Maternal embryonic leucine zipper kinase (MELK), is an adenosine monophosphate-activated protein kinase-related kinase that serves important roles in tumourigenesis in multiple malignant tumours. However, to the best of our knowledge, the effect of MELK in lung adenocarcinoma (LUAD) has not been elucidated. The present study aimed to explore the clinical significance of MELK in the prognosis of LUAD. Data from Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA) and The Cancer Genome Atlas (TCGA) were selected to predict the differential mRNA expression levels of MELK mRNA in LUAD and normal tissues. Subsequently, LUAD and adjacent normal tissue samples were collected from 75 patients with the disease, and immunohistochemistry was used to detect the protein expression of MELK. In addition, the Kaplan-Meier Plotter database, GEPIA and TCGA were used to verify the effect of MELK expression on clinical prognosis in patients with LUAD. MELK was significantly upregulated in LUAD tissues compared with that in normal tissues based on Oncomine, GEPIA and TCGA data (P<0.05). In addition, the results from immunohistochemistry demonstrated that the MELK protein level in LUAD tissues was significantly higher compared with that in matched normal tissues (P<0.05). Prognostic analysis performed using the Kaplan-Meier plotter, GEPIA and TCGA suggested that the expression of MELK was negatively associated with the overall survival time of patients with LUAD (P<0.05). In conclusion, MELK was highly expressed in LUAD based on bioinformatics and immunohistochemistry analysis, and increased expression of MELK was associated with a poor patient prognosis. MELK may serve as a potential diagnostic marker and therapeutic target for LUAD.
Lung cancer is the most common malignancy and the leading cause of death among all types of cancer worldwide, with 11.6% of the total cancer and 18.4% of the total cancer deaths according to the Global Cancer Statistics 2018. Lung cancer contains multiple subtypes, such as small cell lung cancer (SCLC), squamous cell carcinoma and lung adenocarcinoma (LUAD). LUAD is the most common type of lung cancer (
Growing evidence has confirmed that the expression of certain genes to which the cancer has become ‘addicted’ or ‘dependent on’ is necessary for tumour growth (
Therefore, in the present study, the differential expression of MELK in patients with LUAD and its association with prognosis were assessed. Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA) and TCGA data were used to determine the levels of MELK mRNA in malignant tumour and normal lung tissues. In addition, immunohistochemical staining was used to detect the expression of MELK protein in LUAD and adjacent normal tissue. Kaplan-Meier plotter, GEPIA and TCGA survival analyses were conducted to compare the prognostic outcomes in patients with LUAD with different levels of MELK.
Data from the Oncomine (
A total of 75 cancer tissue and 35 normal adjacent tissue samples were obtained from patients diagnosed with LUAD who underwent surgical resection were recruited from Jiangxi Provincial People's Hospital (Nanchang, China) between September 2018 and August 2019. The distance between normal adjacent tissue and tumour tissue was 3 cm. The inclusion/exclusion criteria were as follows: i) Patients who underwent primary surgical resection and had histopathologically confirmed LUAD; ii) patients who were ≥18 years old, iii) patients who had no pulmonary metastases or other concomitant cancers; and iv) patients for whom informed consent was provided for the use of tissues. Detailed patient clinical information was collected retrospectively, including age (46 males and 29 females), sex (mean age, 61.6 years; range 40–83 years), tumour location, tumour size, histological differentiation grade, lymph node metastasis, and Tumour-Node-Metastasis (TNM) stage. All patients were staged according to the 8th edition of the TNM staging system for lung cancer (
Immunohistochemical staining was performed as described previously (
Oncomine, TCGA and GEPIA data were analysed using an unpaired Student's t-test for the comparison of 2 groups or ANOVA followed by a post hoc Tukey's honest significance difference (HSD) test for multiple comparisons conducted to compare the differential mRNA and protein expression of MELK. The patients' clinical data are presented as the mean ± S.E.M. SPSS 19.0 software (IBM Corp.) was used to analyse the clinical data using the unpaired t-test and χ2 tests as appropriate. Kaplan-Meier plotter, GEPIA and TCGA analyses were performed to assess the effect of MELK on the overall survival time of patients with LUAD. Log-rank P-values and HRs with 95% CIs were calculated and displayed on the webpage. P<0.05 was considered to indicate a statistically significant difference.
MELK expression in different cancer types was assessed using the Oncomine database (
A meta-analysis of 7 studies of MELK mRNA levels in LUAD was performed using the Oncomine database. As demonstrated, there was a higher level of MELK in LUAD tissues compared with that in normal tissues in different datasets (
Tumour and matched adjacent normal tissues were collected from 75 patients with LUAD to evaluate the protein expression of MELK. Morphological changes were observed between tumour and matched normal tissues from patients with LUAD using H&E staining. Matched normal tissues showed normal histological status and consolidation and less alveoli were presented in LUAD tissues (
Kaplan-Meier plotter, GEPIA and TCGA were used to analyse the prognosis of patients with LUAD with high and low MELK expression levels. The association between MELK mRNA level and OS in LUAD patients was analysed using the Kaplan-Meier plotter. The analysis demonstrated that high MELK expression was negatively associated with OS time (
MELK is a highly conserved serine/threonine kinase that was originally cloned from mice and is expressed in a wide range of early embryonic cellular stages (
The expression of MELK is negatively associated with the survival rate of patients with cancer (
MELK has been reported to participate in tumour progression via the JNK, p53, Bcl-G and forkhead box protein M1 (FOXM1) signalling pathways, which are all extremely important in multiple human cancer types (
The present study has several limitations. Firstly, only 75 LUAD samples were used and a larger sample size is needed for future research. Secondly, only bioinformatics analysis was used to calculate the survival rate of patients with LUAD with high and low expression of MELK and further studies are required to verify the findings of the present study and increase accuracy. Thirdly, the present study mainly discussed MELK expression and prognosis in patients with LUAD, and the detailed molecular mechanism of MELK involvement in LUAD was not explored. All these limitations need to be investigated in future studies.
In summary, the present study demonstrated that MELK is highly expressed in LUAD and that there is a negative association between MELK expression and prognosis in affected patients. MELK may serve as a potential diagnostic marker and therapeutic target in LUAD; however, more studies are needed to investigate the potential mechanisms of MELK in this disease.
Not applicable.
This study was supported by the Science and Technology Plan Project of Jiangxi Administration of Traditional Chinese Medicine, China (grant no. 2019A067).
The three datasets generated and/or analyzed during the current study are available in the Oncomine databases (
SC and ZX designed the study. ZL, XC, XW, HT and LY collected the data. SC, LY, and HT analysed the data. SC, LY and ZX drafted and revised the manuscript for important intellectual content. All authors have read and approved the manuscript.
The research was approved by the Ethical Committee of Jiangxi Provincial People's Hospital (Nanchang, China) (approval no. 2018070), informed consent was provided by the clinicians and obtained from the patients who provided written informed consent for the usage of the tissues for research.
Not applicable.
The authors declare that they have no competing interests.
lung adenocarcinoma
non-small cell lung cancer
maternal embryonic leucine zipper kinase
Gene Expression Profiling Interactive Analysis
The Cancer Genome Atlas
MELK expression in different cancer types was analysed with Oncomine. The expression of MELK in different cancers is summarized in the figure. The numbers of unique comparisons satisfying the thresholds (P<0.01; fold change ≥2; gene rank ≤10%; and data type, mRNA) are displayed in the coloured cells. Red denotes upregulation of MELK in tumour tissues compared with normal tissues, whereas blue indicates decreased expression of MELK in tumour tissues compared with normal tissues. The gene rank is depicted by the colour gradient in the cells. CNS, central nervous system; MELK, maternal embryonic leucine zipper kinase.
Levels of MELK expression in LUAD based on data from the Oncomine database. (A) Meta-analysis of the 7 analyses on MELK mRNA levels in LUAD identified via Oncomine. (B) mRNA expression of MELK in LUAD using Oncomine database. (a) MELK mRNA expression in LUAD in Hou Lung dataset. (b) MELK mRNA expression in LUAD in Landi Lung dataset. (c) MELK mRNA expression in LUAD in Okayama Lung dataset. (d) MELK mRNA expression in LUAD in Beer Lung dataset. (e) MELK mRNA expression in LUAD in Selamat Lung dataset. (f) MELK mRNA expression in LUAD in Stearman Lung dataset. (g) MELK mRNA expression in LUAD in Su Lung dataset. The X axis of the plot represents the normal vs. cancer group, and the Y axis represents log2-transformed, median/mean centred mRNA expression. The line in the middle represents the median value. LUAD, lung adenocarcinoma; MELK, maternal embryonic leucine zipper kinase.
Levels of MELK mRNA expression in LUAD based on TCGA and GEPIA databases. (A) MELK expression in LUAD in TCGA data. The X axis of the plot represents the normal vs. cancer group and the Y axis represents mRNA expression in transcripts per million. (B) MELK expression in patients with LUAD based on gender in TCGA data. The x-axis of the plot represents the normal vs. cancer group and the y-axis represents mRNA expression in transcripts per million. (C) MELK expression in different age groups of LUAD in TCGA data. The x-axis of the plot represents the normal vs. cancer group, and the y-axis represents mRNA expression in transcripts per million. (D) MELK expression in different N stages of LUAD in TCGA data. The x-axis of the plot represents the normal vs. cancer group and the y-axis represents mRNA expression in transcripts per million. (E) MELK expression in LUAD in GEPIA data. The x-axis of the plot represents the normal vs. cancer group and the y-axis represents mRNA expression in transcripts per million. (F) MELK expression in different stages of LUAD in the GEPIA database. The x-axis of the plot represents different stages of cancer and the y-axis shows mRNA expression in transcripts per million. Unpaired student's t-test or ANOVA with a post hoc Tukey's honest significant difference test was used to compare the differential expression of MELK mRNA in different groups as appropriate. *P<0.05. N, node; LUAD, lung adenocarcinoma; MELK, maternal embryonic leucine zipper kinase; TCGA, The Cancer Genome Atlas; GEPIA, Gene Expression Profiling Interactive Analysis.
Changes in the expression of MELK protein in tumour and normal tissues from patients with LUAD. (A) Haematoxylin and eosin staining of LUAD tissues (original magnifications, ×50 and ×200). (B) MELK expression in LUAD was detected by immunohistochemistry (original magnifications, ×50 and ×200). (C) Quantification of positive staining for MELK in LUAD and normal tissues. (D) High expression of MELK in LUAD (n=75). A Student's t-test was used to compare the expression of MELK between LUAD and normal tissues. *P<0.05 vs. non-cancer. LUAD, lung adenocarcinoma; MELK, maternal embryonic leucine zipper kinase.
OS rate of patients with LUAD with low and high MELK mRNA expression according to Kaplan-Meier plotter, GEPIA and TCGA analyses. (A) GEPIA analysis for OS in patients with LUAD with low and high levels of MELK expression. (B) Kaplan-Meier plots for OS in patients with LUAD according to MELK expression. (C and D) TCGA analysis for OS in patients with low and high levels of MELK expression. Samples with MELK expression values above the median value were categorized into the high MELK group and those with MELK expression values below the median value were categorized into the low MELK group. The red line represents patients with high MELK expression and the blue and black lines represent patients with low MELK expression. The x-axis represents the number of patients at risk at a specific time (in months or days) and the y-axis represents OS time (Kaplan-Meier plotter and GEPIA for moths, TCGA for days). Log-rank P-values and HR with 95% CI were calculated and displayed on the webpage. LUAD, lung adenocarcinoma; MELK, maternal embryonic leucine zipper kinase; TCGA, The Cancer Genome Atlas; GEPIA, Gene Expression Profiling Interactive Analysis; CI, confidence interval; HR, hazard ratio; OS, overall survival.
Association between MELK protein expression and clinicopathological features in patients with lung adenocarcinoma (n=75).
MELK levels, n (%) | ||||
---|---|---|---|---|
Clinicopathological features | Patients, n (%) | Low | High | P-value |
Age, years | >0.999 | |||
<60 | 32 (42.7) | 7 (38.9) | 25 (43.9) | |
≥60 | 43 (57.3) | 11 (61.1) | 32 (56.1) | |
Sex | 0.257 | |||
Male | 46 (61.3) | 9 (50.0) | 37 (64.9) | |
Female | 29 (38.7) | 9 (50.0) | 20 (35.1) | |
Tumour location | 0.480 | |||
Left | 28 (37.8) | 7 (41.2) | 21 (36.8) | |
Right | 46 (62.2) | 10 (58.8) | 36 (63.2) | |
Tumour size, cm | 0.611 | |||
<5 | 31 (59.6) | 9 (60.0) | 22 (59.5) | |
≥5 | 21 (40.4) | 6 (40.0) | 15 (40.5) | |
Histological differentiation | 0.088 | |||
No | 2 (5.0) | 1 (7.7) | 1 (3.7) | |
Low | 16 (40.0) | 2 (15.4) | 14 (51.9) | |
Moderate | 14 (35.0) | 5 (38.5) | 9 (33.3) | |
High | 8 (20.0) | 5 (38.5) | 3 (11.1) | |
T stage | 0.057 | |||
T1-2 | 30 (55.6) | 12 (75.0) | 18 (47.4) | |
T3-4 | 24 (44.4) | 4 (25.0) | 20 (52.6) | |
N stage | 0.382 | |||
N0 | 26 (50.0) | 9 (56.3) | 17 (47.2) | |
N1-3 | 26 (50.0) | 7 (43.8) | 19 (52.8%) | |
M stage | 0.032 |
|||
M0 | 37 (50.7) | 13 (72.2) | 24 (43.) | |
M1 | 36 (49.3) | 5 (27.8) | 31 (56.4) | |
TNM stage | 0.045 |
|||
I–II | 25 (62.5) | 11 (84.6) | 14 (51.9) | |
III–IV | 15 (37.5) | 2 (15.4) | 13 (48.1) |
P<0.05. The χ2 test was used to analyse the data. There was missing data in tumour location, tumour size, histological differentiation, T stage, N stage, M stage, and TNM stage. MELK, maternal embryonic leucine zipper kinase; TNM, Tumour-Node-Metastasis; T, tumour; N, node; M, metastasis.