Raw data

For the gene expression analysis, our researchers evaluated RNA-seq data from 602 ccRCC examples (72 healthy samples and 530 carcinoma samples) as well as directly relevant clinical data from the TCGA database (https://portal.gdc.canceroustumor.gov/). Using R language 3.5.1 and the ESTIMATE algorithm, the proportion of immune-matrix elements in the respective sample TME was assessed using the estimation package, and it was displayed as three scores, i.e., ImmuneScore, StromalScore, and ESTIMATEScore [23]. The above-mentioned scores had a positive correlation with the proportion of immune, matrix, and both, i.e., the proportion rises with the elevation of the respective score.

HK-2 cell line (Pnoxa Bio, China); ACHN cells (Pnoxa Bio, China); 786-O cell line (Pnoxa Bio, China); THP-1 cells (Pnoxa Bio, China); complete MEM medium (KGI Bio, China); complete RPMI-1640 medium (KGI Bio, China); FBS (Gibco, USA). Trypsin–EDTA digest (Solarbio, China); 1 × PBS (0.01 M, pH 7.4) (KGI, China); PMA (MCE, USA); Polyclotamine (Solarbio, China); Lipofectamine 3000 Transfection Reagent (Invitrogen, USA); CCK-8 Assay (KGI Bio, China). Clean bench (BBS-SDC, BIOBASE); medical centrifuge (TD4A, Changsha Yingtai Instruments Co., Ltd.)

Survival analysis and statistical methods

The software programs Survivorship and survminer were used, along with the R programming language. The log-rank test was used to evaluate whether a difference had reached the role of statistics, and a p value of 0.05 indicated that it had. The Kaplan–Meier method was employed to create the survival curves. The clinical markers OS and PFS were employed in this study. In general, oncology OS in clinical practice refers to the OS time of the oncology patient, i.e., the time from the start of the randomization grouping until the death of the patient for any reason. Moreover, the data on oncology OS are usually considered the optimal endpoint for determining efficacy in oncology-associated clinical trials. Whether the sum of the Z values of gene expression in malignant tumor tissue was notably elevated in comparison to adjacent normal tissue was examined through the Wilcoxon logarithm test. The Wilcoxon logarithm test was used to determine whether there was a meaningful distinction between neighboring normal tissue and malignant tissue in terms of the sum of gene expression Z values. The variability of RGS1 expression at several stages of tumor growth was assessed through the Kruskal–Wallis test. Survival was analyzed (log-rank tests, Cox proportional hazards multiple regressions, and KM graphs). Spearman’s test was performed for related research. All study was conducted using the programming language R (model 3.6.0; R Foundation). A difference that achieved statistical significance was indicated by a two-sided p < 0.05.

Enrichment analysis of KEGG and GO

The tools clusterProfiler, enrich the plot, and ggplot2 were used in R to carry out the enrichment analysis of GO and KEGG. A difference that reached statistical significance was only shown by p < 0.05.

Comparison of test results and clinical phases

The surgical and pathological characteristics of the ccRCC cases were extracted using the TCGA. The study employed R and Wilcoxon rank sum or Kruskal–Wallis rank sum tests, depending on how many medical stages were being compared. SURVIVE, a package for the R language, was loaded for COX regression using single and multiple variables.

RGS1 and TMB or MSI correlation was looked into.

The R package "maftools" was used to judge somatic cell information (MAF information) from the TCGA human pan-cancer tumor database. For each corresponding malignant tumor, it was calculated how many exon mutations were required to identify TMBs. Using the TCGA database, MSI scores were computed [24]. The interaction between RGS1 expression and TMB or MSI was explored using Spearman mode.

TIICs and genomic enrichment studies

To comprehend the hyperlink between the expression of RGS1 and immune cell invasion, Spearman correlation analysis was utilized. To estimate TIIC abundance profiles [25], the CIBERSORT computing method was applied for determining all of the tumor samples. Only the NOM p < 0.05 genomes showed a difference with statistical significance after GSEA of the full transcriptome for all tumor samples.

Cell culture and transfection

The HK-2 cell line, ACHN cell line, and 786-O cell line were cultured in MEM complete medium. The cells were subjected to the culturing process in a 5% CO2 incubator at 37 °C. Lentiviral packaging carried RGS1 or RGS1-free plasmids. The cells were transfected following the reagent manufacturer's instructions.

Cell viability

After transfection, cells were divided into normal control, transfected control, and RGS1 overexpressing groups. After the cells had been incubated, 10% CCK-8 was added.

Western blotting analysis

Prepare the collected cells or tissues and extract the total protein using Western and IP Cell lysis buffer (Beyotime, China) and PMSF configuration. The lysis buffer was introduced into the cells or tissues, and the cells or tissues were completely broken using a cell crusher or tissue grinder. The respective sample protein was subjected to SDS-PAGE gel electrophoresis. Next, the separated proteins were placed on PVDF membranes and closed using skimmed milk, followed by three TBST washes and incubation of secondary antibodies for 1 h at ambient temperature, followed by washing and exposure to the ultrasensitive luminescent solution (Thermo Fisher, USA) for development. The obtained bands were analyzed using ImageJ software. The antibodies used were ACTIN, Caspase3, Cleavedcaspase3, Bcl2, Bax, Jagged-1, and Notch1.

Flow cytometric analysis

To detect the cell cycle, cells were first molded, then digested with trypsin and collected, the cell suspension was centrifuged for the removal of the supernatant, then washed with pre-chilled PBS and centrifuged to remove the supernatant, and then protected from light. The sample was incubated for 30 min, an assay was performed on the machine, and the data were analyzed. To detect apoptosis, cells were acquired and cleaned using PBS. The cells were resuspended using 500 μl of pre-cooled 1 × Binding Buffer. Annexin V-APC and 10 μl 7-AAD were introduced into the sample. The sample was mixed lightly without light. The assay was performed on a flowmeter.

Wound-healing assay

Cells were spread evenly in a plate, incubated in an incubator, molded, and continued to be incubated until cells reached 70–80% growth area. Iron-walled cells were administrated with the tip of a 10 μL autoclaved pipette, making an even vertical scratch. Next, the cells were then gently cleaned 3 times using PBS to remove the scratched-down cells. Photographs were recorded at 0 h and 24 h.

IHC

Tumor and para cancer wax blocks originated from the Pathology Department of the Second Affiliated Hospital of An Medical University, and to repair the antigen, paraffin slices were dewaxed in water and treated with sodium citrate. The sample was incubated in normal goat serum (diluted in PBS) for 10 min at ambient temperature, the serum was decanted, and the sample was not washed. The sample was rinsed with PBS. Next, the sample was incubated at 37 °C and then rinsed with PBS. An appropriate amount of horseradish or alkaline phosphatase-labeled streptavidin working solution was added and then rinsed with PBS. Afterward, the sample developed with DAB chromogen, and it was rinsed well with tap water, re-stained, dehydrated, cleared, and then sealed.

Real-time PCR

The cells were collected, an appropriate amount of triazole was added, 1/5 chloroform was added, and the tubes were centrifuged at 4 °C at 12,000 rpm. An equal volume of anhydrous ethanol (pre-cooled at 4 °C) was added to the resulting aqueous phase solution and mixed upside down. The precipitate was washed and air-dried; next, it dissolved into enzyme-free water. The RNA concentration was examined and reverse-transcribed. Primer sequences are presented as follows:

β-actin F TGGCACCCAGCACAATGAA.

β-actin R CTAAGTCATAGTCCGCCTAGAAGCA.

RGS1 F CTTGCCAACCAAACTGGTCAA.

RGS1 R TCTCGAGTGCGGAAGTCAAT.

CD163 F GAAATCCCTGCTACTGAACCCC.

CD163 R CAATGGAAACCAGAGAGGAACCC.

Statistical analysis

All biological experiments were performed at least three times independently. All quantitative data have the expression of mean ± standard deviation. The relevant data were analyzed and then graphed with the use of GraphPad Prism 8.4 software (GraphPad Software, San Diego, CA, USA).