients. Multivariate analysis results suggested that high TRIM66 expression was an independent predictor of prognosis in NSCLC patients, which was similar to the prognosis associated with accompanying lymph node metastasis or more advanced TNM stage. Table 3.

4. Discussion

TRIM family proteins possess three domains: RING finger, 1 or 2 B-box, and 1 coiled-coil region. Of them, B-box represents its characteristic domain, while coiled-coil region mainly plays a connective role [10]. Presently, 70 TRIM family members have been successively discovered [10, 11]. Structures of TRIM C-terminal differ from each other, which makes it possible for TRIM to specifically bind to different substrates [10, 12]. TRIM family proteins are further divided into 11 subfamilies, according to different C-terminal domains of TRIM [13, 14]. Most TRIM proteins mainly function as a ubiquitin E3 ligase, while a few TRIM proteins can serve as SUMO E3 ligase or other ubiquitin-like E3 ligases [15, 16]. Some TRIM proteins do not depend on their RING finger domains while functioning [17]. TRIM proteins are related to multiple human diseases, such as developmental disorders, neurodegenerative diseases, viral infections, and cancer [4, 15, 18, 19, 20, 21, 22, 23]. An increasing number of studies have found that TRIM family proteins are related to the genesis, development, and prognosis of tumors.

Abnormal TRIM expression is closely related to the genesis and development of NSCLC. For instance, expression of TRIM29 is upregulated in NSCLC tissues, and high TRIM29 expression indicates poorer prognosis in NSCLC patients [24]. In human lung squamous carcinoma NCI-H520 cells, silencing TRIM29 expression through siRNA contributes to inhibiting the proliferation and invasion of tumor cells [25]. Zhan et al. discovered that TRIM59 could be highly expressed in multiple lung cancer cell lines, and the knockout of TRIM59 gene affected the expression of cyclin (including CDC25C and CDK1), thus promoting the proliferation and migration of lung cancer cells [26]. Expression of TRIM44 was upregulated in lung cancer A549 and H441 cells, which could remarkably enhance the migration and invasion abilities of tumor cells, as was suggested in a series of experiments carried out by Luo et al. [27]. In addition, they also found that TRIM44 could upregulate the expression of CXCL16 and MMP9 through activating the NF- $\kappa$ B pathway, thus promoting NSCLC development [27]. TRIM11 is verified to promote growth, migration, and invasion of lung cancer cells through the AKT pathway in both in vivo and in vitro studies [28]. Overexpression of TRIM11 suggests a poorer prognosis in NSCLC patients. However, study findings regarding the relation between TRIM66 and NSCLC are lacking.

It was also discovered that, compared with normal human bronchial epithelial cells, expression of TRIM66 is upregulated in all NSCLC cell lines [26]. However, this finding was based on an in vitro study, and the expression of TRIM66 in vivo remains unknown. Taking the previously stated reasons into consideration, expression of TRIM66 in NSCLC tissue is detected by an immunohistochemical method. We observed that TRIM66 expression was mainly limited to the nucleus of NSCLC tumor cells. Moreover, the results of statistical analysis reconfirmed the high expression of TRIM66 in NSCLC tumor cells. Secondly, RT-PCR results also indicated that TRIM66 mRNA content in NSCLC tissue was notably higher than in para-carcinoma tissue. Further, we also found that TRIM66 expression level was closely associated with clinicopathological characteristics such as lymph node metastasis and TNM stage, suggesting that TRIM66 may promote the growth, proliferation, and invasion of NSCLC tumor cells. A long-term follow-up of 130 NSCLC patients was carried out; the results of which demonstrated that NSCLC patients with high TRIM66 expression have shorter survival time than those with low TRIM66 expression. The multivariate analysis showed that high TRIM66 expression is one of the important indicators for independently predicting a poorer prognosis in NSCLC patients. We can speculate, based on these results, that TRIM66 expression is closely associated with the genesis and development of NSCLC and the malignancy grade of NSCLC may be positively correlated with TRIM66 expression.

Taken together, our findings suggest that high TRIM66 expression indicates poorer prognosis in NSCLC patients, and thus, TRIM66 can be serve as an important molecular marker for predicting the prognosis in NSCLC patients. However, this study is limited by its design as a clinical observational study, and the precise mechanism concerning TRIM66 remains unclear, which will require further investigation in future studies. A foundation for the further investigation of the molecular mechanism of TRIM has been established in this study. We believe that blocking TRIM-related signal transduction pathway contributes to achieving the goals of delaying tumor growth, proliferation, invasion, and metastasis.

Conflict of interest

The authors report no conflicts of interest in this work.

Funding statement

This study was funded by grants from Section Development Fund.

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