NFIB promotes the migration and progression of kidney renal clear cell carcinoma by regulating PINK1 transcription

Patients and clinical samples

Thirty pairs of clinical tissue samples of KIRC were collected from patients undergoing surgery at the Department of Urology, Hanyang Hospital (Wuhan, China) from September 2015 to December 2015. The diagnosis of KIRC was confirmed by the original histopathological reports. The tissue samples were rapidly fixed in formalin for paraffin embedding. All clinical samples were collected with written informed consent to participate in this study. The study was approved by the Ethics Committee of the Academic Medical Center of Wuhan University of Science and Technology (No. 2020-IEC1453).

Cell culture

The source of the tubular epithelial cell line (HKC-5; BNCC100598) and KIRC cell lines (LoMet-ccRCC and 786-0) were obtained from the BeNa Culture Collection (Beijing, China). Cell culture methods were performed as previously described (Yuan et al., 2019) .

Transfection

The siRNA and normal control (NC) siRNAs for NFIB and PINK1 were purchased from RiboBio (Guangdong, China). The sequences of siRNAs are as follows: NFIB sense: 5′-CCAGGUGGUGAAGAAUCUATT-3′, antisense: 5′-UAGAUUCUUCACCACCUGGTT-3′; PINK1 sense: 5′- CAAGACUUGGAGCUAAAAATT-3′, antisense: 5′- UUUUUAGCUC CAAGUCUUGTT-3′. The overexpression plasmids of NFIB and PINK1 were constructed and cloned into the CV061 vector (GeneChem, Shanghai, China) between the EcoRI and HindIII sites. Once the cells reached 30–40% confluence, LoMet-ccRCC and 786-0 cells were transfected using Lipofectamine 3000 reagent (Invitrogen, MA, USA) according to the manufacturer’s instructions. Subsequent treatments were performed after the cells were cultured for an additional 48 hours.

CRISPR-Cas9

NFIB-knockout cells were generated using CRISPR-Cas9 gene editing, and the sgRNA targeting NFIB gene was cloned into GV371-U6-NFIB sgRNA-SV40-EGFP and GV371-CMV-hSpCas9-SV40-Puro, respectively. The sgRNA sequence is as follows: GGGAGTGTCTCCTGCCGAGA. In addition, LV-sgRNA and LV-Cas9 lentiviruses were also produced (GeneChem, Shanghai, China). LV-Cas9 was seeded in 786-0 cells with a MOI of 2 after 3 days of puromycin selection with a final concentration of 5 µg/ml. LV-sgRNA was then seeded, and another 3 days later, the cells were harvested for further detection.

qRT-PCR

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed as previously described (Yuan et al., 2019). The following primers were used: NFIB forward, 5′-GTAATTAGAGCTGTGACA-3′; NFIB reverse, 5 ′-TGTATGCCATCTAGTGGAT-3′; PINK1 forward, 5′-CGTGGCGAGCTGTAATTGAC-3′; PINK1 reverse, 5′-GGCTCCGCAGACGTTATCTA-3 ′; GAPDH forward, 5′-GAAGGTGAAGGTCGGAGTC-3′; and GAPDH reverse, 5′-GAAGATGGTGATGGGATT-3′. All reactions were performed in triplicate. The relative expression levels of the genes were calculated using the 2^−ΔΔCq method.

Western blotting

Total protein lysates from the cultured cells were extracted using a lysis buffer containing proteinase inhibitors (Sigma-Aldrich, Darmstadt, Germany). Proteins were denatured, separated in 10% polyacrylamide gels, and transferred to polyvinylidene fluoride (PVDF) membranes for probing with the following primary antibodies: anti-NFIB (1:500, Abcam, ab186738, Cambridge, UK), PINK1 (1:500, Abcam, ab216144, Cambridge, UK) and anti-GAPDH (1:2000, Proteintech, 10494-1-AP, Wuhan, China). For detection, anti-rabbit secondary antibodies conjugated to horseradish peroxidase (1:3000, Proteintech, Wuhan, China) were used. Band signals were visualized by an enhanced chemiluminescence detection kit (Meilunbio, Dalian, China). All western blotting was performed in triplicate.

Cell proliferation assay

Cells were seeded into 96-well plates at a density of 3 × 10³ cells per well and observed for 1 to 5 days. Then, 20 µl of MTT (5 mg/mL) was added to each well, and the media were replaced with 200 mL of DMSO (Sigma) after incubation for 4 h. When the crystals were completely dissolved, a microplate reader was used to determine the resulting absorbance at 570 nm. All MTT assays were performed in triplicate.

Colony formation assay

Cells of treatments and experimental groups were respectively seeded into six-well plates at a density of 500 cells per well and cultured with complete medium at 37 °C in 5% CO₂ for 15 days. The colonies were then stained with 1% crystal violet (Sigma-Aldrich, Darmstadt, Germany) for 30 min and counted. All experiments were performed in triplicate.

Migration and wound-healing assay

Transwell chambers (Beaverbio, Jiangsu, China) were used to assess KIRC cell metastasis ability. Suspensions of 8 × 10⁴ cells in 200 µl of FBS-free medium (Sigma-Aldrich, Darmstadt, Germany) were added to the top chamber, and medium containing 20% FBS (Sciencell, CA, USA) was added to the bottom chamber to serve as the chemotaxin. The cells were cultivated at 37 °C in 5% CO₂ for 48 h, and the cells that failed to migrate were wiped off the top of the membranes. The migrated cells attached to the bottom chamber were fixed and stained. Cells from five random fields were quantified by light microscopy. To determine the invasion ability of KIRC cells, invasion assay was performed by transwell migration chambers coated with Matrigel (Sigma).

For the wound-healing assay, the cells were seeded into 12-well plates (Beaverbio, Jiangsu, China) and grown to 80–90% confluence at 37 °C in 5% CO₂. The cell monolayers were prepared with a 200 µl pipette tip and cultured in serum-free medium. Cell migration was assessed by microscopy at 0 and 48 h and objectively analyzed with ImageJ 1.8.0. All experiments were performed in triplicate.

Immunohistochemical

Paraffin-embedded clinical tissues were cut into 5- µm-thick sections and placed on glass slides. The tissue sections were deparaffinized and subjected to antigen retrieval using 0.01 m citric acid buffer (pH 6.0) for 15 min and incubated overnight at 4 °C with primary antibodies against NFIB (1:200, Abcam, ab186738, Cambridge, UK). After three washes with Tris-buffered saline, the sections were incubated with a horseradish peroxidase-conjugated secondary antibody (1:100, Boster) for 1 hour at room temperature. The immunohistochemical (IHC) staining results were evaluated by two independent observers. When staining on the nucleus and ≥30% positive cells in the section, immunohistochemical staining of NFIB was estimated to be positive.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (CST, #9005, MA, USA) according to the manufacturer’s recommended protocol. Cells were crosslinked with formaldehyde and sonicated to an average size of 150 to 900 bp. Then, chromatin extracts were immunoprecipitated using 4 µg of monoclonal anti-NFIB antibody (Abcam, ab186738, Cambridge, UK). The purified DNA was subjected to qPCR to amplify the binding sites of the PINK1 promoter region. All ChIP assays were performed in triplicate.

Data acquisition and statistical analysis

Raw KIRC data containing mRNA sequencing and clinical information were obtained from the TCGA Genome Data Analysis Center (http://gdac.broadinstitute.org/runs/analyses__latest/reports/cancer/STAD/). In addition, the KIRC mRNA expression array of GSE83999 and GSE53757 were acquired from the NCBI GEO (Perron et al., 2018; Von Roemeling et al., 2014). The data analysis methods were performed as previously described (Yuan et al., 2019). The R ‘limma’ Bioconductor package was used to screen the differentially expressed genes (DEGs) between KIRC and adjacent tissues based on the following criteria: Fold change (FC), —log2(FC)—>1; and false discovery rate (FDR) <0.05. Adjusted P < 0.05 was used to define a gene as a DEG. Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 (david-d. ncifcrf.gov/) was used to analyze functional enrichment among DEGs. In addition, only those Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with P ≤ 0.05 and ≥10 enriched genes were considered significant. The 2000bp sequence upstream of the PINK1 gene was used as the gene promoter sequence. The open database JASPAR (http://jaspar.genereg.net) and PROMO (http://alggen.lsi.upc.es/cgi-bin/ promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) were used to predicate the potential NFIB sites on the PINK1 gene promoter.

The assay data were analyzed by SPSS 13.0 and presented as the mean value ± standard deviation (SD). Depending on the type of experiment performed, the assay results were analyzed through two-tailed unpaired or paired Student’s t test. The relationships between the expression of NFIB and the clinical pathological characteristics of KIRC were analyzed by chi square (X²) test. The statistical significances between the groups are presented as P values. When p < 0.05, the results were considered statistically significant.

NFIB is overexpressed in human KIRC

To identify novel genes that potentially drive KIRC tumorigenesis, we performed differential expression analysis using the dataset from TCGA (Table S1) and the datasets from GEO (Fig. 1A and Tables S2–S3). Hierarchical cluster analysis revealed alterations in the mRNA transcript expression between KIRC and paired noncancerous kidney (NK) tissue among the three datasets (Fig. 1B). NFIB was one of the prominently upregulated mRNAs in KIRC tissues compared with NK tissues (Fig. 1B and 1C). After a literature search, we found that the roles of NFIB in the development of KIRC have not been reported, so we aimed to further investigate its roles in driving KIRC tumorigenesis. To confirm these results, we further tested the expression levels of NFIB in cell lines. Compared with normal human renal tubular epithelial cell lines, KIRC cell lines showed significantly increased mRNA expression levels of NFIB (Fig. 1D), and the western blot results were consistent with the results obtained from RT-qPCR (Fig. 1E). In addition, NFIB expression was detected in 30 paired KIRC and NK clinical samples using IHC analysis. The expression level of NFIB was significantly higher in KIRC tissues than in paired NK clinical tissues (Fig. 1F and 1G). Taken together, these results demonstrate a novel dysregulated gene, NFIB, in KIRC.

NFIB is associated with KIRC progression and worse prognosis

To explore the clinical significance of NFIB, we assessed the correlation between the expression levels of NFIB and the individual clinicopathological features of KIRC patients using the dataset from TCGA (Table S4). As shown in Table 1, the results indicated that the overexpression of NFIB was significantly associated with poor histological grade (P = 0.008), pathological stage (P = 0.011), T stage (P = 0.035), lymphatic invasion (P < 0.001) and distant metastasis (P = 0.019). Moreover, we found that high NFIB expression was significantly associated with worse prognosis in patients (Fig. 1H, HR: 0.175, 95% CI: [0.091, 0.336]), and we obtained consistent results (Fig. 1I) with the online data sites GEPIA (http://gepia.cancer-pku.cn) (Tang et al., 2017).

NFIB promotes the proliferation and migration of KIRC cells.

To investigate the biological roles of NFIB, the mRNA and protein expression of NFIB was upregulated by transfection with plasmids containing the NFIB sequence (NFIB vector) compared to the vector control (NC vector) in the LoMet-ccRCC and 786-0 cell lines (Fig. 2A). The results showed that NFIB overexpression significantly increased the proliferation of the LoMet-ccRCC and 786-0 cell lines (Fig. 2B). We further used colony formation experiments to demonstrate that the overexpression of NFIB promoted the number of tumor cell colonies formed in the LoMet-ccRCC and 786-0 cell lines (Fig. 2C and 2D). In addition, the migration ability of LoMet-ccRCC and 786-0 cells was significantly increased after NFIB overexpression compared with NC vector transfection in transwell, invasion and wound-healing assays (Figs. 2E–2J). Additionally, siRNAs against human NFIB (NFIB-si) were applied to knockdown the expression of NFIB in KIRC cells (Fig. 3A). The knockdown of NFIB significantly repressed the proliferation and colony formation of LoMet-ccRCC and 786-0 cells (Figs. 3B–3E). Furthermore, transwell, invasion and wound-healing assays showed that NFIB depletion caused an obvious suppression of the migration ability of KIRC cells (Figs. 3F–3M). Therefore, the above results suggest that NFIB might be a promoter of KIRC tumorigenesis.

PINK1 is a potentially target of NFIB

To investigate the target genes of NFIB, we first performed functional enrichment analysis of the differentially expressed genes between KIRC and adjacent tissues obtained from three datasets (Fig. 1A and Table S5) using the DAVID online analysis website (https://david.ncifcrf.gov) (Jiao et al., 2012). These genes are significantly enriched in multiple KEGG pathways involved in tumorigenesis (Fig. 4A), such as pathways in cancer (P = 0.001) and MAPK signaling pathway (P = 0.002), associated with tumor cell proliferation, ECM-receptor interaction (P < 0.001) and the calcium signaling pathway (P < 0.001), associated with drug transport and metabolism in tumor cells. Given that NFIB is a transcription factor, we next investigated the possible regulation of NFIB by analyzing the gene promoters enriched in these pathways. The open database JASPAR (http://jaspar.genereg.net) and PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) were used to predicate the potential NFIB sites on the PINK1 gene promoter. Fortunately, we found three potential binding sites on PINK1 gene promoter for NFIB (Fig. 4B) (Khan et al., 2018; Messeguer et al., 2002), and bioinformatics analysis revealed that PINK1 was enriched in focal adhesion and pathways in cancer (Table S5). Hence, we hypothesized that PINK1 is one of the targets by which NFIB promotes KIRC progression and metastasis.

NFIB regulates PINK1 expression

To further verify the role of NFIB, the KIRC cell lines LoMet-ccRCC and 786-0 were infected with NFIB vector or NC vector. Using qRT-PCR analysis, we found that NFIB overexpression significantly induced an increase in the mRNA expression level of PINK1 and that PINK1 expression was not induced by the NC vector (Fig. 4C). Furthermore, we used western blotting to detect the effect of NFIB overexpression on the expression of PINK1 protein. As expected, the protein level of PINK1 was significantly increased by NFIB overexpression compared with NC vector transfection (Fig. 4D). On the other hand, we observed the opposite results in KIRC cell lines after NFIB knockdown. qRT-PCR and western blotting showed that the knockdown of NFIB downregulated the expression levels of PINK1 mRNA and protein (Fig. 4E and 4F), respectively. As shown in Fig. 4B, three putative NFIB transcriptional binding sites in the PINK1 gene promoter region were found via bioinformatics analysis. Next, we performed a ChIP assay in LoMet-ccRCC and 786-0 cells using NFIB-specific antibodies. As shown in Fig. 4G, based on the qRT-PCR reads and the ChIP analysis results, we found that the NFIB-bound complex was remarkably enriched in PINK1 promoter site 2 but not site 1 or site 3. These results strongly implied that NFIB promotes KIRC metastasis and progression by regulating the promoter of PINK1.

PINK1 is the critical downstream of NFIB in KIRC

To confirm that PINK1 is a key factor through which NFIB promotes KIRC progression, we first conducted a rescue experiment. The cotransfection of PINK1 siRNA with NFIB vector dramatically decreased NFIB-upregulated PINK1 mRNA expression (Fig. 5A and 5B), and western blotting also obtained consistent results, and the promotion of PINK1 protein expression by NFIB was inhibited by PINK1 siRNA (Fig. 5C). We further examined the effect of PINK1 siRNA on the cell proliferation and progression induced by the overexpression of NFIB using LoMet-ccRCC cells. As expected, under the action of PINK1 siRNA, the increased proliferation and migration abilities of the LoMet-ccRCC cell line after NFIB overexpression were inhibited again (Figs. 5D–5L). On the contrary, we used CRISPR-Cas9 to knockout NFIB and express exogenous PINK1 under a constitutive expression promoter to observe the effects of KIRC cell migration, invasion and reproduction. We found that NFIB knockout resulted in a significantly down-regulation of PINK1 (Fig. 6A), and the proliferation and migration ability of KIRC cells was significantly reduced (Figs. 6B–6J). Then, after the exogenous overexpression of PINK1, the proliferation and migration ability of KIRC cells were significantly restored (Figs. 6A–6J). Therefore, PINK1 was the key downstream of abnormal expression of NFIB leading to abnormal biological behavior of KIRC cells. Taken together, these results suggested that PINK1 is a critical target of NFIB and that PINK1 exerts tumor-promoting roles in KIRC.