miR-1247-3p targets STAT5A to inhibit lung adenocarcinoma cell migration and chemotherapy resistance

Abstract

Background: Although several advancements have been achieved in research and treatment of lung adenocarcinoma in the past few years, the mechanism concerning cancerous cell migration and the cause of chemoresistance remains ambiguous. This research aimed to explore the impact of miR-1247-3p in lung adenocarcinoma. Methods: The mRNA expression of miR-1247-3p and STAT5A were conducted with qRT-PCR. Lentiviral vectors containing miR-1247-3p mimics and inhibitors were constructed. Cell migration were examined using Transwell assay. To observe chemotherapy resistance, Docetaxel, Doxorubicin, and Gefitinib were used. DIANA, miRDB, and TargetScan databases were applied to detect target genes. The binding sites were verified by double luciferase assay. Results: Low expression of miR-1247-3p was observed in lung adenocarcinoma tissues and cell lines. Its expression was lower in advanced stages. Cell migration of lung adenocarcinoma was inhibited by miR-1247-3p, and it could negatively regulate the process of chemoresistance. miR-1247-3p directly binds to 3' UTR of STAT5A mRNA, and it functions via targeting STAT5A. Conclusions: miR-1247-3p acted as a potential governor monitoring cell migration and chemotherapy resistance of LUAD by interacting with STAT5A. It has the potential to be exploited as novel therapeutic target for LUAD in the future.

Keywords: STAT5A; cell migration; chemoresistance; lung adenocarcinoma; miR-1247-3p.

Figure 1

Low expression of miR-1247-3p was found in lung adenocarcinoma tissues. A:162 pairs from adenocarcinoma tissues of lung were examined and miR-1247-3p expression was observed using qRT-PCR. Unlike adjacent normal tissue cells, a reduced miR-1247-3p expression was found in these tissues. B: Among these 162 pairs, 113 tumor tissues showed low and 7 showed high expressions of miR-1247-3p. C: miR-1247-3p expression in T1+T2 vs T3+T4 in adenocarcinoma tissues of lung was observed. Results revealed a higher expression in T1+T2 as compared to T3+T4. D: The expression of miR-1247-3p was also detected in adenocarcinoma tissue of lung with or without lymph nodes tumors. E: The expression of miR-1247-3p was also detected in adenocarcinoma tissue of lung showing distant metastasis and also in the tissues without showing any symptom of distant metastasis. F: miR-1247-3p expression was also detected in normal epithelial cells of lung and cell lines of adenocarcinoma tissue cells. The expression was higher in normal epithelial cells of lung. ***p<0.001 compared with group normal, T1+T2, N0, M0, or HNBE. Lentiviruses-inhibitor normal control (LV-INC), Lentiviruses-inhibitor (LV-inhibitor), Lentiviruses-mimics normal control (LV-mNC), Lentiviruses- mimics (LV-mimics). The experiments were repeated at least 3 independent times.

Figure 2

Adenocarcinoma cell proliferation of lung was inhibited by miR-1247-3p. A: We identified the best mimic and inhibitor sequences. B: As compared to control groups, a lower expression of miR-1247-3p was observed in inhibitor group and higher in mimic group. C: Reduced expression of miR-1247-3p resulted in an increased cell proliferation and invasion. D: Suppression process can result in increment of cell number as shown. LV-mimics group was found to be decreased in cell number of A459 cell lines whereas it was higher in LV-mNC group. E-F: Cell number percent in A549 cell lines was raised by inhibitor group when comparing to LV-INC group. G-H: Cell apoptosis was measured. ***p<0.001 compared with group INC, mNC, LV-INC, or LV-mNC. Lentiviruses-inhibitor normal control (LV-INC), Lentiviruses-inhibitor (LV-inhibitor), Lentiviruses-mimics normal control (LV-mNC), Lentiviruses- mimics (LV-mimics). The experiments were repeated at least 3 independent times.

Figure 3

Chemotherapy resistance of adenocarcinoma cells of lung was inhibited by miR-1247-3p. A: When treated with Docetaxel, in the cell lines of A549, the mimics group was found to be in reduced survival rate as compared to the group of LV-mNC. B: When administered Doxorubicin, in the cell lines of A549, the mimics group was found to be in reduced survival rate when comparing with the LV-mNC group. C: When treated with Gefitinib, in A549 cells lines, LV-mimics group was lower in survival rate than LV-mNC group. D: In the cell lines of NCI-H1395, when treated with Docetaxel, a higher survival rate of inhibitor group was observed as compared to LV-INC group. E: In NCI-H1395 cell lines, when treated with Doxorubicin, LV-inhibitor group was increased in survival rate than LV-INC group. F: In NCI-H1395 cell lines, when treated with Gefitinib, a higher survival rate of inhibitor group was observed as compared to group of LV-INC. ***p<0.001 compared with group LV-INC or LV-mNC. Lentiviruses-inhibitor normal control (LV-INC), Lentiviruses-inhibitor (LV-inhibitor), Lentiviruses-mimics normal control (LV-mNC), Lentiviruses- mimics (LV-mimics). The experiments were repeated at least 3 independent times.

Figure 4

miR-1247-3p regulated STAT5A. A. Three databases (DIANA, miRDB, and TargetScan) were applied to prognosticate miR-1247-3p target genes. B. A negative correlation between relative microRNA-1247-5p expression and STATA5A expression (r=-0.314, p=0.001). C. Relative microRNA-1247-5p expression and KCTD 17 expression negatively correlated (r=-0.116, p=0.227). D. Relative microRNA-1247-5p expression and GNLY expression negatively correlated (r=-0.035, p=0.714). E. Relative microRNA-1247-5p expression and LUC7L expression negatively correlated (r=-0.059, p=0.539). F. A positive correlation between relative microRNA-1247-5p expression and SMIM7 expression (r=0.023, p=0.813). G. Relative microRNA-1247-5p expression and B4GALT3 expression negatively correlated (r=-0.113, p=0.241). H. A positive correlation between relative microRNA-1247-5p expression and ZNF234 expression (r=0.049, p=0.612). I. Relative microRNA-1247-5p expression and ALKBH4 expression negatively correlated (r=-0.126, p=0.190). J. A positive correlation between relative microRNA expression and SFRP5 expression (r=0.003, p=0.976). K. The expression of STAT5A was examined in sable transfected cell lines and was observed to be more in LV-mNC group than in mimics group (p<0.001). An enhanced expression of STAT5A was found in inhibitors group than the group of LV-INC. ***p<0.001 compared with group LV-INC or LV-mNC. Lentiviruses-inhibitor normal control (LV-INC), Lentiviruses-inhibitor (LV-inhibitor), Lentiviruses-mimics normal control (LV-mNC), Lentiviruses- mimics (LV-mimics). The experiments were repeated at least 3 independent times.

Figure 5

miR-1247-3p directly binds to STAT5A mRNA 3' UTR. A. Plasmids, both mutant type and wild-type, of STAT5A mRNA 3' UTR were constructed on the active binding sites of miR-1247-3p and mRNA of STAT5A gene. B-C. The binding of miR-1247-3p with wild type plasmid and not the mutant type was verified by the dual luciferase assay. D. Sequence of siRNA of STAT5A exhibiting the best interference effect was identified and separated for further experiments. E. Higher survival rate of inhibitor group as compared to LV-INC group upon treatment with Docetaxel. No statistical significance between LV-INC+STAT5A siRNA and LV-inhibitor+STAT5A siRNA group was observed. F. Higher survival rate of inhibitor group as compared to the LV-INC group upon treatment with Doxorubicin. No statistical difference between LV-INC+STAT5A siRNA and LV-inhibitor+STAT5A siRNA groups was observed. G. Higher survival rate of inhibitor group as compared to the LV-INC group upon treatment with Gefitinib. No statistical difference between LV-INC+STAT5A siRNA and LV-inhibitor+STAT5A siRNA groups was observed. ***p<0.001 compared with group NC, LV-INC, or LV-mNC. Lentiviruses-inhibitor normal control (LV-INC), Lentiviruses-inhibitor (LV-inhibitor), Lentiviruses-mimics normal control (LV-mNC), Lentiviruses- mimics (LV-mimics). The experiments were repeated at least 3 independent times.

Figure 6

Down-regulated STAT5A significantly promoted tumor growth. A: Transplanted tumor model was established to investigate the role of STAT5A on tumor growth. B: Down-regulation of STAT5A significantly increased the tumor size. C: The levels of c-myc, AXIN, and β-catenin were measured using IHC. D: The expression of c-myc, AXIN, and β-catenin were remarkably increased by STAT5A siRNA. *<0.05 Compared with group control. *p<0.05 compared with group control or sham. The animal experiment was performed for one time, and the IHC staining was repeated 3 independent times.