miR-1251-5p Overexpression Inhibits Proliferation, Migration, and Immune Escape in Clear Cell Renal Cell Carcinoma by Targeting NPTX2

Background miR-1251-5p was identified as a tumor suppressor in a variety of malignancies; however, its biological function in clear cell renal cell carcinoma (ccRCC) is unknown. Methods The Cancer Genome Atlas (TCGA) database was used to download expression information, including miR-1251-5p, in 521 ccRCC tissues and 71 ordinary tissues, and bioinformatics was used to explore possible target mRNAs. The relationship between miR-1251-5p, target mRNA activity, and clinical factors was examined. To estimate the biological activity of miR-1251-5p and target mRNA in ccRCC cells, we used MTT, colony formation, enzyme-linked immunosorbent, and Transwell assays. We employed a dual-luciferase reporter assay and a western blot to examine the molecular mechanisms of miR-1251-5p in ccRCC cells. In addition, the expressions of miR-1251-5p and target mRNA were further verified in the GEO database. Results Our findings revealed that miR-1251-5p binds with NPTX2's 3′-UTR. In TCGA and GEO datasets, miR-1251-5p activity is found to be lower in ccRCC tissues than that in nearby conventional tissues, although NPTX2 activity is higher. In ccRCC sufferers, miR-1251-5p and NPTX2 act as biomarkers that indicate a bad prognosis. Meanwhile, in miR-1251-5p tissues, NPTX2 expression and multiple clinical variables (survival status, grade, T staging, N staging, M staging, and clinical stage) had significant differences (p < 0.05). Structurally, miR-1251-5p inhibited proliferation, migration, and immune escape of ccRCC cells by targeting NPTX2. Conclusion Our findings indicate that miR-1251-5p constrained ccRCC cell advancement, migration, and immune evasion via targeting NPTX2, providing novel insights into ccRCC target treatment.


Introduction
In China, the most frequent malignant tumor of a urinary scheme is renal carcinoma, whereas the RCC is the next most common. RCC occurrence is rising in most Asian nations. For example, RCC is increasing in men in China at 7.7 percent yearly growth. Affected regions in China are Hong Kong and Shanghai. Meanwhile, women's RC prevalence has increased by 2.0% annually in India, with Chiang Mai and Mumbai being the worst affected. e National Cancer Spectrum of China shows 6.6 percent annual growth in RC prevalence in the last 20 years and 40 percent of people dying from RCC. It is estimated that 20 to 30 percent of RCC sufferers have the recurring or metastatic disease after their first trip to the hospital, and this malignancy is responsible for over 1 million fatalities each year. e ccRCC has been the 13th commonly diagnosed cancer all over the world in 2020, which brings deep influence on human health [1]. At present, the biomarker study about ccRCC has made great progress, and micro-RNAs (miRNAs) are increasingly being discovered for having a function in the occupancy and growth of ccRCC [2]. miRNAs are a kind of noncoding RNAs with more than 20 nucleotides that have no potential to encode proteins [3]. Several miRNAs have been known to have a great influence on proliferation and metastasis of ccRCC cells, such as miR-5710, miR-335, and miR-155. miR-5710 binds transcription factor PDE1B, thus inactivating transcriptional activity of complexes, which inhibits apoptosis of ccRCC cells [4]. miR-335 upregulates the expression of KDM3A and YAP1, which shows carcinogenic effect in ccRCC [5]. In ccRCC lines, miR-155 suppresses FOXO3 expression, accelerating cellular tumor growth [6]. miR-1251-5p was found on chromosome 12q23.1, but there were few studies about this miRNA in ccRCC. Liang et al. [7] and Zhou et al. [8] have confirmed that miR-1251-5p is a prognostic factor of ccRCC based on database analysis, but no experimental verification has been carried out and only TCGA database has been used for analysis. e activity of miR-1251-5p is upregulated with hepatocellular carcinoma cells, according to a microarray study of miRNAs, and could represent a viable therapeutic target for therapy [9]. miR-1251-5p acts as an oncogene in ovarian cancer, suppressing TBCC and α/β-tubulin expression [10]. However, the potential molecular system of miR-1251-5p in ccRCC was never explored and not confirmed by functional experiments.
NPTX2 binds to its receptor, NPTXR, as an extracellular ligand [11]. e importance of NPTX2/NPTXR axis in nervous system disorders [12], which entails the activation of glutamate receptors as well as the formation of synapses, has been discovered via extensive research. In female mice having anxiety and depression, NPTX2 expression has been reduced [13]. NPTX2 was shown to be overexpressed in colorectal cancer tissues in this investigation, and T stages were connected to increased NPTX2 expression, lymph node invasion, distant metastases, clinical step, and bad prognosis in colorectal cancer patients [14]. von Roemeling et al. [15] revealed that NPTX2 promotes cell migration by interacting with the GluR4 subunit of the -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor. In addition, Xiang et al. [16] discovered a new regulatory system for miR96 regulating NPTX2 expression in ccRCC cases. e molecular basis of NPTX2 with miR-1251-5p in ccRCC, however, was never investigated.
We discovered that miR-1251-5p has been downregulated in ccRCC cell lines in this investigation. miR-1251-5p acted as a tumor inhibitor by inhibiting cell proliferation, migration, and immune evasion by targeting NPTX2.

Microarray and Clinical
Data. TCGA database was used to obtain RNA-sequence data and clinical data from ccRCC patients, comprising 521 ccRCC tissues and 71 surrounding normal tissues. After incomplete information on clinical factors was deleted, 511 patients having ccRCC have been included in the study for further investigation. e gene expression characteristics and clinical information (gender, age, and surviving condition) of patients with ccRCC were added to TCGA database. e dual-luciferase reporter assay and western blot methods have been employed to analyze the immune and survival values in ccRCC cases. All of the patients were separated into two groups: cancerous and healthy (Table 1).

Exploring Clinical
Benefit. NPTX2 and miR-1251-5p for predicting survival were assessed by the area under the curve (AUC) and receiver operating characteristic (ROC) curve. To evaluate the cutoff value within the ROC curve, we computed individual patients' risk score, which was used to identify "high-risk" and "low-risk" groups. e Kaplan-Meier survival test suggests survival differences between high-risk and low-risk groups.

Cell
Culture. Human ccRCC cell lines 786-O, A498, ACHIN, and Caki-2 and also human ordinary epithelial cell line HK-2 were supplied by the Chinese Academy of Sciences in Shanghai. Every one of the cells being cultured in DMEM media contained a lot of glucose (Gibco, USA). Penicillin (100 U/ml) and streptomycin (100 g/ml) had been mixed with 10% fetal bovine serum (PS; Servicebio, China) (FBS; Gibco, USA). 0.25% trypsin-EDTA solution (Servicebio, China) was employed for cell passage cultivation. e cells were cultured in a cell culture incubator at 37°C and 5% CO 2 .

MTT Assay and Colony Formation Assay.
For the MTT test, transfected A498 and Caki-2 cells were served in 96-well plates at the density of 1000 cells per well; thereafter, the growth of the cell was estimated at 24 h, 48 h, 72 h, 96 h, and 120 h. e supernatants were collected after 6 h of observation at 37°C using 5% CO 2 , and 150l DMSO was injected into every well to disintegrate formazan (Sangon, China), with the absorbance reading recorded at 490 nm. Again for colony formation assay, transfected A498 and Caki-2 cells were served in 6-well plates at a density of 500 cells per well, and cells were observed for 7-14 days at 37°C using 5% CO 2 and refreshed per 3 days. e medium was replaced after the clones were detectable, and the cells were rinsed 3 times using phosphate buffer saline (PBS, Servicebio, China), preserved for 10 min with 95% ethanol, and stained with 0.1% crystal violet (YEASEN, China). Relevant photographs were taken using the camera.

Western Blot Analysis. A BCA Protein Quantification
Kit was used to assess protein content and RIPA lysis solution (YEASEN, China) (YEASEN, China). For polyacrylamide gel electrophoresis (PAGE), 30 g total protein was used and nitrocellulose membranes were used to transmit the proteins identified inside the gel (Pall, USA). Upon the transfer, the membrane was incubated using 5% nonfat milk for 1 hour until being incubated with primary antibodies to NPTX2 for 12-18 hours at 4°C (CST, USA) and GAPDH (Abclonal, China). en, membranes were rinsed 3 times using Tris-buffered saline Tween and blocked with secondary antibody IRDye ® 800CW goat anti-rabbit IgG for one hour at room temperature (LICOR, USA).

Dual-Luciferase Reporter Assay.
e expression of firefly luciferase and Renilla luciferase was identified with dualluciferase reporter gene assay kit methodology (Promega, USA).

Enzyme-Linked Immunosorbent Assay (ELISA).
Nanjing Biotech provided activated peripheral blood mononuclear cells (PBMCs). Using phytohaemagglutinin, generated PBMCs were cocultured with A498 and Caki-2 cells over 24 hours in a humidified incubator (PHA; Invitrogen). e concentration of interferon (IFN-α) and tumor necrosis factor (TNF-c) were determined employing IFN-ELISA kit as well as the TNF-ELISA kit (Invitrogen) as directed by the manufacturer.

Statistical Analysis.
e information investigation was done using GraphPad Prism, which was given as mean-± standard deviation (SD). Regarding two-group comparisons, Student's t-test is applied. p < 0.05 is viewed as a crucial difference. All of the trials were done in triplicate.

Expression, Survival Prediction, and Correlation of Clinical Parameters of miR-1251-5p in ccRCC Patients.
To examine the expression of miR-1251-5p in breast cancer, we downloaded microarray data including 521 ccRCC tissues and 71 adjacent ordinary tissues from TCGA database and computed the expression of miR-1251-5p. As shown in our results, in most ccRCC tissues, expression of miR-1251-5p is minimum compared to ordinary tissues (Figures 1(a) and S1B). Meanwhile, Kaplan-Meier survival findings indicate the groupings with low expression possess short survival periods than those with high risk (p � 0.004), as shown in Figure 1 Our outcomes depict such an amount of miR-1251-5p expression is linked to survival prediction as well as clinical factors.

Expression, Survival Prediction, and Correlation of Clinical Parameters of NPTX2 in ccRCC Patients.
To locate the objective gene of miR-1251-5p, we analyzed TCGA dataset and screened the expression of 1283 genes that were negatively tested with the expression of miR-1251-5p in ccRCC. Meanwhile, we searched the miRDB database, StarBase database, and TargetScan database for potential regulatory factors. Finally, we identified 10 genes from the intersection as potential targets of miR-1251-5p, including NPTX2, GJC1, BTN3A2, TENM1, TMEM92, PSMB9, FCGR1B, CXCL11, FCGR1A, and NETO1 (Figure 2(b)). Among the 10 genes, only NPTX2 expression was most significantly associated with survival status (p < 0.001, as shown in Figure 2(c)). As a result, we concentrated on NPTX2 with molecular mechanisms of miR-1251-5p . Using a luciferase reporter assay, we verified the association of miR-1251-5p with NPTX2 in the following part (Figure Journal of Oncology 3 3(c)). In ccRCC tissues, NPTX2 expression should be greater than in healthy tissues (Figures 2(a) and S1A). NPTX2 is negatively tested with the expression of miR-1251-5p, as shown in Figure 2(b). As with miR-1251-5p, the area under the ROC curve analysis indicated that NPTX2 could predict OS in ccRCC patients (0.691 at 1 year, 0.655 at 3 years, and 0.680 at 5 years), as depicted in Figure 2(d). Besides, NPTX2 expression is significantly greater in T4 cases (Figure 2(e)), N1 cases (Figure 2(f )), M1 cases (Figure 2(g)), advancedstage cases (Figure 2(h)), and advanced-grade cases (Figure 2(i)).
Our outcomes depict such NPTX2 is a possible target gene for miR-1251-5p and also NPTX2 expression levels are directly related to clinical features as well as survival conditions.

Overexpression of miR-1251-5p Inhibits Proliferation,
Migration, and Immune Escape of ccRCC Cells. In a normal cell line (HK-2) and four cancer cell lines, we found expression of miR-1251-5p (786-O, A498, ACHIN, and Caki-2); similarly, expression of miR-1251-5p is minimum in cancer cell lines compared to ordinary epithelial cell line (Figure 4(a)). We also transfected A498 and Caki-2 cells using a miR-1251-5p mimic to achieve the gain function of miR-1251-5p, and the effectiveness of overexpression is determined by real-time PCR. Proliferation (Figures 4(b) and 4(c)) and migration (Figure 4(d)) of ccRCC cells were inhibited after the overexpression of miR-1251-5p. Furthermore, coculturing with mimic-NC-transfected cells decreased cell supernatant levels of IFN-α and TNF-c upregulated in PHA-stimulated PBMC, but coculturing with mimic-transfected cells blocked this immune-suppressive action (Figure 4(e)).
Our outcomes imply that miR-1251-5p inhibited ccRCC cell growth, migration, and immune evasion in vitro.

Knockdown of NPTX2 Inhibits Proliferation, Migration, and Immune Escape of ccRCC Cells.
e NPTX2 expression level was downregulated dramatically in ccRCC cell lines  Journal of Oncology transfected with three sh-NPTX2 (p < 0.01), as shown in Figure 3(a). We carried out further studies using sh-NPTX2-3 because it was able to significantly downregulate NPTX2 expression in A498 and Caki-2. To verify the association between miR-1251-5p and NPTX2, we used a luciferase reporter experiment with a pmirGLO-NPTX2 3′-UTR-wild vector as well as a pmirGLO-NPTX2 3′-UTR-mutant vector. Our findings revealed that miR-1251-5p can bind to the 3′-UTR of NPTX2 mRNA, increasing its degradation (Figures 3(c) and 3(d)). Furthermore, we used western blot to identify changes in NPTX2 protein expression, and the findings revealed that miR-629-5p may suppress NPTX2 expression ( Figure 5(b)). Finally, we marked the biological function of NPTX2 in ccRCC, as our findings showed shRNA-induced knockdown of NPTX2 constrained proliferation, migration, and immune escape of ccRCC cells ( Figures 5(e)), which indicated that NPTX2 was a tumor driver in ccRCC. Taken together, our results revealed that NPTX2 could combine with miR-1251-5p and downregulation of NPTX2 constrains proliferation, migration, and immune escape of ccRCC in vitro.

NPTX2 Can Reverse the Effects of miR-1251-5p
Overexpression. We investigated the effect of mimic-miR-1251-5p and oe-NPTX2 on ccRCC based on the previously mentioned negative correlation between miR-1251-5p and NPTX2. Firstly, the effectiveness of miR-1251-5p and NPTX2 overexpression was investigated ( Figure 5(a)). e consequences of miR-1251-5p on A498 and Caki-2 cells might be mitigated by NPTX2 overexpression, according to (f ) After knocking down NPTX2, the "colony formation assay" has been executed to compute cell's capability in A498 and Caki-2 cells. (g) "Transwell assay" is used to explore the potential of A498 and Caki-2 cells to migrate after knockdown of NPTX2 (100×). (h) An ELISA kit was used to assess the levels of IFN-α and TNF-c in the supernatants of A498 and Caki-2 cells. * p < 0.05 and * * p < 0.01.

Discussion
miRNAs play a major role in the development and advancement of several malignancies, renal cell carcinoma, and kidney disease. e function of miR-1251-5p is the only establishment. In ovarian cancer cells, by addressing the tumor suppressor TBCC, Zhou et al. [8] found that miR-1251-5p increased carcinogenesis and autophagy. However, it is unknown if miR-1251-5p performs a function in developing HCC [7]. Several functional assays were carried out in this work, and the findings revealed that miR-1251-5p suppressed ccRCC cell proliferation, migration, and immune escape in vitro. Our outcomes imply that miR-1251-5p operations as an antioncogene in ccRCC. In this work, there was enough evidence to conclude that NPTX2 is a specific functional goal of miR-1251-5p in ccRCC. In ccRCC tissues, miR-1251-5p was shown to be negatively linked with NPTX2 mRNA levels. Second, miR-1251-5p had a substantial impact on the luciferase expression of vectors containing the wt-3′-UTR of NPTX2, but not the mut-3′-UTR. Finally, miR-1251-5p relaxed the expression of NPTX2 in ccRCC cells on the mRNA and protein levels. Critically, restoring NPTX2 expression in ccRCC cells may be able to reverse the action of miR-1251-5p.
C-Met, NPTX2, NEUROD6, and hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 have all been found to be HCN1 in a study comparing gene expression in Alzheimer's and nondisordered Alzheimer's diseases [12]. Furthermore, we discovered that NPTX2 expression in ccRCC was greater than in the normal tissue. NPTX2 was shown to improve cell migration and proliferation, according to our findings. ese findings demonstrated that NPTX2-expressing cells had more ability to migrate to the ccRCC, ensuring increased NPTX2 expression in ccRCC tissues.   In conclusion, our findings highlighted the bioactivity and molecular mechanism of miR-1251-5p in ccRCC, revealing new information about ccRCC target treatment.

Conclusions
e bioactivity of miR-1251-5p in ccRCC was recognized in the research. TCGA search was performed for miR-1251-5p data including target mRNAs. Our study looked at miR-1251-5p, target mRNA expression, and clinical factors. e bioactivity of miR-1251-5p and target mRNA has been estimated in ccRCC cells using MTT, colony formation, enzyme-linked immunosorbent, and Transwell assays. is miR-1251-5p was also studied utilizing the dual-luciferase reporter assay with western blot. e GEO data were used to confirm miR-1251-5p with target mRNA activity. e 3′-UTR of NPTX2 binds miR-1251-5p. In TCGA and GEO databases, miR-1251-5p activity was found less in ccRCC versus normal tissues. As a result of our findings, we now know that miR-1251-5p can reduce ccRCC cell proliferation, migration, and immune evasion via target NPTX2.

Data Availability
e data used to support the findings of the study were obtained from TCGA database and the Kaplan-Meier Plotter database.
Ethical Approval e authors are responsible for the overall management of the project, including assuring that any queries about the work's validity or authenticity were thoroughly explored and rectified. e ethics committees of the ird Affiliated Hospital of Wenzhou Medical University accepted this research, which was based on the Declaration of Helsinki.