miR-4521-FAM129A axial regulation on ccRCC progression through TIMP-1/MMP2/MMP9 and MDM2/p53/Bcl2/Bax pathways

Abstract

Clear cell renal cell carcinoma (ccRCC) is the most aggressive RCC subtype with high metastasis, chemotherapy and radiotherapy resistance, and poor prognosis. This study attempted to establish the deregulations of miR-4521 and FAM129A together with their correlation to and mechanism of regulation of ccRCC development and progression. FAM129A acted as tumor promotor and miR-4521 acted as a suppressor in ccRCC. As measured in surgical tumorous tissues from ccRCC patients, FAM129A overexpression and miR-4521 deficiency together contributed to ccRCC progression by promoting advances in patients' TNM stage and Fuhrman grade. Both the FAM129A knockdown and miR-4521 overexpression could reduce the in vitro migration and invasion abilities of renal cancer cells 786-O and ACHN, through the TIMP-1/MMP2/MMP9 pathway and could decrease their proliferation by promoting their apoptosis through the MDM2/p53/Bcl2/Bax pathway. By directly targeting the 3'-UTR domain of FAM129A, miR-4521 was negatively correlated with FAM129A/FAM129A levels in ccRCC progression and renal cancer cell malignancies. This work establishes the miR-4521-FAM129A axial regulation mechanism in ccRCC. Micro-4521 deficiency leads to FAM129A/FAM129A upregulation, which synergistically enhances the migration and invasion of renal cancer cells due to the induced decrease of TIMP-1 and increases of MMP2 and MMP9, and increases their growth through escaping apoptosis by suppressing p53 by way of upregulation of induced MDM2. The current work provides new clues to assist fundamental research into the diagnosis and treatment of ccRCC.

Fig. 1. Expression of miR-4521 in ccRCC surgical tumorous tissues and its link with TNM stage and Fuhrman grade of patients.

a miR-4521 expression levels were significantly downregulated in ccRCC cancer tissues compared with paracancerous nontumor renal tissues. b The downregulation efficiency of miR-4521 in tissue samples is shown. Correlations between miR-4521 expression level and the TNM stage (c) and Fuhrman grades (d) of ccRCC are shown, respectively. ccRCC clear cell renal cell carcinoma. *P < 0.05, ****0.0001

Fig. 2. Expression of FAM129A in ccRCC patients’ tumorous tissues and associated with TNM stage and Fuhrman grade.

a Upregulation of FAM129A mRNA levels in ccRCC cancer tissues; normal tissues served as controls. b The upregulation efficiency of FAM129A in tissue samples. c, d Correlations between the FAM129A mRNA expression level and the TNM stage and Fuhrman grades of ccRCC. e Representative images of FAM129A IHC in ccRCC cancer tissues and normal tissues. f, g Western blots of FAM129A showed alterations in protein levels consistent with variations in mRNA levels in clinical samples. h Protein expression of FAM129A is high compared with normal tissues. Correlations between FAM129A protein levels and the TNM stage (i) and Fuhrman grades (j) of ccRCC are shown. ccRCC clear cell renal cell carcinoma, IHC immunohistochemistry. *P values < 0.05, **0.01, ****0.0001

Fig. 3. miR-4521 deficiency is negatively correlated with FAM129A upregulation in clinical tumorous tissues of ccRCC patients.

a 45 cases in 55 samples showed high mRNA expression of FAM129A when miR-4521 expression was low. Inverse correlations of FAM129A mRNA (b) and protein (c) levels with miR-4521 levels in ccRCC tissue are shown. ccRCC clear cell renal cell carcinoma

Fig. 4. miR-4521 downexpression accompanied with FAM129A overexpression in RCC cell lines through binding with the latter’s 3′-UTR domain.

a miR-4521 and FAM129A expressions in RCC cell lines (786-O and ACHN) compared with that in HK-2. b Sequence alignment of the FAM129A 3′UTR with wild-type (WT) versus mutant (MUT) potential miR-4521 targeting sites. c Dual-luciferase reporter assays showed decreased reporter activity after transfection of the wild-type FAM129A 3′UTR reporter construct in 786-O cells overexpressing miR-4521. The FAM129A 3′UTR MUT and control constructs showed no effect on reporter activity. d Alteration of the miR-4521 expression levels of 786-O and ACHN cells 24 h after transfection with the miR-4521 mimics or NC mimics. mRNA (e) and protein (f) levels of FAM129A were examined by qRT-PCR and Western in 786-O and ACHN cells that were transfected with miR-4521/NC mimics. *P values < 0.05, **0.01, ***0.001, ****0.0001

Fig. 5. FAM129A knockdown reduces the in vitro malignant behaviors of 786-O and ACHN cells.

mRNA (a) and protein (b) levels of FAM129A were examined by qRT-PCR and western blot in 786-O and ACHN cells that were transfected with si-NC /si-FAM129A. MTT assays revealed that transfection of the siRNA of FAM129A can remarkably attenuate the proliferation of 786-O (c) and ACHN (d) cells compared with siNC. Representative photographs are shown of transwell assays of 786-O (e) and ACHN (f) cells identifying FAM129A as oncogenic. g Flow cytometry was performed to reveal that FAM129A downregulation largely induces the cell apoptosis of 786-O and ACHN cells. Each experiment was performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 6. The influence of miR-4521 overexpression in vitro malignant properties of 786-O and ACHN cells.

MTT assays showed that transfection of the miR-4521 mimic can significantly attenuate the proliferation velocity of 786-O (a) and ACHN (b) cells. Representative photographs are shown of migration and invasion assays of 786-O (c) and ACHN (d) cells to determine the tumor suppressor function of miR-4521. e Flow cytometry was performed to reveal that the miR-4521 mimic induced the cell apoptosis of 786-O and ACHN cells. *P values < 0.05, **0.01, ***0.001, ****0.0001

Fig. 7. FAM129A and miR-4521 mediate the malignant behaviors of via TIMP-1/MMP2/MMP9 and MDM2/p53/Bcl2/Bax pathways in 786-O cells.

The protein levels of FAM129A, TIMP-1, MMP2, MMP9, MDM2, p53, Bcl2 and Bax were examined using western blot analysis in 786-O cells treated with si-NC /si-FAM129A (a) or NC mimic/miR-4521 mimic (b). GAPDH was used as an internal control. *P < 0.05, **P < 0.01

Fig. 8. A schematic model of miR-4521-FAM129A axis on ccRCC cell malignant behaviors.

miR-4521 induces cell migration and invasion via TIMP-1/ MMP2/MMP9 pathways and also suppresses cell apoptosis by the MDM2/p53/Bcl2/Bax pathway by targeting FAM129A mediating ccRCC malignant behaviors. ccRCC clear cell renal cell carcinoma