miR-93, miR-373, and miR-17-5p Negatively Regulate the Expression of TBP2 in Lung Cancer

Recently, several miRNAs have been revealed to play critical roles in oncogenesis and tumor progression of many cancers. Thioredoxin-1 (Trx-1) binding protein-2 (TBP-2) is an internal inhibitor of Trx-1, which plays the role in regulating oxidative stress, inhibiting cell growth, and promoting apoptosis. The expression of TBP-2 is usually decreased in cancer tissues. However, whether the miRNAs regulate the TBP-2 expression in lung cancer is still unclear. In this study, we examined the levels of TBP-2, miR-93, miR-373, and miR-17-5p in lung cancer tissues and their adjacent normal lung tissues of 36 patients. We found that the expressions of miR-93, miR-373, and miR-17-5p were higher, whereas the expression of TBP-2 mRNA and protein was significantly lower in lung cancer tissues compared with adjacent normal lung tissues. After the three miRNA mimics were transfected in the lung cancer cells, NCI-H460, the level of TBP-2 mRNA and TBP-2 protein was decreased. Then, the anti-cancer drug 5-fluorouracil was used to stimulate the NCI-H460 cells; the mRNA levels of miR-93, miR-373, and miR-17-5p were decreased, and the level of TBP-2 mRNA and protein was increased. Collectively, the above results suggest that miR-93, miR-373, and miR-17-5p negatively regulate the TBP-2 expression in lung cancer. This study may provide therapeutic targets with lung cancer.


INTRODUCTION
Lung cancer is the most common malignant tumor in the world. The therapies for lung cancer have been improved much, such as surgical treatment, molecule-targeted therapy, and chemotherapy; however, the prognosis of patients with lung cancer is still unsatisfactory due to the high rate of metastasis and recurrence and the low rate of 5-years survival (1,2). Therefore, developing novel therapeutic methods and understanding the mechanisms on lung cancer are very important.
In this study, we examined the levels of miR-93, miR-373, and miR-17-5p and the expression of TBP-2 in lung cancer tissues. We found that levels of miR-93, miR-373, and miR-17-5p were higher, whereas the expression of TBP-2 was lower in lung cancer tissues compared with adjacent normal tissues. We further found that miR-93, miR-373, and miR-17-5p could negatively regulate the expression of TBP-2 in NCI-H460 lung cancer cells.

Patients and Samples
Fresh lung cancer and adjacent normal tissues from 36 patients were collected. All the samples were residual specimens after diagnostic sampling. The primary tumor and the adjacent normal tissues from the same patient were separated and then immediately stored at −80 • C. All lung cancer patients were hospitalized in the Department of Thoracic Surgery, the First People's Hospital of Yunnan province and treated with radical operation, and none of them received any preoperative treatments. All patients signed separate informed consent forms for sampling and molecular analysis. This study was approved by the Ethics Committee of the First People's Hospital of Yunnan Province, China.

Total RNA Extraction and Real-Time PCR Assay
Total RNA was isolated from NCI-H460 cells by using TRIzol (CWBIO Corporation, Beijing, China) as described by the manufacturer and converted to cDNA using the RevertAid TM First Strand cDNA Synthesis Kit (Fermentas, Walldorf Baden, Germany).
Real-time PCR was performed to detect the relative level of TBP-2 expression. The PCR reactions were performed in a total volume of 25 µl, including 12.5 µl of Real-time PCR Master Mix, 1 µl of cDNA (5 ng/µl), and 1 µl of primer mix (10 µM each). PCR amplification and detection were performed by using the Prism 7300 Sequence Detection System (Applied Biosystems, Foster, CA, USA) as follows: an initial denaturation at 95 • C for 2 min; 40 cycles of 95 • C for 30 s, 59 • C for 30 s, and 70 • C for 1 min. The relative gene expression was calculated by using the comparative CT method. GAPDH was used as an internal standard for all samples. The sequences of primers are as follows: TBP-2 forward primer: 5 ′ -GGATCCCAGCAGTGCAAAC-3 ′ ; TBP-2 reverse primer: Frontiers in Oncology | www.frontiersin.org 5 ′ -AAGCCGAACTTGTACTCATATTTGT-3 ′ ; GAPDH forward primer: 5 ′ -CAAGG CTGAGAACGGGAAG-3 ′ ; GAPDH reverse primer: 5 ′ -GGTGAAGACGCCAGTGGACT-3 ′ .
Hairpin-it TM qRT-PCR Primer Set (GenePharma) was used for the measurement of the relative levels of miR-93, miR-373, and miR-17-5p. The mRNA expressions of miR-93, miR-373, and miR-17-5p were normalized to the expression of U6.

Western Blot Analysis
Cells were detached with trypsin, centrifuged, and washed twice with pre-chilled phosphate buffer saline (PBS). Cell lysis

Statistical Analysis
The data in this study are presented as mean ± standard deviation (SD) and statistically analyzed by using SPSS software (version 18.0, SPSS Inc., Chicago, USA). Two-tailed unpaired Student's t test was used to analyze the difference between two groups, normal non-cancerous tissues, and lung cancer tissues as indicated in corresponding figure legends. The one-way ANOVA followed by a Bonferroni's multiple comparisons test was used to compare the control and treated groups as indicated in corresponding figure legends.

RESULTS
The mRNA Levels of miR-93, miR-373, and miR-17-5p in Lung Cancer and Adjacent Normal Non-cancerous Tissues To investigate which miRNAs are involved in the development of lung cancer, we first detected the mRNA levels of miR-93, miR-373, and miR-17-5p in lung cancer and adjacent normal tissues of 36 samples by using Real-time qPCR. The mRNA levels of miR-93, miR-373, and miR-17-5p were higher in most carcinoma of the lung tissues compared with adjacent normal non-cancerous tissues. The folds of relative mRNA levels of miR-93, miR-373, and miR-17-5p in normal non-cancerous tissues and lung cancer tissues are shown in Figures 1A,C,E.

TBP-2 Expression in Lung Cancer and Adjacent Tissue
To investigate TBP-2 expression in lung cancer, we further detected the level of mRNA and protein of TBP-2 in lung cancer tissues and their compared normal non-cancerous lung Frontiers in Oncology | www.frontiersin.org tissues of 36 lung cancer samples by using Real-time qPCR and Western blot. The folds of relative mRNA level of TBP-2 in normal non-cancerous tissues and lung cancer tissues as shown in Figure 2A. The fold of cancer tissue to normal non-cancerous tissue in each sample was calculated by normal value as one. TBP-2 mRNA level was significantly lower in lung cancer tissues compared with non-cancerous tissues, 27 of 36 samples, which was 75% (n = 36, * * P < 0.01) (Figure 2B). The blots and folds of TBP-2 protein expression in normal non-cancerous tissues and lung cancer tissues are shown in Figures 2C,D. The fold of cancer tissue to normal non-cancerous tissue in each sample was calculated by normal value as one. The level of TBP-2 protein was remarkably lower in lung cancer tissues compared with the normal non-cancerous tissues, 30 of 36 samples, which was 83.3% (n = 36, * * * P < 0.001) (Figure 2E). Contrary to the expressions of miR-93, miR-373, and miR-17-5p, the expression of TBP-2 was lower in lung cancer tissues.
To compare the correlation between the mRNA levels of miR-93, miR-373, and miR-17-5p and the level of TBP-2 mRNA and protein, we organized change trends of the above results in Table 1.

DISCUSSION
Increased levels of miR-93, miR-373, and miR-17-5p were found in various types of cancers including lung cancer. In NSCLC, miR-93 expression was significantly correlated with survival rate, and silencing miR-93 expression might inhibit cell proliferation, migration, and invasion of lung cancer cells by targeting gene of phosphate and tension homology deleted on chromosome ten (PTEN) and retinoblastoma 1 (RB1) (14). It has been reported that miR-93 activated the PI3K/AKT signaling pathway and promoted tumorigenesis and metastasis of NSCLC via targeting liver kinase B1 (LKB1) (15). Qu et al. reported that miR-93 could down-regulate NEDD4L and increase TGF-induced EMT in lung cancer (9). MiR-93 regulates translocation of liposarcoma 1 (TLS1, FUS1), transducin repeat containing 2 (TRCP2), and disabled homolog 2 (DAB2), and plays an oncogenic role in lung cancer carcinogenesis. Gao et al. reported that miR-373 expression was decreased in glioma tissue specimens and cell lines and was involved in growth of cancer cells (16).
Trx-1 is a redox-regulating protein and plays a role in promoting growth of cells and inhibiting apoptosis. It has been reported that Trx-1 is increased and TBP-2 is decreased in various cancers. TBP-2 is the inhibitor of Trx-1 and inhibits the activity of Trx-1 by binding to its catalytic active center (17). TGF-beta1 and 1,25-dihydorxyvitamin D3 block the cancer cell cycle to inhibit cancer cell growth by increasing TBP-2 expression (18). In pancreatic tumor cells, miR-224 could enhance the proliferation and migration via directly targeting TBP-2 and activating downstream hypoxia-inducible factor-1 (HIF-1) signaling pathway (19). Park et al. found that downregulation of TBP-2 promoted the tumor growth by reducing p27 expression in vitro and in vivo in breast cancer (20). Interestingly, the bromodomain-containing protein 4 (BRD4) inhibitor, JQ1, induced apoptosis in acute myeloid leukemia cells by reactivating TBP-2 (21). Combination of mammalian target of rapamycin (mTOR) and histone deacetylase (HDAC) inhibitors could kill the NF-kB mutant and kirsten rat sarcoma viral oncogene (KRAS) mutant NSCLC cells by regulating the TBP-2/Trx-1 signaling pathway (22). Tyrosine kinase inhibitors (TKIs) inhibit the PI3K/Akt pathway in NSCLC cells and increase TBP-2 expression (23). It has been reported that Insulin-like growth factor 1 (IGF1) negatively regulates the expression of TBP-2 (24). Thus, TPB-2 plays an important role in inhibiting the growth of cancer cells.
MiR-373 negatively regulated TBP-2 expression and increased the expressions of HIF-1α and twist family basic helix-loop-helix transcription factor (TWIST). The miR-373/TBP-2/HIF1α/TWIST signaling axis was related to a poor prognosis of patients with breast cancer (25). MiR-17-5p inhibited TBP-2 expression during the senescence process. In senescent fibroblasts, TBP-2 expression is regulated by forkhead box O3 (FOXO3) and miR-17-5p (26). In the present study, we found that miR-93, miR-373, and miR-17-5p were higher and TBP-2 was lower in lung cancer tissues (Figures 1, 2). However, our results showed that the correlation between miRs and TBP-2 was not always direct in lung cancer tissues ( Table 1). There are several possibilities: miR-93, miR-373, or miR-17-5p could, on their own, suppress the expression of TBP-2. The mechanisms on miRs regulate the expression of TBP-2 via different or complexed pathways. The reasons of the lung cancers are different. We further demonstrated that miR-93, miR-373, and miR-17-5p negatively regulated the expression of TBP-2 in NCI-H460 cells (Figure 3). To our knowledge, our results firstly revealed that miR-93 negatively regulated TBP-2 expression in lung cancer tissues and cells.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of First People's Hospital of Yunnan Province, China. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
JB and ZS conceived the idea and planned the study, wrote and reviewed the main manuscript. YL produced the figures and did statistical analysis. ML performed PCR and Western blot experiments. BY and YZ collected the samples of patients. WG performed cell experiments. All authors reviewed the manuscript.