Based on Bioinformatics Studies on the Effect of a lpha1H on Key Genes, Pathways in Bladder Cancer and lncRNA on Patient Prognosis

Objective: To analyze the differential genes, lncRNA, signaling pathway and patient prognosis of bladder cancer after alpha1H treatment. Methods: Sequencing data GSE172112 for patients in clinical trials with a new bladder cancer drug were downloaded from the GEO database, The bladder cancer tissues using the new drug alpha1H (alpha1-oleate) and placebo were analyzed in the R language, The differentially expressed genes were selected; Differential genes were analyzed for KEGG pathway enrichment using the DAVID database, To explore the effect of a lpha1H treatment on pathways in patients with bladder cancer; at the same time, lncRNA expression data from the Bladder Cancer (BLCA) dataset were also downloaded through the TCGA database, First, screening for differential lncRNA expression, The screening results were then analyzed by univariate Cox regression to initially screen for lncRNA associated with prognosis, The key lncRNA affecting the prognosis were further screened out, The prognostic model was also constructed using multivariate Cox regression analysis. Results: There yielded 394 signicantly upregulated genes and 385 signicantly downregulated genes in bladder cancer tissue after a lpha1H treatment. Through gene signaling pathway enrichment analysis, the upregulated genes were mainly enriched in the signaling pathways regulating the pluripotent line of stem cell cells, the TGF-signaling pathways, and the cell cycle. The downregulated genes were mainly enriched in the MAPK signaling pathway, phagosomes, and the TNF signaling pathway. After a lpha1H treatment, of 119 differentially expressed lncRNA, from the lnc2cancer database, two genes were found to be potentially associated with bladder cancer prognosis, and the nal analysis conrmed the key lncRNA affecting prognosis. The results of survival analysis showed that high expression of LINC00152 unfavorable unfavorable patient prognosis and the new drug alpha1H reduces LINC00152 favors patient prognosis. Conclusion: Alpha1H can cure cancer by regulating hallmark signaling, TGF-beta signaling, and LINC00152.


Background
Bladder cancer (Bladder cancer, BC) is a common malignancy in the male genitinary system, and a 2018 global cancer statistics reported that the incidence of BC was 4.5% in men and a mortality rate of 2.8% [1] .The main clinical presenting symptom of BC is hematuria [2] .90% of BC occurrence is caused by the malignant proliferation of urothelial cells, most of which are located in the bladder [3] .BC can be classi ed into non-muscle-invasive bladder cancer and muscle-invasive bladder cancer [2] .Currently, more than 80% of bladder tumors are non-muscle-invasive papillary tumors, and although their 5-year survival rate is 90%, approximately 70% of these patients will relapse [4] .
In 2002, BCG rst reported the effective use of BCG in the treatment of super cial BC by Morales et al [5] .During the subsequent decades, the researchers made great clinical progress in treating BC by surgical resection, chemotherapy, and immunocheckpoint inhibitors. Transurethral bladder tumor resection is often used clinically for the treatment of non-muscle-invasive bladder cancer [6] .Platinumbased chemotherapy is often used for patients with muscle-invasive bladder cancer [7] .In 2020, the European Urology Guidelines Association reported on treatment for muscle invasive and metastatic bladder cancer, they clearly stated that cisplatin-based chemotherapy remains preferred in patients with BC metastatic. Secondly, immunotherapy is recommended for patients positive for programmed death ligand 1 (PD-L1), or with carboplatin for PD-L1-negative patients. For second-line therapy of BC metastatic disease, Pymtuzumab therapy is recommended [8] .It is well known that immunotherapy has better effects and less side effects compared with the strong toxic side effects and high recurrence rate of traditional chemotherapy therapy. In recent years, despite the success of immunosuppressive therapy in BC, the objective response to immunosuppressive monotherapy remains within the 20% range, suggesting a lower response to immunotherapy in some patients [9] .Therefore, developing a less toxic antitumor complex for BC would be very popular. Human milk albumin is exible and able to form stable protein-fatty acid complexes with fatty acids. Among them, the alpha1-oleate generated by its binding to oleic acid has been con rmed by many researchers to have antitumor effects. It has been shown that alpha1H shows better therapeutic effects in the mouse MB49 bladder cancer model [10] .In an animal study, mice found a dose-dependent reduction in tumor size, bladder size and bladder weight in alpha1H treated mice compared to the counterfeit drug group, and increased alpha1H dose had no toxic side effects on healthy tissues [6] .In a single-center, placebo-controlled, double-blind phase I / II interventional clinical trial of nonmuscle invasive bladder cancer, the infusion of alpha1H in the bladder resulted in massive cell loss of tumor cells, smaller tumors, apoptosis after treatment, and suppressed expression of cancer-related genes [11] .
As bioinformatics developed, researchers found that lncRNA expression is closely related to tumor genesis and progression [12] .This suggests that lncRNA can serve as a tumor biomarker as well as as a therapeutic target. In recent years, many studies have found lncRNA to be associated with the diagnosis and prognosis in BC patients. Li Gang et al [13] found that lncRNA TUC338 expression was upregulated in early BC patients and showed early diagnostic value. After surgical resection of the BC tumours, the plasma lncRNA TUC338 levels were signi cantly downregulated.Luo Huarong et al [14] found that plasma lncRNA CASC11 was upregulated in early BC patients as compared with healthy controls.MA ZHIPENG et al [15] found that lncRNA SNHG5 was upregulated in BC tissues and promoted BC cell proliferation by targeting p27, which subsequently had adverse effects on patient prognosis. In addition, lncrna UCA1 was found in a meta-analysis to be a diagnostic biomarker for BC [16] .
In this study, we screened differential genes for BC patients after alpha1H treatment and placebo-treated BC patients, BC patients and placebo-treated BC patients, followed by KEGG signaling enrichment analysis of the above differential genes, and con rmed prognostic key LncRNA for alpha1H treatment of bladder cancer.

alpha1H treatment affects protein-coding gene expression in patients with bladder cancer
We collected gene expression sequencing data from clinical experimental patients with alpha1H, a new bladder cancer drug, GSE172112, with simple preliminary preprocessing and statistics, in which 39 patients using the new drug and 42 patients using placebo were tested, and gene expression data from a total of 81 samples were tested. Subsequently, using the R package "DESeq2", we transformed the gene read count data detected by the samples into the corresponding expression values, combined with the RNA classi cation information in the R package "biomaRt" package, yielding a total of 20,356 PCG and 12,382 lncRNA (including 7,109 lincRNA and 5,273 antisense).
First, for 20,356 protein-coding genes, using the patient drug pro le, we performed differential expression analysis between patients using the new drug alpha1H versus patients using placebo. We counted the mean expression of each protein-coding gene in both patient groups, calculated the corresponding FC values, and recorded the Wilcoxon rank-sum test p-values, nally obtaining 781 differentially expressed genes with a p-value <0.01, containing 396 signi cantly up-regulated genes and 385 signi cantly downregulated genes (Table 1, Figure 1). Furthermore, we distributed differentially expressed protein-coding genes to functionally annotated signi cantly up-and signi cantly down-regulated genes.among, For the 394 genes that were signi cantly upregulated, We found that their function is mainly associated with the following KEGG pathway: "hsa04550: Signaling pathways regulating pluripotency of stem cells" regulates signaling in stem cells, "hsa04350: TGF-beta signaling pathway" TGF-beta signaling pathway, "hsa04110: Cell cycle" cell cycle, et al. (Table 1, Figure 2).
But for the 385 signi cantly downregulated genes, we found that their function was mainly with (Table 2, Figure 3). like the following KEGG pathway-associated: "hsa 04010:MAPK signaling pathway" MAPK signaling pathway, "hsa04145:Phagosome" phagosomal, "hsa04668:TNF signaling pathway" TNF signaling pathway In addition, for the non-coding RNA molecule lncRNA, the protein detected in the GSE172112 dataset, we also performed a differential expression analysis. As with the analysis of protein-coding genes, for 12,382 lncRNA, we nally obtained 119 signi cantly differentially expressed lncRNA, including 53 signi cantly upregulated lncRNA and 66 signi cantly downregulated lncRNA (Table 2, Figure 4).
Further, we obtained from the lnc2cancer database (http: / / biobigdata.hrbmu.edu.cn/lnc2cancer/index.The 369 bladder cancer-related lncRNA entries were downloaded from html) and two of these lncRNA were closely associated with bladder cancer and LINC00152 with TUG1 (Table 3).We found that both lncRNA were upregulated in bladder cancer patients in most literature reports, and in the GSE172112 dataset, we also compared the expression of patients using new drugs and placebo. Combined with the two lncRNA analysis described above, we suggest that the new drug alpha1H most likely caused a series of gene expression changes by reducing LINC00152 expression and ultimately played a good role in killing tumor cells.
2.3 alpha1H treatment altered part of the hallmark pathway activation in patients with bladder cancer Using differential expression analysis between the two classes of patients in GSE172112, we found that many differentially expressed genes were generated in alpha1H-treated patients, and that treatment of alpha1H at a single gene level had a huge effect on patient gene expression.Further, we evaluated whether both types of patients have a huge impact on the activation of certain pathways from the perspective of the overall pathways.
Therefore, we collected and collated gene members of the 50 hallmark pathways from the MsigDB database, rated each sample in GSE172112 for pathway activation using the "ssgsea" algorithm in the R package "GSVA", and assessed score differences between samples using the Wilcoxon rank-sum test. nal, We found that in alpha1H treatment compared with placebo, Activation of the following pathways in patients: where "REACTIVE_OXYGEN_SPECIES_PATHWAY", "P53_PATHWAY", "UV_RESPONSE_UV", "HYPOXIA", "APOPTOSIS", "TNFA_SIGNALING_VIA_NFKB", "COMPLEMENT", "IL6_JAK_STAT3_SIGNALING", "INFLAMMATORY_RESPONSE" is all downregulated (Table 4). Based on the above results, we found that the downregulation of "P53_PATHWAY" indicates the decreased proliferation capacity of tumor cells; while "TNFA_SIGNALING_VIA_NFKB", "IL6_JAK_STAT3_SIGNALING" and "INFLAMMATORY_RESPONSE" pathways indicate the alleviated immunoin ammatory response in patients after using the new drug alpha1H, indicating the removal of tumor cells ( Figure 5).

alpha1H and the TGF-beta signaling pathway
Combined with single gene-level differential analysis of overall pathway activation, we found that alpha1H affected some of gene members in TGF-beta signaling (including SMAD4, ACVR1C, ID4, SMAD9, SMAD6 and ACVR2B), but the activation of overall TGF-beta signaling was not signi cantly changed with Wilcoxon rank sum test p-value of 0.7528. However, SMAD4, SMAD9, SMAD6 Three SMAD family members also have important effects on TGF-beta signaling. Therefore, we focused on the expression of these three genes and found that all three SMAD family members were relatively highly expressed (Table  5) in patients treated with alpha1H.  (Figure 7). For SMAD9, we divided the patients into high expression groups (expression above 11.3209) and low expression group (expression below 11.3209) according to their expression value, and the HR [95%CI] of the nal constructed Cox proportional-risk model was 1.521[1.064-2.174], and the log-rank test p-value was 0.021, indicating that the worse the prognosis of patients with higher SMAD9 expression (Figure 8).
The above results show that the upregulation of SMAD6 gene expression after alpha1H treatment contributes to patient prognosis, but the upregulation of SMAD4 and SMAD9 genes is instead unfavorable to patient prognosis.

LINC00152 and patients with bladder cancer
In the analysis of differentially expressed lncRNA in the GSE172112 dataset, we found signi cantly downregulated LINC00152 expression in patients treated with alpha1H, and the results collected in the lnc2cancer database also showed generally high expression of this lncRNA in bladder cancer patients, we used this lncRNA to assess the prognostic value using the TCGA BLCA dataset, A total of 252 patients who examined lncRNA expression and recorded clinical prognostic information were analyzed.
We found that, given as the expression values of the LINC00152, Patients were divided into high expression groups (expression above 1.130234) and low expression groups (expression below 1.130234), The HR [95%CI] of the nal constructed model of the Cox scale analysis was 3.681 [1.354-10.01], The logrank test p-value was 0.0062, It shows that the high expression of LINC00152 is unfavorable to the patient prognosis, The new drug alpha1H reduces LINC00152 and has a positive effect on curing cancer ( Figure 9).

Discussion
In this study, by comparing the BC tissues after alpha1H treatment and after placebo treatment, a total of 781 differential genes were selected, including 396 upregulated genes and 385 downregulated genes. Upregulated genes are mainly involved in seven pathways, including the signaling pathway regulating stem cell pluripotency, TGF-signaling pathway, cell cycle, RNA degradation, nucleotide excision repair, valine, leucine, and isoleucine degradation, and Notch signaling pathway. The downregulated genes are mainly involved in 12 pathways, including Salmonella infection, MAPK signaling, pulmonary tuberculosis, phagosomes, cytokine receptor interactions, tumor necrosis factor signaling, insulin signaling, NF-B signaling, chemokine signaling, amoebosis, NOD-like receptor signaling, and prion disease. Signaling pathways associated with bladder cancer (BC) progression may be important targets for systemic therapy. Manal Elmasry et al [17] found that mitogen-activated protein kinase (MAPK / ERK) signaling is associated with BC progression, and the results showed that MAPK signaling can control BC cell activity, and that inhibition of M A P C signaling in BC xenografts would inhibit BC cell growth and reduce BC cell subsets. In this study, the genes signi cantly downregulated after treatment with the new drug alpha1H were associated with MAPK signaling.
In this study, we found that alpha1H can have a positive effect on the prognosis of BC through intervention in related signaling pathways, mainly including P53 signaling, IL-6 / Stat3 signaling, TNF-/ NF-B signaling, etc.YE GUOMEI et al 26 found that puerurin inhibited bladder cancer T24 cell proliferation and induced apoptosis by inhibiting the SIRT1 / p53 signaling pathway. Jelena Korac-Prlic et al [18] found that blocking the IL-6 / Stat3 axis alone inhibits bladder cancer progression.Chen Jia-Liang et al [19] found that mechanical ectopic pain, depression-like behavior in cyp-induced in a mouse model of TNF-/ NFinduced cystitis by inhibition of P-κB signaling. This study found that P53 pathway downregulation after alpha1H treatment indicates the decreased proliferative capacity of tumor cells and the IL-6 / Stat3 axis and TNF-/ NF-κB signaling pathway, indicating that the immunoin ammatory response was alleviated in vivo in patients after the use of the new drug alpha1H.
This study found that some of the genes in the TGF-signaling pathway changed treatment with the new drug alpha1H.Hung Tzong-Tyng et al [20] found that the TGF-pathway is involved in BC progression. BIAN JING et al 24 showed that BC cells utilize mutant TGF-receptors for TGF-signaling, leading to their enhanced migration and invasion, and avoiding growth arrest. Shi Heng et al [21] found that LINC01451 promotes epithelial-mesenchymal transformation through the activation of the LIN28 / TGF-/ Smad signaling pathway, thereby aggravating BC progression. Zhu Feng et al [22] found that LncRNA AWPPH can inhibit SMAD4 through EZH2, promote BC cell proliferation, migration, and inhibit apoptosis.SMAD4 levels were downregulated in BC tissues as compared to normal bladder tissues. The results of this study show that some of the genes in the TGF-signaling pathway have changed after the new drug alpha1H treatment, and that the upregulation of SMAD6 gene expression contributes to patient prognosis, but the upregulation of SMAD4 and SMAD9 genes is instead unfavorable to patient prognosis.
LINC00152 may be involved in cell cycle arrest, apoptosis, epithelial to mesenchymal transformation, cell migration, and may serve as reliable biomarkers for the diagnosis of some cancer types. Tang Xian-li et al [23] found that Linc00152 is highly expressed in bladder cancer patients, and that the possible oncogenic role of Linc00152 in bladder cancer is achieved through the activation of Wnt / b-Catenin signaling, suggesting that Linc00152 may be a novel biomarker for the diagnosis and prevention of bladder cancer. In this study, the constructed Cox proportional analysis model found that high LINC00152 expression unfavorable patient prognosis, while the new drug alpha1H reduced LINC00152 has a positive effect on the cancer cure.
In this comparative study, differentially expressed genes between BC tissues and BC tissues after placebo treatment. Functional and pathway enrichment analyses of differential genes revealed potential molecular pathways underlying alpha1H in multiple signaling for BC. Further combined with analyses, obtaining key LncRNA closely related to the prognosis of BC patients provide potential targets for subsequent studies on BC treatment, while elucidating the molecular mechanisms of cancer suppressor in BC patients after alpha1H treatment.

Conclusions
In Conclusion, based on the GEO and lnc2cancer databases, we used bioinformatics methods to screen the differentially expressed genes of bladder cancer after alpha1H treatment, which are mainly enriched in the TGF-signaling pathways, the MAPK signaling pathway, and the TNF signaling pathway and other pathways, and veri ed The high expression of SMAD4, SMAD9 and LINC00152 is not conducive to the prognosis of patients, and the low expression of SMAD6 is not conducive to the prognosis of patients. It points out the diagnostic and therapeutic value of LINC00152 in bladder cancer. However, the molecular mechanism of LINC00152 affecting prognosis needs further study.

Data collection and preprocessing
We are working from the GEO (https: // www.ncbi.nlm.nih.Sequencing data from patients with clinical trials of a new bladder cancer drug were collected in the gov / geo /) database, GSE172112, in which some patients used the new drug alpha1H (alpha1-oleate), some patients used placebo (placebo), including several tissues using the new drug alpha1H, and several tissues using placebo (placebo).We are also quoted from the UCSC Xena (https://rowser.The Bladder Cancer (BLCA) dataset from the TCGA plan was collected and downloaded from the net / datapages /) database, including the gene RNA-seq sequencing results (after quantile standardization) of the sample, and the clinical information of the patient. In addition, we also from TANRIC (https:// www.tanric. RNA-seq Level 2 data and their clinical information from the Bladder Cancer (BLCA) dataset were collected and downloaded from the org) database, with total case samples, where several were organized after the new drug alpha1H (alpha1oleate) and several after placebo (placebo).
The TCGA database provides the raw counts and E n t r z e I D for the RNA-seq Level 2 mRNA. The annotation of the most complete human lncRNA was included and analyzed in the GENCODE v7 database, and the lncRNA dataset is more comprehensive than other databases. According to the chromosomal location of the exon provided by the TCGA database, the raw counts, and the reads number per thousand base from the map to the exon in the reads per 1 million map (reads per kilobase of exon per million mapped reads, RPKM) Information, Alignment to the chromosomal locations of the lncRNA in the GENCODE v7 database, If the chromosome position, the information is consistent, The exon is de ned as a lncRNA. Using the R package "DESeq2", we transformed the read count values of the raw sequencing data into the gene expression values t by the "DESeq2" package, and used the R package "biomaRt" to classify the detected RNA molecules into PCG (Protein Coding Gene) as well as lncRNA classes.

Gene differential expression analysis
For the gene expression dataset for new drug treatment, GSE172112, we divided the patient's medication pro le into two groups, one using the new drug alpha1H and the other using a placebo. The data chips in the gene expression data pro ling were normalized in R 3.6.1 using the limma software package, Screening for differentially expressed genes, The Fold Change values were calculated, follow, Differential expression analysis was performed using the Wilcoxon rank-sum test, Record the test p-value, | FC (fold change) | 2 and P <0.01 were used as screening criteria for differential genes. 5.3 KEGG pathway enrichment analysis DAVID 6.8 database (https://david.ncifcrf.The gov /) is a commonly used database for gene enrichment and functional annotation analysis. The KEGG pathway was analyzed for the differentially expressed genes using DAVID. The selection conditions were: human gene, P <0.05.

GSVA pathway difference analysis
To assess the impact of new drug use on overall pathway activation in patients, we obtained from the MsigDB database (http://www.gsea-msigdb.org/gsea/index.Gene sets for downloading 50 hallmark signaling pathways were collected in jsp).Using the "ssgsea algorithm" in the R package "GSVA", combined with a gene set of 50 hallmark signaling pathways, we scored pathway activation for each sample and subsequently scored difference comparison using Wilcoxon's rank-sum test.

Screening of the L ncRNA
Transcripptomic sequencing data of BC and detailed clinical data of BC patients were downloaded from the TCGA database, Expression matrix of the long-chain non-coding RNA data was extracted from the transcriptome sequencing data; The threshold was set as a corrected P <0.05 with a differential expression multiple> 4 (FDR <0.05 and | log FC |> 2), Differentially expressed lncRNA were screened using the R software edgeR package, The expression data of the lnc RNA were merged with the downloaded survival data, The lnc RNA related to patient outcome after alpha1H treatment was screened by univariate Cox regression analysis; LASSO (Least Absolute Shrinkage and Selection Operator through the glmnet package and the survival package in R, LASSO) Regression analysis screening for lnc RNA with more critical prognosis with alpha1H treatment. Finally, the lnc RNA model associated with BC prognosis after alpha1H treatment was established by multivariate Cox regression analysis. K-M survival analysis was performed on lncRNA with statistically differences in Cox multivariate regression analysis to determine prognostic biomarkers.

survival analysis
To assess the impact of gene expression on patient prognosis, we combined the cox proportional hazards survival model in TCGA BLCA patients using the R package "survival" and performed log-rank test, record HR [95%CI] of survival model and test p-values to assess the prognostic value of genes.
Kaplan-Meier curves were drawn for the genes, lncRNA using Graphpad Prim 7 statistical software and whether statistical differences between the two groups were determined by Log-rank test, considered by P <0.05 as statistically signi cant.

Fundings
Not applicable Authors' contributions Zhou HY designed the study. Wang X, Yu JD, Wen HL, Wang RJ and Peng K collected and analyzed the data. Wang X wrote the manuscript.  396 KEGG pathway associated with signi cantly upregulated genes