Insulin-Like Growth Factor Binding Protein 5—A Probable Target of Kidney Renal Papillary Renal Cell Carcinoma

Kidney renal papillary renal cell carcinoma (KIRP) accounts for 10–15% of renal cell carcinoma (RCC). The need to find more therapeutic targets for KIRP is urgent because most targeted drugs have limited effects on advanced KIRP. Insulin-like growth factor (IGF) binding protein 5 (IGFBP5) is a secreted protein related to cell proliferation, cell adhesion, cell migration, the inflammatory response and fibrosis; these functions are independent of IGF. In our study, we determined the expression and functions of IGFBP5 with data from the database of The Cancer Genome Atlas (TCGA). We found that IGFBP5 is down regulated in KIRP kidney tissues compared to its expression in control tissues and that the expression of IGFBP5 is negatively related to patient survival. Bioinformatic analysis showed the probable processes and pathways involved in altered IGFBP5 expression, including blood vessel development, the cellular response to growth factor stimulus, the response to transforming growth factor β (TGF-β), and extracellular matrix organization. We proposed that VEGFA and TGF-β act as upstream regulatory factors of IGFBP5 and verified this in the Caki-2 cell line. Based on our results, we suggest that IGFBP5 might be a therapeutic target of KIRP.


Introduction
Renal cell carcinoma (RCC) is a common kind of malignant tumor originating from the epithelium of renal tubules. e most frequent forms of RCC are clear cell renal cell carcinoma (ccRCC), kidney renal papillary renal cell carcinoma (KIRP), and kidney renal chromophobe renal cell carcinoma (KICH). ccRCC accounts for 60-70% of RCC, and KIRP accounts for 10-15% of RCC. Treatment of advanced RCC rely on targeted drugs, such as sorafenib [1], which targets the RAF/MEK/ ERK-induced signal transduction pathway and VEGFR, and sunitinib [2], which is a targeted receptor tyrosine kinase inhibitor. ese targeted drugs have been approved as firstline drugs for metastatic RCC. However, most of these drugs are targeted on ccRCC but have limited effects on advanced KIRP. Because of the different mechanisms of KIRP and ccRCC and the low proportion of KIRP in RCC, KIRP patients have been excluded from large clinical trials of these targeted drugs [3], and research on KIRP progresses slowly. Although some KIRP patients can be diagnosed by ultrasonography and receive surgery at an early stage, a large number of advanced KIRP patients miss the opportunity due to the low efficiency of targeted drugs. us, the need to find more therapeutic targets in KIRP is urgent.
In this study, we found that insulin-like growth factor binding protein 5 (IGFBP5) is associated with KIRP patient survival and is a probable therapeutic target in KIRP. IGFBP5 is a secreted protein with a molecular weight of 30.57 kDa and it is an IGF-binding protein which is belonged to IGFBPs family. IGFBPs family is a group of proteins that are capable to bind IGF and have the two-way effects on IGF I and IGF II. e family consists of six identified proteins named IGFBP1 to IGFBP6. ese proteins, in addition to being as the binding protein of IGF, have very important functions independent of IGF, especially in the progression of carcinoma. e main function of IGFBP5 is to bind circulating IGF and prolong its half-life [4]. Furthermore, an increasing number of studies have shown that IGFBP5 is related to cell proliferation, cell adhesion, cell migration, the inflammatory response and fibrosis independent of IGF [5][6][7][8]. is study focused on the relationship between IGFBP5 and KIRP determined from data from e Cancer Genome Atlas (TCGA) and describes the primary verification of this relationship.

Clinical Cohorts and RNA-Seq Data.
Clinical cohort and RNA-seq data were downloaded from TCGA (http://www.tcga. org//). A total of 290 KIRP patients and 32 normal controls were included in the analysis. e clinical data included the patients' age, gender, race, neoplasm staging and survival time.

qPCR Verification.
A First Strand cDNA Synthesis Kit (New England Biolabs, E6560S) was used for cDNA synthesis with a standard protocol. First, the RNA sample (up to 1 µg) was mixed with the d(T) 23 VN primer (2 µl) and nuclease-free H 2 O (to a total volume of 8 µl) in a sterile RNase-free microfuge tube. Second, the RNA/d(T) 23 VN sample was denatured for 5 minutes at 65°C, spun briefly and promptly placed on ice.
ird, 10 µl reaction mix (2×) and 2 µl enzyme mix (10×) were added to the microfuge tube. Finally, the 20 µl cDNA synthesis reaction was incubated at 42°C for one hour, and the enzyme was inactivated at 80°C for 5 minutes.
A SYBR S elect Master Mix kit (Applied Biosystems, 4472908) was used for PCR with the following protocol. cDNA (1 µl), 8 µl nuclease-free H 2 O, 10 µl reaction mix, and 1 µl primer were added to 8-strip PCR tubes, and PCR was conducted through a standard procedure by an Applied Biosystems 7500 Real-Time PCR system.

Western Blot Verification.
Western blotting was used to detect the expression of IGFBP5 in kidney tissues from KIRP patients. Protein lysates were extracted from 50 mg each paracarcinoma and carcinoma tissue with cell lysis buffer (Beyotime, P0013B) by a tissue homogenizer. e protein concentration was detected by a BCA assay ( ermo Pierce, 23225), and proteins were denatured with LDS sample buffer T 1: SiRNA and primer sequences.
(Invitrogen, NP0007). A total of 30 μg protein was subjected to electrophoresis on SDS-PAGE gels and transferred onto nitrocellulose (NC) membranes (Pall BioTrace, 66485). e membranes were blocked with 5% milk, incubated with primary antibody overnight at 4°C and incubated with a secondary antibody a er washing with TBST 3 times for 7 minutes each. Finally, proteins on the membranes were detected with a chemiluminescent reagent. e following antibodies were used for Western blotting: primary antibodies against IGFBP5 (Proteintech, 55205-1-AP, rab) and GAPDH (CST, 2118, rab) and a goat anti-rab IgG-HRP secondary antibody (Beyotime, A0208). Proteins were detected with the BeyoECL Plus (Beyotime, P0018s) chemiluminescent reagent.

Cell Proliferation Assay.
e MTT assay (Beyotime, C0009) was used to determine cell proliferation according to the manufacturer's instructions. Cells were seeded onto 96-well plates and divided into normal control, IGFBP5 knockdown, IGFBP5 stimulation, VEGFA stimulation and TGF-β stimulation group. e MTT reagent was added to the medium at 0 h, 12 h, 24 h, and 48 h a er intervention respectively and then incubated for 4 hours. e formazan precipitate was dissolved in solving reagent. e optical density was measured at a wavelength of 570 nm.

Statistical Analysis of Verification.
Experimental Data were presented as the mean ± standard deviation (SD). e two-tailed Student's t-test was used to analyze the difference of the experimental data between two groups, including qPCR, Western blot and cell proliferation. All calculations were performed with the Graphpad Prism 5.0 so ware. 푝 < 0.05 was considered statistically significant.

Clinical KIRP Patient Data.
Clinical data from KIRP patients ( Table 2) were downloaded from TCGA and supplemented with the Human Protein Atlas and UALCAN analysis tools. A total of 290 patients were included in the study, of which 214 were male and 76 were female. e ratio of males to females was approximately 3:1, the average age was 61.67 ± 11.89, and 58.6% (170/290) of the patients had stage I KIRP with a median survival time of 768.5 days. is is close to the natural population morbidity of KIRP, so the data represented the essential features of KIRP.

IGFBP5 Is Downregulated in KIRP.
IGFBP5 was significantly downregulated in KIRP patients compared with IGFBP5 levels in normal controls (Figure 1 en, we classified IGFBP5 expression in KIRP patients by patient gender, age, race, histologic subtype and cancer stage and found significant differences in IGFBP5 expression between the normal controls and all the subgroups (Figures 1(b)-1(f)). IGFBP5 expression levels were significantly different among the 41-60 year and 81-100 year age subgroups, and the 61-80 year and 81-100 year age subgroups ( Figure 1(b)). IGFBP5 expression levels were significantly different among subgroups with KIRP at different stages, including stage I and stage II, stage II and stage III, and stage II and stage IV (Figure 1(e)), but we do not think these differences in IGFBP5 expression have clinical implications because the number of samples was limited, and the expression levels of IGFBP5 in different groups were very similar. A larger number of samples are needed to confirm the differences within subgroups of KIRP patients classified by age, gender, race, weight, subtype and stage. In conclusion, IGFBP5 is stably expressed at low levels in KIRP patients and therefore may be explored as a biomarker or target of KIRP.    Table 3. en, we analyzed IGFBP5 and its related genes by Metascape analysis tools and found that the main GO biological processes for these genes were blood vessel development, cellular response to growth factor stimulus, response to transforming growth factor β, and extracellular matrix organization (Figure 3(a)). e PPI networks determined by function cluster analysis showed similar results (Figures 3(b) and 3(c)). ese functions are well-known and active biological processes that take part in carcinoma. is indicates that the IGFBP5 protein is active in the progression of KIRP.

IGFBP5 and Key Proteins in KIRP.
To determine the complex mechanism by which one cytokine is involved in the progression of carcinoma and to explore the possible participants were lost). e median fragments per kilobase of transcript per million mapped reads (FPKM) in patients with low and high levels of IGFBP5 were 10.85 vs 56.1, respectively. erefore, we suggest that low IGFBP5 expression might be a protective factor in KIRP patients. TGF-β is another canonical cytokine involved in carcinoma, and the Pearson correlation coefficient between TGF-β and IGFBP5 was 0.41 in our study. GO and PPI analyses of the genes most positively related to IGFBP5 showed the response to transforming growth factor β as the active biological process most enriched in these genes. Many studies have shown that excessive expression of TGF-β can promote the development and metastasis of carcinoma via stimulating the formation of peripheral blood vessels, inhibiting the immune system, forming the extracellular matrix, and promoting epithelial to mesenchymal transition (EMT). Based on these studies, we first examined the expression of TGF-β in normal samples and KIRP patients but found no significant difference. However, within the KIRP patients, patients with low TGF-β expression had a better prognosis than those with high TGF-β expression, as shown by survival curve analysis (푝 = 0.0017) (Figure 2(d)).
us, we hypothesized that VEGFA and TGF-β are key proteins that may regulate IGFBP5 expression.

Verification
Verification in Kidney Tissue. To verify the RNA-seq data, IGFBP5 expression in three pairs of paracarcinoma and carcinoma samples was determined by qPCR and Western blotting at the mRNA and protein levels, respectively. To verify differences in IGFBP5 protein expression, IGFBP5 was detected in 30 µg of total protein lysate per sample by Western blotting, with GAPDH as a reference protein.
Western blotting also showed significantly less IGFBP5 expression in carcinoma tissues than in paracarcinoma tissues (Figure 4(a)). Semiquantitative analysis by ImageJ showed IGFBP5 to be decreased by 2.5-fold, 100-fold, and 40-fold in samples No. 101, No. 226 and No. 246, respectively, compared to its expression in paracarcinoma tissue (Figure 4(b)). qPCR showed that the expression of IGFBP5 mRNA was significantly lower in carcinoma samples than in paracarcinoma samples among all three pairs, with an average difference in expression of approximately 5.22-fold (푝 < 0.001) (Figure 4(c)).

Verification in the Caki-2 Cell Line.
To demonstrate the relationship of IGFBP5 with VEGFA and TGF-β in tumor cells, we detected the expression of IGFBP5 in Caki-2 cells knocked down with VEGFA and TGF-β. VEGFA and TGF-β were successfully and efficiently inhibited by siRNA (Figures 5(a)  and 5(b)). IGFBP5 was significantly downregulated a er VEGFA and TGF-β were blocked by siRNA ( Figure 5(c)). To confirm the interaction between IGFBP5 with VEGFA and TGF-β, we stimulated the Caki-2 cell line with animal-free recombinant human VEGF 165 and TGF-β1. Not surprisingly, as shown by qPCR, IGFBP5 was upregulated ( Figure 5(d)).
To explore the probable function of IGFBP5, we detected cell proliferation rate of Caki-2 by MTT assays. e results showed that cell proliferation levels of cells stimulated by IGFBP5 and VEGFA groups are higher than control group (푝 < 0.05) (Figures 5(e) and 5(f)). In contrast, the cell proliferation levels of cells knocked down of IGFBP5 is lower mechanism of IGFBP5 in KIRP, we identified two key proteins, VEGFA and TGF-β, which are highly related to IGFBP5 and actively participate in carcinoma.
VEGFA is a well-known cytokine that stimulates capillary proliferation in carcinoma. e Pearson correlation coefficient between VEGFA, which was in our list of genes positively correlated with IGFBP5, and IGFBP5 was high at 0.71. is suggests an interaction between IGFBP5 and VEGFA. GO and PPI analyses of the genes most positively related to IGFBP5 showed blood vessel development to be the most active biological process enriched in these genes. We compared the expression of VEGFA in normal control samples and samples from KIRP patients; the expression of VEGFA is significantly lower in KIRP patients than in normal controls (Figure 2(c)). Moreover, low VEGFA expression indicates a better KIRP prognosis (푃 < 0.0001) (Figure 2(b)). ese trends are consistent with those observed for IGFBP5.

Discussion
As IGF-binding proteins, the main function of members of the IGFBP family is to bind IGF in the circulation to prolong its half-life and regulate the bidirectional function of IGF [15]. In addition, the independent function of IGFBPs cannot be (푝 < 0.05) ( Figure 5(e)). In addition, the cell proliferation level of cells stimulated by TGF-β is lower than normal control group (푝 < 0.05) ( Figure 5(f)). Based on all of these data, we confirmed that IGFBP5 is a protective factor of KIRP and that VEGFA and TGF-β are upstream regulators of IGFBP5.  progression by stimulating blood vessel development [18]. However, VEGFA is downregulated in KIRP, and most large clinical studies of VEGF/VEGFR-targeted drugs in RCC patients showed unsatisfactory results in KIRP patients, unlike ccRCC patients [3]. A previous study showed that VEGFA is able to augment the expression of IGFBP5 in bovine aortic endothelial cells [19]. us, we hypothesized that VEGFA downregulation could downregulate the expression of IGFBP5 to inhibit tumorigenesis. Expression level of IGFBP5 in Caki-2 can be regulated by VEGFA siRNA and cytokine stimulation experiments and cell proliferation detection by MTT assays confirmed our hypothesis. Based on this evidence, we suggest that downregulated IGFBP5 expression induced by the downregulation of VEGFA might act as a protective factor in KIRP. TGF-β is another canonical cytokine involved in carcinoma. Previous studies showed that TGF-β has the two-way effects on tumors. It was recognized as the suppress factor in carcinoma which can induce tumor cell apoptosis at first, but it's function of promoting tumor has been revealed by various studies [20]. Previous study showed that TGF-β is a key protein that induces tumor cell infiltration and metastasis via ignored. IGFBP5 has a unique function and mechanism of action and participates in several biological processes in carcinoma, such as cell proliferation, angiogenesis, cell migration and cell-cell adhesion, but these functions are bidirectional [6,14,16,17].
In our study, IGFBP5 was downregulated in KIRP, and the survival curve showed a negative correlation between IGFBP5 expression and survival time. is suggests that the downregulation of IGFBP5 might be a protective factor of KIRP. What is the possible mechanism by which IGFBP5 participates in KIRP? GO and PPI analyses of genes most positively related to IGFBP5 showed active biological processes that including blood vessel development, cellular response to growth factor stimulus, response to transforming growth factor β, and extracellular matrix organization. In addition, the VEGFA and TGF-β genes were highly correlated with IGFBP5. Based on our data and those of previous studies, we analyzed VEGFA and TGF-β expression in KIRP.
EMT, but has less effect on cell proliferation. EMT causes epithelial cells to lose their epithelial phenotype, including a loss of polarity and basal membrane rupture, and gain a mesenchymal phenotype, including migration and invasion, anti-apoptosis and enhanced extracellular matrix degradation [21]. MTT tests in our study also showed that cell proliferation level of Caki-2 cells in TGF-β stimulation group is lower than that in the normal control group. e results are in accord with previous studies. However, few studies have investigated the interaction between IGFBP5 and TGF-β in carcinoma. A study targeting IGFBP5 and TGF-β in NMuMG cells revealed that IGFBP5 enhances epithelial cell adhesion and protects epithelial cells from TGF-β-induced mesenchymal invasion [22]. Several studies of idiopathic pulmonary fibrosis (IPF) [8] and rheumatoid arthritis [23] suggested a correlation between IGFBP5 and TGF-β in fibrosis and autoimmune disease, but the precise interaction between IGFBP5 and TGF-β is still unclear. GO and PPI network analyses showed that IGFBP5 functions in response to TGF-β, and expression and prognosis analysis showed the correlation between IGFBP5 and TGF-β in KIRP. TGF-β knockdown by siRNA and TGF-β cytokine stimulation experiments confirmed that IGFBP5 responds to TGF-β in the Caki-2 cell line. Based on our study, we propose that IGFBP5 is regulated by TGF-β and plays a role in tumor suppression.
In addition to VEGFA and TGF-β, TNF-α is another important cytokine correlated with IGFBP5, although TNF-α expression and survival prognosis were not significant different between KIRP patients and controls. ere has been reported that IGFBP-5 inhibits TNF-α by competitively binding to TNFR1 [24,25]. We hypothesize that downregulated IGFBP5 expression might release the binding domain of TNFR1 to restore apoptosis induced by TNF-α in cancer cells.

Conclusion
In conclusion, IGFBP5 is downregulated in KIRP kidney tissues, and low levels of IGFBP5 are correlated with a longer survival time in KIRP patients rather than high IGFBP5 levels. How IGFBP5 is involved in KIRP is unclear, but bioinformatics analysis has identified proteins, such as VEGFA, TGF-β, and TNF-α, and pathways, including blood vessel development, cell apoptosis, and the cellular response to transforming growth factor β, potentially central to the role of IGFBP5 in KIRP. Based on bioinformatics analysis and subsequent primary verification, we suggest that IGFBP5 is regulated by VEGFA and TGF-β and that IGFBP5 may play a protective role and be a potential drug target in KIRP. We are eager to determine the exact mechanism of IGFBP5 in more rigorous studies.
Data Availability e data sources used to support the findings of this study are included within the article.