Polypyrimidine tract-binding protein 3/insulin-like growth factor 2 mRNA-binding proteins 3/high-mobility group A1 axis promotes renal cancer growth and metastasis

Summary Polypyrimidine tract-binding protein 3 (PTBP3) plays an important role in the post-transcriptional regulation of gene expression, including mRNA splicing, translation, and stability. Increasing evidence has shown that PTBP3 promotes cancer progression in several tumor types. However, the molecular mechanisms of PTBP3 in renal cell carcinoma (RCC) remain unknown. Here, tissue microarrays (TMAs) suggested that PTBP3 expression was increased in human RCC and that high PTBP3 expression was correlated with poor five-year overall survival and disease-free survival. We also showed that PTBP3 binds with HMGA1 mRNA in the 3′UTR region and let-7 miRNAs. PTBP3 interacted with IGF2BP3, and the PTBP3/IGF2BP3 axis prevented let-7 mediated HMGA1 mRNA silencing. PTBP3 promotes renal cancer cell growth and metastasis in vitro and in vivo. Taken together, our findings indicate PTBP3 serves as a regulator of HMGA1 and suggest its potential as a therapeutic agent for RCC.


INTRODUCTION
RNA-binding proteins (RBPs) regulate RNA metabolism at the physiological and pathological levels and are involved in the occurrence and development of various diseases. 1RBPs engage in almost every step of the post-transcriptional regulation of RNA, including transcription, splicing, modification, intracellular trafficking, translation, and decay. 2 RBPs are rapidly becoming potential therapeutic targets for various diseases. 3,4Therefore, dissecting RBP-RNA networks may provide new targets for cancer therapy.
Polypyrimdine Tract Binding Protein 3 (PTBP3) belongs to the PTB family.PTBP3 has a structure similar to that of PTBP1, with four RNA recognition domains (RRM1-4). 5PTBP3 was originally found to be primarily expressed in hematopoietic cells. 6PTBP3 is a cofactor of activation-induced cytidine deaminase (AID) in B cells, PTBP3 can regulate the recognition of AID to the target sites and further affect hyper-IgM syndrome type 2 caused by AID mutation. 7PTBP3 is highly expressed in the breast and promotes breast cancer metastasis by increasing the stability of ZEB1 mRNA. 8In colorectal cancer, PTBP3 promotes metastasis by enhancing internal ribosome entry segment (IRES)-mediated HIF-1a translation 9 and promotes cell proliferation by maintaining UBE4A mRNA stability. 10The carcinogenic effect of PTBP3 on the cell cycle and growth of gastric cancer cells has also been reported. 11PTBP3 promotes hepatocellular cancer by disrupting the splicing balance of NEAT1 and pre-miR-612. 12However, the functions and molecular mechanisms by which PTBP3, an RNA-binding protein (RBP), regulates RNA metabolism remain unclear.
High-mobility group A1 (HMGA1) is a nucleoprotein that binds to a ditch in AT-rich DNA strands and regulates the transcriptional activity of genes. 13High HMGA1 protein expression has been observed in all neoplastic tissues analyzed, including colon, kidney, breast carcinomas, myeloproliferative neoplasms, and so forth. 14,15HMGA1 regulates malignant features of tumors and is related to resistance to antineoplastic therapy. 168][19] Insulin-like growth factor 2 (IGF2) mRNA-binding proteins (IGF2BPs) can prevent let-7 miRNA target gene silencing, such as HMGA1/2, and so forth., [20][21][22] suggesting that RBP-miRNA ribonucleoproteins (RNPs) are one of the mechanisms by which RBPs regulate gene expression.
In this study, to determine the mechanism of PTBP3 in RCC, we investigated the expression of PTBP3 in clinical samples of patients with renal cell carcinoma, and analyzed its role and molecular mechanism in regulating the malignant characteristics of RCC.We discovered that PTBP3 was dramatically upregulated in RCC tissues of patients compared to adjacent normal renal tissues, and high PTBP3 expression was correlated with poor five-year overall survival and disease-free survival.Overexpression of PTBP3 promotes renal cancer cell proliferation, ll OPEN ACCESS migration, and invasion in vitro and increases tumor growth and metastasis in vivo.Importantly, we found that PTBP3 regulated HMGA1 mRNA and protein expression by increasing the stability of HMGA1.We also showed that IGF2BP3 interacts with PTBP3 and mediates the PTBP3-induced malignant features of RCC.These data provide new insights into the mechanism of RCC tumorigenesis, and support PTBP3 as a potential target for RCC treatment.

Polypyrimidine tract-binding protein 3 was upregulated in patients with renal cancer and poor 5-year survival
To investigate its role in RCC development, we assessed PTBP3 protein expression by IHC in patients with RCC using tissue microarray (TMA) slides, which contained 68 normal renal tissues and 302 renal cancer tissues.The results showed that PTBP3 expression was dramatically upregulated in cancerous tissues compared to normal tissues in patients with RCC (Figures 1A and 1B) (p < 0.001).In the training cohort, TMA slides containing 74 cases of RCC tissues with paired non-tumor tissues, we observed significantly higher PTBP3 expression in tumor tissues than in paired adjacent non-tumor tissues (Figures 1C and 1B).The correlation between PTBP3 expression and clinicopathological characteristics of RCC tissue samples was analyzed (Table 1).Compared with histological grades I and II, the expression of PTBP3 was dramatically increased in stages III and IV (p = 0.036).In addition, high PTBP3 expression positively correlated with distant metastasis (p = 0.028) (Table 1).Kaplan-Meier survival analysis was performed to evaluate the impact of PTBP3 expression on the overall survivals and disease-free survivals in patients with RCC based on the IHC staining scores of PTBP3 in the TMAs.The results revealed that high PTBP3 levels correlated with poor overall survival (p < 0.001, log rank test, Figure 1D) and disease-free survival (p < 0.001, log rank test, Figure 1E).
To further examine whether PTBP3 expression was an independent prognostic factor for RCC, we used univariate and multivariate Cox regression models to confirm the prognostic value of PTBP3 expression in RCC.Univariate Cox regression analysis suggested that PTBP3 expression was a significant prognostic factor for the overall ratio (HR 0.487, 95% confidence interval [CI], 0.361-0.656;p < 0.001) and disease-free survival (HR 0.486, 95%CI, 0.354-0.669;p < 0.001) in patients with RCC (Table S1).In multivariate Cox regression analysis, we also found that PTBP3 expression was an independent prognostic marker for both overall survival (HR 0.549, 95%CI, 0.401-0.751;p < 0.001) and disease-free survival (HR 0.524, 95%CI, 0.375-0.732;p < 0.001) in patients with RCC (Table S2).Therefore, we assessed the biological functions and molecular mechanisms of PTBP3 in RCC.

Polypyrimidine tract-binding protein 3 promotes growth, migration, and invasion of renal cell carcinoma in vivo
As our RCC cohort showed that PTBP3 expression was associated with RCC progression, we investigated the biological function of PTBP3 in RCC.Firstly, we detected PTBP3 expression levels in different RCC cancer cell lines, data showed PTBP3 was higher expressed in 786-O, ACHN and Ketr3 RCC cell lines than normal kidney derived HK2 cell (Figure S1) To investigate the role of PTBP3 in RCC progression, 786-O and ACHN cells were stably infected with lentivirus-mediated PTBP3 shRNA and control shRNA or transfected with PTBP3 overexpression and control lentivirus (Figures 2A and 2B).To determine whether PTBP3 regulates the activity of renal cell carcinoma cells, we measured the viability of stable knockdown (KD) and overexpression (OE) of PTBP3 in 786-O and ACHN cells using cell counting kit-8 (CCK8).CCK8 assays showed that PTBP3 KD inhibited the proliferation of 786-O and ACHN cells (Figures 2C and 2D).In contrast, 786-O and ACHN cells overexpressing PTBP3 exhibited significantly higher proliferation (Figures 2E and 2F).To investigate whether PTBP3 contributes to RCC a Two -sided Fisher's exact tests.
invasiveness of renal cell carcinoma, we tested the effect of PTBP3 on cell migration and invasion in vitro, which are two key features of the metastatic phenotype. 23Transwell assays showed that PTBP3 KD and OE inhibited and increased the migration and invasion abilities, respectively (Figures 2G-2K).

Polypyrimidine tract-binding protein 3 binds to high-mobility group A1 mRNA
To investigate the possible molecular mechanism by which PTBP3 regulates renal cancer malignancy, we analyzed eCLIP-SEQ data on genome-wide PTBP3-RNA interactions in HCT116 cells.We identified multiple sites of association between PTBP3 and HMGA, particularly in the 3 0 -UTR (Figure 3A).The interaction between PTBP3 and HMGA1 mRNA was subsequently confirmed in 786-O and ACHN cells (Figures 3B and 3C) by RNA immunoprecipitation.To determine the effect of PTBP3 on HMGA1 mRNA, we assessed the expression of HMGA1 in renal cancer cells with PTBP3 overexpression or knockdown.The mRNA levels of HMGA1 were determined by real-time quantitative PCR, which showed that PTBP3 knockdown significantly repressed HMGA1 mRNA expression, whereas PTBP3 overexpression significantly increased HMGA1 mRNA expression in 786-O and ACHN cells (Figures 3D and 3E).
Polypyrimidine tract-binding protein 3 promotes HMGA1 expression and regulating renal cancer cells malignant through high-mobility group A1 HMGA1 plays an important role in tumor metastasis and EMT progress. 14We showed that the protein levels of HMGA1 were also downregulated or upregulated by PTBP3 knockdown or overexpression in 786-O and ACHN cells (Figures 3Fand 3G).In addition, the known EMT markers regulated by HMGA1 changed according to PTBP3 knockdown or overexpression; E-cadherin protein levels were upregulated and N-cadherin and Fibronectin were downregulated in 786-O and ACHN cells with PTBP3 knockdown (Figure 3F), E-cadherin protein levels were downregulated, and N-cadherin and Fibronectin were upregulated in 786-O and ACHN cells with PTBP3 overexpression (Figure 3G).Realtime PCR experiments also revealed the same mRNA expression changes in the EMT markers (Figure S2).
Our results demonstrate that PTBP3 promotes the growth and metastasis of renal cell carcinoma cells.Furthermore, we assessed the role of HMGA1 in PTBP3 induced migration and invasion of RCC cells.We ectopically expressed HMGA1 in the 786-O and ACHN PTBP3 KD cells (Figure 3H).The data showed that the ectopic expression of HMGA1 restored the growth, migration, and invasion capacity of PTBP3 KD cells (Figures 3I-3K), which further supports HMGA1 as a critical target of PTBP3.

Knockdown of polypyrimidine tract-binding protein 3 decrease high-mobility group A1 mRNA stability
It has been reported that PTBP3 regulates mRNA turnover through binding to mRNA 3 0 UTR transcripts. 8,10Our data showed that PTBP3 binds to HMGA1 mRNA 3 0 -UTR and passively regulates HMGA1 mRNA levels (Figure 3).We hypothesized that PTBP3 stabilizes HMGA1 mRNA expression.To verify this, we first used dual-luciferase reporter assays to determine the role of PTBP3 in the regulation of HMGA1 stability.HMGA1 3 0 UTR cDNA was cloned into the 3 0 UTR of luciferase cDNA to simulate the regulation of mRNA stability, and it was found that PTBP3 overexpression enhanced luciferase activity in 786-O and ACHN cells (Figures 4A and 4B), suggesting that PTBP3 may stabilize HMGA1 mRNA through HMGA1 3 0 UTR.Actinomycin D (Act.D) was used to block transcription in shCtrl and shPTBP3 cells, and the half-life of HMGA1 mRNA was assessed.Our data demonstrated that PTBP3 knockdown decreased the half-life of HMGA1 mRNA in 786-O and ACHN cells (Figures 4C and 4D).We used cycloheximide (CHX) to block protein synthesis and measured HMGA1 decay curves.We measured HMGA1 protein levels at different time points and found that PTBP3 knockdown had little effect on HMGA1 protein half-life (Figures 4E-4H).These data strongly suggested that PTBP3 increased HMGA1 mRNA levels by enhancing HMGA1 mRNA stability.
1][22] Therefore, we aimed to determine whether PTBP3 regulates HMGA1 by interacting with IGF2BP3 and let-7 miRNAs.The interaction between PTBP3 and IGF2BP3 was determined by co-immunoprecipitation (Co-IP).Co-IP results clearly indicated that IGF2BP3 interacts with PTBP3 in 786-O and ACHN cells (Figures 5A and 4B).Next, using PTBP3 FLAG tag pull-down followed by Co-IP, the data suggested that IGF2BP3 were associated with PTBP3 in 786-O and ACHN cells (Figure 5C).Then, we downregulated IGF2BP3 in PTBP3 OE 786-O and ACHN cells using IGF2BP3 siRNA, we noticed that IGF2BP3 KD can partially decrease HMGA1 expression (Figure 5D), as well as repressing cell migration and invasion induced by PTBP3 overexpression (Figures 5E and 5F).We also repressed IGF2BP3 in PTBP3 KD 786-O and ACHN cells, data showed siIGF2BP3 could further downregulate HMGA1 expression, we also noticed a repression of PTBP3 when silencing IGF2BP3 (Figure S3), it seems IGF2BP3 may be a regulator of PTBP3.These results provided remarkable evidence that PTBP3 and IGF2BP3 may form a complex to regulate HMGA1 transcription in renal cancer.We further investigated the interactions between PTBP3 and let-7.We checked the PTBP3 eCLIP-seq data, and the HOMER motif analysis results showed that several binding consensus sequences of PTBP3 were similar to those of let-7 miRNAs (Figure 5G), suggesting a possible interaction between PTBP3 and let-7 miRNAs.To test this, we used RNA pull-down assays to verify the interaction between PTBP3 and let-7 miRNAs, and the results showed that let-7a RNA Oligos interacted with endogenous PTBP3 protein (Figure 5H).Furthermore, let-7a directly interacted with purified GST-PTBP3 protein (Figure 5I), suggesting a direct interaction between PTBP3 and let-7 miRNAs.We co-transfected PTBP3 and let-7a to 786-O and ACHN cells, western blot assays showed that PTBP3 could partly restore let-7a induced HMGA1 repression (Figure 5J).Dual-luciferase reporter assays were used to investigate the effects of PTBP3 on let-7 mediated HMGA1 3 0 UTR activity.It was found that let-7a repressed HMGA1 3 0 UTR luciferase activity, whereas PTBP3 overexpression restored the luciferase activity repression caused by let-7a (Figure 5K).These results suggested that PTBP3 interacts with let-7 miRNAs and prevents let-7 miRNA-mediated HMGA1 silencing.

Polypyrimidine tract-binding protein 3 promoted renal cancer growth and metastasis through high-mobility group A1
To observe the effect of xenografts on tumor growth in vivo, we used a nude mouse model to evaluate the effect of 786-O PTBP3 KD cells and HMGA1 overexpression in 786-O PTBP3 KD cells on tumor growth.Compared to the control group, mice with xenografts of PTBP3 KD tumor cells formed smaller tumors in terms of tumor size and weight in vivo (Figures 6A and 6B).Re-expression of HMGA1 completely rescued the growth capacity of PTBP3 KD in vivo (Figure 6C).Western blotting and IHC staining of tissue sections from nude mice were performed to evaluate PTBP3 and HMGA1 expression (Figures 6D, 6E, and S4).These results suggested that PTBP3 promotes RCC cell growth via HMGA1.
In the present study, PTBP3 promoted HMGA1 expression and renal cancer cell migration and invasion.It is well known that metastasis and invasion are essential for cancer metastasis, 24 we investigated the role of PTBP3 in RCC metastasis in vivo.786-O-Luc control, 786-O-Luc PTBP3 KD, and HMGA1 overexpression in 786-O-Luc PTBP3 KD cells were injected separately into the tail veins of nude mice.Interestingly, 786-O-Luc control and HMGA1 overexpression in 786-O-Luc PTBP3 KD cells significantly enhanced lung metastasis in nude mice, whereas 786-O-Luc-PTBP3 KD cells metastasized to the lungs of nude mice, as confirmed by bioluminescence imaging (Figures 6F and 6G).The survival of mice was also determined.PTBP3 KD increased survival rates, whereas the re-expression of HMGA1 decreased survival (Figure 6H).In addition, in 201 RCC tissue samples, the relative protein expression levels of PTBP3 were concordant to HMGA1 (Figures 6I and 6J).Therefore, HMGA1 may be responsible for the metastasis of RCC induced by PTBP3.
In conclusion, our study reveals an important posttranscriptional mechanism of PTBP3.Our results suggested a model in which PTBP3 prevents let-7 miRNA-mediated HMGA1 silencing during RCC progression.We showed PTBP3 is an important regulator of HMGA1 and promotes RCC growth and metastasis (Figure 6K).

DISCUSSION
The PTB family is comprised of three members: PTBP1, PTBP2, and PTBP3.RBPs bind to RNA directly through one or more RNA-binding domains, including RNA recognition motifs, K-homology domains, and zinc fingers. 25,26RBPs regulate RNA expression by affecting various aspects of gene metabolism. 27The functions and mechanisms of PTBP1 and PTBP2 have been studied extensively.PTBP1 and PTBP2 interact with RNAs to regulate splicing, stabilization, localization, and translation. 28As an important member of the PTB family, PTBP3 has been reported to be overexpressed and play a role in the progression of various cancers. 8,9,11,12However, the function and mechanism of action of PTBP3 in renal cell carcinoma remains unclear.
In this study, we explored the biological function of PTBP3 in RCC progression, and investigated its potential molecular mechanisms.Our results showed that PTBP3 expression was increased in human RCC, and high PTBP3 expression was correlated with clinicopathological parameters such as lymph node metastasis, distant metastasis, TNM stage and poor five-year overall survival, and disease-free survival.In addition, univariate and multivariate Cox regression analyses showed that high PTBP3 expression was an independent adverse prognostic factor in patients with RCC.These results suggest that PTBP3 plays a significant role in RCC progression and could function as a potential clinical prognostic predictor in patients with RCC.
The HMGAs protein has been reported to be overexpressed in various human cancers, including epithelial cancers such as breast cancer, 29 colorectal cancer, 30,31 lung cancer 32,33 uterine cancer, 34 and mesenchymal tumors such as glioma/glioblastoma, 35 lipoma/liposarcoma, 36 leiomyoma, 37 fibroma/fibrosarcoma 38 and osteosarcoma, 39 and is often associated with metastases and a poor prognosis. 14HMGA1 and HMGA2 are two important members of it and play an important role in tumor progression. 14,40Our data demonstrated that PTBP3 promotes the proliferation of renal cell carcinoma cells.Additionally, PTBP3 promotes the migration and invasion abilities of renal cancer cells by regulating HMGA1 expression in vivo.HMGA1 overexpression restored the effect of PTBP3 downregulation on the proliferation, migration, and  invasion of renal cell carcinoma in vivo.In addition, PTBP3 promotes the proliferation and pulmonary metastasis of renal cancer cells in an animal xenograft model.Similarly, HMGA1 overexpression restored the effect of PTBP3 downregulation on proliferation and pulmonary metastasis of renal cell carcinoma cells in vitro.The survival time of mice in the HMGA1 high expression group was significantly prolonged.These results suggested that we explore the potential mechanism by which PTBP3 regulates HMGA1 expression and activity.
HMGA1 proteins do not have transcriptional activity; however, by interacting with transcriptional mechanisms, they alter chromatin structure, thereby negatively or positively regulating the transcriptional activity of several genes. 41,42HMGA1 acts as an oncogene through transcriptional regulation and protein-protein interactions. 43For example, HMGA1 protein expression indicates adverse clinical outcomes in breast cancer. 29HMGA1 is also a target gene for let-7 miRNAs. 44,451][22] Enhanced CLIP (eCLIP) is critical for revealing the binding profiles of RBPs, and eCLIP-seq data showed the binding of PTBP3 to HMGA1 3 0 -UTR and let-7 transcripts.Moreover, PTBP3 prevents let-7 mediated HMGA1 silencing in RCC and promotes RCC growth and metastasis through HMGA1.We previously showed that PTBP3 interacts with AGO2 and prevents the miRNA-mediated degradation of ZEB1 mRNA. 8These results suggested that PTBP3 regulates target gene expression by forming complexes with mRNAs, miRNAs, and RBPs.
Insulin-like growth factor 2 (IGF2) mRNA-binding protein 3 (IMP3, IGF2BP3) is a member of the RNA binding protein family (IMP1, IMP2, and IMP3) involved in RNA localization, translation, and stability. 46In this work, our data showed that PTBP3 protein and IGF2BP3 protein could bind to each other, both in exogenous or endogenous.IGF2BP3 silencing decreased HMGA1 protein levels accordingly in PTBP3 overexpressing renal cell carcinoma cells.Hence, in our study, we demonstrated that PTBP3 interact with IGF2BP3 to regulate the stability of HMGA1 mRNA by acting on the 3 0 UTR region of HMGA1.We also found a downregulation of PTBP3 when the silencing of IGF2BP3 (Figures 5D and S3), it suggested a potential role of IGF2BP3 in regulating PTBP3 expression, it would be further investigated in the future.IGF2BPs worked as m 6 A readers and regulating mRNA stability and translation. 47Considering that the RNA binding protein PTBP3 interacted with IGF2BP3 and regulated mRNA stability, it suggested that PTBP3 might be involved in the m 6 A regulating pathway, but it needs further studies in this area.
In summary, this study provides new evidence of the clinical and biological significance of PTBP3 in RCC.Our data showed that PTBP3 expression was increased in human RCC, and high PTBP3 expression was correlated with poor five-year overall survival and disease-free survival.Furthermore, PTBP3 promotes renal cancer cell proliferation, migration, and invasion in vitro as well as tumor growth and metastasis in vivo.Importantly, we discovered that PTBP3/IGF2BP3 prevents let-7 miRNA-mediated HMGA1 silencing during RCC progression.Our data showed that PTBP3 is an important regulator of HMGA1 and promotes RCC growth and metastasis.These findings shed light on the potential of PTBP3 as a biomarker and target for RCC prognosis and therapy.

Limitations of the study
The protein level of PTBP3 was upregulated in RCC tissues, while the mRNA level of PTBP3 was downregulated in RCC tissues.It seems PTBP3 may be regulated at a post-transcriptional level, but we did not study the mechanisms of the upregulation of PTBP3 in RCC.Our study also showed a regulation of IGF2BP3 in PTBP3 expression, but the mechanisms were unclear, further research is needed to fully understand the molecular mechanism by which IGF2BP3 regulates PTBP3 expression.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Figure 1 .
Figure 1.PTBP3 was upregulated in patients with renal cancer and poor 5-year survival (A) Representative immunohistochemical images of PTBP3 protein expression in patients with renal cancer and adjacent non-cancerous tissues.(B) Staining intensities of PTBP3 in 302 cases renal cancer tissues compared with 68 cases normal tissues.Statistical analysis was conducted using a paired t test, p = 0.0001.(C) Staining intensities of PTBP3 in renal carcinoma tissues compared with paired adjacent non-cancerous tissue.N, paired adjacent non-cancerous tissues.C, renal carcinoma tissues (n = 74).(D) Staining intensities of PTBP3 in 74 paired cases of renal cancer tissues and normal tissues.(p < 0.0001).(E and F) Kaplan-Meier survival curves depicting overall survival (n = 243, p < 0.0001) or disease-specific survival of patients with RCC (n = 222, p < 0.0001) stratified by PTBP3 protein expression levels in RCC tissues.Statistical analysis was conducted using log rank test.

Figure 2 .
Figure 2. PTBP3 promotes growth, migration, and invasion of renal cell carcinoma in vivo (A and B) Detection of PTBP3 protein level in PTBP3 knockdown (KD) and over-expression (OE) 786-O and ACHN stable cell lines.GAPDH was used as a loading control.(C-F) Effect of PTBP3 KD or OE on 786-O and ACHN cells proliferation as assessed by Cell Counting Kit-8 (CCK8) assays.*p < 0.05, **p < 0.01, ***p < 0.001.(G-K) Relative migration and invasion fold changes in 786-O and ACHN cells G PTBP3 KD/OE.Statistical analysis was conducted using unpaired t test; Data are presented as the means G SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3 .
Figure 3. PTBP3 promotes HMGA1 expression and regulating renal cancer cells malignant through HMGA1 (A) The eCLIP-seq mapped PTBP3 binding events are shown on the HMGA1 transcript.(B and C) RNA immunoprecipitation with PTBP3 antibody in 786-O and ACHN cells, the co-immunoprecipitated HMGA1 RNA was detected by qRT-PCR, 18s RNA was used as a negative control, ***p < 0.001.

Figure 3 .
Figure 3. Continued (D and E) HMGA1 mRNA expressions were analyzed in 786-O and ACHN cells G PTBP3 KD/OE by qRT-PCR.The relative mRNA expression levels were normalized to GAPDH.**p < 0.01, ***p < 0.001.(F and G) Western blots of EMT markers and HMGA1 in 786-O and ACHN cells G PTBP3 KD/OE.GAPDH was used as a loading control.(H) 786-O and ACHN cells with PTBP3 KD were co-transfected with expression vectors encoding either empty vector, HMGA1 and assessed by Western blot for HMGA1 and PTBP3 expression.GAPDH was used as a loading control.(I) Effects of HMGA1 expression on the proliferation of 786-O and ACHN cells with PTBP3 KD by Cell Counting Kit-8 (CCK8) assays.**p < 0.01, ***p < 0.001.(J and K) Effects of HMGA1 expression on the migration and invasion of 786-O and ACHN cells with PTBP3 KD.Statistical analysis was conducted using unpaired t test; Data are presented as the means G SD. **p < 0.01, ***p < 0.001.

Figure 4 .
Figure 4. PTBP3 regulates HMGA1 mRNA stability and does not regulate HMGA1 protein stability (A and B) Effects of PTBP3 on HMGA1 3 0 UTR luciferase activity.PTBP3 and HMGA1 3 0 UTR Renilla Luciferase reporter plasmid were co-transfected to 786-O and ACHN cells, the relative luciferase activity was analyzed 24 h after transfection and normalized to Renilla activity.Data are presented as the means G SD, n = 3. ***p < 0.001.(C and D) Relative HMGA1 mRNA remaining were calculated versus the time of incubation with 10 mg/mL actinomycin D (Act.D) by qRT-PCR.Data are presented as the means G SD, n = 3. ***p < 0.001.(E and F) Western blot showing HMGA1 protein levels in 786-O and ACHN cells G PTBP3 KD at the indicated time points after cyclohexamide (CHX).GAPDH was used a loading control.(G and H) Graphical representation of HMGA1 protein levels from (E and F) based on densitometry and normalized to GAPDH.Statistical analysis was conducted using unpaired t test; Data are presented as the means G SD.

Figure 5 .
Figure 5. PTBP3 interacts with IGF2BP3 and prevents let-7 mediated HMGA1 silencing (A and B) Immunoprecipitation with anti-PTBP3 or anti-IGF2BP3 antibody in 786-O and ACHN cells, anti-IgG antibody as a negative control.(C) Immunoprecipitation with anti-Flag antibody and anti-IgG antibody as a negative control in RCC cells.(D) Effect of IGF2BP3 KD on the expression of HMGA1 protein level, as assessed by Western blot.(E and F) Effects of IGF2BP3 KD in cell migration and invasion (G) HOMER motif analysis results showed several binding consensus sequences of PTBP3 were similar to the let-7 miRNAs, PTBP3 eCLIP-seq data was analyzed.(H) RNA pull down assays were used to test the interactions between PTBP3 and let-7.Biotin labeled let-7a was used to perform RNA pull down assays using 786-O and ACHN cell lysates and western blots were performed to test PTBP3 and IGF2BP3, IGF2BP3 was used as a positive control.(I) Western blot of PTBP3 using RNA pull down samples by biotin labeled let-7a and PTBP3 GST fusion protein.(J)Western blots of HMGA1 in PTBP3 and let-7a co-transfected 786-O and ACHN cells.PTBP3 plasmids and let-7a mimics were co-transfected to 786-O and ACHN cells, cells were harvested at 24 h after transfection and western blots were performed to calculate HMGA1 and PTBP3 expressions, GAPDH was used as a loading control.(K) Effects of PTBP3 on let-7a mediated HMGA1 3 0 UTR luciferase activity silencing.PTBP3 plasmid and let-7a minics and HMGA1 3 0 UTR reporter plasmid were cotransfected to 786 and ACHN cells, the relative luciferase activities were analyzed 24 h after transfection.Statistical analysis was conducted using unpaired t test; Data are presented as the means G SD, n = 3. **p < 0.01, ***p < 0.001.

Figure 6 .
Figure 6.PTBP3 promoted renal cancer growth and metastasis in vivo (A-C) Effect of PTBP3 KD and PTBP3 KD with overexpression HMGA1 in 786-O cells on the xenograft model was assessed by evaluating tumor volume and tumor weight.5310 6 786-O PTBP3 Con/KD cells and PTBP3 KD with overexpression HMGA1 and Matrigel (Corning; 1:1 ratio) were subcutaneously injected into the abdominal flanks of each mice, respectively.***p< 0.001.(D) Western blot of HMGA1 expression in tumor xenografts formed.GAPDH was used as a loading control.(E) The tumor sections were performed immunochemistry staining by antibody against PTBP3 and HMGA1, representative images were shown.(F) Representative bioluminescence images of lung metastases in mice via tail vein injection of indicated cells.(G) The metastases were quantified by measuring the photo flux.Data are presented as the mean G standard deviation,**p < 0.01,***p < 0.001.(H) Kaplan-Meier survival curves depicting overall survival of the mice with the injection of 786-O PTBP3 Con/KD cells and PTBP3 KD with overexpression HMGA1, n = 10.(I and J) Analysis of the correlation coefficients between PTBP3 and HMGA1 in 201 RCC tissue samples.The correlation coefficients were calculated by Pearson test, p < 0.0001, representative images were shown.(K) A cartoon summarizing our findings.PTBP3 prevents let-7 mediated HMGA1 mRNA degradation in RCC, thereby promoting RCC growth, and metastasis.

Table 1 .
Relationship between PTBP3 expression and clinicopathological features of patients with renal cancer

TABLE
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