Correlation analysis of RBM3 expression in tumor stroma and prognosis of patients with non-small cell lung cancer

Objective: To investigate the correlation between the expression of RNA binding motif protein 3 (RBM3) in non-small cell lung cancer (NSCLC) pathological tissue and known risk factors. Methods: A retrospective analysis of the clinical and pathological features of 128 patients with NSCLC from August 1, 2014 to August 1, 2015 was performed. The expression of RBM3 and protein kinase B (AKT) in the tumor stroma was examined by immunohistochemistry and pathological enumeration, followed by analysis of its correlation with prognosis. The survival status of patients was followed up. The relationships between RBM3 and AKT protein expression in tumor stroma with overall survival (OS) and progression-free survival (PFS) were evaluated. Results: The expression of RBM3 was signicantly correlated with tumor differentiation ( P =0.012), lymph node metastasis ( P =0.02), T staging ( P =0.041), and AKT expression ( P =0.021). Univariate Kaplan-Meier analysis showed that high expression of RBM3, lymph node metastasis, T staging, and high expression of AKT were prognostic factors in NSCLC ( P <0.05). Multivariate analysis of Cox proportional hazard model showed that high RBM3 expression, lymph node metastasis, and high AKT expression were independent risk factors for prognosis ( P <0.05). Conclusion: RBM3, AKT, and lymph node metastasis are independent prognostic factors for NSCLC, signicantly affecting the prognosis of patients possibly through the classical signaling pathway AKT. RBM3 may be a prognostic treatment of NSCLC,


Background
Lung cancer is one of the most malignant tumors with the highest morbidity and mortality [1], which seriously endangers human health, of which non-small cell lung cancer (NSCLC) accounts for about 85% [2]. The main treatment methods include surgery, radiotherapy, chemotherapy, and combined chemotherapy with radiotherapy and chemotherapy. Molecularly targeted therapies are now being accepted. However, the 5-year survival rate of NSCLC is only 15%. Therefore, further study on the occurrence, development, and mechanism of lung cancer is the focus of current research.
In recent years, studies have demonstrated that the cold shock protein family plays an important role in the occurrence and development of tumors, including RNA binding motif protein 3 (RBM3). RBM3 is highly expressed in most human tumors [3,4], and the earliest gene-based approach clari ed the role of RBM3 as a tumor marker. The involvement of RBM3 in tumorigenesis and clinical outcome prediction has been extensively studied and has reached different conclusions in many cancers, including breast cancer, urothelial cancer, prostate cancer, colorectal cancer, and gastric cancer [5][6][7][8][9]. However, the role of RBM3 in NSCLC has not been reported. Besides, over-activation of protein kinase B (AKT) pathway was found to promote proliferation of lung cancer cells. This study focused on the analysis of the relationship between the expression of RBM3 in NSCLC pathological tissues and known high-risk factors (including AKT), providing a theoretical basis for the clinical treatment of NSCLC patients.

Clinical data
In this study, 128 non-small cell lung cancer (NSCLC) patients treated in the Department of Thoracic Surgery, Qingdao City Hospital from August 1, 2014 to August 1, 2015 were enrolled. All patients were followed up. None of the selected patients had any anti-tumor treatment before surgery. There were 34 males and 94 females, aged 42 to 75 years old. RNA binding motif protein 3 (RBM3) was highly expressed in 55 cases and lowly expressed in 73 cases. Staging was performed according to the 7th Edition tumor node metastasis (TNM) staging criteria from the American Joint Committee on Cancer (AJCC).

Specimen examination
Immunohistochemistry two-step method for detecting gene expression in lung cancer tissues All para n sections were made into 4-um pieces and then stained in strict accordance with the reagent instructions. The EDTA repair solution was used for antigen retrieval. Main reagents: rabbit anti-human RBM3 monoclonal antibody (working concentration: 1:100, purchased from Beijing Boaosen Biotechnology Co., Ltd.); mouse anti-human AKT monoclonal antibody (working concentration: 1:400, purchased from Abcam).

Immunohistochemical staining procedures
The negative control group was also immunohistochemically stained. The procedure was the same as the experimental group, and only the primary antibody was replaced with PBS buffer. The counting process was performed by two pathologists, and the tissue sections were counted separately by doubleblind method. When the results of the grouping were inconsistent, the third pathologist recounted and grouped. According to the result determination method, all the slices were grouped, and the obtained data was used for statistical analysis.

Result judgment
For RBM3 staining, 5 high-magni cation elds were randomly selected, and 100 cells were counted. The depth of coloration and the number of positive cells were determined. Staining intensity scores: 3 for sepia, 2 for brown, 1 for pale yellow, and 0 for colorless. Positive cell percentage scoring criteria: 25% of positive staining cells were the low expression, and >25% were the high expression. RBM3 immunostaining was independently evaluated by two pathologists in a blinded way.
For AKT staining, 5 high-magni cation elds were randomly selected, and 200 cells were counted. The staining intensity of cancer cells and the proportion of stained cells were comprehensively scored. The lm was read by a pathologist in a blinded way. Once the cytoplasm and/or nucleus were stained, it was judged to be a positive cell. Staining intensity: 3 points for sepia, 2 points for brown yellow, 1 point for pale yellow, 0 for unresponsive cells. The percentage of stained cells indicated: ≥50% was 3 points, <10% was 0 points, <50% was 2 points, <20% was 1 point. The staining intensity multiplied by the percentage of the stained cells was used to evaluate the AKT expression. The integral 0 to 2 and 3 to 9 were classi ed into negative and positive expression, respectively.

Statistical methods
Statistical analysis was performed using SPSS 21.0 software. Correlation comparisons were performed using a chi-square test. Survival analysis was performed using univariate Kaplan-Meier analysis and multivariate Cox regression analysis. The difference was statistically signi cant at P < 0.05.

Relationship between RBM3 expression and clinicopathological parameters
The results of immunohistochemical staining are shown in Figure 1.There were 31 cases of high expression of AKT and 24 cases of low expression of AKT in 55 patients with high expression of RBM3, and 47 cases of low expression of AKT and 26 cases of high expression of AKT in 73 cases of low RBM3 expression. The difference was statistically signi cant (P = 0.021), indicating that the expression of AKT in most patients with high expression of RBM3 was also highly expressed, and the expression of RBM3 was signi cantly correlated with the expression of AKT (Table 1). Therefore, RBM3 may promote the proliferation of lung cancer cells through the PI3K/AKT signaling pathway.

Multivariate analysis of RBM3 and OS, PFS
We then performed the multivariate analysis of Cox proportional hazard model and found signi cant differences in overall survival (OS) and progression-free survival (PFS) between patients with high expression of RBM3 and those with low expression of RBM3 (P = 0.000). In other words, patients with high RBM3 expression had a worse prognosis than those with low RBM3 expression, indicating that high expression of RBM3 is an independent risk factor for the prognosis of NSCLC, which was not affected by age, gender, lymph node metastasis, smoking or not, and the degree of differentiation.
There were signi cant differences in OS and PFS between patients with lymph node metastasis and those without lymph node metastasis (P = 0.000). Therefore, patients with lymph node metastasis had a worse prognosis than those with non-lymph node metastasis, suggesting that lymph node metastasis is an independent risk factor for NSCLC prognosis, which was not affected by age, gender, smoking or differentiation.
There were signi cant differences in OS (P = 0.003) and PFS (P = 0.005) between the patients with low AKT expression and those with high AKT expression. Patients with low AKT expression had a better prognosis when comparing to those with high AKT expression, indicating that high expression of AKT is also an independent risk factor for NSCLC prognosis.
Taken together, we found that high RBM3 expression, lymph node metastasis, and high AKT expression were independent risk factors for prognosis (Table 3).

Discussion
Lung cancer is one of the most common malignant tumors in China, ranking rst in cancer death worldwide [1]. Among them, NSCLC accounts for about 85% [2]. In the treatment of NSCLC, it can be surgically removed at an early stage, but approximately 70% of the patients are diagnosed as locally advanced or metastatic when initially diagnosed [10]. The prognosis of advanced NSCLC is extremely poor, with a median survival time of about 1 year and a 5-year survival rate of less than 20% [11][12][13][14]. In this study, we investigated the relationship between RBM3 expression in the pathological tissues of NSCLC patients and OS, PFS, and indicated that RBM3 may promote the proliferation of lung cancer cells via the PI3K/AKT signaling pathway. Therefore, it is possible to further understand or clarify the possible mechanisms of tumor development to nd more effective methods of diagnosis and treatment to improve the therapeutic effect of lung cancer.
RBM3 is a highly conserved cold-inducing RNA-binding protein that is one of three X-chromosomeassociated RBM genes (RBMX, RBM3, RBM10), localized to Xp11.23, and transcriptionally upregulated in stress reaction [15]. RBM3 is involved in the biological action of mRNA, stimulates protein synthesis, promotes cell proliferation, and exerts anti-apoptotic effects. RBM3 is expressed in various tissues such as the cerebellum, lung, spleen, small intestine, and uterus, and its expression is increased in response to cold shock. With the deepening of RBM3 research, the role of RBM3 protein in the occurrence and development of tumors has been paid more and more attention. More and more immunohistochemical studies have shown that RBM3 acts as a proto-oncogene and is associated with the progression and metastasis of various cancers [16]. The main reasons for RBM3 acting as a proto-oncogene were listed as follows. First, the expression level of RBM3 was related to the stage of the tumor, suggesting that it may be related to tumorigenesis. Second, RBM3 silencing increased the number of G2/M-phase cells, and ultimately led to apoptosis; G2 phase control by RBM3 was found in RBM3-silencing A2780 cells [17] and RBM3 knockout mice [18]. Finally, RBM3 can improve the stabilization and translation of mRNAs of cyclooxygenase-2, IL-8, and vascular endothelial growth factor [3,19,20]. Recently, some studies suggest that RBM3 may in uence tumor progression by altering the level of cellular microRNA control of globulin expression [21]. Although it has been con rmed that the carcinogenic factors of lung cancer include smoking, air pollution, environmental factors, ionizing radiation, ras, myc gene family, etc., there are few studies on the role of RBM3 in the occurrence, development, and mechanism of lung cancer. In this study, we found that the high expression of RBM3 was associated with a poor prognosis of NSCLC patients, making itself an independent prognostic marker for NSCLC.
AKT is a central mediator that inhibits the sensitivity of chemotherapeutic drugs that induce apoptosis. A growing number of experiments have shown that inhibition of Akt expression can block the PI3K/Akt classical signaling pathway to improve the treatment effect. In a recent study of nasopharyngeal carcinoma (NPC) [22], shRNA-mediated knockdown of RBM3 was found to reduce AKT phosphorylation, and direct interaction between RBM3 and PI3K was also detected.
Furthermore, they demonstrated that RBM3 was involved in NPC radioresistance via the PI3K/AKT/Bcl-2 signaling pathway. Therefore, RBM3 may be a new candidate for predicting the development of radioresistance and a potential therapeutic target for NPC. In this study, we also found that RBM3 might affect the prognosis of patients through the AKT signaling pathway.
The invasion and metastasis of malignant tumors is an extremely complex multi-step, multi-stage development process, including degradation of extracellular matrix, invasion, circulating diffusion, distant cloning, and angiogenesis. The expression of RBM3 was detected in 88 cases of benign and malignant prostate tumors by immunohistochemistry and tissue microarray and it was found that RBM3 was rarely expressed in the tissues of surgically resected benign bladder tumors but signi cantly increased in bladder malignant tumors, with higher expression in distant invasive bladder cancer [23]. These ndings indicate that RBM3 is highly expressed in bladder transitional cell carcinoma and is more closely related with distant metastasis than in situ invasiveness. In this study, RBM3 was highly expressed in NSCLC with lymph node metastasis, while in NSCLC without lymph node metastasis, RBM3 expression was low, also suggesting that RBM3 plays a role in distant metastasis of the malignant tumor.
In summary, the expression of RBM3 was signi cantly correlated with the degree of tumor differentiation, lymph node metastasis, T staging, and AKT expression. High RBM3 expression, high AKT expression, and lymph node metastasis were independent risk factors for the prognosis of NSCLC. RBM3 indirectly affects the survival prognosis of NSCLC by affecting the classical AKT signal transduction pathway and

Consent for publication
Written informed consent was obtained from all participants.
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Availability of data and material
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.  Figure 1 Immunohistochemical staining of RBM3 and AKT in lung cancer tissues.