Analysis of TMEM174 gene expression in various renal cancer types by RNA in situ hybridization

Transmembrane protein 174 (TMEM174) mRNA is easily detectable in human kidney tissues and activates AP-1 and promotes 293T cell proliferation. In the present study, RNA in situ hybridization was used to detect TMEM174 gene expression in various malignant renal cancer and normal renal tissues. The results showed that TMEM174 exhibits differential expression in renal tissues, with a high positive rate of expression in squamous cell carcinoma with necrosis, papillary renal cell carcinoma and transitional cell carcinoma, and a low positive rate of expression in clear cell carcinoma, interstitial nephritis, undifferentiated carcinoma, retroperitoneal metastatic clear cell carcinoma, adrenal gland metastatic clear cell carcinoma, pelvic cavity metastatic chromophobe carcinoma, severe atypical hyperplasia of transitional epithelium and hyperplasia. Extremely weak expression was exhibited in collecting duct carcinoma, Wilms’ tumor, chronic pyelonephritis, acute pyelonephritis, cancer adjacent normal renal tissue and normal renal tissue. In conclusion, the TMEM174 gene exhibited high expression levels in certain renal carcinomas, which may indicate that TMEM174 may have a significant role in the development and progression of these renal carcinomas.


Introduction
Transmembrane protein 174 (TMEM174) is a type III transmembrane protein that lacks a clear signal peptide. The N and C terminals are located inside of the cell. TMEM174 was originally identified from a large gene pool by high-throughput cell screening technology. This technique is used for the isolation of functional genes and for the analysis into the mechanisms of gene function (1). Dependent on its transmembrane helices, TMME174 overexpression is able to promote the transcriptional activity of activator protein 1 (AP-1), which is partly mediated by the ERK pathway (2). Our previous study demonstrated high TMEM174 expression in the kidney and also revealed its potential involvement with renal cancer based on its capacity to stimulate cell proliferation (2). The kidney is vital for the maintenance of the salt and water balance within the body, in order to keep a stable internal environment, i.e., for homeostasis. However, the expression of TMEM174 in renal cancer and normal renal tissues remains to be elucidated. In the present study, RNA in situ hybridization was used to detect the TMEM174 gene expression in various malignant renal cancer and normal renal tissues. The aim of this study was to provide a theoretical basis for the molecular mechanisms of the development of kidney cancer.

Materials and methods
Tissue microarray. Tissue microarrays were purchased from Shaanxi Chao Ying Biotechnology, Ltd., Co. (Xi'an, China). Information on each specimen, consisting of patient age, gender, organization, pathological diagnosis, clinical grade, tumor-node-metastasis classification, clinical stage, specimen type and result information, was available. TNM staging, clinical staging and pathological grading were determined based on the American Joint Committee on Cancer manual (3). Specimens were used from a total of 208 cases, including 178 cases of renal cancer and nephritis, 20 adjacent tissues and 10 normal tissues.
Preparation of digoxigenin-labeled probes for RNA in situ hybridization. The sense and anti-sense probes that matched the TMEM174 core responding sequence were as follows: Anti-sense, 5'-GAGCATTGTGTTATTATATCAG*AATA GCCTCTAGCGAGGGAGAGAGTATATT-3'DIG and sense, 5'-ATATACTCTCTCCCTCGCTAGAGGC*TATTCTGATA TAATAACACAATGCTCA-3'DIG. The asterisks indicate that the 3' terminals were labeled with digoxigenin. All probes were synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China).

Results
Association between TMEM174 expression and renal pathological cell types. TMEM174 gene expression in the various malignant renal cancer and normal renal tissues was detected by RNA in situ hybridization. As shown in Table Ⅰ   Table I. Association between TMEM174 expression and renal pathological cell type. Staining was scored using a 0-3+ scale. 0 indicates no staining; 0-1+, indicates trace staining that is weaker than 1+, but stronger than 0. Scores of 1+, 2+ and 3+ indicate an increased intensity of staining. Sub-regions excluding necrosis, macrophages and infiltrated neutrophils and lymphocytes were selected and scored. The intensity score for an array spot is the average of all its sub-regions. TMEM174, transmembrane protein 174.  I  108  58  II  26  50  III  14  71  IV  4  50   TMEM174, transmembrane protein 174. and Fig. 1, TMEM174 exhibits differential expression in renal tissues. TMEM174 had a high positive rate of expression in squamous cell carcinoma with necrosis, papillary renal cell carcinoma and transitional cell carcinoma, and a low positive rate of expression in clear cell carcinoma, interstitial nephritis, undifferentiated carcinoma, retroperitoneal metastatic clear cell carcinoma, adrenal gland metastatic clear cell carcinoma, pelvic cavity metastatic chromophobe carcinoma, severe atypical hyperplasia of transitional epithelium and hyperplasia. Extremely weak expression was observe in collecting duct carcinoma, Wilms' tumor, chronic pyelonephritis, acute pyelonephritis, cancer adjacent normal renal tissue and normal renal tissue.
Association between TMEM174 expression and renal pathological clinical stage. The association between TMEM174 expression and renal pathological clinical stage was analyzed. As shown in Table II, the THEM174 expression rate was 58% in phase I tissue samples, 50% in phase II tissue samples, 71% in phase III tissue samples and 50% in phase IV tissue samples.

Discussion
Each year in the United States, nearly 55,000 individuals are diagnosed with kidney cancer. In order to be effectively treated, an early diagnosis and effective surgical therapy are required. In more advanced-stage cases with involvement of the renal vein and lymph nodes or invasion through the renal cortex, surgical therapy frequently fails (4)(5)(6)(7)(8). Kidney cancer cell progression is a coordinated process that comprises cell cycle dysregulation and a specific gene expression program to determine tissue identity (9). The TMEM174 gene is highly expressed in the kidney tissues. The AP-1 transcription factor is a heterodimeric protein formed from proteins of the c-Fos, ATF, c-Jun and JDP families. In response to a range of stimuli, including cytokines, stress, growth factors and viral and bacterial infections, AP-1 regulates gene expression (10). The cellular transcription factor cAMP-response element binding protein (CREB) binds to specific DNA sequences referred to as cAMP response elements (CREs), thereby regulating downstream gene transcription (11). TMEM174 overexpression has been shown to enhance the transcriptional activity of AP-1 and promote cell proliferation (2). In addition, our recent studies demonstrated that the CREB and AP-1 transcription factors are involved in the transcriptional regulation of the TMME174 gene (12).
When antibodies are not available, RNA in situ hybridization is a useful method that allows the determination of the transcriptional expression pattern of a gene (13). With this technique, the expression of multiple RNA species may be assayed using distinct RNA-labeled probes, or the RNA and protein localization within larval tissues may be examined. In the present study, RNA in situ hybridization analysis was used to detect the expression of TMEM174, and the results showed that its expression is varies among differing renal tissues. These results indicate that TMEM174 may have a significant role in the development of renal cancer. 81302323) and the Natural Science Foundation of Hebei Province (nos. C2009001260, C2014209140 and C2013209024).