MiR‐149 sensitizes esophageal cancer cell lines to cisplatin by targeting DNA polymerase β

Abstract Human DNA polymerase β (polβ) is a small, monomeric protein essential for short‐patch base excision repair (BER). polβ plays an important role in the regulation of chemotherapy sensitivity in tumour cells. In this study, we determined that the expression levels of polβ mRNA and miR‐149 in tumour tissues were significantly higher than in adjacent non‐tumour tissues. We also found that the expression level of miR‐149 in EC tumour tissues was inverse to that of polβ expression. Bioinformatics analysis and dual‐luciferase reporter assay predicted that miR‐149 negatively regulates polβ expression by directly binding to its 3′UTR. CCK‐8 assay indicated that miR‐149 could enhance the anti‐proliferative effects of cisplatin in EC1 and EC9706 cell lines. Flow cytometry, caspase 3/7 activity, and immunofluorescence microscopy results indicated that miR‐149 could enhance the apoptotic effects of cisplatin in EC1 and EC9706 cell lines. We also showed that the expression of polβ lacking the 3′UTR sequence could override the proliferative and apoptotic functions of miR‐149, suggesting that miR‐149 negatively regulates polβ expression by binding to its 3′UTR. Surface plasmon resonance results also showed that miR‐149 could bind with wild‐type polβ. In addition, we identified a new variant of polβ (C1134G). In conclusion, this study confirms that miR‐149 may enhance the sensitivity of EC cell lines to cisplatin by targeting polβ, and that miR‐149 may be unable to regulate the C1134G variant of polβ. Based on these findings, potential drugs could be developed with a focus on enhanced sensitivity of EC patients to chemotherapy.

Therefore, aberrant expression of miRNAs may lead to the development and progression of cancer, and have prognostic significance for several tumour types. [13][14][15][16] DNA polymerase b (polb) is a member of the DNA polymerase family and is essential for base excision repair (BER), one of the major pathways of DNA repair. [17][18][19][20][21] Thirty percent of all tumours reported to date harbour mutations in the polb gene. 22 Aberrant polb expression results in an increased rate of spontaneous mutagenesis, and a highly mutagenic phenotype. 23,24 Studies have reported polb mutations in various cancer types, and have shown that this may play a role in mediating tumour sensitivity to cisplatin. [25][26][27][28][29] Esophageal cancer (EC) is a major cause of cancer-related deaths worldwide. Many previous studies have reported that the polb gene is often mutated in primary EC tissues. EC also exhibits varying degrees of sensitivity to chemotherapy in the clinic. Previously, we performed miRNA chip-based expression analysis of EC tissues and found that the expression of miR-149 in EC tissues was aberrant.
Based on bioinformatic analyses, we hypothesized that the human polb 3 0 UTR contains the putative binding sites for miR-149, and that miR-149 may affect the sensitivity of EC cell lines to cisplatin.
In this study, we first investigated whether miR-149 modulates polb expression, and then examined the influence of miR-149 on cisplatin sensitivity in EC cell lines. We identified a novel homozygous C to G point mutation at nucleotide 1134 (C1134G) in the polb gene of EC patient tissues, and analysed the relationship between C1134G polb and miR-149.

| Patients and tissue specimens
Specimens were collected from a total of 82 EC patients with TNM stage III between 2011 and 2015, from the First Affiliated Hospital of Zhengzhou University and the Oncology Hospital of Linzhou City.
All specimens were obtained using endoscopy and biopsy assays.
Patients received chemotherapy with cisplatin (100 mg/m 2 body surface area; Day 1) and 5-FU (1000 mg/m 2 body surface area; Days 1-5), repeated every 28 days; none had received chemotherapy or radiotherapy prior to surgery. The patients were followed for a minimum of 36 months. All patients were informed in advance and signed explicit informed consent forms. This study was approved by the ethics committee of Zhengzhou University.

| RNA extraction and quantitative real-time PCR
Total RNA was isolated from biopsy EC tissues and adjacent nontumour tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. miR-149 expression level was obtained using quantitative real-time PCR (qRT-PCR) assay with high-specificity miR-149 qRT-PCR Detection Kit (Stratagene Corp, La Jolla, CA). U6 snRNA was used as normalization control for miR-149. To determine polb expression level, b-actin was used as normalization control. HET-1A cell line was used as untreated control to make the different groups comparable. The qRT-PCR results were expressed as threshold cycle (Ct) and were converted to the fold change (2 ÀDDCt ).

| Cell lines
EC1, EC9706, and HET-1A cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were maintained in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD, USA) and incubated at 37°C and 5% CO 2 .

| CCK-8 assay
Cells were transferred to 96-well plate at a density of 1 9 10 4 cells/ well, with five replicate wells per group. Next, the cells were treated with 0-30 lmol/L cisplatin (dissolved in NaCl, stored at À20°C).
After 48 hours, the relative numbers of viable cells were detected using Cell Counting Kit-8 reagents (CCK-8; Dojindo, Japan). Results were recorded using a microplate reader (Elx800; BioTek, VT, USA), with the absorbance optical density at 450 nm, and the IC50 was calculated. The experiments were done in triplicate.

| Flow cytometry assay
The cells were washed once with PBS, and then cultured in fresh medium containing 5 lg/mL 5-FU or 0.5 lg/mL cisplatin. Cells were harvested at 48 hours post-transfection by trypsinization, and resus-

| Caspase3/7 activity assay
Cells from each treatment group were harvested at 48 hours posttransfection and caspase activity was measured using a caspase activity assay kit (Beyotime, Haimen, China). Cellular extracts and substrates (Ac-DEVD-pNA) were kept in 96-well plate for 2 hours at 37°C. Absorbance values were measured using a microplate reader at 405 nm (Infinite M200, Tecan, Switzerland).

| Immunofluorescence microscopy
Immunofluorescence was performed to observe the expression level of c-H2AX in the nuclei of cells in different treatment groups. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.1% Triton X-100. After blocking in PBS containing 5% BSA for 1 hours, the cells were incubated with anti-c-H2AX primary antibodies and stained with FITC-conjugated secondary antibodies. Nuclei were stained with DAPI for 15 minutes. The mean number of immunostained nuclei per high-power field was determined using immunofluorescence microscopy. Results are presented as the average of at least three fields.

| DNA sequencing analysis
PCR-amplified fragments were cloned into pGEM-T vectors and transformed into Escherichia coli DH5a, which were grown at 37°C to mid-log phase. The DH5a transformants were subjected to sequencing analysis at Sangon Biotech (Shanghai).

| Surface plasmon resonance
The binding ability of miR-149 and polb was tested by Surface Plasmon Resonance (SPR) using a Biacore T200 instrument (Biacore, GE Healthcare). Biotinylated miR-149 and miR-NC were pur-   Figure 1A). In addition, the expression levels of polb mRNA in the tumour tissues of cisplatin insensitive patients was higher than that of cisplatin sensitive patients (P < .05, Figure 1B). Conversely, the expression levels of miR-149 in tumour tissues were significantly lower than in adjacent non-tumour tissues (P < .05, Figure 1C). Furthermore, the expression levels of miR-149 in the tumour tissues of cisplatin in-sensitive patients were lower than that of cisplatin sensitive patients (P < .05, Figure 1D). We also investigated the correlation between polb and miR-149 expression, and found that polb mRNA expression levels were increased in the EC tumour tissues, whereas miR-149 expression was reduced, demonstrating a significant negative correlation (R 2 = 0.623, P < .05, Figure 1E).

| Polb is identified as a target gene of miR-149 in EC1 and EC9706 cell lines
Bioinformatics analysis by TargetScan and miRanda predicted that the 3 0 UTR of polb contained binding sites for miR-149 (Figure 2A). polb protein expression levels in miR-149 group cell was significant lower than that in Blank and miR-NC groups (P < .05).
To verify whether polb is a direct target of miR-149, we used a Dual-Luciferase reporter system containing either the wild-type or the mutant 3 0 UTR of polb. There was no significant difference in luciferase activity between cells transfected with miR-NC and those co-transfected with miR-149 mimic and mutant-type 3 0 UTR of polb ( Figure 2C,D). However, the luciferase activity of cells co-transfected with miR-149 mimic and wild-type 3 0 UTR of polb was significantly decreased (P < .05, Figure 2C,D). These results indicate that miR-149 negatively regulates polb expression by directly binding to the putative binding sites in the 3 0 UTR.

| Cisplatin-induced proliferation inhibition was enhanced by miR-149 in EC1 and EC9706 cell lines
The relative expression of miR-149 in different cell lines is presented in Figure 3A. The expression of miR-149 was higher in the miR-149 group compared to the Blank and miR-NC groups (P < .05). The cell survival rate curves are presented in Figure 3B,C.
Cell viability in all groups decreased with increasing doses of cisplatin. The viability of cells in the miR-149 group was significantly lower than that of the Blank and NC groups, across cisplatin doses (P < .05, Figure 3B,C). The IC50 of cisplatin in EC1 and EC9706 cells was calculated, and this is shown in Figure 3D. The IC50 of cisplatin in the miR-149 cells was lower than that of the Blank and NC groups (P < .05).

| Cisplatin-induced apoptosis was enhanced by miR-149 in EC1 and EC9706 cell lines
Flow cytometry results indicated that treatment with cisplatin increased apoptosis in both the miR-NC and miR-149 cells. In addition, apoptosis in the miR-149 cells was significantly greater than that in the miR-NC cells (P < .05; Figure 4A). In addition, after treatment with cisplatin, caspase 3/7 activity in the miR-149 cells was significantly higher than in the miR-NC cells (P < .05; Figure 4B).
Western blotting showed that the expression of cleaved PARP was also significantly increased in miR-149 cells treated with cisplatin (P < .05; Figure 4C). Immunofluorescence microscopy results indicated that the expression level of c-H2AX in the nuclei of miR-149 cells treated with cisplatin was significantly higher than that of miR-NC cells treated with cisplatin, and miR-NC or miR-149 cells without cisplatin (P < .05; Figure 4D). These results indicated that miR-149 could enhance the apoptotic effects of cisplatin in EC1 and EC9706 cell lines.

| Expression of polb restored the function of miR-149
As discussed above, the exogenous expression of miR-149 enhanced the anti-proliferative and pro-apoptotic effects of cisplatin in EC1 and EC9706 cell lines (Figures 3 and 4). Therefore, we exogenously expressed recombinant polb lacking the 3 0 UTR miR-149 on EC1 and EC9706 cell lines that treated with cisplatin (P < .05, Figure 5B).

| miR-149 did not regulate the C1134G variant of polb in EC tissues
We have shown that the expression level of miR-149 in the tumour tissues was significantly lower than in adjacent non-tumour tissues, and that polb mRNA expression in tumour tissues was significantly higher than in adjacent non-tumour tissues (P < .05, Figure 1A,C).
Interestingly, in one sample (No. 22), the expression levels of miR-149 in the tumour tissue were not lower, and polb expression in the tumour tissue was higher, compared to the adjacent non-tumour tissue ( Figure 6A-C). Using a DNA sequencing assay, we analysed the polb DNA sequence of sample No. 22 and identified a novel homozygous C to G point mutation at nucleotide 1134 ( Figure 6D).
To further analyse the binding properties of polb with miR-149, surface plasmon resonance analysis was performed. BIAcore analysis confirmed that the binding affinity of miR-149 and wild-type polb is stronger than that of the miR-149 and C1134G variants of polb, and much stronger than that of the miR-149 and mutant-type polb, or the miR-NC and polb variants ( Figure 6E).
To verify the relationship between the C1134G variant of polb and miR-149, we conducted a Dual-Luciferase reporter assay in polb À/À EC9706 cells. The luciferase activity of cells co-transfected with miR-149 mimic and wild-type 3 0 UTR of polb was significantly decreased (P < .05, Figure 6F). Conversely, there was no significant change in the luciferase activity of cells co-transfected with miR-149 and mutant-type polb, or the miR-NC and polb variants. These results indicate that miR-149 may be unable to regulate the C1134G variant of polb in EC tissues and EC9706 cells.

| DISCUSSION
EC is a major cause of cancer-related deaths worldwide. Although methods for the diagnosis and treatment of EC have advanced, the disease continues to have a poor prognosis due to invasion and early stage metastasis. 30,31 Due to the difficulty of surgical intervention and its potential complications, most patients choose to undergo palliative treatments such as chemotherapy. However, the clinical efficacy of chemotherapy is inadequate; the 5-year survival rate is only 10%-30%, and the rates of local uncontrolled tumour growth and recurrence range from 60%-80%. 32,33 Therefore, researchers are currently focusing on increasing the efficacy of chemotherapy in EC. In recent years, studies have identified a variety of genes whose expression products may affect tumour response to chemotherapy; examples include cell cycle regulatory genes, apoptotic genes, and DNA damage repair proteins.
DNA polymerase b (polb) is a key enzyme in the DNA damage repair system. This system serves as a crucial factor in maintaining genome integrity and stability, in addition to modulating the chemotherapy sensitivity of tumour cells. Many studies demonstrated that the polb is required for cisplatin sensitivity. 34,35 Not surprisingly, polb has become a research hot spot worldwide. miRNAs have been estimated to regulate up to 30% of human genes and control a variety of cellular processes. 36 Recent studies have shown that miRNAs are dysregulated in various cancers, and that their expression is relevant to a diverse array of tumours. 37,38 In this study, we determined that the expression levels of polb mRNA in tumour tissues were significantly higher than that in adjacent non-tumour tissues. Interestingly, the expression levels of polb mRNA in the tumour tissues of cisplatin insensitive patients were higher than in cisplatin sensitive patients, and the expression level of miR-149 in the EC tumour tissues was inverse to that of polb. Our study revealed that polb mRNA expression levels were increased, where miR-149 mRNA expression levels were reduced, demonstrating a negative correlation. Kaplan-Meier method results showed that patients with low expression levels of polb mRNA or high miR-149 survived longer than patients with high polb mRNA or low miR-149.
Bioinformatics analysis and dual-luciferase reporter assay predicted that the 3 0 UTR of polb contained binding sites for miR-149, and that miR-149 negatively regulates polb expression by directly binding to the 3 0 UTR. CCK-8 assay indicated that miR-149 could enhance the anti-proliferative effects of cisplatin in EC1 and EC9706 cell lines.
Flow cytometry, caspase 3/7 activity, PARP cleavage, and immunofluorescence microscopy results of c-H2AX in the nucleus indicated that miR-149 could enhance the pro-apoptotic effects of cisplatin in EC1 and EC9706 cell lines. In addition, we also showed that the expression of polb lacking the 3 0 UTR sequence could override the anti-proliferative and pro-apoptotic functions of miR-149, suggesting that miR-149 negatively regulates polb expression by binding to its 3 0 UTR. Surface plasmon resonance results also showed that the miR-149 could bind with wild-type polb.
Interestingly, the expression levels of miR-149 and polb in sample No. 22 were abnormal. The miR-149 expression level in the tumour tissue was not lower, and polb expression in the tumour tissues was higher, compared to the adjacent non-tumour tissue.
DNA sequencing assay was used to analyse the polb DNA sequence of sample No. 22, and revealed a novel homozygous C to G point mutation at nucleotide 1134 in polb DNA. Dual-luciferase reporter assay results indicated that miR-149 may be unable to regulate the C1134G variant of polb in EC tissue and EC9706 cells.
In conclusion, this study confirms that miR-149 may enhance cisplatin sensitivity in EC cell lines by targeting polb. In addition, we identified a new variant of polb (C1134G) and found that miR-149 may be unable to regulate the C1134G variant of polb. Based on these findings, potential drugs could be developed with a focus on enhanced sensitivity of EC patients to chemotherapy.

CONFLI CTS OF INTEREST
The authors declare no conflict of interest.