Comprehensive Analysis of Prognostic Signicance and Immune Inltration for FAM83 Family in Cervical Cancer

Background: This study aimed to explore the expression of Family with sequence similarity 83 (FAM83) members in cervical cancer, its prognostic value, related signaling pathways, regulatory mechanisms, and immune inltration. It’s of great value to explore the potential role of FAM83 family in cervical cancer and provide a new scientic basis for targeted therapy. Methods: The expression, gene mutations and prognostic value of FAM83 family members in cervical cancer were analyzed by various bioinformatics tools and databases. We further explored the interaction regulation network and immune inltration between FAM83 family members and their closely related genes through a series of databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) enrichment were also conducted. Results: This study showed that the expression levels of FAM83A/B/C/D/E/F/G/H gene were upregulated in cervical cancer, the expression of FAM83B/C/D/E/F/G/H were related to tumor stages of cervical cancer, and the promoter methylation of FAM83A/D/E/F/G genes in cervical cancer were lower than those in normal tissues. What’s more, high expression of FAM83A, FAM83B, and FAM83H mRNA was associated with shortened overall survival. GO results showed that FAM83A, FAM83B, and FAM83H and their closely related genes can play an important role in cell-cell junction, calcium-dependent protein binding, regulation of peptidase activity, inammatory response. KEGG analysis results showed that FAM83A, FAM83B, and FAM83H and their closely related genes were signicantly enriched in cancer pathways, estrogen signaling pathway, MAPK signaling pathway. Furthermore, FAM83A, FAM83B, and FAM83H are all closely related to lymphocytes (Tcm_CD4, Tcm_CD8, and neutrophils) and immunomodulators (TGFBR1, TGFB1, and TNFSF9). Conclusions: multiple databases, we found that the high expression of FAM83A, FAM83B, and FAM83H were associated with the shortened survival time and poor prognosis in patients with cervical cancer, and also closely correlated with lymphocytes and immune inltration, suggesting that FAM83A, FAM83B, and FAM83H played an important role in the occurrence, development, malignant biological behavior, and immune inlatration of cervical cancer, which provides an important theoretical basis for early diagnosis and targeted therapy for cervical cancer. Oncomine database collected a total of eight FAM83 family members in different tumor types. The Oncomine database was used to compare the transcription levels of FAM83 family members in cervical cancer tissues and normal tissue. The results showed that compared to normal cervical the mRNA levels of FAM83A, FAM83D, FAM83E, and FAM83G in P In the Biewenga Cervix dataset, the expression level of FAM83A in CESC was signicantly higher than that in normal tissues (fold change = 1.795, P = 0.001). In the two datasets, the expression level of FAM83D mRNA in cervical cancer patients was signicantly higher than that in the normal group. In the Biewenga Cervix dataset, the expression level of FAM83D in CESC was higher than that in normal tissues (fold change = 2.95, P = 1.09E-04). In the Pyeon multi-cancer dataset, the expression level of FAM83D in the cervical cancer group was higher than that in normal tissues (fold change = 1.742, P = 1.1E-02). In the Scotto Cervix 2 dataset, the expression level of FAM83E in CSCC was higher than that in normal cervical tissue (fold change = 2.298, P = 1.00E-03). In addition, the expression level of FAM83G in CSCC in the Biewenga Cervix dataset was higher than that in normal tissues (fold change = 1.889, P = 1.00E-03). and In order to better clarify the molecular mechanism, function, and related signal regulation network of FAM83A/B/H in cervical cancer, GO enrichment analysis on FAM83A/B/H and their closely related genes was performed, which revealed that the above genes were involved in the formation of cell-substrate junction, membrane raft, desmosome, and regulated various biological behaviors, including lipid binding, cadherin, and calcium-dependent protein binding, and regulating skin and epidermis development, inammatory response and cell junction organization. At the same time, KEGG enrichment analysis results showed that FAM83A/B/H and their closely related genes were signicantly enriched in cancer pathways, estrogen signaling pathway, MAPK signaling pathway, Rap1 and Hippo signaling pathway. We speculate that FAM83A/B/H can regulate the occurrence, development, and malignant biological behavior of cervical cancer by regulating the above-mentioned tumor-related pathways. This study provides more supporting evidences for FAM83A/B/H and the related signaling pathways and provides clues for the rational development of FAM83A/B/H-mediated multi-target therapy.


Results
The mRNA levels of FAM83 family members in patients with cervical cancer Oncomine database collected a total of eight FAM83 family members in different tumor types. The Oncomine database was used to compare the transcription levels of FAM83 family members in cervical cancer tissues and normal tissue. The results showed that compared to normal cervical tissues, the mRNA levels of FAM83A, FAM83D, FAM83E, and FAM83G were increased in cervical cancer tissues ( Fig. 1 and Table 1) (all P < 0.05). In the Biewenga Cervix dataset, the expression level of FAM83A in CESC was signi cantly higher than that in normal tissues (fold change = 1.795, P = 0.001). In the two datasets, the expression level of FAM83D mRNA in cervical cancer patients was signi cantly higher than that in the normal group. In the Biewenga Cervix dataset, the expression level of FAM83D in CESC was higher than that in normal tissues (fold change = 2.95, P = 1.09E-04). In the Pyeon multi-cancer dataset, the expression level of FAM83D in the cervical cancer group was higher than that in normal tissues (fold change = 1.742, P = 1.1E-02). In the Scotto Cervix 2 dataset, the expression level of FAM83E in CSCC was higher than that in normal cervical tissue (fold change = 2.298, P = 1.00E-03). In addition, the expression level of FAM83G in CSCC in the Biewenga Cervix dataset was higher than that in normal tissues (fold change = 1.889, P = 1.00E-03). Table 1 The transcription levels of FAM83 family members in different types of cervical cancer and normal tissues (Oncomine). The GEPIA online website was used to analyze the expression of FAM83 family members in CESC and normal cervical tissues. The results showed that the expression levels of FAM83A, FAM83B, FAM83C, FAM83D, FAM83E, FAM83F, FAM83G, and FAM83H in CESC were signi cantly higher than those in normal tissues (all P < 0.05) ( Fig. 2A-H). We further con rmed that the expression level of FAM83A, FAM83B, FAM83C, FAM83D, FAM83F, FAM83G, and FAM83H in CESC were signi cantly higher than those in normal tissues in UALCAN (all P < 0.05). The expression level of FAM83E in CESC was also higher than that in normal tissues, but the difference was not statistically signi cant ( Fig. 2I-P and Additional le: Table S1)(P > 0.05).
The relationship between the expression of FAM83 family members and tumor stage in 298 cases of CESC was further explored using the UALCAN online website (Fig. 3C). The results showed that in CESC, the expression levels of FAM83B and FAM3C in stage 3 were signi cantly higher than those in stage 1 (both P < 0.05) ( Fig. 3B). The expression levels of FAM83D and FAM83G in stages 2 and 3 were higher than those in stage 1 (all P < 0.05) ( Fig. 3D and Fig. 3G). The expression level of FAM83E in stages 2 and 3 were lower than those in stage 1 (all P < 0.05) (Fig. 3E). The expression level of FAM83F in stage 3 was lower than that in stage 1 (P < 0.05) (Fig. 3F). The expression level of FAM83H in stages 3 and 4 were higher than that in stage 1 (all P < 0.05) (Fig. 3H). There was no signi cant difference in FAM83A expression among various tumor stages (P > 0.05) ( Fig. 3A and Additional le: Table S2). The relationship between the expression of FAM83 family members and promoter methylation were further analyzed in 307 cases of CESC (Fig. 3C). The results showed that in CESC, the promoter methylation levels of FAM83A, FAM83D, FAM83E, FAM83F, and FAM83G were lower than those in normal tissues (P < 0.05)  Table S3).
Genomic alteration and gene interaction network of FAM83 family members in patients with cervical cancer The cBioPortal was used to analyze the mutation frequency of FAM83 family members in cervical cancer.  4A-B). Co-expression networks of eight FAM83 family members was constructed by GeneMANIA. The results showed that the above family members are closely associated with shared protein domains and co-expression. Furthermore, the changes and functions of genes, such as MYEOV (myeloma overexpressed), POF1B (POF1B actin binding protein), IPPK (c inositol-pentakisphosphate 2-kinase), KDF1 (keratinocyte differentiation factor 1) LAD1 (ladinin 1) were signi cantly correlated with FAM83 family members (Fig. 4C).
Prognostic values of FAM83 family members in patients with CESC The prognostic values of FAM83 family members in CESC patients were explored by GEPIA. The survival signi cance map showed that FAM83A, FAM83B, FAM83G, and FAM83H in CESC have important prognostic values (Fig. 5A). The KM plotter was further used to explore the relationship between There were 4,106 genes that were signi cantly positively correlated with FAM83B (dark red dots), and 5,459 genes that were signi cantly negatively correlated with FAM83B (dark green dots; FDR < 0.01) (Fig.  6C). The heat map showed the top 50 gene sets that were signi cantly positively and negatively correlated with FAM83B (Fig. 6C). The statistical scatter plot of a single gene showed that the expression of FAM83B was correlated with DSP (Pearson correlation = 0.7682, P = 1.928e-60), DENND2C (Pearson correlation = 0.7085, P = 1.233e-47) and FAM83G (Pearson correlation = 0.7017, P = 2.249e-46) (Fig. 6D). There were 2,294 genes that were signi cantly positively related to FAM83H (dark red dots), and 5,317 genes that are signi cantly negatively related to FAM83H (dark green dots, FDR < 0.01) (Fig. 6E). The heat map showed the top 50 gene sets that were signi cantly positively and negatively correlated with FAM83H (Fig. 6E). The statistical scatter plot of a single gene showed that the expression of FAM83H is related to PLEC (Pearson correlation = 0.6789, P = 2.005e-42), PLEKHN1(Pearson correlation = 0.6599,P = 2.22e-39) and PKP3 (Pearson correlation = 0.6532, P = 2.337e-38) (Fig. 6F).
Functional and pathway enrichment analysis of FAM83A/B/H in patients with cervical cancer GO and KEGG enrichment analysis of FAM83A, FAM83B, and FAM83H and their associated genes were performed by Metascape. GO showed that FAM83A and its related genes were mainly located in cellsubstrate junction, membrane raft, desmosome, and basal part of the cell, and these molecules could bind cadherin and participate in biological processes, such as skin development, regulation of peptidase activity, in ammatory response, wound healing and cell junction organization ( Fig. 7A and Table 2). KEGG enrichment analysis showed that the signaling pathways involved in FAM83A and its related genes include axon guidance, regulation of actin cytoskeleton, pathways in cancer, estrogen signaling pathway, NOD-like receptor signaling pathway, and MAPK signaling pathway ( Fig. 7B and Table 3). GO results showed that FAM83B and its related genes are mainly located in cell-cell junction, desmosome, and polymeric cytoskeletal ber. They could participate in lipid binding, epidermis development, wound healing, establishment of skin barrier, and involved in biological processes, such as desmosome and cell junction organization ( Fig. 7C and Table 4). KEGG enrichment analysis showed that the signaling pathways involved in FAM83B and its related differential genes include adherens junction, pathways in cancer, Rap1 signaling pathway, Hippo signaling pathway, and central carbon metabolism in cancer ( Fig.  7D and Table 5). GO results showed that FAM83H and its related differential genes were mainly located in cell-cell junctions, and invlved in cadherin-binding and calcium-dependent protein-binding and participated in biological process including epidermis development, regulation of endopeptidase activity, intermediate lament cytoskeleton, and desmosome organization ( Fig. 7E and Table 6). KEGG enrichment analysis showed that the signaling pathways involved in FAM83H and its related differential genes included axon guidance, MAPK signaling pathway, estrogen signaling pathway, pathogenic infection, and regulation of actin cytoskeleton ( Fig. 7F and Table 7). The above-mentioned signaling pathways can participate in the occurrence and development of a variety of tumors including cervical cancer.

Discussion
In recent years, many studies have shown that FAM83 family members are potential oncogenes, which are abnormally expressed in a variety of tumors, and affect the occurrence and development of tumors through different signaling pathways, such as EGFR, MAPK, and PI3K-AKT [7,9,30] . Abnormal expression or activation of FAM83 family members is a common phenomenon in malignant tumors, and is associated with tumor occurrence or progression. However, the expression and mechanism of different FAM83 family members in cervical cancer are still unclear. In this study, we investigated the expression, prognostic value, immune in ltration, and potential molecular functions of different FAM83 family members in cervical cancer for the rst time.
FAM83A is the smallest protein in the FAM83 family, which was initially considered as a potential tumor biomarker [31] . Researchers have found that FAM83A mRNA is highly expressed in circulating tumor cells of patients with lung adenocarcinoma by PCR [31] . High expression of FAM83A was correlated with poor prognosis of patients in breast cancer [32] . Several studies have shown that FAM83A is highly expressed in lung cancer and liver cancer, and related to advanced satges and poor prognosis, and also can promote the proliferation and invasion of lung cancer cells [33][34][35][36] . In this study, we found that the expression of FAM83A mRNA were upregulated in cervical cancer, while methylation levels of FAM83A gene decreased. The OS of cervical cancer patients with high expression of FAM83A was shortened and signi cantly related to poor prognosis. The above results are consistent with those from Liu et al. and Rong et al [15,32] . However, other studies have shown that high expression of FAM83A was negatively correlated with shortened survival time in patients with cervical cancer, and that FAM83A has a tumor-suppressing effect on cervical cancer by inhibiting integrin expression [38] , which may be due to the number of cervical cancer patient samples and the interaction of other related factors. Nonetheless, the above studies are su cient to show that FAM83A has important value in the occurrence, development, and prognosis evaluation of cervical cancer.
FMA83B is another important member of the FAM83 family. Jackson et al. rst discovered that FAM83B can act as a driving factor and induce malignant transformation for human breast epithelial cells [9] .
Many studies have reported that FAM83B is overexpressed in several malignant tumors, including endometrial cancer, lung cancer, pancreatic cancer, and ovarian cancer [39][40][41]13] , and promotes tumor proliferation, invasion, and migration. Studies have shown that FAM83B reversed the inhibitory effect of 14-3-3 protein on CRAF and promotes cell membrane localization of CRAF, thereby activating the MAPK signaling pathway and promoting tumor growth [9] . The interaction between FAM83B and EGFR was necessary to promote the activation of EGFR and phospholipase D1 [42] . In this study, we found for the rst time that the expression level of FAM83B mRNA in cervical cancer was increased and correlated with tumor stages, while the OS in cervical cancer patients with high expression of FAM83A was shortened, which was signi cantly related to poor prognosis, indicating that FAM83B plays an important role in the occurrence, development, invasion, metastasis, and prognosis of cervical cancer.
At present, there are few studies on FAM83C, and only a few have shown that FAM83C is highly expressed in lung cancer and is related to poor prognosis [43] . Thus, relevant experimental con rmation is still lacking. Researchers have performed proteomics to determine that FAM83G can participate in the BMP signaling pathway and affect the expression and function of SMAD1 [44] . In addition, FAM83G can form a complex with Axin1, CK1α, and GSK3β in the β-catenin degradation complex to promote the phosphorylation of CK1α and GSK3β, thereby activating Wnt signaling [45] . Brown et al. found that FAM83G could interact with Smad2 and Smad3 to regulate the TGF-β signaling pathway by mass spectrometry [46] . The present study found that the expression of FAM83C and FAM83G gene were upregulated in cervical cancer and correlated with tumor stages, but has no correlation with prognosis of patients with cervical cancer. However, additional experiments are still necessary for further con rmation of these research.
FAM83D was originally thought to be a protein correlated with the spindle [47] , which can interact with proteins in the spindle, such as chromokinesin, dynein light chain 1, and HMMR [48][49] . Recent studies have shown that FAM83D was highly expressed in pancreatic cancer, lung cancer, and ovarian cancer and involved in regulating tumor cell proliferation, apoptosis, invasion, and metastasis [50][51][52] . Furthermore, FAM83D could promote tumor cell proliferation by regulating multiple signaling pathways, such as MAPK and Notch [53] , indicating that FAM83D played an important role in the occurrence and development of malignant tumors. There were also reports in the literature that the high expression of FAM83E in pancreatic cancer was related to the shortened survival time [54] . However, relevant experimental evidence is lacking. It has been shown that FAM83F enhanceed its stability by inhibiting the ubiquitination and degradation of p53 [55] . FAM83F could activate the MAPK signaling pathway by interacting with BRAF and RAF in thyroid cancer [10] . Studies have shown that miR-1827 and miR-940 inhibited the malignant progression of lung cancer by targeting FAM83F [56] . The present study revealed for the rst time that the expression levels of FAM83D, FAM83E, and FAM83F in cervical cancer were upregulated and correlated with tumor stages, while the high expression of FAM83D and FAM83F were negatively correlated with the shortened OS in cervical cancer patients, showing that FAM83D, FAM83E, and FAM83F played a potentially important role in the occurrence, development, and prognosis values of cervical cancer, although the speci c functions and mechanisms need to be further explored.
Abnormal expression of FAM83H was initially con rmed to be related to calcium-de cient enamel hypoplasia, FAM83H mutants can destroy the subcellular structure of tooth enamel and related proteins [57] . Recent studies have reported that FAM83H was overexpressed in a variety of malignant tumors, including gastric cancer, osteosarcoma, and renal clear cell carcinoma [11,58−59] , and associated with the shortened survival time, and regulated the malignant biological behaviors. Kim et al. [60] reported that FAM83H was highly expressed in liver cancer cells and correlated with the shortened OS in patients, and could promote the expression of CCND1 and CCNE1 and inhibit the expression of P53, thereby affecting the proliferation and invasion of liver cancer cells. In our study, we found that the expression level of FAM83H mRNA in cervical cancer was increased, while the OS in cervical cancer patients with high expression of FAM83H was shortened and related to poor prognosis. The above results were consistent with the research by Chao et al. [16] . This shows that FAM83H is also signi cantly associated with the occurrence, development, and prognosis values of cervical cancer.
The above research showed that the high expression of FAM83A/B/H in cervical cancer had important value in the occurrence, development, and prognosis evaluation of cervical cancer. In recent years, many studies have shown that FAM83A/B/H can activate MAPK, Wnt, and PI3K/AKT/mTOR signaling pathways in lung and pancreatic cancers [13,30,[34][35][36][37][61][62][63] and regulate tumor occurrence, development, and biological behavior. In order to better clarify the molecular mechanism, biological function, and related signal regulation network of FAM83A/B/H in cervical cancer, GO enrichment analysis on FAM83A/B/H and their closely related genes was performed, which revealed that the above genes were involved in the formation of cell-substrate junction, membrane raft, desmosome, and regulated various biological behaviors, including lipid binding, cadherin, and calcium-dependent protein binding, and regulating skin and epidermis development, in ammatory response and cell junction organization. At the same time, KEGG enrichment analysis results showed that FAM83A/B/H and their closely related genes were signi cantly enriched in cancer pathways, estrogen signaling pathway, MAPK signaling pathway, Rap1 and Hippo signaling pathway. We speculate that FAM83A/B/H can regulate the occurrence, development, and malignant biological behavior of cervical cancer by regulating the above-mentioned tumor-related pathways. This study provides more supporting evidences for FAM83A/B/H and the related signaling pathways and provides clues for the rational development of FAM83A/B/H-mediated multitarget therapy.
The main cause of cervical cancer is HPV infection, a very small number of infected people will develop immune tolerance and tumors, and the immunogenicity of tumor patients may be higher [2] . Therefore, immunotherapy plays a very important role in the pathogenesis and disease progression of cervical cancer. In recent years, the treatment of cervical cancer has not been limited to mature surgical treatment or radiotherapy or chemotherapy. Immunotherapy has also become a new type of treatment for advanced and recurrent cervical cancer, mainly including PD1/PD-L1 immune checkpoint suppression (Pembrolizumab), adoptive T lymphocyte therapy, and therapeutic tumor vaccines [64][65] . Immune in ltration in the tumor has important value in the survival, prognosis, and immunotherapy of patients with cervical cancer. In the present study, we assessed the relationship between FAM83A/B/H and immune in ltration in cervical cancer using the TISIDB database. The results showed that FAM83A/B/H were signi cantly correlated with lymphocytes (neutrophils, Tcm_CD4, and Tcm_CD8), immunoinhibitors (TGFB1, TGFBR1, and CD274), immunostimulators (RAET1E and TNFSF9), and MHCs (TAP1, TAP2, and HLA-E). Studies have shown that the high expression of FAM83A in lung cancer was negatively correlated with the level of B cells and dendritic cell in ltration [66] . In pancreatic cancer, FAM83H was associated with the in ltration of CD8 + T cells, gamma delta T cells, and CD4 + T cells and immunomodulators [62] .
Therefore, the above research shows that FAM83A/B/H has important value in the immunoregulatory response of cervical cancer and is expected to become a new target for improving the prognosis of cervical cancer patients.
However, there exist some limitations in our research. All of the data came from different online databases, which may cause background heterogeneity, therfore, a larger clinical sample size is necessary to further con rm the above research. Furthermore, in vitro and in vivo experiments are necessary to validate the present study and explore the underlying mechanisms.

Conclusion
In summary, with various bioinformatics tools, we determined that the high expression of FAM83A/B/H in cervical cancer were correlated with shortened OS and poor prognosis, indicating that FAM83A/B/H were involved in the occurrence and development of cervical cancer and played an important role in prognosis assessment. The gene regulatory network, tumor-related signaling pathways, and immune in ltration of FAM83A/B/H were also illuminated, which will help to clarify the function and mechanism of FAM83A/B/H in cervical cancer. Therefore, FAM83A/B/H can potentially be identi ed as tumor biomarker for cervical cancer and is valuable in the pathogenesis, early diagnosis, prognostic evaluation, and immunotherapy of cervical cancer. However, the speci c functions and molecular mechanisms of The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.
Ethics approval and consent to participate No ethical approval nor informed consent was required in this study due to the public-availability of the data used.
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