PPP1R14B is a diagnostic prognostic marker in patients with uterine corpus endometrial carcinoma

Abstract Uterine corpus endometrial carcinoma (UCEC) is one of the most common malignancies of the female genital tract. A recently discovered protein‐coding gene, PPP1R14B, can inhibit protein phosphatase 1 (PP1) as well as different PP1 holoenzymes, which are important proteins regulating cell growth, the cell cycle, and apoptosis. However, the association between PPP1R14B expression and UCEC remains undefined. The expression profiles of PPP1R14B in multiple cancers were analysed based on TCGA and GTE databases. Then, PPP1R14B expression in UCEC was investigated by gene differential analysis and single gene correlation analysis. In addition, we performed gene ontology term analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, gene set enrichment analysis, and Kaplan–Meier survival analysis to predict the potential function of PPP1R14B and its role in the prognosis of UCEC patients. Then, a tool for predicting the prognosis of UCEC, namely, a nomogram model, was constructed. PPP1R14B expression was higher in UCEC tumour tissues than in normal tissues. The results revealed that PPP1R14B expression was indeed closely associated with tumour development. The results of Kaplan–Meier plotter data indicated that patients with high PPP1R14b expression had poorer overall survival, disease‐specific survival, and progression‐free interval than those with low expression. A nomogram based on the results of multifactor Cox regression was generated. PPP1R14B is a key player in UCEC progression, is associated with a range of adverse outcomes, and can serve as a prognostic marker in the clinic.

UCEC in recent years, there is still a dearth of viable therapeutic options for advanced recurrent UCEC. 6 As a result, discovering and identifying new compounds that may be used as prognostic biomarkers and therapeutic targets in the treatment of UCEC is critical.
The PPP1R14B gene, also known as PLCB3N, SOM172, or PNG, is the protein-encoding gene located on chromosome region 11q13 close to the human phosphatidylinositol-specific phospholipase Cβ3 gene (PLCB3). 7 The PPP1R14B protein is capable of inhibiting protein phosphatase 1 (PP1) as well as different PP1 holoenzymes. 8,9 PP1, a widespread Ser-/Thr-specific phosphatase in organisms, plays a key role in numerous biological processes, such as RNA splicing, protein synthesis, control of cell cycle progression, promotion of apoptosis, and glycogen metabolism. [10][11][12] These biological processes are critical in the development of tumorigenesis and influence numerous functions, such as tumour growth, invasion, and metastasis. Studies have shown that PP1 cannot only inhibit the mitosis of tumour cells but also promote cell apoptosis when the cells are damaged beyond repair. 11 Upregulation of PPP1R14B inhibits the expression of protein phosphatase 1 (PP1) 13 and inhibits the function of PP1 to regulate cell growth and the cell cycle and promote apoptosis by inhibiting the myosin, glycogen-related holoenzyme, and monomeric catalytic subunits of PP1. 8 These effects may further lead to the proliferation, metastasis, and invasion of tumour cells.
A previous study found that PPP1R14B was significantly overexpressed in ovarian clear cell carcinoma (OCCC) and endometriosis. 14 Another study found that the mRNA expression of PPP1R14B was significantly higher in the plasma of patients with prostate cancer. 15 A recent study also showed that PPP1R14B was highly expressed in tumour tissues, and its high expression predicted a shorter survival time for patients. 16 To date, although one study has demonstrated the role of high expression of PPP1R14B in pancancer, 17 no results have revealed the specific mechanism of PPP1R14B in UCEC, and its function in the development of UCEC remains unclear. This study was based on the analysis of online data without relevant experimental verification. Using the cervical cancer HeLa cell line and endometrial cancer HEC-1-A cell line, Xiang Nan et al. 18 demonstrated that PPP1R14B knockdown could inhibit the activation of the Akt signalling pathway, thereby inhibiting cell proliferation and promoting cell death, but this study did not reveal a correlation of PPP1R14B with the clinical characteristics of tumours.
In this paper, we further verified the differential expression of the Kaplan-Meier survival analysis were used to predict PPP1R14B's role in UCEC patient prognosis. On the basis of a previous study, 17,18 the clinical characteristics of 105 clinical samples were used for correlation analysis, and we found that PPP1R14B expression was significantly correlated with FIGO grade and differentiation degree.
Finally, we constructed a nomogram plot as a tool for clinicians to predict the prognosis of UCEC patients and help clinicians develop more suitable treatment plans for UCEC patients.

| Data sources and preprocessing
The differential RNAseq expression data of PPP1R14B in pancancer were obtained from UCSC XENA (https://xenab rowser.net/datap ages/) in the TPM format of the TCGA and GTEx processed uniformly by the Toil process. 19 The differential RNAseq expression data of PPP1R14B in unpaired and paired samples were in level 3 HTSeq-FPKM format from the TCGA (https://portal.gdc.cancer.gov/) UCEC project. FPKM (Fragments Per Kilobase per Million) format RNAseq data were converted to TPM (transcripts per million reads) format and log2 transformed. All final analyses were performed using data in TPM format. The differential analysis data for PPP1R14B in dataset GSE17025 20,21 were downloaded from the GEO database using the GEOquery package (version 2.54.1). 22 These data were obtained by removing probes corresponding to multiple molecules, and when probes corresponding to the same molecule were encountered, only the probe with the largest signal value was retained, and then the data were normalized again by the normalize Between Arrays function of the limma package (version 3.42.2). 23 All statistical analyses and visualizations were performed using R (version 3.6.3).

| Study population
The protocol of this retrospective study was approved by the Ethics Committee of the School of Nursing of Jilin University (Changchun, China) and was consistent with the principles of the Declaration of Helsinki. All enrolled patients were informed and agreed to participate in the present study and gave written informed consent. The paraffin-embedded specimens of a total of 105 patients, all female, with UCEC who were diagnosed between 1 January 2019 and 31 May 2019 were collected from The Second Hospital of Jilin University (Changchun, China). The inclusion criteria were as follows: The first surgery was performed at the Second Hospital of Jilin University, and the pathological diagnosis was UCEC. The exclusion criteria were as follows: diagnosis of other malignant tumours; intrauterine device (IUD) and/or hormone therapy were used within 6 months before surgery. Ten specimens from UCEC patients of fresh-frozen tumours and adjacent noncancerous tissue were collected between May 2021 and June 2022. Baseline patient characteristics and pathological data, including age, menopause status, differentiation degree, and FIGO stage, were extracted from the database of The Second Hospital of Jilin University.

| Differential expression analysis of PPP1R14B
The expression profiles of PPP1R14B across cancers were analysed for differences using the Mann-Whitney U test (Wilcoxon rank sum test). The Shapiro-Wilk normality test was used to test the normality of the PPP1R14B expression data in paired samples, unpaired samples, and GSE17025, and the independent samples t test was used to analyse the differences in the data in unpaired samples. The paired samples t test was used to analyse the differences in the data in paired samples, and the Mann-Whitney U test (Wilcoxon rank sum test) was used for analysis of variance of data in GSE17025. The results of all the above analyses were visualized using ggplot2 (version 3.3.3) and were considered statistically significant when p < 0.05.

| Differential analysis of PPP1R14B protein expression levels in UCEC
Immunohistochemical staining images of PPP1R14B in UCEC and normal tissue sections were downloaded using the HPA database (https://www.prote inatl as.org/), where these sections were stained using the same antibodies and experimental methods.

| Single-gene differential analysis and correlation analysis of PPP1R14B
Single-gene differential analysis of RNAseq data in level 3 HTSeq-Counts format from the UCEC (endometrial cancer) project of TCGA was performed using the DESeq2 package (version 1.26.0). 27 Single-gene correlation analysis was performed on expression profile data in TPM format using the STAT package (version 3.6.3). The target molecule in the above analysis was PPP1R14B. Volcano plots were drawn using the results of single-gene differential analysis, setting a threshold of |log2(FC)| > 1 and p.adj <0.05. These differentially expressed genes were entered into the STRING database, 28 and protein-protein interaction (PPI) of differentially expressed genes was determined using Cytoscape software network analysis. Then, the HUB genes were identified using the MCODE plugin.
Finally, using the results of single-gene correlation analysis, the results were sorted by |Pearson value| in descending order, the genes whose correlations were in the top 50 were extracted, and the single-gene coexpression heatmap of PPP1R14B was drawn using these genes and the HUB gene. Volcano plots and coexpression heatmaps were both generated by ggplot2 (version 3.3.3).

| Functional enrichment analysis of PPP1R14B in UCEC
By using the clusterProfiler package (version 3.14.3), GO, KEGG, and GSEA functional enrichment analyses were performed on the results of single-gene differential analysis. 29

| Immunoinfiltration analysis of PPP1R14B
The relative infiltration levels of 24 immune cells were analysed using the GSVA package (version 1.34.0). 32 ssGSEA was performed for the algorithm of immune infiltration, and the chosen correlation analysis method was Spearman. Markers for 24 immune cells were obtained from an Immunity study. 33  plot was drawn to check the accuracy of the rainfall prediction. All prognostic data for the above survival analysis were obtained from a paper in Cell. 35

| Statistical analysis
Data are expressed as the mean ± standard deviation (mean ± SD).
The difference in the expression of PPP1R14B in UCEC tumour tissues and adjacent tissues was analysed by Student's t test.
One-way analysis of variance (anova) was used for comparisons between multiple groups. The association between the expression of PPP1R14B and the clinical data of UCEC patients was analysed by Mann-Whitney U test analysis. The statistical graph was completed using GraphPad Prism 8, and p < 0.05 was regarded as statistically significant.

| Differential expression of PPP1R14B in pancancer and UCEC
The results of differential expression analysis of PPP1R14B across carcinomas are shown in Figure 1A (T = 7, N = 7) was higher than that in the adjacent tissue, and the difference was statistically significant (p < 0.05). In both paired and unpaired samples of UCEC, the expression of PPP1R14B was significantly different compared with that in normal samples, and the results are shown in Figure 1C,D. We then used the GSE17025 dataset from the GEO database to verify the results in the TCGA database, and the results are shown in Figure 1E; the results were

| Single-gene differential analysis and correlation analysis of PPP1R14B
Single gene differential analysis of PPP1R14B was performed in UCEC, and the results are shown in Figure 2A Figure 2C. These hub genes were all keratin-associated proteins or keratins, and based on these genes, we drew their gene coexpression heatmap with PPP1R14B, as shown in Figure 2D. Finally, we performed single gene correlation analysis of PPP14R14B and selected the top 50 most strongly correlated genes to draw a correlation heatmap with PPP1R14B, and the results are shown in Figure 2E

| Functional enrichment analysis of PPP1R14B in UCEC
GO, KEGG, and GSEA enrichment analyses were performed using the results of single-gene differential analysis, and the results are shown in Figure 3. Figure 3A,C and Table 1 show the results of GO analysis, which revealed that PPP1R14B is functionally related to epidermal cell differentiation, endopeptidase activity regulation, blood microparticles, and hormone activity. Figure 3B,D and Table 1 show the results of KEGG analysis, which revealed that PPP1R14B was associated with neuroactive ligand-receptor interaction, retinol metabolism, chemical carcinogenesis, tyrosine metabolism, steroid hormone biosynthesis, oestrogen signalling pathway, etc. The Z score reflects the correlation of PPP1R14B with these pathways to some extent. A negative Z score indicates a negative correlation, and a positive Z score indicates a positive correlation. Figure 3E,F show the enrichment and grading results of GSEA, which suggested that there was significant enrichment in kinsey targets of ewsr1 flii fusion up, hsiao liver specific genes, benporath es 1, vart kshv infection angiogenic markers up, sabates colorectal adenoma up, heller hdac targets silenced by methylation up, and other genes related to tumorigenesis, invasion, and angiogenesis, suggesting that PPP1R14B was indeed closely related to cancer.

| Immunoinfiltration analysis of PPP1R14B
To determine the effect of PPP1R14B expression on the tumour microenvironment, immune infiltration analysis was performed using the ssGSEA method. The correlation between immune cell

| Prognostic analysis of survival of PPP1R14B expression
First, we divided the expression profile data into high and low ex- . These results suggested that high expression of PPP1R14B was associated with poor prognosis and was closely related to tumour development. Furthermore, these findings indicated that high expression of PPP1R14B is a risk factor for patients.

| Clinical correlation analysis of PPP1R14B expression
To further verify that high expression of PPP1R14B is associated with poor prognosis and is closely related to tumour development, we

| Construction and verification of the nomogram based on PPP1R14B
To construct an easy-to-use nomogram graph as a tool for clinicians to judge prognosis, we first performed univariate and multivariate COX regression analyses using different clinicopathological factors with PPP1R14B expression values to find independent prognostic factors for UCEC patients. The results are shown in Figure 7 and reveal that high expression of PPP1R14B is an independent prognostic risk factor for UCEC patients. The nomogram we constructed is shown in Figure 8A, and the factors used for prediction included F I G U R E 6 Clinical correlation analysis of PPP1R14B expression. (A-J) Expression levels of PPP1R14B in different groups of patients with distinct clinicopathological factors. (K) ROC curve, the area under the ROC curve is between 0.5 and 1. The closer the AUC is to 1, the better the diagnosis is. The AUC is between 0.5 and 0.7 with low accuracy, the AUC is between 0.7 and 0.9 with some accuracy, and the AUC is above 0.9 with high accuracy.
clinical stage, histologic grade, histological type, primary therapy outcome, tumour invasion, and radiation therapy with the expression of PPP1R14B. To verify the accuracy of this prediction tool to predict prognosis, we constructed a calibration plot, as shown in Figure 8B, with a concordance index (C index) of 0.83, indicating a moderate accuracy of predictive ability, and the calibration plot was very close to the diagonal, which indicated good calibration performance.

| Evaluation of the expression of PPP1R14B in clinical samples of UCEC
Next, we evaluated the expression of PPP1R14B in 10 UCEC tumour tissues and 10 adjacent tissues by Western blot analysis. As shown in Figure 9 A and B, the expression of PPP1R14B was higher in UCEC tumour tissues than in adjacent tissues. Additionally, the results of IHC staining confirmed the upregulated expression of PPP1R14B in low-grade UCEC ( Figure 9B-D

| DISCUSS ION
Uterine corpus endometrial carcinoma is one of the most common gynaecological malignancies. In recent years, the incidence and related mortality of UCEC has been increasing year by year, and a trend toward younger age-of-onset has also emerged. 36 Epidemiological results showed that 6.5% of patients with UCEC were younger than 45 years of age, and nearly 70% of them were diagnosed before first pregnancy. 37 How to develop personalized treatment programs to preserve fertility, reduce mortality, and improve life quality will be the focus and theme of more research in the future.
Therefore, further exploration of more molecular biomarkers is of great importance to detect the occurrence and prognosis of UCEC and develop more reasonable treatment plans. In this study, UCEC expression profile data from the TCGA and GEO databases were used to screen differentially expressed genes between UCEC and normal tissues. PPP1R14B expression in UCEC was higher than that in normal tissues, suggesting that PPP1R14B may be involved in the occurrence and development of tumours. Similar to this study, Mingxia Deng et al. 17 found that PPP1R14B in pancancer was a type of diagnostic molecular marker associated with immune infiltration.
To further predict the molecular mechanism by which PPP1R14B promotes the occurrence and development of endometrial cancer, single-gene differential analysis and correlation analysis were performed, and a PPI protein interaction network was constructed.
Fifteen HUB genes most related to PPP1R14B expression and 50 genes closely related to PPP1R14B expression were found to predict the function of the PPP1R14B gene. Among them, keratin-related  the prognosis of tumour patients and can be used as a prognostic marker. [40][41][42][43] In addition, keratin 82 (KRT82) mutations have been shown to be prevalent in gastric, colorectal, and endometrial cancers. 44,45 Keratin-associated protein 6-3 (KRTAP6-3) mutations have been found in patients with aggressive brain tumours. 46 Therefore, we hypothesized that PPP1R14B may play a role in promoting endometrial cancer by promoting the function of keratin-related proteins and keratin. In addition, among the genes closely associated with PPP1R14B expression, UBE2S, JPT1, and PTTG1 were associated with abnormal expression of endometrial cancer desiccations, proliferation, migration, methylation, and prediction of response to metformin therapy. [47][48][49][50] Our study suggested that the high expression of PPP1R14B may be related to gene mutation and tumour proliferation, invasion, and metastasis in UCEC patients. In addition, the enrichment of retinol metabolism and tyrosine metabolism has been observed, and retinol and its derivatives can effectively delay or prevent precancerous lesions and induce tumour cell differentiation and apoptosis. 53 Abnormal tyrosine metabolism also plays a very important role in the occurrence and development of tumours. 54  been shown to be a promising molecular marker for predicting prognosis in a high-risk "no specific molecular profile" (NSMP) subgroup of patients with UCEC. 62 Meanwhile, abnormal expression of noncoding RNAs such as CCAT2, DLEU1, PVT1, LINC01170, MEG3, and FER1L4 in UCEC has also been proven to be related to the occurrence, development, and prognosis of UCEC. These non-coding RNAs could also be used as prognostic molecular markers to guide the risk stratification of UCEC patients. 63

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated and/or analysed during the current study are available in the GEO (https://www.ncbi.nlm.nih.gov/geo/), TCGA (https://portal.gdc.cancer.gov), and HPA (https://www.prote inatl as.org/) repositories.