CircPDK1 promotes RCC cell migration and invasion through the miR-377-3p/NOTCH1 axis

Circular RNAs (circRNAs) are novel clusters of endogenous noncoding RNAs (ncRNAs) that are widely expressed in various tissue types. Recent studies have indicated that circRNAs involved in multiple tumorigenesis processes, and the potential significance of circRNAs remains to be explored. However, the roles roles and underlying mechanisms of circRNAs in clear cell renal cell carcinoma (RCC) remain unclear. We found that CircPDK1 is highly expressed in RCC by circRNA-seq assay. Moreover, the functional enrichment experiments showed that CircPDK1 is significantly associated with RCC metastasis. Eighty pairs of clinical samples were analyzed, and the results showed a correlation between CircPDK1 and lymph node and distant organ metastases in RCC. RNA-sequencing following knockdown of CircPDK1 revealed significant changes in the expression of many molecules in the metastasis-associated pathway. After bioinformatics prediction, we verified the relationships between CircPDK1 and miR-377-3p and between miR-377-3p and NOTCH1 by a luciferase reporter assay. Furthermore, the effect of CircPDK1 on RCC metastasis can be functionally influenced by miR-377-3p and NOTCH1 . Altogether, our study shows that the CircPDK1 - miR-377-3p - NOTCH1 axis plays an important role in RCC metastasis, suggests CircPDK1 might serve as a potential therapeutic target for RCC treatment. its biological function and mechanism.

for the paired-end libraries was 300 bp (± 50 bp). Then, we performed paired-end sequencing on an Illumina X Ten at LC Bio, China, in accordance with the vendor's recommended protocol. Raw data were normalized by the quantile algorithm and the limma packages in R, and we used Poisson distribution to determine the read number for specificity to clarify the specific circRNAs in each cancerous aged-matched normal tissue.
4. The analysis of CircRNA-seq data Firstly, Cutadapt(v1.9)was used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We used HISAT2 to map reads to the genome of (species). Remaining reads (unmapped reads) were still mapped to genome using tophatfusion (v2.0.10). CIRCExplorer was used to denovo assemble the mapped reads to circRNAs at first, then, back splicing reads were identified in unmapped reads by tophat-fusion and CIRCExplorer. All samples were generated unique circRNAs. The differentially expressed circRNAs were selected with log2 (fold change) > 1 or log2 (fold change) <-1 and with statistical significance (p value < 0.05) by R package-edgeR.

Quantitative real-time PCR analysis
Total RNA was extracted from tissues with TRIzol Reagent (TaKaRa, Japan) according to the manufacturer's protocol. The isolate RNA was transcribed into cDNA using a reverse transcription kit (TaKaRa, Japan). qRT-PCR was performed to quantify the RNA expression using a standard protocol from SYBR Green PCR Kit (Roche, USA) on the StepOne plus qRT-PCR System (ABI, USA). All PCR primers were purchased from RiboBio Co., Ltd. (Guangzhou, China) (primer list in Table 1). PCR was conducted at 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Each sample was analyzed in triplicate, and the relative expression was calculated using the 2 −ΔΔCt method relative to that of GAPDH.

Fluorescence in situ hybridization (FISH)
To locate the CircPDK1 distribution in RCC cells, we designed the FISH assay to detect the details. A FISH-specific target CircPDK1 RNA probe (RiboBio Co., Ltd., China) was labeled by CY3, and we chose U6 and 18S as references for nuclear localization and cytoplasmic localization, respectively. The cell slides were placed in the bottom of a 24-well plate and incubated the appropriate amount of cells (6 × 10 4 cells/well, the cell fusion degree was 60%-70% before the experiment), then the cells were washed with 1x PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing the cells with 1 x PBS, 200 µL of prehybridization solution (mixed with blocking solution and prehybridization buffer at a ratio of 1:99) and 2.5 µL of 20 µM FISH Probe Mix or internal reference FISH Probe Mix was added. After discarding the prehybridization solution, an appropriate amount of probe-containing hybridization solution containing the probe was added, and the samples were hybridized overnight at 37 °C protected from light. The slides were washed with washing solution and 1 x PBS the next day and then stained with DAPI staining solution for 10 min before mounting. Images were obtained using a laser scanning confocal microscope (Leica, Germany) at 400x magnification.
The transfection efficiency was tested by qRT-PCR. For transient transfection, the cell lines were seeded in 6-well plates at a concentration of 2.5 × 10 5 cells/well and transfected with miR-377-3p mimics, miR-377-3p inhibitors or pDONR-NOTCH1 (Addgene, USA) as indicated in the manuscript. The medium was replaced after 24 h and incubated for up to 48 h. Each experiment was repeated three times independently. 8. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay The MTT kit was purchased from RiboBio Co., Ltd. (Guangzhou, China). After the cells were seeded and grown in 96-well plates for a certain time with 200 µl medium, 10 µl of MTT reagent was added to each well at the appropriate concentration. The absorbance at 0, 24, 48, 72, 96 h in 469 mm was measured by a microplate reader. 9. Cell migration and invasion experiment BD Matrigel and serum-free medium were diluted and mixed at a ratio of 1:8; 80 µl of the mix were pipetted into the upper chamber of the transwell and placed in the incubator for at least 2 h for the Matrigel invasion assay. RCC cell suspensions of 2.5 × 10 5 cells cells/ml were seeded into the transwell plate (for migration assay without BD Matrigel in the plate), and the chamber was placed in the plate. A total of 500 µl of complete medium containing 20% FBS was added to the transwell plate.
Plate was cultured in a CO 2 (content 5%) incubator at 37 °C for 24 h, then the cells were stained with crystal violet for 10 min. Images were obtained using a microscope (Leica, Germany) 10. Wound-healing assay RCC cells were seeded into 6-well plates at a density of 80%-90% and then scratched perpendicularly to the bottom of the plate. After washing with PBS and adding serum-free medium, the plates were cultured in a 37 °C incubator containing 5% CO 2, and the pictures were taken at the scheduled time.

Flow cytometry assay for apoptosis and cell cycle identification
The Annexin V-FITC PI Apoptosis Detection Kit and Cell Cycle Detection Kit were purchased from Solarbio (Beijing, China). 786-0 and ACHN cell lines were treated with plasmid for more than 48 h and stained with Annexin V-FITC/ PI for cell apoptosis; all the procedures followed the manufacturer's instructions. Imagines obtained by using flow cytometry (Leica, Germany) and analysis with ModFit LT software.

In vivo mouse experiments
Six-week-old male BALB/c nude mice were purchased from Charles River Laboratories (Beijing, China).
CircPDK1-OE or the control vector was stably transfected into 786-0 cells that were harvested until the total cell number was approximately 1*10 6 . The mice were divided into two groups, and the cells were injected in the tail vein of each mouse in the two groups (each group had 10 mice). One month later, the mice were sacrificed, their lung tissues were removed and fixed with paraformaldehyde, and the number of metastatic nodules in the lung tissue was counted. All animal studies were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University.
13. RNA-seq RNA was extracted as previously described using the circRNA extraction procedure from sh-CircPDK1 AND sh-control 786-0 and ACHN cell lines. RNA-seq was performed at Novogene Co., Ltd. (Beijing, China). Raw data were normalized by the Quantile algorithm, limma packages in R. The differentially expressed mRNAs were selected with log2 (fold change) > 1 or log2 (fold change) <-1 and with statistical significance (p value < 0.05) by the R package Ballgown.

Western blotting
The cells were lysed using RIPA, and the supernatant was retrieved after centrifugation. Then, the protein concentration was measured using a DC protein detection kit. After the protein was detached from the fillister and transferred to the PVDF membrane, it was incubated with a primary antibody overnight following incubation with 5% (5 g/100 mL) nonfat dry milk (Bio-Rad, USA) blocking for one hour. One hour after incubation in the HRP-conjugated secondary antibody, images were taken on the machine with ECL Luminescent liquid. The results were analyzed by Immobilon™ Western Chemiluminescent HRP Substrate (Millipore). β-Tubulin was used as a control. The primary antibodies used were anti-NOTCH1 (#3608, Cell Signaling Technology), anti-β-Tubulin (#2128, Cell Signaling Technology).

Luciferase reporter assays
The potential interactions between CircPDK1 and miR-337-3P or miR-337-3P with NOTCH1 were predicted by circRNA-seq data. By using pSI-Check2 as a template, we constructed hsa-NOTCH1-3'UTR-wt, hsa-NOTCH1-3'UTR-mu, hsa-CircPDK1-wt and has-CircPDK1-mu plasmids and transfected them into a 293T cell line. Then, a Promega Dual-Luciferase System (Hanbio Co., LTD., China) was used to detect the Renilla luciferase score in different groups, which can reflect the interaction between miR-377-3p with CircPDK1 and with NOTCH1. 16. Statistical analysis All data are indicated as the means ± standard error of the mean (SEM) processed by GraphPad Prism 8.0 (La Jolla, USA) and IBM SPSS Statistics 20.0 (IBM, USA). Student's t-test, one-way ANOVA, Cox regression, and LSD-t-test, Pearson chi-square test, log-rank test, and linear regression analyses were used to evaluate the group differences. P < 0.05 was considered to have a significant change.

CircRNA expression profiles analysis
To detect the circRNAs associated with RCC, we acquired five pairs of RCC tissues and paired paratumor tissues and performed circRNA-seq analysis. We detected 201821 circRNAs and 20809 of these exhibited differential expression between RCC and normal tissues (fold change > 2.0, P < 0.05); among these, 11220 circRNAs were upregulated and 9589 were downregulated in cancer tissues, and each differentially expressed circRNA displayed as a red dot in the volcano plot and separately as either a red dot or green dot in the scatter plot ( Fig. 1A, B). The top 100 differentially expressed circRNAs are shown separately in the heat map (Fig. 1C). We used R package ggplot2 and local Perl scripts to analyze the statistical enrichment OD of the host genes of those differentially expressed protein-coding transcripts in the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig. 1D, E). To confirm the expression of circRNAs and the RNAseq data, qRT-PCR analysis was performed for 20 circRNAs randomly selected from the top 100 circRNAs of circRNA-seq results ( Table 3). The results demonstrated that the qRT-PCR results were consistent with the RNA-seq results; this result confirmed that the circRNA-seq data are reliable.
Among these, the magnitude of fold change was found in many circRNAs, such as circ12132, circRNA2976, circRNA1526, and circRNA2326 ( Fig. 1F-I). Other circRNAs expression data showed in Table 3.

CircPDK1 is upregulated in RCC and is mainly located in the cytoplasm
In all circRNAs, we selected CircPDK1 (circ12132) for further study for its biggest fold change among the 20 selected CircRNAs, CircPDK1 is a circular RNA derived from the PDK1-201 transcript (Transcript ID: ENST00000282077.7) (sequences suppled in Supplemental 1), which contains 5 exons. CircPDK1 is formed by the middle part of the PDK1-201 transcript, which contains 5 exons (from the 2nd to 6th exon) ( Fig. 2A, Supplemental 1). We designed primers to amplify the circular junctions, and qRT-PCR was implemented to further validate the expression of CircPDK1 in 30 pairs of RCC tissues and para tumor tissues. Among which, CircPDK1 was significantly overexpressed in 24 pairs of tumor tissues  (Table 2).

CircPDK1 promotes RCC cell invasion and metastasis
Because CircPDK1 is overexpressed in RCC, We further investigated the role of CircPDK1 in tumorigenesis and tumor metastasis in RCC by wound-healing, migration and Matrigel invasion assays. We found that the migration and invasion abilities of RCC cells were significantly inhibited after knockdown of CircPDK1 in both 786-0 and ACHN cell lines, although MTT proliferation assay and apoptosis assay showed that CircPDK1 did not greatly affect the proliferation and apoptosis of RCC cell lines after knock down of CircPDK1 (Fig. 3A-E). Futhermore, the effects of CircPDK1 on tumor metastasis in vivo was measured in a tumor xenograft model by injecting 786-0 cells overexpressing CircPDK1 or vector control via the tail vein. The results indicated that overexpressed CircPDK1 significantly promoted metastatic lung tumors compared with control group, suggesting that CircPDK1 is an independent factor that could promote RCC metastasis. (Fig. 3F). To explore the potential mechanism of CircPDK1, we used sh-CircPDK1 and sh-non-specific (NS) cells to perform RNA-sEq. The GO and KEGG analyses results showed that CircPDK1 strongly affected the process, such as extracellular interstitial component matrix interactions with the receptor and extracellular components, which are deeply involved in tumor metastasis. These results are also consistent with our clinical data and functional experimental results (Fig. 3G, H).

CircPDK1 directly binds to miR-377-3p in RCC
In tumor research, the use of circRNAs as miRNA "sponges" to regulate downstream target genes has been widely reported. We searched the RNA-seq data and bio-information tool to predict the potential miRNAs for CircPDK1. We found two complementary sequences between CircPDK1 and the miR-377-3p seed region (Fig. 4A). Then, we used a luciferase reporter assay to verify the potential influence between them. As we predicted, the wild-type CircPDK1 luciferase activity was significantly curbed by miR-377-3p, while the mutated CircPDK1 luciferase activity had no influence on miR-377-3p, which means that miR-377-3p can directly bind to the CircPDK1 3'UTR region in these sites (Fig. 4B). Next, we explored the important role of miR-377-3p in RCC. qRT-PCR showed that miR-377-3p expression was decreased in RCC tumors or cell lines ( Fig. 4C-D). Then, we treated 786-0 and ACHN cells with miR-377-3p-specific inhibitor or mimics, the RT-qPCR results showed that miR-377-3p was downregulated or upregulated compared with the NS groups, while it had no effect on CircPDK1 expression. These data suggest that CircPDK1 is upstream of miR-377-3p (Fig. 4E-G).

MiR-377-3p suppresses RCC cell invasion and metastasis by targeting NOTCH1
Based on the bio-information prediction results of our RNA-seq, we speculate that miR-377-3p may play a role by serving NOTCH1 as a substrate. To understand the mechanism, we transfected miR-377-3p mimics into 786-0 and ACHN cell lines. The qRT-PCR and Western blotting results showed that the RNA and protein expression levels of NOTCH1 were significantly decreased (Fig. 5A-C). In contrast, the RNA and protein expression levels of NOTCH1 were significantly increased after the miR-377-3p inhibitor was transfected into both 786-0 and ACHN cell lines (Fig. 5A-C). The clue that may further confirm its relationship in our hypothesis was that two direct potential target sites that may serve as the intermediates of miR-377-3p and NOTCH1 were identified. To further verify whether there is a direct effect between NOTCH1 and miR-377-3p, we designed clones of the NOTCH1 wildtype and mutant-type 3'UTR region (Fig. 5D). The dual-luciferase reporter assay showed that miR-377-3p mimics significantly downregulated the fluorescence of the NOTCH1 wild-type region and had no significant effect on the fluorescence of the NOTCH1 mutant-type region (Fig. 5E). To examine the role of miR-377-3p and NOTCH1 in in renal tumor cell lines, we performed a Matrigel invasion assay and a wound-healing assay after transfecting miRNA mimics or pDONR-Notch1 in 786-0 and ACHN cell lines, respectively. miR-377-3p mimic significantly decreased migration and invasion in RCC cells compared with control cells, and these effects were reversed by overexpression of NOTCH1 (Fig. 5F-G). Wound healing assay indicated that the miR-377-3p mimic led to slower closing of scratch wounds compared with the control group, while overexpression of NOTCH1 reversed these results (Fig. 5H-I).
These results suggested that miR-377-3p involvement in RCC cells invasion and metastasis is mediated by the modulation of NOTCH1.
CircPDK1 promotes RCC cell invasion and metastasis by the sponge activity of miR-377-3p and the upregulation of NOTCH1 Since we had confirmed the relationship between miR-377-3p and NOTCH1, we were curious whether CircPDK1 could regulate the expression of NOTCH1 via miR377-3p. After knocking down CircPDK1, we used qRT-PCR and western blotting to detect the expression of NOTCH1 at the RNA and protein levels.
The RT-PCR and western blot results showed that silencing CircPDK1 significantly downregulated NOTCH1 expression and miR377-3p inhibitor upregulated NOTCH1 expression (Fig. 6A-B). These results indicate that CircPDK1 and miR-377-3p are involved in the regulation of NOTCH1 in the same pathway. We found that CircPDK1 silencing significantly decreased the migration and invasion in RCC cells compared with control cells, and these effects were reversed by inhibition of miR-377-3p or overexpression of NOTCH1 (Fig. 6D-E). A wound healing assay indicated that CircPDK1 silencing led to slower closing of scratch wounds compared with the control group, while inhibition of miR-377-3p or overexpression of NOTCH1 reversed these results (Fig. 6F-G).
These results confirmed that CircPDK1 could directly affect the invasion and migration of RCC cells by sponging miR377-3p.

Discussion
Although the treatment of RCC had made great progress with the advancement of diagnostic techniques and treatments, the significant characteristic of advanced RCC was often accompanied by the shedding of tumor thrombi and distant metastasis; once metastasis occurs, the 5-year survival rate drops significantly [6, 20, 21]. Therefore, preventing RCC metastasis has always been the focus of RCC treatment. In our study, we found a significant upregulation of CircPDK1 in RCCs. While in our chosen 30 paired RCC tissues, we didn't find its host gene PDK1 overexpression, which means CircPDK1 overexpression is not correlated with the PDK1 expression level. What's more, after overexpress or knockdown CircPDK1, PDK1 expression also not change significantly and those experiments confirm the CircPDK1 play its role independently. At the same time, clinical data analysis showed that although the expression of CircPDK1 was not correlated with the proliferation and apoptosis of RCC, it was obviously associated with lymph node metastasis and distant metastasis of renal tumors. To further explore the role of CircPDK1 in RCC, we performed a series of experiments after silencing CircPDK1 in RCC cell lines. The results of the wound-healing assay, migration assay, Matrigel invasion assay or mouse experiments in vivo were consistent with the clinical data. Furthermore, RNA-seq of sh-CircPDK1 indicated that CircPDK1 was mainly involved in the interaction between extracellular matrix components and their receptors. Mechanistically, CircPDK1 could function as a sponge by harboring miR377-3p and thereby abolishing the suppressive effect on the target gene NOTCH1 in RCC. Thus, our data suggest that CircPDK1 plays an important role in RCC metastasis.
CircRNAs are recently discovered as a special novel type of noncoding RNA whose function has not yet been elaborated and characterized by its highly conserved sequence and strong stability, which is considered by many researchers and doctors to be an important clinical diagnostic marker or therapeutic target, while the study of the role of circRNA in RCC is still unclear. The most common mechanism of action of circRNA includes 1) acting on miRNA through the sponge effect, thereby reducing the number of active miRNAs and inhibiting their effects on downstream target genes; 2) directly encoding the protein to function; and 3) directly functioning with its parental mRNA to prevent or promote its expression. In view of the obvious role of CircPDK1 in RCC metastasis, we hope to actively explore its mechanism of metastasis. Through our RNA-seq data and bioinformation prediction, we found that CircPDK1 potentially targets miR-377-3p. When we mutated their potential binding sites, their binding was significantly inhibited, which indicated a direct correlation between them. miR-377-3p had been reported to be low-express in non-small-cell lung cancer (NSCLC), glioma cancer and breast cancer, in which miR-377-3p could inhibit the proliferation and metastasis of NSCLC and promote apoptosis of NSCLC by targeting E2F3 or HOXC6 [22-25], but its role in RCC still remains unclear. In our study, we first investigated miR-377-3p expression levels in renal tumor tissues and RCC cell lines. Similar to our expectation, miR-377-3p was significantly decreased in renal tumor tissue and renal tumor cell lines compared with the HK-2 cell line, which was regarded as normal renal cell. A wound-healing assay and Matrigel invasion assay showed that miR-377-3p can also inhibit the metastasis of RCC. Website prediction tools indicated that miR-377-3p possibly targets NOTCH1, which was one of the receptors involved in the Notch signaling pathway. NOTCH1 plays a role in preventing Notch signaling with N-terminal EGF-like repeats followed by LNR domains, which form a complex with ligands. The Notch signaling pathway is involved in processes such as cell proliferation, differentiation, and survival. The activation of NOTCH1 has been widely shown to be correlated with mammary tumorigenesis in animal models, and the upregulation of Notch receptors has been frequently observed in many cancer types. Furthermore, NOTCH1 has been shown to promote metastasis in many cancer types, including RCC, liver cancer, head and neck cancer, and gastric cancer [26][27][28][29]. It correlated with the formation of vasculogenic mimicry (VM) and the expression of epithelial-to-mesenchymal transition (EMT) biomarkers to be involved in the process of tumor metastasis [30,31]. In our study, we found that miR-377-3p can interact with the 3'UTR of NOTCH1. Overexpression NOTCH1 reversed the decrease in cell invasion and metastasis induced by the m miR-377-3p mimic in the RCC cell lines, which confirmed that miR-377-3p can affect the metastasis of RCC by NOTCH1.
Furthermore, our study confirmed miR-377-3p acts as an intermediary of CircPDK1 and can also regulate NOTCH1 to affect RCC metastasis. In normal condition, miR-377-3p can binding the 3'UTR region of NOTCH1 and inhibit its expression, but when RCC occurs, overexpressed CircPDK1 could function as a sponge by harboring miR-377-3p and induce less available miR-377-3p to binding to the 3'UTR region of NOTCH1 which may lead to the activation of NOTCH1 and promote the tumor metastasis (Fig. 7). Therefore, CircPDK1 can be used as a new biomarker and potential therapeutic target for predicting RCC metastasis. At the same time, in our study, we found that many of the differentially expressed circRNAs in RCC are involved in tumor metastasis and EMT, which makes us think whether the formation of circRNA is closely related to renal cancer and other tumor metastasis processes, and this is worth exploring further.

Conclusions
Our data revealed that CircPDK1 was significantly correlated with tumor metastasis of RCC patients and may function through CircPDK1-miR-377-3p-NOTCH1 axis to promote tumor metastasis process. Therefore, CircPDK1 could be used as a new biomarker for predicting RCC metastasis and a potential target for the tumor therapy.   MiR-377-3p suppresses RCC cell invasion and metastasis by targeting NOTCH1. A&B. 786-0 and ACHN cells were transfected with miR-377-3p mimics and inhibitors or their negative controls, respectively, and the expression level of NOTCH1 was verified in each group. Data are shown as the means ± SD.
The P value was calculated between the indicated groups (N=3). C. 786-0 and ACHN cells were transfected with miR-377-3p mimics and inhibitors or their negative control, respectively, and Western blotting was used to verify the expression level of each group. β-tubulin was used as a control. D.
Predicted potential binding sites of hsa-miR-377-3p to hsa-NOTCH1 and hsa-NOTCH1 mutation site positions. E. has-miR-377-3p plasmid was constructed and cotransfected with wild-type or mutant NOTCH1 plasmid into 293T cells as shown in Fig. 5D. The cells were treated using the method in Fig. 4F to verify the normalized Rluc/Fluc values for each well representing the level of mutual binding. The P value was calculated between the indicated groups (N=3). F&G. 786-0 and ACHN cells were transfected with miR-377-3p negative control (NS mimics), miR-377-3p or miR-377-3p with pDONR-Notch. The cells were treated using the method in Fig. 3B to verify the migration ability of each group. Data shown as the means ± SD. The P value was calculated between the indicated groups (N=3). H&I. 786-0 and ACHN cells were transfected with the miR-377-3p negative control (NS mimics), miR-377-3p or miR-377-3p with pDONR-Notch, and the cells were treated by using the method in Fig. 3C to verify the invasion ability of each group. Data are shown as the means ± SD. The P value was calculated between the indicated groups (N=3).

Figure 6
CircPDK1 promotes RCC cell invasion and metastasis by sponging miR-377-3p and upregulating NOTCH1. A. sh-NS or sh-CircPDK1 was transfected with or without miR-377-3p inhibitors into 786-0 and ACHN stable cell lines, and the cells were seeded into 6-well plates; qRT-PCR was used to verify the expression level of NOTCH1 in each group. The P value was calculated between the indicated groups (N=3). B. sh-NS or sh-CircPDK1 was transfected with or without miR-377-3p inhibitors into 786-0 and ACHN stable cell lines, and the cells were seeded into 6-well plates; Western blotting was used to verify the expression level of NOTCH1 in each group. β-tubulin was used as a control. C&D. sh-NS or sh-CircPDK1 was transfected with or without miR-377-3p inhibitors and pDONR-NOTCH1 into 786-0 and ACHN stable cell lines, and the cells were seeded into 6-well plates and treated by using the method in Fig. 3D&E to verify the migration ability of each group. Data are shown as the means ± SD. The P value was calculated between the indicated groups (N=3). E&F. sh-NS or sh-CircPDK1 was transfected with or without miR-377-3p inhibitors and pDONR-NOTCH1 into 786-0 and ACHN stable cell lines to verify the invasion ability of each group. The cells were treated using the method in Fig. 3B to verify the migration ability of each group. Data shown as the means ± SD. The P value was calculated between the indicated groups (N=3). G. sh-NS or sh-CircPDK1 was transfected with or without miR-377-3p inhibitors and pDONR-NOTCH1 into 786-0 and ACHN stable cell lines, cells were treated by using the method in  Working model. In normal condition, miR-377-3p binds on the 3'UTR region of NOTCH1 mRNA and inhibits its function. While in the RCC, circPDK1 is overexpressed and adsorbs miR-377-3p by the sponge effect, which decreases the binding of miR-377-3p on the 3'UTR region of NOTCH1 mRNA and promotes NOTCH1 function in return.

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