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Expression of pre-selected TMEMs with predicted ER localization as potential classifiers of ccRCC tumors

View Article: PubMed Central - PubMed

ABSTRACT

Background: VHL inactivation is the most established molecular characteristic of clear cell renal cell carcinoma (ccRCC), with only a few additional genes implicated in development of this kidney tumor. In recently published ccRCC gene expression meta-analysis study we identified a number of deregulated genes with limited information available concerning their biological role, represented by gene transcripts belonging to transmembrane proteins family (TMEMs). TMEMs are predicted to be components of cellular membranes, such as mitochondrial membranes, ER, lysosomes and Golgi apparatus. Interestingly, the function of majority of TMEMs remains unclear. Here, we analyzed expression of ten TMEM genes in the context of ccRCC progression and development, and characterized these proteins bioinformatically.

Methods: The expression of ten TMEMs (RTP3, SLC35G2, TMEM30B, TMEM45A, TMEM45B, TMEM61, TMEM72, TMEM116, TMEM207 and TMEM213) was measured by qPCR. T-test, Pearson correlation, univariate and multivariate logistic and Cox regression were used in statistical analysis. The topology of studied proteins was predicted with Metaserver, together with PSORTII, Pfam and Localizome tools.

Results: We observed significant deregulation of expression of 10 analyzed TMEMs in ccRCC tumors. Cluster analysis of expression data suggested the down-regulation of all tested TMEMs to be a descriptor of the most advanced tumors. Logistic and Cox regression potentially linked TMEM expression to clinical parameters such as: metastasis, Fuhrman grade and overall survival. Topology predictions classified majority of analyzed TMEMs as type 3 and type 1 transmembrane proteins, with predicted localization mainly in ER.

Conclusions: The massive down-regulation of expression of TMEM family members suggests their importance in the pathogenesis of ccRCC and the bioinformatic analysis of TMEM topology implies a significant involvement of ER proteins in ccRCC pathology.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-015-1530-4) contains supplementary material, which is available to authorized users.

No MeSH data available.


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Differential expression of TMEMs in tumor tissue. (a) A comparison of TMEM expression between a tumor tissue and healthy kidney tissue. (b) TMEM expression in metastatic and non-metastatic tumors. (c) TMEM expression in organ-confined and advanced stage tumors (d) TMEM expression in low Fuhrman grade and high Fuhrman grade samples. Average log2 relative expression data in each sample group ± standard error of mean is shown in each chart. FC – fold-change. n – number of samples. T – tumor tissue samples. C – healthy tissue samples. M0 – non-metastatic ccRCC. M1 – metastatic ccRCC tissues. pT1/pT2 – organ-confined tumors, as assessed by TNM staging system. pT3/pT4 – advanced tumors, as assessed by TNM staging system. G1/G2 – low Fuhrman grade samples. G3/G4 – high Furhman grade samples. * - q < 0.05. ** - q < 0.01. *** - q < 0.001. **** - q < 0.0001
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Fig1: Differential expression of TMEMs in tumor tissue. (a) A comparison of TMEM expression between a tumor tissue and healthy kidney tissue. (b) TMEM expression in metastatic and non-metastatic tumors. (c) TMEM expression in organ-confined and advanced stage tumors (d) TMEM expression in low Fuhrman grade and high Fuhrman grade samples. Average log2 relative expression data in each sample group ± standard error of mean is shown in each chart. FC – fold-change. n – number of samples. T – tumor tissue samples. C – healthy tissue samples. M0 – non-metastatic ccRCC. M1 – metastatic ccRCC tissues. pT1/pT2 – organ-confined tumors, as assessed by TNM staging system. pT3/pT4 – advanced tumors, as assessed by TNM staging system. G1/G2 – low Fuhrman grade samples. G3/G4 – high Furhman grade samples. * - q < 0.05. ** - q < 0.01. *** - q < 0.001. **** - q < 0.0001

Mentions: In line with our previous observations, we detected statistically significant differences in expression of all ten TMEMs in tumor tissue and healthy kidney tissue (Fig. 1a) [20]. The down-regulation varied between the genes, with 1758.34 fold for TMEM213 (q < 0.0001) and 3.06 fold for TMEM116 (q < 0.05). Both, SLC35G2 and TMEM45A were found to be up-regulated with 5.33 (q < 0.01) and 6.75 fold change (q < 0.0001), respectively. We investigated if analyzed TMEMs displayed differential expression in metastatic and non-metastatic tumors. As shown in Fig. 1b, we detected significant difference in expression of TMEM72 and TMEM116: 39.78 fold and 6.65 fold down-regulation in metastatic tissue (n = 12 and n = 14, respectively) as compared to 5.13 fold and 2.57 fold in the non-metastatic samples (n = 58, q = 0.051 and n = 62, q = 0.056, respectively). Next, we subdivided samples into organ-confined tumors (pT ≤ 2) and advanced tumors (pT ≥ 3). A decrease of TMEM30B (q = 0.106) and TMEM45B (q = 0.097) expression was found in advanced-stage samples, with −2.21 and −2.71 fold-change, as compared to early-stage tumors, respectively (Fig. 1c). T-test was used to compare TMEM expression in well differentiated tumors (Fuhrman grade, G ≤ 2) with undifferentiated tissues (G ≥ 3, Fig. 1d) and we observed a stronger down-regulation of TMEM30B in high Fuhrman grade samples (fold-change −31.18, n = 36) in contrast to low Fuhrman grade samples (fold-change −12.29, n = 39, q < 0.05). Expression comparisons of all analyzed 10 TMEM genes in metastatic and non-metastatic tumors, early- and late stage samples, and tumors with undifferentiated and well-differentiated tissues are shown in Additional file 4, 5 and 6: Figures S1-S3, respectively.Fig. 1


Expression of pre-selected TMEMs with predicted ER localization as potential classifiers of ccRCC tumors
Differential expression of TMEMs in tumor tissue. (a) A comparison of TMEM expression between a tumor tissue and healthy kidney tissue. (b) TMEM expression in metastatic and non-metastatic tumors. (c) TMEM expression in organ-confined and advanced stage tumors (d) TMEM expression in low Fuhrman grade and high Fuhrman grade samples. Average log2 relative expression data in each sample group ± standard error of mean is shown in each chart. FC – fold-change. n – number of samples. T – tumor tissue samples. C – healthy tissue samples. M0 – non-metastatic ccRCC. M1 – metastatic ccRCC tissues. pT1/pT2 – organ-confined tumors, as assessed by TNM staging system. pT3/pT4 – advanced tumors, as assessed by TNM staging system. G1/G2 – low Fuhrman grade samples. G3/G4 – high Furhman grade samples. * - q < 0.05. ** - q < 0.01. *** - q < 0.001. **** - q < 0.0001
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Fig1: Differential expression of TMEMs in tumor tissue. (a) A comparison of TMEM expression between a tumor tissue and healthy kidney tissue. (b) TMEM expression in metastatic and non-metastatic tumors. (c) TMEM expression in organ-confined and advanced stage tumors (d) TMEM expression in low Fuhrman grade and high Fuhrman grade samples. Average log2 relative expression data in each sample group ± standard error of mean is shown in each chart. FC – fold-change. n – number of samples. T – tumor tissue samples. C – healthy tissue samples. M0 – non-metastatic ccRCC. M1 – metastatic ccRCC tissues. pT1/pT2 – organ-confined tumors, as assessed by TNM staging system. pT3/pT4 – advanced tumors, as assessed by TNM staging system. G1/G2 – low Fuhrman grade samples. G3/G4 – high Furhman grade samples. * - q < 0.05. ** - q < 0.01. *** - q < 0.001. **** - q < 0.0001
Mentions: In line with our previous observations, we detected statistically significant differences in expression of all ten TMEMs in tumor tissue and healthy kidney tissue (Fig. 1a) [20]. The down-regulation varied between the genes, with 1758.34 fold for TMEM213 (q < 0.0001) and 3.06 fold for TMEM116 (q < 0.05). Both, SLC35G2 and TMEM45A were found to be up-regulated with 5.33 (q < 0.01) and 6.75 fold change (q < 0.0001), respectively. We investigated if analyzed TMEMs displayed differential expression in metastatic and non-metastatic tumors. As shown in Fig. 1b, we detected significant difference in expression of TMEM72 and TMEM116: 39.78 fold and 6.65 fold down-regulation in metastatic tissue (n = 12 and n = 14, respectively) as compared to 5.13 fold and 2.57 fold in the non-metastatic samples (n = 58, q = 0.051 and n = 62, q = 0.056, respectively). Next, we subdivided samples into organ-confined tumors (pT ≤ 2) and advanced tumors (pT ≥ 3). A decrease of TMEM30B (q = 0.106) and TMEM45B (q = 0.097) expression was found in advanced-stage samples, with −2.21 and −2.71 fold-change, as compared to early-stage tumors, respectively (Fig. 1c). T-test was used to compare TMEM expression in well differentiated tumors (Fuhrman grade, G ≤ 2) with undifferentiated tissues (G ≥ 3, Fig. 1d) and we observed a stronger down-regulation of TMEM30B in high Fuhrman grade samples (fold-change −31.18, n = 36) in contrast to low Fuhrman grade samples (fold-change −12.29, n = 39, q < 0.05). Expression comparisons of all analyzed 10 TMEM genes in metastatic and non-metastatic tumors, early- and late stage samples, and tumors with undifferentiated and well-differentiated tissues are shown in Additional file 4, 5 and 6: Figures S1-S3, respectively.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: VHL inactivation is the most established molecular characteristic of clear cell renal cell carcinoma (ccRCC), with only a few additional genes implicated in development of this kidney tumor. In recently published ccRCC gene expression meta-analysis study we identified a number of deregulated genes with limited information available concerning their biological role, represented by gene transcripts belonging to transmembrane proteins family (TMEMs). TMEMs are predicted to be components of cellular membranes, such as mitochondrial membranes, ER, lysosomes and Golgi apparatus. Interestingly, the function of majority of TMEMs remains unclear. Here, we analyzed expression of ten TMEM genes in the context of ccRCC progression and development, and characterized these proteins bioinformatically.

Methods: The expression of ten TMEMs (RTP3, SLC35G2, TMEM30B, TMEM45A, TMEM45B, TMEM61, TMEM72, TMEM116, TMEM207 and TMEM213)&nbsp;was measured by qPCR. T-test, Pearson correlation, univariate and multivariate logistic and Cox regression were used in statistical analysis. The topology of studied proteins was predicted with Metaserver, together with PSORTII, Pfam and Localizome tools.

Results: We observed significant deregulation of expression of 10 analyzed TMEMs in ccRCC tumors. Cluster analysis of expression data suggested the down-regulation of all tested TMEMs to be a descriptor of the most advanced tumors. Logistic and Cox regression potentially linked TMEM expression to clinical parameters such as: metastasis, Fuhrman grade and overall survival. Topology predictions classified majority of analyzed TMEMs as type 3 and type 1 transmembrane proteins, with predicted localization mainly in ER.

Conclusions: The massive down-regulation of expression of TMEM family members suggests their importance in the pathogenesis of ccRCC and the bioinformatic analysis of TMEM topology implies a significant involvement of ER proteins in ccRCC pathology.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-015-1530-4) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus