The present study included 212 patients who underwent partial or radical nephrectomy at Yamaguchi University Hospital (Ube, Japan) between October 2005 and December 2022. Tumor tissue samples were collected from all patients, while adjacent normal tissue samples were collected from a subset of patients (n=25) for further analysis. All patients were pathologically diagnosed with ccRCC. Blood samples were collected preoperatively and 7 days postoperatively for the analysis of clinical data, such as neutrophil-to-lymphocyte ratio (NLR). NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count. Among these patients, 10 underwent ICI therapy, with renal cancer tissue samples from five effective and five ineffective patients available for analysis. The detailed patient characteristics are presented in Table I.

To identify lncRNAs that may predict the efficacy of ICI therapy in patients with ccRCC, a Human V5.0 LncRNA Array Service (cat. no. AS-S-LNC-H; ArrayStar Inc.) was performed on formalin-fixed paraffin-embedded (FFPE) samples (4 µm; fixed in 10% neutral-buffered formalin at room temperature for 48 h) from patients who continued ICI therapy for 2 years (n=2) and those who experienced disease progression within 6 months after ICI therapy (n=2). After nephrectomy, samples of renal cancer tissues were collected from all 212 patients, while adjacent normal renal tissues were obtained from a subset of 25 patients. One part was stored at −80°C in RNAlater to stabilize and protect RNA through immediate RNase inactivation, while the other part was processed into FFPE blocks. The FFPE sections were stained with hematoxylin and eosin using the Tissue-Tek Prisma Plus system (Sakura Finetek Japan). Hematoxylin staining was performed at room temperature for 8 min, followed by eosin staining for 6 min, according to the standard protocol. The sections were then microdissected to identify cancerous and normal tissues. An optical microscope (Nikon Corporation) was used for the observation. RNA was extracted from each of these microdissected tissues using the Maxwell^® RSC simplyRNA Tissue Kit (cat. no. AS1340; Promega Corporation). The total RNA concentration was measured using NanoDrop One (Thermo Fisher Scientific, Inc.). Total RNA purity was confirmed by measuring the RNA integrity number equivalent. Using 100 ng total RNA, the samples were processed for hybridization onto the Arraystar Human LncRNA Expression Array V5.0 platform. This platform covers 39,317 well-annotated lncRNAs. The extracted RNA was labeled with Cy3 using the Arraystar RNA Labeling Kit (Arraystar Inc), and then hybridized onto the lncRNA microarray slides according to the manufacturer's protocol. After hybridization, the slides were washed and scanned using the Agilent G2565CA Microarray Scanner System (Agilent Technologies, Inc.). The raw signal intensities were extracted using Agilent Feature Extraction software (v11.0.1.1; Agilent Technologies, Inc.) and data analysis was performed using GeneSpring GX software (v12.1; Agilent Technologies, Inc.). The raw data were normalized using the quantile normalization method, and differentially expressed lncRNAs were identified by comparing the two patient groups. Statistical significance was determined using an unpaired Student's t-test, and lncRNAs with a fold change >1.8 and a P<0.5 were considered significantly differentially expressed.

Renal cancer cell lines [769-P (ATCC no. CRL-1,933), 786-O (ATCC no. CRL-1,932), ACHN (ATCC no. CRL-1,611) and A498 (ATCC no. HTB-44)] were purchased from American Type Culture Collection. The 769-P, 786-O, ACHN and A498 cell lines were cultured in Roswell Park Memorial Institute 1640 medium (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (cat. no. 172012; Nichirei Biosciences Inc.), and penicillin G (100 U/ml) and streptomycin sulfate (0.1 mg/ml) (cat. no. A5955; Sigma-Aldrich; Merck KGaA), and were maintained in a humidified incubator at 37°C with 5% CO₂.

Total RNA was extracted from human renal cancer tissues and adjacent non-cancerous normal renal tissues using the miRNeasy FFPE Kit (Qiagen GmbH) after pathological examination and the FFPE method. Additionally, RNA (miRNA and total RNA) was extracted from frozen tissues and RCC cell lines using the PureLink^® RNA Mini Kit (Invitrogen; Thermo Fisher Scientific Inc.) according to the manufacturer's protocol.

cDNA was synthesized from the extracted RNA by reverse transcription (RT) using the PrimeScript RT Reagent Kit (Takara Biotechnology Co., Ltd.), following the manufacturer's instructions, and was subsequently used for quantitative (q)PCR.

The 769-P and 786-O cells were transfected with LRRC75A-AS1 small interfering (si)RNA (si-LRRC75A-AS1; cat. no. n547418) or a negative control siRNA [si-negative control (si-NC); cat. no. 4390843] (both from Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Briefly, the cells were grown in six-well plates at a density of 0.25–1×10⁶ cells/well and transfected individually with si-LRRC75A-AS1 at a concentration of 50 pmol/well. The transfection was performed at 37°C and incubated for 72 h. The effect of si-LRRC75A-AS1 knockdown was examined by reverse transcription (RT)-qPCR using RNA extracted 48 h after transfection. Cell viability was assessed 24, 48, and 72 h after transfection, and invasion 48 h after transfection. Transfection was performed using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

Cell viability was assessed using the MTS assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega Corporation) according to the manufacturer's protocol. Measurements were taken at 24, 48 and 72 h after plating by determining the optical density (OD) at 490 nm. The OD measurements were conducted in triplicate.

Cell invasion was assessed using the CytoSelect 24-well Cell Invasion Assay Kit (Cell Biolabs Inc.) on a 24-well Transwell plate (pore size, 8 µm). The chambers were coated with Matrigel at room temperature for 1 h. The transfected cells were transferred to the upper chamber in triplicate at a cell density of 0.5–1.0×10⁶ cells/ml; a total of 300 µl cell suspension was added to each well, resulting in 1.5–3.0×10⁵ cells/well. After 48 h of incubation at 37°C (5% CO₂), the cells that migrated through the membrane were stained. Extraction was performed using the Extraction Solution from the kit, and the results were expressed as the number of invaded cells. Quantification was performed by measuring the OD at 560 nm using a plate reader, according to the manufacturer's instructions.

qPCR was performed in triplicate using the Applied Biosystems StepOnePlus and TaqMan Universal PCR Master Mix (both from Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. TaqMan probes and primers were also purchased from Applied Biosystems; Thermo Fisher Scientific, Inc. Human β-actin (Assay ID: Hs01060665_g1) and human RNU48 (Assay ID: 001006) were used as endogenous controls. RNU48 was used as a control for miRNA, while human β-actin was used as a control for LRRC75A-AS1 and ADAMTS5. The expression levels of lncRNA LRRC75A-AS1 (Assay ID: Hs00415106_m1), hsa-miR-370-5p (Assay ID: 462392_mat) and ADAMTS5 (Assay ID: Hs01095518_m1) were determined using StepOnePlus software (version 2.1; Applied Biosystems; Thermo Fisher Scientific, Inc.) and the 2^−ΔΔCq method (33). The following thermocycling conditions were used: For lncRNA and ADAMTS5, initial denaturation at 95°C for 20 sec, followed by 40 cycles at 95°C for 1 sec and 60°C for 20 sec; for miRNA, initial denaturation at 95°C for 20 sec, followed by 40 cycles at 95°C for 3 sec and 60°C for 30 sec.

To identify miRNAs with strong binding affinity to LRRC75A-AS1, the miRNA target scanner miRanda (http://www.microrna.org/) was used (34). The Ensembl Canonical cDNA sequence (https://rapid.ensembl.org/index.html) for LRRC75A-AS1 (ENST05220043563.1; 3,310 bp) and mature miRNA sequences for Homo sapiens from miRBase (35) were used as input for miRanda. The mature miRNA sequences for Homo sapiens used as input of miRanda were extracted from the mature miRNA sequences file downloaded from the miRBase (https://www.mirbase.org/download/). Pairing score ≥160 and energy score ≤-20 were used as cutoff thresholds for miRanda analysis. Target genes for miRNAs were searched through TargetScanHuman (https://www.targetscan.org/vert_80/) and miRDB (https://mirdb.org/mirdb/index.html).

Continuous variables were compared using the unpaired Student's t-test, the paired Student's t-test for matched samples, or the Mann-Whitney U test. Survival analysis was performed using the Kaplan-Meier method, and comparisons were made using the log-rank test. A Cox proportional hazards regression model was employed in both univariate and multivariate analyses to identify the risk factors for recurrence and progression. Statistical significance for functional in vitro analysis was determined using an unpaired Student's t-test for comparing two groups, or one-way analysis of variance (ANOVA) followed by the Tukey post hoc test for more than two groups. Statistical analyses were performed using JMP software (Pro.16; SAS Institute, Inc.). All numerical data are presented as the mean ± standard deviation. P-values were two-sided, and P<0.05 was considered to indicate a statistically significant difference.

The lncRNA microarray analysis identified several lncRNAs that were highly expressed in patients who were unresponsive to ICI therapy (Fig. 1A). However, the present study focused on lncRNA LRRC75A-AS1 due to the limited existing literature on this particular lncRNA.

RT-qPCR was performed to determine whether LRRC75A-AS1 expression was upregulated in human ccRCC tissues. Although the sample size was small, the expression of LRRC75A-AS1 was significantly higher in ccRCC tissues from the ICI ineffective group (n=5) compared with that in the effective group (n=5) (P<0.01; Fig. 1B).

The present study compared LRRC75A-AS1 expression in 25 matched normal renal tissues and renal cancer tissues (all ccRCC) by RT-qPCR. LRRC75A-AS1 expression was significantly higher in all renal cancer tissues compared with that in the matched normal renal tissues (P=0.0016; Fig. 2A). Subsequently, the expression levels of LRRC75A-AS1 were assessed in 212 ccRCC samples, and the samples were divided into two groups based on the median expression of LRRC75A-AS1. The high LRRC75A-AS1 group exhibited significantly shorter progression-free survival (PFS) compared with the low LRRC75A-AS1 group (log-rank P=0.0196) (Fig. 2B). However, no significant difference was found in the overall survival rate between the groups (P=0.08; data not shown). Subsequently, the present study analyzed the association between LRRC75A-AS1 expression and various clinicopathological parameters (Table II). In addition, prognostic factors for PFS, including sex, age, body mass index, neutrophil-to-lymphocyte ratio, stage, Fuhrman grade, sarcomatoid features and LRRC75A-AS1 expression levels were assessed in patients with ccRCC (Table II). Elevated LRRC75A-AS1 expression emerged as a significant independent risk factor for PFS in the multivariate analysis, similar to other clinical prognostic factors (hazard ratio=2.88; P=0.006).

The present study analyzed and compared the expression levels of LRRC75A-AS1 in four RCC lines (769-P, 786-O, ACHN and A498) using RT-qPCR. Among these, 769-P and 786-O cells exhibited the highest expression of LRRC75A-AS1 and were selected for further experiments (Fig. S1). In 769-P and 786-O cells, LRRC75A-AS1 expression was significantly decreased after si-LRRC75A-AS1 transfection (P<0.0001; Fig. 3A). The MTS assay results revealed that the knockdown of LRRC75A-AS1 significantly suppressed cell proliferation in both cell lines 48 h post-transfection, compared with that in the si-NC group (P<0.01 and P<0.001, respectively; Fig. 3B). In the invasion assay, LRRC75A-AS1 knockdown significantly reduced the invasive capacity of 769-P and 786-O cells compared with that in the si-NC group (P<0.01; Fig. 3C). These results indicated that the knockdown of LRRC75A-AS1 expression significantly inhibited the proliferation and invasion of RCC cell lines in vitro.

The present study investigated the potential relationship between LRRC75A-AS1 and miRNAs. Cytoplasmic lncRNAs act as miRNA sponges; therefore, it was hypothesized that LRRC75A-AS1 may function as a miRNA regulator. A total of 66 miRNAs identified by bioinformatics analysis as having potential binding sites on LRRC75A-AS1 were selected (Table SI). Among these, miR-370-5p functions as a tumor suppressor in renal cancer (36). Therefore, the present study focused on miR-370-5p and investigated its interaction with LRRC75A-AS1 in a renal cancer cell line. LRRC75A-AS1 knockdown significantly increased the expression of miR-370-5p in the 769-P and 786-O cell lines (P<0.01; Fig. 4A). These results suggested a reciprocal interaction between LRRC75A-AS1 and miR-370-5p. To identify the potential target genes of miR-370-5p, public databases such as miRDB and TargetScanHuman were utilized. Based on its high TargetScores and relevance to cancer, particularly as an unfavorable prognostic marker of RCC, ADAMTS5 was selected for further investigation. The expression levels of ADAMTS5 were compared between si-NC- and si-LRRC75A-AS1-transfected 769-P and 786-O cell lines. The results demonstrated a significant decrease in ADAMTS5 expression in the si-LRRC75A-AS1 group (P<0.01; Fig. 4B). These findings suggested that LRRC75A-AS1 may regulate ADAMTS5 expression through a sponge effect on miR-370-5p, thereby contributing to RCC progression. Specifically, LRRC75A-AS1 acted as a molecular sponge for miR-370-5p, preventing miR-370-5p from binding to ADAMTS5 mRNA. This inhibition could lead to increased ADAMTS5 expression, which is implicated in RCC progression and metastasis. The present results demonstrated that LRRC75A-AS1, through its sponging effect on miR-370-5p, may serve a critical role in the regulation of these target genes, contributing to the pathogenesis of RCC.