Prognostic significance of NDRG2 combined with EGFR patients with lung adenocarcinoma

Background: N-myc downstream-regulated gene 2 (NDRG2) plays a substantial role in lung adenocarcinoma (LUAD). Epidermal growth factor receptor (EGFR) mutation could significantly improve prognosis in patients with LUAD. In this study, we aimed to elucidate the prognostic value of NDRG2/EGFR in patients with LUAD. Methods: Immunohistochemistry, western blotting, and real-time polymerase chain reaction (RT-PCR) were conducted to detect the expression levels of NDRG2 protein. Associations between NDRG2/EGFR expression and clinicopathological characteristics of patients with LUAD were examined as well. Serum levels of carcinoembryonic antigen (CEA) were tested prior to treatments. Patients’ overall survival (OS) was assessed by the Kaplan-Meier method. Multivariate Cox regression analysis was carried out to investigate the effects of patients’ demographic characteristics on overall survival . Results: The expression of NDRG2 was significantly decreased in patients with LUAD. The expression of NDRG2 was positively correlated with the levels of CEA and EGFR. Advanced stages were significantly associated with low expression of NDRG2. We found that the patients in the NDRG2-high/EGFR(+) group had the best outcomes, while the patients in the NDRG2-low/EGFR(-) group had the worst outcomes. Cox regression analysis showed that NDRG2-low/EGFR(+), NDRG2-high/EGFR(+), and vascular invasion were independent prognostic factors of LUAD. Conclusion: NDRG2 and EGFR should be considered in patients with LUAD. Total The sulfate-polyacrylamide transferred mouse Cell at overnight after for β-actin was used internal The membranes washed secondary The blots were visualized using enhanced kit according manufacturer’s instructions. Each experiment was performed in triplicate.

chi-square test or Fisher's exact test. The differences in means between groups were analyzed using the Mann-Whitney U-test or Student's t-test. Associations between two variables were quantified using Spearman's rank correlation coefficient. The OS rate was evaluated using the Kaplan-Meier method, and differences between the groups were assessed using the log-rank test. Multivariable analysis was conducted using Cox's proportional hazards regression model to investigate the effects of patients' demographic characteristics on OS. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.

Results
NDRG2 was downregulated in LUAD, SOCS1, and CyclinD3 compared with normal tissues NDRG2 protein was mainly found in the cytoplasm, and a weak expression could be found in a limited Relationship between the expression of NDRG2 and clinicopathological features of patients with LUAD As shown in Figure 4, the expression level of NDRG2 was associated with CEA (P < 0.001). Table 2, the expression level of NDRG2 was notably higher in LUAD tissues in stages I-II than that in stage III-IV (P<0.001). In addition, the frequencies of no vascular invasion and EGFR positivity (+) were significantly higher in patients with high expression of NDRG2 than that in patients with low expression of NDRG2 (P<0.001 and 0.001, respectively). There were no associations between expression levels of NDRG2 and other clinicopathological features, including age, sex, smoking history, and blood type (P>0.05).

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Regarding the 34 patients who underwent surgery, the expression level of NDRG2 was significantly higher in stage I-II than that in stage III-IV (P=0.028). The frequencies of no vascular invasion and research, we reported the EGFR mutation status in patients with adenocarcinoma and its correlation with the expression level of NDRG2. Mutant EGFR expression was positively correlated with higher OS in the presence of a high expression level of NDRG2.
MYC and EGFR have been identified as potential biomarkers that can predict the efficacy of targeted therapy [26,27]. We, herein, analyzed the prognostic value of the combined detection of NDRG2 and EGFR for LUAD. We found that the patients in the NDRG2-high/EGFR(+) group had the best outcome, while the patients in the NDRG2-low/EGFR(-) group had the worst. Cox regression analysis revealed that NDRG2-low/EGFR(+), NDRG2-high/EGFR(+), and vascular invasion were independent prognostic factors of OS (Table 3).
This study has several limitations. First, it was a retrospective study performed at a single center.
Second, the sample size was very limited. The present study, in itself, cannot be used to change clinical practice and to propose the use of a new test. Those results have to be validated using a large sample size. Hence, further study is required to explore the putative association between NDRG2/EGFR level and OS in patients with NSCLS.

Conclusions
In summary, this study reported the different expression levels of NDRG2 in patients with LUAD. In addition, for the first time, the relationship between NDRG2/EGFR expression and clinicopathological characteristics of patients with LUAD, especially prognosis status, was investigated. NDRG2/EGFR can be used as a novel prognostic biomarker for patients with LUAD.    Figure 2 The expression level of NDRG2 in LUAD patients and normal tissues (Figure 2A-E).
Immunohistochemistry showed the expression level of NDRG2 in normal lung tissues ( Figure   2A), LUAD patients ( Figure 2B), and negative control ( Figure 2C) (200× magnification). The expression level of NDRG2 protein was determined by western blot assay ( Figure 2D) and RT-PCR ( Figure 2E). It was significantly downregulated in patients with LUAD compared with that in normal tissues at both protein and mRNA levels (*P < 0.001). Abbreviations: NDRG2, N-Myc downstream-regulated gene 2; LUAD, lung adenocarcinoma; RT-PCR, reverse transcription-polymerase chain reaction. Associations between the expression levels of NDRG2 and CEA levels of patients with LUAD.
The CEA levels were notably lower in patients with a high expression level of NDRG2 than that in patients with the low expression level of NDRG2 (P < 0.001). Abbreviations: CEA, carcinoembryonic antigen; NDRG2, N-Myc downstream-regulated gene 2; LUAD, lung adenocarcinoma.

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