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

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.


Introduction
Renal cell carcinoma (RCC) comprises up to 85% of kidney cancer cases 1 . As one of the most common malignant urological tumors, RCC is characterized by a high mortality-to-incidence ratio and poor prognosis for late-stage patients [2][3][4] . Clear cell RCC (ccRCC), accounting for~80% of RCCs, is the most aggressive RCC subtype. Accompanied by extremely high rates of local invasion, metastasis, and resistances to chemotherapy and radiotherapy, the 5-year survival rate of the over 30% of ccRCC patients with metastasis was below 20% [5][6][7] . The pathogenesis, diagnosis and treatment of ccRCC deserves more attention.
MicroRNAs (miRNAs) play important roles in the pathogenesis, development and prognosis of various major diseases by degrading mRNA or by inhibiting the translation processes of target genes by binding their 3′ UTRs [8][9][10][11][12] . As a member of tRNA-derived small RNAs (tsRNAs), miR-4521 is involved in breast cancer, chronic lymphocytic leukemia (CLL), lung cancer, pancreatic ductal adenocarcinoma (PDAC) and esophageal adenocarcinoma [12][13][14][15] . Its deregulation in MCF-7 cells is accompanied by hypoxic hypoxia induction 13 and in enhanced malignancy of CLL and lung cancer 14 . It was one of 11 joint miRNAs implicated in the prognosis of PDAC patients with pancreaticoduodenectomy 12 . A higher miR-4521/miR-340-5p ratio was implicated in better disease-free survival of esophageal adenocarcinoma patients with neoadjuvant chemoradiotherapy and esophagectomy 15 . Except for the finding that miR-4521 was downregulated in the sunitinib-resistant ACHN and RCC23 cell lines, SR-ACHN and SR-RCC23 16 , no study has addressed its role or regulation mechanism in ccRCC. In this study miR-4521 acted as a tumor suppresser in ccRCC. Its deficiency negatively correlated with FAM129A upregulation and prompted clinical development and progression of ccRCC. It mediated ccRCC carcinogenesis by affecting cancer cells' malignant invasiveness via the TIMP-1/MMP2/MMP9 and MDM2/p53/ Bcl2/Bax pathways.
The member A of family with sequence similarity 129 (FAM129A), also known as Niban, was originally identified in Eker rats with hereditary renal carcinoma induced by tuberous sclerosis 2 gene mutation (Tsc2) 17 . It is commonly overexpressed in patients with thyroid cancer [18][19][20] , head and neck squamous cell carcinoma (HNSCC) 21 and sporadic renal carcinomas 22 . Although highly expressed in early stages of cancer development and remaining overexpressed throughout cancer progression, its function and mechanism of action remain unclear. FAM129A was detected in sporadic RCCs including clear cell, granular cell and spindle cell carcinomas. Its common expression might be an indicator for renal carcinogenesis 17,22 . FAM129A has been linked to renal interstitial fibrosis by increasing renal tubular cell apoptosis 23 ; however, its function and mechanism in ccRCC remain unclear.
Current work shows FAM129A is a promotor and miR-4521 is a suppressor in ccRCC. FAM129A overexpression positively correlates with advances in TNM stage and Fuhrman grade of ccRCC patients. miR-4521 deficiency contributed to enhanced TNM stage and Fuhrman grade of ccRCC. FAM129A knockdown, miR-4521 overexpression with its induced FAM129A downregulation decreased the proliferation, migration and invasion, and increased the apoptosis of renal carcinoma cells through the TIMP-1/MMP2/MMP9 and MDM2/p53/Bcl2/Bax pathways.

Discussion
As the highest mortality urological malignancy 24,25 , ccRCC accounts for~80% of RCC incidence 4 . The prognosis for cases of advanced ccRCC is poor 26 . A 5-year survival rate of the over 30% of patients with metastatic ccRCC is below 20% 5,6 . Better understanding on the pathogenesis, diagnosis and treatment of ccRCC 7 is urgently needed.
Binding to 3′UTR regions of targeting genes, miRNAs cause degradation of mRNA or inhibit their translation processes. miRNAs play important roles in a variety of human major diseases including cancer [9][10][11][12] . miR-4521 dysexpression is involved in some cancers. Its deficiency is  14 . It was one of 11 miRNAs found to be prognostic indicators for PDAC patients 12 . The miR-4521/miR-340-5p ratio was regarded as a prediction factor for disease-free survival of esophageal adenocarcinoma patients 15 . miR-4521 has been linked to drugresistance of RCC cells. In sunitinib-resistant ACHN and RCC23 cell lines, its expression level decreased 16 . Some miRNAs were reported as sensitive and specific indicators for the pathologic stage, recurrence, metastasis and survival of ccRCC patients 27 . The role and mechanism of action of miR-4521 in ccRCC has not been reported.
Our current work shows miR-4521 is a suppressor in ccRCC progression, interacting with FAM129A via the TIMP-1/MMP2/MMP9 and MDM2/p53/Bcl2/Bax pathways. miR-4521 downregulation was inversely correlated with the TNM stage and Fuhrman grade (Fig. 1, Table 1) of ccRCC patients. miR-4521 was also downexpressed in RCC cell lines 786-O and ACHN compared with HK-2 cells (Fig. 4a). 786-O and ACHN are appropriate cell models for studying the role and cellular regulation mechanism of miR-4521 in ccRCC malignancy. Since it is downregulated in both ccRCC patients' tumorous tissues and renal cancer cells, its overexpression should antagonize the malignant behaviors of cancer cells. miR-4521 upregulation (Fig. 4d) resulted in significant reduced in vitro proliferation, migration and invasion capacities of 786-O (Fig. 6a, c) and ACHN cells (Fig. 6b, d). miR-4521 overexpression might affect 786-O and ACHN growths by promoting their apoptosis. The apoptotic rates of miR-4521 overexpressing 786-O and ACHN cells increased by 48.9% and 62.7% (Fig. 6e). This demonstrates that miR-4521 downexpression increases malignant properties of RCC cells, which might contribute to enhanced development and progression of ccRCC patients. This work also showed miR-4521 level was negatively correlated with FAM129A level in ccRCC progression.
FAM129A (Niban or Clorf24) is liked to thyroid cancer 20 , HNSCC 21 and sporadic renal carcinomas 22 , acting in all as a tumor promoter. FAM129A, ITM1 and PVALB were the three earliest genes used for distinguishing benign thyroid nodules from malignant ones [18][19][20] . FAM129A was absent in specimens from normal thyroid, benign follicular thyroid adenoma and thyroid hyperplasia, but was present and upregulated in papillary thyroid carcinoma and follicular thyroid carcinoma 20 tissues. It was consistently found to be more highly expressed in FTC cell lines FTC133, FTC236, FTC238 and WRO, and in the TPC cell line, TPC1, in comparison with a normal thyroid cell line, PCCL3 20 . FAM129A knockdown antagonized the in vitro proliferation, migration and invasion capacities of WRO and TPC1. FAM129 upregulation promoted the carcinogenic process of HNSCC and head and neck dysplastic lesions. Compared with its absence in normal HNS epithelia, IHC assays indicated that FAM129A was positively detected in 42 out of 43 HNSCCs (97.6%) and 20 of 30 (66.6%) dysplastic lesions. FAM129A expression frequently began at HNSCC early stages and continued to be upregulated throughout the carcinogenesis 21 . FAM129A expression was detected in Tsc1 and Tsc2 knockout mice, in sporadic human RCC including clear cell carcinomas, granular cell carcinomas and spindle cell carcinomas. Its common expression might be a marker for renal carcinogenesis 17,22 . We propose that FAM129A acts in renal carcinoma by influencing tumor cell apoptosis based on its involvement in renal interstitial fibrosis via promoting renal tubular cells apoptosis 23 . This work revealed FAM129A as a promotor for ccRCC malignancy. FAM129A upregulation (Fig. 2a, b) was positively correlated with advances in TNM stage (Fig. 2c, Table 1) and Fuhrman grade (Fig. 2d, Table 1) of ccRCC patients. Both IHC and WB assays indicated FAM129A protein expression level was increased in patients' tumorous tissues (Fig. 2e, f, Table 2). The positive immunoreactivity against FAM129A was 70% higher, the positive detection rates of FAM129A with ++ and +++ degrees were 4fold and 17-fold higher (Table 2), and the overall level determined by WB was 214.7% higher (Fig. 2g, h, Table 2) in patients' tumorous tissues than in nontumorous tissues. Both IHC and WB assays indicated that FAM129A upregulation was positively correlated with TNM advance (Fig. 2i) and tended to be associated with Fuhrman grade advance (Tables 1 and 2) among patient's clinicopathological parameters. FAM129A was consistently more highly expressed in renal cancer cells, 786-O and ACHN, than in the normal renal cell, HK-2 (Fig. 4a). Consequently, FAM129A knockdown (Fig. 5a, b) resulted in ameliorated proliferation (Fig. 5c, d), migration and invasion (Fig. 5e, f)
Matrix metalloproteinases (MMPs) play important roles in tumor cell proliferation, apoptosis, invasion and metastasis. We investigated changes in the levels of MMP2 and MMP9, two key metalloproteinases, and TIMP1, a key tissue inhibitor of metalloproteinase that regulates most MMPs 28,29 , by following changes in the levels of FAM129A and miR-4521 in RCC cell lines. TIMP-1 was upregulated by 45.3%, and MMP2 and MMP9 were downregulated by 42.4% and 49.2%, respectively, in 786-O cells following FAM129A knockdown (Fig. 7a). These also implicated, acting as a promotor, the upregulation of FAM129A promoted ccRCC progression through enhancing MMP2 and MMP9 activation by suppressing TIMP-1. On the basis of its direct binding to and inverse correlation with FAM129A, we propose miR-4521 should also affect renal cancer cell proliferation, migration and invasion via the above-mentioned molecules. We tried to overexpress and downexpress miR-4521 by mimic transfection and inhibitor transfection in 786-O and ACHN but were unsuccessful. Only miR-4521 overexpression succeeded (Fig. 4d). Its overexpression induced FAM129A downregulation (Figs. 4e, f and 7b), TIMP-1 increased by 68.4%, and MMP2 and MMP9 decreased by 55.1% and 72.5%, respectively (Fig. 7b). As shown in Fig. 8, the above results indicate FAM129A upregulation and/or miR-4521 deficiency contributed FAM129A upregulation affects ccRCC malignancy via the TIMP-1/MMP2/MMP pathway. Interestingly, although FAM129A was more reduced in si-FAM129A-transfected 786-O and ACHN cells (Figs. 4f, 5a, b and 7), more reductions on the invasion and slightly higher reductions on the migration capacities were observed in miR-4521overexpressing 786-O and ACHN cells (Figs. 5e, f and 6c,  d), which was also consistent with more increased TIMP-1 upregulation and MMP2 and MMP9 downregulations resulting in better suppressed invasiveness and metastases of tumor cells. Except for its direct targeting with FAM129A, which has been established, the role of miR-4521 and its detailed action mechanism in ccRCC deserve more attention.
Collectively, FAM129A and miR-4521 act as a tumor promotor and suppressor in ccRCC. Negatively correlated, FAM129A upregulation and miR-4521 deficiency contribute to ccRCC clinical progression with enhanced TNM stage and Fuhrman grade. Binding to 3′UTR of FAM129A, miR-4521 retroregulates FAM129A/FAM129A in mediating ccRCC progression and renal cancer cell malignant properties. FAM129A knockdown or miR-4521 upregulation decreases renal cancer cells' invasiveness via TIMP-1/MMP2/MMP9 and MDM2/p53/Bcl2/Bax. miR-4521 deficiency with its potential contribution to FAM129A upregulation might synergistically promote renal cancer cells' malignant behaviors by enhancing MMP2/MMP9 through suppressing TIMP-1 and increasing their growth by escaping apoptosis through suppressing p53 by way of upregulation of MDM2 via a decreased Bax/Bcl2 ratio. The miR-4521-FAM129A axial regulation pathway provides clues useful to the fundamental research, diagnosis and treatment for ccRCC.

Materials and methods
Collection and treatment of tumorous and paired paracancerous normal tissues from ccRCC patients Fifty-five pairs of tumorous and matched paracancerous nontumoral renal tissues from ccRCC patients were collected at the Urology Department of the Second Affiliated Hospital of Dalian Medical University, Dalian. The paracancerous normal renal tissue was taken >5 cm away from the edge of the tumor. No patients received radiotherapy or chemotherapy before surgery. After cleaning, the surgical tissues for western blotting and qRT-PCR assays were immediately snap-frozen in liquid nitrogen and stored at −80°C. Thirty pairs of sliced surgical tumorous and paracancerous tissues were fixed in 10% neutralbuffered formalin and blocked in paraffin for IHC assay. The ccRCC specimens were classified according to the age, gender, tumor position, TNM stage and Fuhrman grade of patients. The histological subtypes and tumor stages were assessed according to the 2016 WHO Classification of Tumors of the Urinary System of the Tumor, Nodes and Metastasis (TNM) system. Specimen use and investigation protocols were approved by the Medical Ethics Committee of Dalian Medical University. Informed consents were obtained from the patients.

Quantitative real-time polymerase chain reaction (qRT-PCR) assay
Total RNA was extracted either from each group of specimens or cells by using Trizol™ reagent (Invitrogen, USA) according to the instruction manual. A piece of tissue of~50 mg was cut into 4 μm slices by a freezing microtome for RNA extraction. Then the EasyScript One- Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen, China) was used for cDNA reverse transfection. qRT-PCR was carried out on a StepOnePlus™ Real-Time PCR system (ThermoFisher, USA) using FastStart Universal SYBR Green Master (ROX) reagent (Roche, Germany). SnRNA U6 and β-actin were used as internal standards for miR-4521 and FAM129A. miR-4521 and U6 primers were purchased from RiboBio Company (Guangzhou, China). Primers were designed as below for FAM129A, F: 5′-CTCAGCCCTTTGTGGTCCT-3′, R: 5′-CTC CTGTCGGAAGAATTGCAC-3′ and for β-actin, F: 5′-AGGCCAACCGCGAGAAG-3′, R: 5′-AGAGCCTG GATAGCAACGTACA-3′. The 2 −ΔΔCT method was performed for quantification analysis. Finally, 100 μL of the above mixture was added into each of the already prepared group cells, gently mixed well, and incubated at 37°C with 5% CO 2 for 24 h. The cells from each group were trypsinized with trypsin-EDTA (0.25%, Gibco, USA) and centrifuged at 1000 rpm for 5 min. Cell pellets were collected for further experiments.

SDS-PAGE and western blotting assay Protein extraction
For protein extraction from specimens,~50 mg of tumorous or paracancerous tissue was washed with PBS (4°C), cut into small pieces, ground under liquid nitrogen into powder and suspended in 300-600 μL ice-cold RIPA buffer (50 mM pH 8.0 Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na 3 VO 4 , 1 μg/mL leupeptin and 0.5 mM PMSF). The tissue mixture was then ground well with a pestle for 3 × 10 min on ice. Supernatant protein was collected by 12,000 rpm centrifugation at 4°C for 15 min. For protein extraction from cells, each cell pellet per well obtained from six-well plates of 786-O/ACHN or miRNA mimictransfected or siRNA-FAM129A-transfected 786-O/ ACHN cells was suspended in about 50 μL of the RIPA buffer and lysed on ice for 30 min with suspension using a pipet every 10 min. Supernatant protein was collected by centrifugation at 12,000 rpm at 4°C for 15 min.

Immunohistochemistry (IHC assay)
The paraffin blocks of tumorous and paracancerous renal tissues were cut into 4-μm slices. The slices were boiled in citrate buffer (0.01 M, pH 6.0) in a microwave oven using high power for 4 min, and cooled to RT. After doing this four times, the slices were washed clean with PBS for 3 × 5 min, blocked in 3% H 2 O 2 for 20 min and in 10% nonimmune goat serum for 15 min at RT, and incubated with FAM129A antibody (1:200) at 4°C overnight. The tissue slices were then warmed at 37°C for 30 min, treated with biotin-streptavidin HRP detection kit (ZSGB-BIO, China), and imaged with 3,3′-diamino-benzidine (DAB) development kit (ZSGB-BIO, China) under an upright light BX3-CBH microscope (Olympus, Japan).

Dual-luciferase reporter assay
PsiCHECK™2.0 dual-luciferase expression vector (Promega, USA) was used as expression vector in a dualluciferase reporter assay as we previously reported 36 . Briefly, one potential binding site of miR-4521 was revealed in the 3′-UTR region of FAM129A with the miRDB and TargetScan platforms. The wild-type (WT) and mutant (MUT) binding site sequences in FAM129A 3′-UTR were amplified and cloned into psiCHECK™2.0 vectors. The correct recombinant plasmids, validated by nucleotide sequencing (Invitrogen, USA), were named as wt-FAM129A-3′UTR and mut-FAM129A-3′UTR, respectively. 5 × 10 4 786-O cells in 2 mL of RPMI-1640 with 15% FBS were seeded into each well of a six-well plate and incubated at 37°C, 5% CO 2 for 24 h. Then, 786-O cells were cotransfected with the additions of 4 μL of miR-4521 mimic/NC mimic (20 μM) and 4 μL (3 μg) of wt-FAM129A-3'UTR/mut-FAM129A-3′UTR at 37°C with 5% CO 2 for 24 h, respectively. A cell pellet from each group, obtained by washing with PBS and centrifuging at 1000 rpm for 5 min, was lysed in 200 μL of passive lysis buffer at 4°C for 15 min. Then 20 μL of supernatant lysate was loaded into a luminometer tube, mixed well with 100 μL of Luciferase Assay ReagentII (LAR II) for Firefly luciferase activity detection, then mixed with 100 μL of Stop & Glo Reagent for Renilla luciferase activity assay using a GloMax fluorescence reader (Promega, USA).

MTT assay for cell proliferation
The influences of changes in the levels of miR-4521 and FAM129A on 786-O and ACHN proliferation were determined by MTT assay. The cells from each 786-O group were seeded into a 96-well plate at the density of 5000 cells in 200 μL of RPMI-1640 with 15% FBS per well. ACHN group cells were seeded at the density of 10,000 cells/well. The cells were continuously incubated at 37°C, 5% CO 2 for 24, 48, 72, 96 and 120 h, separately, then incubated with 0.5 mg/mL MTT working solution (Sigma, USA) by replacing culture medium at 37°C, 5% CO 2 for 4 h in darkness. After the removal of the supernatant, 150 μL DMSO (Sigma, USA) was added into each well to dissolve formazan crystals. The absorbance at 490 nm was measured using a microplate reader (Thermo, USA) for cell density quantification. Triplicate experiments were performed for each assay.

Boyden transwell-chamber assay for cell migration and invasion
The 24-well transwell units with 8 mm I.D. polyester membrane with 8 μm pore size polycarbonate filters (Corning, USA) were employed to investigate the influences of miR-4521 upregulation and FAM129A downregulation on the in vitro migration and invasion properties of 786-O and ACHN cells. For migration assays, 600 μL of RPMI-1640 with 15% FBS was loaded into each lower chamber. 10,000 and 2000 cells from each of the 786-O and the ACHN groups were separately loaded into one upper chamber in 200 μL of RPMI-1640 medium and incubated at 37°C with 5% CO 2 for 24 h. The cells on the upper surface of the insert that did not migrate were carefully wiped off using cotton swabs. The cells that migrated to the lower surface of the filter were fixed in methanol (AR, Sigma, USA) for 30 min, dried for 5 min at RT, stained in 0.1% crystal violet for 40 min, washed with PBS (200 μL), and counted by randomly selecting five fields per well using an upright light microscope (Olympus, Japan) at a magnification of ×200.
For invasion assays, the filter surface of an insert transwell unit was first coated with 50 μL ice-cold ECM gel (1:5 dilution with RPMI 1640, Sigma, USA) by incubating at 37°C for 8 h. The loading numbers for the 786-O and the ACHN group cells were 7500 and 15,000, respectively, in 200 μL of RPMI-1640. The remaining steps were the same as for the migration assays.

Flow cytometry
Flow cytometry was performed to investigate miR-4521 upregulation and FAM129A downregulation on the apoptosis of 786-O and ACHN cells using the Annexin V Apoptosis Detection Kit APC (Affymetrix eBioscience, USA). Following transfection, 1 × 10 6 cells from each of 786-O and ACHN groups were harvested, washed once with ice-cold PBS, washed once with the binding buffer, centrifuged at 1000 rpm for 5 min, resuspended in 100 μL binding buffer with the addition of 5 μL of fluorochromeconjugated Annexin V, and incubated in the dark for 15 min at RT. Finally, the cells from each of 786-O and ACHN groups were washed again with 100 μL binding buffer, resuspended in 200 μL of binding buffer with the addition of 5 μL of propidium iodide (PI) staining solution, and analyzed using a flow cytometer (BD Biosciences, USA). Each assay was replicated for four times.

Data processing and statistical analysis
The data were expressed as mean ± SD of at least triplicate independent experiments. SPSS17.0 was used for statistical analysis. The difference between two groups were evaluated by Student's t test and chi-square test analyses. One-way ANOVA analysis was used to evaluate the difference between different groups. Results with P < 0.05 were statistically significant.
Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.