MiRNA-148a inhibits cell growth and drug resistance by regulating WNT10a expression in renal cell carcinoma

Abstract

Background: We aimed to explore miR-148a exerts a tumor suppressor effect and arsenic trioxide (As₂O₃) sensitivity on renal cell carcinoma (RCC).

Methods: We performed polymerase chain reaction (PCR) on 42 pairs of tumor and paracancerous samples collected from RCC patients to investigate the miR-148a expression; meanwhile, we analyzed the interplay between clinical indicators and miR-148a expression of RCC. Then, the influence of miR-148a overexpression on the functions of RCC cells were analyze using transwell migration assay, Cell Counting Kit-8 (CCK-8), and cell wound healing assay. Furthermore, the ability of miR-148a to sensitize Caki-1 cells treated with As₂O₃ were detected using flow cytometry. Finally, the relevant mechanism of miR-148a on the downstream gene Wnt family member 10A (WNT10a) was explored by cell reverse method.

Results: The results from RCC patients indicated a significantly lower miR-148a level than adjacent tissues. The low miR-148a expression increased prevalence of distant metastasis and decreased survival rate compared to those with high expression in patients. In the RCC cell lines, the proliferation and metastasis ability of the miR-148a mimic group was remarkably lower than the miR-NC group. At the same time, it was verified that WNT10a was remarkably higher cell lines and RCC tissues; and negatively related to miR-148a expression. In addition, miR-148a mimics were found to remarkably reduce the protein expression of WNT10a. In the cell reverse experiment, overexpression of WNT10a was confirmed to offset the miR-148a mimics effect on metastasis and proliferation of RCC cells. In addition, an increase in relative apoptosis was detected in As₂O₃ treated with/without miR-148a mimics for 48 hours, and apoptosis was significantly reduced after transfection with WNT10a in the Caki-1 cell line and significantly reduced after combined treatment.

Conclusions: The study revealed that miR-148a is associated with distant metastases and leads to poor prognosis in RCC patients. Moreover, miR-148a inhibit the malignant progression and increase the sensitivity of RCC cells to As₂O₃ by regulating WNT10a.

Keywords: MiR-148a; WNT10a; arsenic trioxide; malignant progression; renal cell carcinoma.

Figure 1

MiR-148a is under expressed in RCC. (A) The miR-148a expression in tumor and paracancerous tissues of RCC was detected by RT-PCR. (B) Cell lines from RCCs were tested for miR-148a expression using RT-PCR. (C) The miR-148a expression was detected in distant metastasis specimens from patients with RCC. (D) Kaplan-Meier survival curve of RCC patients. Data are expressed as mean ± SD, *, P<0.05, **, P<0.01, ***, P<0.001. RCC, renal cell carcinoma; RT-PCR, real-time polymerase chain reaction; SD, standard deviation.

Figure 2

MiR-148a mimics inhibit RCC cell metastasis and proliferation. (A) RT-PCR was performed in 786-O and Caki-1 cell lines to verify the efficiency of transfection of miR-NC and miR-148a mimics. (B) An assay that measures the effect of miR-148a mimic on proliferation of cells was performed using CCK-8. (C) An assay for miR-148a mimicking on RCC cell migration (crystal violet staining, magnification: ×20) was performed. (D) The cell wound healing assay detected the effect of miR-148a mimic on invasive ability of RCC cells (magnification: ×20). Data are expressed as mean ± SD, *, P<0.05. RCC, renal cell carcinoma; RT-PCR, real-time polymerase chain reaction; CCK-8, Cell Counting Kit-8; SD, standard deviation.

Figure 3

The miR-148a and WNT10a proteins have direct targeting in RCC tissues and cell lines. (A) The dual-luciferase reporter assays demonstrate direct targeting of WNT10a and miR-148a. (B) A RT-PCR assay verified WNT10a expression in 786-O and Caki-1 cells after transfection of miR-148a mimics. (C) The differential expression of WNT10a between tumors and adjacent tissues was detected using RT-PCR. (D) The expression of WNT10a in RCC cell lines was detected using RT-PCR. (E) The expression of miR-148a was negatively correlated with that of WNT10a in urinary cancer tissues. Data are expressed as mean ± SD, *, P<0.05, **, P<0.01, ***, P<0.001. RCC, renal cell carcinoma; RT-PCR, real-time polymerase chain reaction; SD, standard deviation.

Figure 4

MiR-148a inhibits renal cell carcinoma development via regulating WNT10a expression. (A) RT-PCR analysis of the miR-148a and WNT10a co-transfected Caki-1 cell line revealed the expression level of WNT10a. (B) The CCK-8 assay was used to detect proliferation of Caki-1 cells after miR-148a and WNT10a co-transfection. (C) MiR-148a and WNT10a were co-transfected into Caki-1 cells and then tested for migration ability using the transwell migration assay (crystal violet staining, 20× magnification). (D) A cell wound healing assay detected that miR-148a enhanced RCC cell invasion. Data are expressed as mean ± SD, *, P<0.05. RCC, renal cell carcinoma; RT-PCR, real-time polymerase chain reaction; CCK-8, Cell Counting Kit-8; SD, standard deviation.

Figure 5

MiR-148a combined with As₂O₃ promoted Caki-1 cell apoptosis but WNT10a reversed this trend at tolerated concentration. After Caki-1 cells were treated with miR-148a or As₂O₃, cells were transfected with WNT10a, and apoptosis was detected by flow cytometry. Data are expressed as mean ± SD, *, P<0.05. As₂O₃, arsenic trioxide; SD, standard deviation.