Identification of key molecules and significant signaling pathways in LINC00958 mediated endometrial cancer cells

doi:10.21203/rs.3.rs-1530254/v1

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Identification of key molecules and significant signaling pathways in LINC00958 mediated endometrial cancer cells

https://doi.org/10.21203/rs.3.rs-1530254/v1

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Endometrial cancer is the most common gynecologic cancer in the United States. Since the cancers of the same stage and histology have very distinct molecular and genomic profiles, the therapy is quite limited. Current Long noncoding RNAs (lncRNAs) LINC00958 showed some benefit to this cancer, but the mechanism is unknown. In this study, we aim to identify the key molecules and signaling pathways by analyzing the RNA-seq data. The GSE198619 was created by the DNBSEQ-T7 (Homo sapiens). The KEGG and GO analyses indicated "Human T − cell leukemia virus 1 infection" and "Hippo signaling pathway" were the main affected biological processes. Moreover, we further figured out several interactive genes including CDK1, CDC42, JUN, FOS, CCNA2, KIF11, CCND1, KAT2B, MAD2L1, and NRAS. Our study may benefit the study of endometrial cancer treatment.

Endometrial cancer is the most common gynecological tumor in developed countries, and its prevalence is developing. Since the disease is frequently symptomatic at an early stage, endometrial cancer is commonly diagnosed at stage I¹. Classically, standard treatment consisted of hysterectomy, bilateral salpingo-oophorectomy, and pelvic lymph node dissection². Endometrial cancer is the fifth most common cancer in women, who have a cumulative risk of 1% of developing the disease by age 75 years³. Though endometrial cancer is commonly thought to be a cancer of the postmenopausal period, 14% of cases are diagnosed in premenopausal women, 5% of whom are younger than 40 years⁴. The main risk factor is exposure to endogenous and exogenous estrogens associated with obesity, diabetes, and the use of tamoxifen⁵.

Long noncoding RNAs (lncRNAs) regulate a number of physiological and pathological processes, including gene transcription and translation, chromatin modification, cell cycle progression, cell proliferation, and oncogenic and tumor-suppressive signals in cancer⁶. For example, increased expression of LINC00958 attributes to cell metastasis in gastric cancer⁷. LINC00958 mediates pancreatic cancer by controlling the miR-330-5p/PAX8 axis. LINC00958 promotes gliomagenesis through the miR-203/CDK2 axis⁸.

In our study, we analyzed the effects of the knockdown of LINC00958 in endometrial cancer cells by using the RNA-seq data. We figured out a number of DEGs and significant signaling pathways. We then performed the gene enrichment and protein-protein interaction (PPI) network analysis to obtain the interacting map. These genes and biological processes may help understand the mechanism for inhibiting LINC00958 in cancers.

Data resources

Gene dataset GSE198619 was downloaded from the GEO database. The data was produced by the DNBSEQ-T7 (Homo sapiens) (China Medical University, Sanhao Street Shenyang, Liaoning, China). The analyzed dataset includes three shRNA controls and three LINC00958 shRNA-treated groups.

Data acquisition and processing

The data were organized and conducted by the R package as previously described^9–12. We used a classical t-test to identify DEGs with P < 0.05 and fold change ≥ 1.5 as being statistically significant.

The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO)

KEGG and GO analyses were performed by the R package (ClusterProfiler) and Reactome. P < 0.05 was considered statistically significant.

Protein-protein interaction (PPI) networks

The Molecular Complex Detection (MCODE) was used to create the PPI networks. The significant modules were produced from constructed PPI networks and String networks (https://string-db.org/). The biological processes analyses were performed by using Reactome (https://reactome.org/), and P < 0.05 was considered significant.

Identification of DEGs in endometrial adenocarcinoma cells (Ishikawa cells) after the knockdown of LINC00958

To determine the effects of the knockdown of LINC00958 in Ishikawa cells, we analyzed the RNA-seq data from the GEO database. A total of 981 genes were identified with a threshold of P < 0.001. The top up- and down-regulated genes were shown by the heatmap and volcano plot (Figure 1). The top ten DEGs were listed in Table 1.

Enrichment analyses of DEGs in Ishikawa cells after the knockdown of LINC00958

To further identify the potential signaling pathways after the knockdown of LINC00958 in Ishikawa cells, we performed the KEGG and GO analyses (Figure 2). We identified the top ten KEGG items including “Human T−cell leukemia virus 1 infection”, “Hippo signaling pathway”, “Thyroid hormone signaling pathway”, “Viral life cycle − HIV−1”, “Renal cell carcinoma”, “Glycerophospholipid metabolism”, “PPAR signaling pathway”, “Fatty acid metabolism”, “Mannose type O−glycan biosynthesis”, and “Galactose metabolism”. We identified the top ten biological processes of GO including “positive regulation of cell cycle”, “mitotic nuclear division”, “positive regulation of cell cycle process”, “Golgi organization”, “spindle organization”, “vesicle budding from membrane”, “regulation of ubiquitin−protein transferase activity”, “organelle inheritance”, “Golgi inheritance”, and “Golgi vesicle budding”. We identified the top ten cellular components of GO, including “cell−cell junction”, “spindle”, “ubiquitin ligase complex”, “mitotic spindle”, “trans−Golgi network”, “midbody”, “cullin−RING ubiquitin ligase complex”, “microtubule associated complex”, “cell division site”, and “cleavage furrow”. We identified the top ten molecular functions of GO, including “molecular adaptor activity”, “protein−macromolecule adaptor activity”, “protein serine kinase activity”, “phosphatase regulator activity”, “protein phosphatase regulator activity”, “phosphatase inhibitor activity”, “ubiquitin ligase−substrate adaptor activity”, “core promoter sequence−specific DNA binding”, “steroid hormone receptor activity”, and “cAMP response element binding”.

PPI network and Reactome analyses

To explore the potential associations among the DEGs, we created the PPI network by using 711 nodes and 1669 edges. The combined score > 0.2 was set as a cutoff by using the Cytoscape software. Table 2 indicated the top ten genes with the highest scores. The top two significant modules were presented in Figure 3. We further analyzed the PPI and DEGs with a Reactome map (Figure 4) and figured out the top ten biological processes including "Estrogen-dependent nuclear events downstream of ESR-membrane signaling", "Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1", "Regulation of cholesterol biosynthesis by SREBP (SREBF)", "Defective HDR through Homologous Recombination (HRR) due to PALB2 loss of function", "Defective HDR through Homologous Recombination (HRR) due to BRCA1 loss-of-function", "Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function", "Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function", "Diseases of DNA Double-Strand Break Repair", and "Defective binding of RB1 mutants to E2F1,(E2F2, E2F3)" (Supplemental Table S1).

Endometrial cancer is the most common gynecologic malignancy¹³. There is a great needy for the treatment of endometrial cancer in the world. LncRNAs are currently identidied noncoding endogenous RNAs that critically regulates the development, invasion, and metastasis of cancer cells¹⁴. Thus, our study is mainly focused on the mechanisms of lncRNAs controlled cancer progression.

By analyzing the KEGG and GO profiles, we found that the "Human T − cell leukemia virus 1 infection" and "Hippo signaling pathway" are the main signaling pathways during the knockdown of LINC00958 in endometrial cancer cells. Interestingly, Mickey V Patel et al found that endometrial Cancer can inhibit CD8 + T cell-mediated cytotoxicity in postmenopausal women¹⁵. Xiaoli Wen et al found that P190A mutations inhibit RhoA and promote malignant transformation via the Hippo-YAP signaling pathway in endometrial cancer¹⁶.

Additionally, we identified several association genes by constructing the PPI network. Jin Huang et al found the inhibition of CDK1 improves the IFNG-mediated adaptive immune resistance in cancer¹⁷. Circadian clocks regulate the pathological processes in different diseases such as diabetes, arthritis, and metabolism disorder^{11, 12, 18–30}. CDK1 was reported to be correlated with the expression of clock gene cry1, suggesting the multiple roles of CDK1 in cancer development³¹. Natalya Chernichenko et al found that cdc42 regulates cancer cell chemotaxis in cancer invasion³². GPCR and RGS signaling pathways are related to various biological disorders including inflammation, metabolism, and aging^{10, 11, 33–54}. Interestingly, Alejandro Castillo-Kauil et al found that Gαs directly promotes PDZ-RhoGEF signaling to cdc42⁵⁵. Evangelos Tsiambas found that JUN/FOS are biomarkers for oral cancer⁵⁶. Rui Yang et al found that CCNA2 acts as a treatment target for prostate cancer via inhibiting cell cycle⁵⁷. Dang Wei et al found that KIF11 enhances cell proliferation through PI3K/ATK signaling in gallbladder cancer⁵⁸. Yan Liu et al found that microRNA-374b represses breast cancer via controlling CCND1 gene⁵⁹. Xue Zhou et al found KAT2B is an immune biomarker for non-small-cell lung cancer⁶⁰. Yu Wang et al found that miR-20a-3p regulates MAD2L1 to further control the proliferation of gastric cancer cells⁶¹. Vlad-Adrian Afrăsânie et al found that NRAS is a key biomarker for the metastatic colorectal cancer⁶².

In summary, this study identified a number of significant genes and biological processes after the knockdown of LINC00958 in endometrial cancer cells. "Human T − cell leukemia virus 1 infection" and "Hippo signaling pathway" are the main signaling pathways. Our study may provide new knowledge for the treatment of endometrial cancer.

Author Contributions

Xiu-ying Wang, Tingxiang Chang: Methodology and Writing. Mengshan Wang: Conceptualization, Writing- Reviewing and Editing.

Funding

This work was not supported by any funding.

Declarations of interest

There is no conflict of interest to declare.

Bell DW, Ellenson LH: Molecular Genetics of Endometrial Carcinoma. Annu Rev Pathol 2019, 14:339–67.
Sonoda Y: Surgical treatment for apparent early stage endometrial cancer. Obstet Gynecol Sci 2014, 57:1–10.
Jiang Y, Li J, Sang C, Cao G, Wang S: Diagnostic and prognostic value of HABP2 as a novel biomarker for endometrial cancer. Ann Transl Med 2020, 8:1164.
Braun MM, Overbeek-Wager EA, Grumbo RJ: Diagnosis and Management of Endometrial Cancer. Am Fam Physician 2016, 93:468–74.
Dignam JJ, Wieand K, Johnson KA, Fisher B, Xu L, Mamounas EP: Obesity, tamoxifen use, and outcomes in women with estrogen receptor-positive early-stage breast cancer. J Natl Cancer Inst 2003, 95:1467–76.
De Martino M, Esposito F, Pallante P: Long non-coding RNAs regulating multiple proliferative pathways in cancer cell. Transl Cancer Res 2021, 10:3140–57.
Chen M, Xu Z, Zhang Y, Zhang X: LINC00958 Promotes The Malignancy Of Nasopharyngeal Carcinoma By Sponging microRNA-625 And Thus Upregulating NUAK1. Onco Targets Ther 2019, 12:9277–90.
Yang L, Li L, Zhou Z, Liu Y, Sun J, Zhang X, Pan H, Liu S: SP1 induced long non-coding RNA LINC00958 overexpression facilitate cell proliferation, migration and invasion in lung adenocarcinoma via mediating miR-625-5p/CPSF7 axis. Cancer Cell Int 2020, 20:24.
Yu G, Wang LG, Han Y, He QY: clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 2012, 16:284–7.
Hanming G: nuotrophils arthritis. Research Square 2021.
Jing L, Letian W, Hanming G: Identification of driver genes and biological signaling for alcoholic myopathy. Research Square 2021.
Yuan G: Identification of biomarkers and pathways of mitochondria in sepsis patients. bioRxiv 2021:2021.03.29.437586.
Bruggmann D, Ouassou K, Klingelhofer D, Bohlmann MK, Jaque J, Groneberg DA: Endometrial cancer: mapping the global landscape of research. J Transl Med 2020, 18:386.
Dong P, Xiong Y, Yue J, S JBH, Kobayashi N, Todo Y, Watari H: Exploring lncRNA-Mediated Regulatory Networks in Endometrial Cancer Cells and the Tumor Microenvironment: Advances and Challenges. Cancers (Basel) 2019, 11.
Patel MV, Shen Z, Rodriguez-Garcia M, Usherwood EJ, Tafe LJ, Wira CR: Endometrial Cancer Suppresses CD8 + T Cell-Mediated Cytotoxicity in Postmenopausal Women. Front Immunol 2021, 12:657326.
Wen X, Wan J, He Q, Wang M, Li S, Jiang M, Qian Z, Liu B, Lu W, Wang K, Gao K, Wan X: p190A inactivating mutations cause aberrant RhoA activation and promote malignant transformation via the Hippo-YAP pathway in endometrial cancer. Signal Transduct Target Ther 2020, 5:81.
Huang J, Chen P, Liu K, Liu J, Zhou B, Wu R, Peng Q, Liu ZX, Li C, Kroemer G, Lotze M, Zeh H, Kang R, Tang D: CDK1/2/5 inhibition overcomes IFNG-mediated adaptive immune resistance in pancreatic cancer. Gut 2021, 70:890–9.
Reppert SM, Weaver DR: Coordination of circadian timing in mammals. Nature 2002, 418:935–41.
Yuan G, Hua B, Yang Y, Xu L, Cai T, Sun N, Yan Z, Lu C, Qian R: The Circadian Gene Clock Regulates Bone Formation Via PDIA3. J Bone Miner Res 2017, 32:861–71.
Zhu Z, Xu L, Cai T, Yuan G, Sun N, Lu C, Qian R: Clock represses preadipocytes adipogenesis via GILZ. J Cell Physiol 2018, 233:6028–40.
Fan XF, Wang XR, Yuan GS, Wu DH, Hu LG, Xue F, Gong YS: [Effect of safflower injection on endoplasmic reticulum stress-induced apoptosts in rats with hypoxic pulmonary hypertension]. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2012, 28:561–7.
Xu L, Cheng Q, Hua B, Cai T, Lin J, Yuan G, Yan Z, Li X, Sun N, Lu C, Qian R: Circadian gene Clock regulates mitochondrial morphology and functions by posttranscriptional way. bioRxiv 2018:365452.
Yuan G, Hua B, Cai T, Xu L, Li E, Huang Y, Sun N, Yan Z, Lu C, Qian R: Clock mediates liver senescence by controlling ER stress. Aging 2017, 9:2647–65.
Zhu Z, Hua B, Xu L, Yuan G, Li E, Li X, Sun N, Yan Z, Lu C, Qian R: CLOCK promotes 3T3-L1 cell proliferation via Wnt signaling. IUBMB Life 2016, 68:557–68.
Mao SZ, Fan XF, Xue F, Chen R, Chen XY, Yuan GS, Hu LG, Liu SF, Gong YS: Intermedin modulates hypoxic pulmonary vascular remodeling by inhibiting pulmonary artery smooth muscle cell proliferation. Pulm Pharmacol Ther 2014, 27:1–9.
Yuan G, Xu L, Cai T, Hua B, Sun N, Yan Z, Lu C, Qian R: Clock mutant promotes osteoarthritis by inhibiting the acetylation of NFkappaB. Osteoarthritis Cartilage 2019, 27:922–31.
Gu H, Yuan G: Identification of potential key genes for SARS-CoV-2 infected human bronchial organoids based on bioinformatics analysis. bioRxiv 2020:2020.08.18.256735.
Zhu Z, Hua B, Shang Z, Yuan G, Xu L, Li E, Li X, Sun N, Yan Z, Qian R, Lu C: Altered Clock and Lipid Metabolism-Related Genes in Atherosclerotic Mice Kept with Abnormal Lighting Condition. Biomed Res Int 2016, 2016:5438589.
Li J, Wang W, Gu H: Identification of biological processes and signaling pathways for the knockout of REV-ERB in mouse brain. bioRxiv 2021:2021.11.22.469579.
Cai T, Hua B, Luo D, Xu L, Cheng Q, Yuan G, Yan Z, Sun N, Hua L, Lu C: The circadian protein CLOCK regulates cell metabolism via the mitochondrial carrier SLC25A10. Biochim Biophys Acta Mol Cell Res 2019, 1866:1310–21.
Huang Y, Jiang X, Yan Y, Liu G, Liu C: Expression of cell proliferation regulatory factors bricd5, tnfrsf21, cdk1 correlates with expression of clock gene cry1 in testes of Hu rams during puberty. Mol Biol Rep 2021, 48:7379–85.
Chernichenko N, Omelchenko T, Deborde S, Bakst RL, He S, Chen CH, Gusain L, Vakiani E, Katabi N, Hall A, Wong RJ: Cdc42 Mediates Cancer Cell Chemotaxis in Perineural Invasion. Mol Cancer Res 2020, 18:913–25.
Stewart A, Fisher RA: Introduction: G Protein-coupled Receptors and RGS Proteins. Prog Mol Biol Transl Sci 2015, 133:1–11.
Yuan G, Yang S, Yang S: Macrophage RGS12 contributes to osteoarthritis pathogenesis through enhancing the ubiquitination. Genes & Diseases 2021.
Hanming G, Wei W, Gongsheng Y: Identification of potential biomarkers and inhibitors in SARS-CoV-2 infected macaques. Research Square 2020.
Fu C, Yuan G, Yang ST, Zhang D, Yang S: RGS12 Represses Oral Cancer via the Phosphorylation and SUMOylation of PTEN. J Dent Res 2020:22034520972095.
Yuan G, Fu C, Yang ST, Yuh DY, Hajishengallis G, Yang S: RGS12 Drives Macrophage Activation and Osteoclastogenesis in Periodontitis. J Dent Res 2022, 101:448–57.
Jing W, Hanming G: Identification of biological processes and potential inhibitors for aging skin. Research Square 2021.
Gu H, Yuan G: Identification of specific biomarkers and pathways in the synovial tissues of patients with osteoarthritis in comparison to rheumatoid arthritis. bioRxiv 2020:2020.10.22.340232.
Xie K, Martemyanov KA: Control of striatal signaling by g protein regulators. Front Neuroanat 2011, 5:49.
Yuan G, Yang S, Ng A, Fu C, Oursler MJ, Xing L, Yang S: RGS12 Is a Novel Critical NF-kappaB Activator in Inflammatory Arthritis. iScience 2020, 23:101172.
Gu H, Wang W, Yuan G: Identification of biomarkers and candidate inhibitors for multiple myeloma. bioRxiv 2021:2021.02.25.432847.
Rosenbaum DM, Rasmussen SG, Kobilka BK: The structure and function of G-protein-coupled receptors. Nature 2009, 459:356–63.
Yuan G, Huang Y, Yang ST, Ng A, Yang S: RGS12 inhibits the progression and metastasis of multiple myeloma by driving M1 macrophage polarization and activation in the bone marrow microenvironment. Cancer Commun (Lond) 2022, 42:60–4.
Gu H: Identification of biomarkers and pathways of mouse embryonic fibroblasts with the dysfunction of mitochondrial DNA. bioRxiv 2021:2021.04.05.438453.
Yuan G, Yang S, Liu M, Yang S: RGS12 is required for the maintenance of mitochondrial function during skeletal development. Cell Discov 2020, 6:59.
Zhang M, Wang J, Gu H: Identification of biological processes and signaling pathways in the stretched nucleus pulposus cells. bioRxiv 2021:2021.11.23.469730.
Yuan G, Yang S, Gautam M, Luo W, Yang S: Macrophage regulator of G-protein signaling 12 contributes to inflammatory pain hypersensitivity. Ann Transl Med 2021, 9:448.
Hanming G, Gongsheng Y: Identification of key genes and pathways in the hPSC-derived lungs infected by the SARS-CoV-2. Research Square 2021.
Gu H, Yuan G: Identification of key genes in SARS-CoV-2 patients on bioinformatics analysis. bioRxiv 2020:2020.08.09.243444.
Yuan G, Yang S, Yang S, Ng A, Oursler MJ: RGS12 is a critical proinflammatory factor in the pathogenesis of inflammatory arthritis via acting in Cox2-RGS12-NF kappa B pathway activation loop. J Bone Miner Res: WILEY 111 RIVER ST, HOBOKEN 07030 – 5774, NJ USA, 2019. pp. 147-.
Spurney RF, Flannery PJ, Garner SC, Athirakul K, Liu S, Guilak F, Quarles LD: Anabolic effects of a G protein-coupled receptor kinase inhibitor expressed in osteoblasts. J Clin Invest 2002, 109:1361–71.
Gu H, Yuan G: Identification of potential biomarkers and inhibitors for SARS-CoV-2 infection. medRxiv 2020:2020.09.15.20195487.
Nakagawa T, Minami M, Satoh M: Up-regulation of RGS4 mRNA by opioid receptor agonists in PC12 cells expressing cloned mu- or kappa-opioid receptors. Eur J Pharmacol 2001, 433:29–36.
Castillo-Kauil A, Garcia-Jimenez I, Cervantes-Villagrana RD, Adame-Garcia SR, Beltran-Navarro YM, Gutkind JS, Reyes-Cruz G, Vazquez-Prado J: Galphas directly drives PDZ-RhoGEF signaling to Cdc42. J Biol Chem 2020, 295:16920–8.
Tsiambas E, Mastronikolis N, P PF, Kyrodimos E, Chrysovergis A, Papanikolaou V, Mastronikolis S, Peschos D, Roukas D, Ragos V: c-Jun/c-Fos complex in laryngeal squamous cell carcinoma. J BUON 2020, 25:618–20.
Yang R, Du Y, Wang L, Chen Z, Liu X: Weighted gene co-expression network analysis identifies CCNA2 as a treatment target of prostate cancer through inhibiting cell cycle. J Cancer 2020, 11:1203–11.
Wei D, Rui B, Qingquan F, Chen C, Ping HY, Xiaoling S, Hao W, Jun G: KIF11 promotes cell proliferation via ERBB2/PI3K/AKT signaling pathway in gallbladder cancer. Int J Biol Sci 2021, 17:514–26.
Liu Y, Zhang A, Bao PP, Lin L, Wang Y, Wu H, Shu XO, Liu A, Cai Q: MicroRNA-374b inhibits breast cancer progression through regulating CCND1 and TGFA genes. Carcinogenesis 2021, 42:528–36.
Zhou X, Wang N, Zhang Y, Yu H, Wu Q: KAT2B is an immune infiltration-associated biomarker predicting prognosis and response to immunotherapy in non-small cell lung cancer. Invest New Drugs 2022, 40:43–57.
Wang Y, Wang F, He J, Du J, Zhang H, Shi H, Chen Y, Wei Y, Xue W, Yan J, Feng Y, Gao Y, Li D, Han J, Zhang J: miR-30a-3p Targets MAD2L1 and Regulates Proliferation of Gastric Cancer Cells. Onco Targets Ther 2019, 12:11313–24.
Afrasanie VA, Marinca MV, Alexa-Stratulat T, Gafton B, Paduraru M, Adavidoaiei AM, Miron L, Rusu C: KRAS, NRAS, BRAF, HER2 and microsatellite instability in metastatic colorectal cancer - practical implications for the clinician. Radiol Oncol 2019, 53:265–74.

Tables 1-2 are available in the Supplementary Files section.

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Identification of key molecules and significant signaling pathways in LINC00958 mediated endometrial cancer cells

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