Investigating the Prognostic and Oncogenic Roles of Membrane-Associated Ring-CH-Type Finger 9 in Colorectal Cancer

Backgroundsand Aims. Colorectal cancer (CRC) represents a major global health challenge, necessitating comprehensive investigations into its underlying molecular mechanisms to enhance diagnostic and therapeutic strategies. This study focuses on elucidating the oncogenic role of Membrane-Associated Ring-CH-Type Finger 9 (MARCHF9), a RING-Type E3 ubiquitin transferase, in CRC. We aim to assess MARCHF9's clinical significance, functional impact on CRC progression, and its potential as a prognostic biomarker. Methods. We leveraged data from the Cancer Genome Atlas (TCGA) cohort to evaluate MARCHF9 expression profiles in CRC. In vitro experiments involved siRNA-mediated MARCHF9 knockdown in COAD cell lines (SW480 and LoVo). Cell proliferation and invasion assays were conducted to investigate MARCHF9's functional relevance. Survival analyses were performed to assess its prognostic role. Results. Our analysis revealed significantly elevated MARCHF9 expression in CRC tissues compared to normal colorectal tissues (P < 0.05). High MARCHF9 expression correlated with advanced clinical stages, distant metastases, and the presence of residual tumors in CRC patients. Survival analyses demonstrated that high MARCHF9 expression predicted unfavorable overall and disease-free survival outcomes (P < 0.05). In vitro experiments further supported its oncogenic potential, with MARCHF9 knockdown inhibiting COAD cell proliferation and invasion. Conclusions. This study unveils the oncogenic role of MARCHF9 in CRC, highlighting its clinical relevance as a potential biomarker and therapeutic target. MARCHF9's association with adverse clinicopathological features and its functional impact on cancer cell behavior underscore its significance in CRC progression. Further research is essential to elucidate precise mechanisms by which MARCHF9 enhances tumorigenesis and to explore its therapeutic potential in CRC management.


Introduction
Colorectal cancer (CRC) is a major global health concern and a leading cause of cancer-related deaths, accounting for over 1.8 million new cases and approximately 880,000 deaths worldwide in 2018 alone, according to the World Cancer Research Fund International [1].Tis malignancy is characterized by the uncontrolled growth of cells in the colon or rectum, with a multifaceted etiology that includes genetic, environmental, and lifestyle factors [2].Despite signifcant advances in the understanding of CRC pathogenesis and treatment modalities, there remains a pressing need to unravel the intricate molecular mechanisms underlying this disease [3].
In recent years, the E3 ubiquitin ligases have emerged as critical regulators of cancer development and progression, ofering new insights into potential therapeutic targets [4].Tese enzymes, which mediate the attachment of ubiquitin moieties to target proteins, play a pivotal role in the regulation of protein stability, intracellular trafcking, and signaling pathways [5].MARCHF9 (Membrane-Associated Ring-CH-Type Finger 9), a RING-Type E3 ubiquitin transferase, has gained attention due to its implications in diverse physiological processes, including immune regulation, cellular signaling, and protein turnover [6][7][8].While our understanding of MARCHF9's functions in normal physiology has expanded, its role in carcinogenesis, particularly in colorectal cancer, remains poorly elucidated.
Te purpose of this study is to investigate the oncogenic role of MARCHF9 in colorectal cancer.Understanding the molecular mechanisms by which MARCHF9 may contribute to CRC development and progression holds signifcant promise for the development of novel diagnostic markers and targeted therapies.In this paper, we provide a comprehensive overview of colorectal cancer and highlight the signifcance of MARCHF9 in cellular processes.Trough this investigation, we hope to contribute valuable insights to the feld of cancer biology and potentially pave the way for more efective treatment strategies for colorectal cancer patients.

Online Dataset.
Te online dataset for this study was obtained from the Cancer Genome Atlas (TCGA), a comprehensive resource that provides genomic and clinical data for various cancer types, including colorectal adenocarcinoma (COAD) and rectum adenocarcinoma (READ).We also enrolled data from the Gene Expression Omnibus (GEO) datasets.

Western Blotting.
After siRNA transfection, total protein was extracted from COAD cells using RIPA lysis bufer, supplemented with protease and phosphatase inhibitors.Ten protein concentrations were determined using a BCA protein assay kit (Pearson).Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difuoride (PVDF) membranes, and blocked with 5% nonfat milk in Tris-bufered saline with Tween 20 (TBST).
Membranes were probed with primary antibodies against MARCHF9 (PA5-103817; Termo Fisher Scientifc, PA, USA) and GAPDH (loading control) and subsequently incubated with appropriate secondary antibodies.Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system, and images were captured and semiquantifed [11].

Matrigel-Transwell Assay.
To assess cell invasion, transfected COAD cells were subjected to Matrigel-Transwell assays [13].Cells were seeded in the upper chamber of Transwell inserts coated with Matrigel (356234, Corning, NY, USA).Te lower chamber contained culture medium with 10% FBS as a chemoattractant.After incubation, non-invading cells were removed from the upper surface of the membrane, while invading cells on the lower surface were fxed, stained, and quantifed using microscopy.

Survival Analyses and Statistics.
Survival analyses were conducted to assess the prognostic role of MARCHF9 in COAD patients.Kaplan-Meier survival curves were established for overall survival and disease-specifc survival.Logrank test was applied to evaluate signifcance of survival diferences.Cox hazard regression test was conducted to identify independent risk factors.Data from in vitro experiments were analyzed using Student's t-test.Te significance level was set at P < 0.05.Statistical analyses were performed using R software, SPSS 24.0 software, and GraphPad Prism 7.0 Software.

Ethics.
Tis study involving human cell lines did not require ethical approval as it does not involve human subjects or patient data.Cell lines used in this study were obtained from established cell repository (ATCC) and maintained according to standard laboratory protocols.All experiments were conducted in compliance with relevant ethical guidelines and biosafety regulations.

Expression Diference of MARCHF9 in Colorectal Cancers and Pan-Cancers.
To gain insights into the potential role of MARCHF9 in CRC and various other cancer types, we conducted a comprehensive analysis of MARCHF9 expression using data from the Cancer Genome Atlas (TCGA) cohort.Initially, we observed signifcant variations in MARCHF9 mRNA levels in pan-cancer tissues compared to normal tissues.Tis analysis, performed using an unpaired To further refne our analysis, we performed a paired analysis to compare MARCHF9 expression in cancer tissues with their corresponding paired normal tissues.Across pancancer samples, this analysis revealed a consistent upregulation of MARCHF9 expression in cancer tissues, emphasizing its potential as a candidate oncogene (Figure 1(c)).Similarly, when focusing on COAD and READ samples, the paired analysis highlighted a signifcant increase in MARCHF9 expression in cancer tissues relative to paired normal colorectal tissues (Figure 1(d)).Tese fndings, determined using a paired Student's t-test, reinforce the notion that MARCHF9 may play a pivotal role in the development and progression of colorectal cancer.

Clinical Characteristics of Colorectal Cancer Patients
Stratifed by MARCHF9 Expression.Our study next delved into the clinical implications of MARCHF9 expression in CRC by stratifying patients into low and high MARCHF9 expression groups (Table 1).While gender and age distributions were consistent between the two groups, we observed intriguing associations with several clinicopathological features.
For example, high MARCHF9 expression was signifcantly associated with advanced disease characteristics.Patients with high MARCHF9 expression exhibited a higher prevalence of distant metastases (pathologic M stage) compared to those with low MARCHF9 expression (P � 0.007).Furthermore, elevated MARCHF9 expression correlated with advanced pathologic stages, with a signifcantly larger proportion of patients diagnosed at Stage IV in the high MARCHF9 expression group compared to the low expression group (P � 0.034).Additionally, our analysis unveiled a signifcant association between elevated MARCHF9 expression and the presence of residual tumors (R1-R2 resection) (P � 0.010).Tis fnding indicates a potential link between high MARCHF9 expression and more aggressive disease phenotypes, underscoring its clinical relevance in CRC, particularly in advanced stages.
Te overall survival analysis of COAD and READ cases from the TCGA cohort revealed intriguing fndings.Patients with high MARCHF9 expression displayed signifcantly poorer overall survival compared to those with low MARCHF9 expression (Figure 2(a), P < 0.05).To further dissect the impact of MARCHF9 on patient outcomes, we assessed disease-specifc survival.Similar to overall survival, high MARCHF9 expression was associated with a signifcantly worse disease-specifc survival outcome for COAD and READ cases in the TCGA cohort (Figure 2(b), P < 0.05), strengthening the evidence for its prognostic signifcance in CRC.Expanding our analysis to a mixed cohort that included both TCGA and GEO data, we evaluated the overall survival of COAD cases (Figure 2(c)) and READ cases (Figure 2(d)) [15].Tese analyses further supported the notion that elevated MARCHF9 expression is associated with diminished overall survival in both COAD and READ patients, reinforcing its potential as a prognostic marker for CRC across diferent datasets (P < 0.05).
To assess the prognostic signifcance of MARCHF9 expression and other clinicopathological factors in CRC, we conducted a comprehensive disease-specifc survival analysis (Table 2).In our univariate analysis, gender did not demonstrate a signifcant association with disease-specifc survival.Both female and male patients exhibited comparable hazard ratios, with no statistically signifcant diferences observed (P � 0.412).Age, another potential prognostic factor, also did not show a signifcant impact on disease-specifc survival in the univariate analysis (P � 0.137).Patients aged 65 years or older displayed a hazard ratio of 1.421 (95% CI: 0.894-2.257)compared to younger patients.
Crucially, MARCHF9 expression emerged as a robust and independent prognostic factor in both univariate and multivariate analyses.High MARCHF9 expression was associated with a signifcantly increased hazard ratio for Genetics Research disease-specifc survival (HR � 2.077, 95% CI: 1.302-3.312,P � 0.002) in the univariate analysis, and this signifcance persisted in the multivariate analysis (HR � 2.510, 95% CI: 1.171-5.382,P � 0.018).Tese fndings underline the prognostic relevance of MARCHF9 in CRC, suggesting its potential as a valuable biomarker for predicting diseasespecifc survival outcomes in CRC patients.

Subgroup Overall Survival Analyses of COAD Cases according to MARCHF9 Expression.
To gain a more nuanced understanding of the prognostic role of MARCHF9 expression in COAD, we conducted subgroup survival analyses (Figure 3).Tese analyses provide insights into how MARCHF9 expression impacts overall survival within specifc subgroups of COAD patients.
For example, we explored the infuence of MARCHF9 expression on survival within gender subgroups.Interestingly, female patients displayed no statistical survival diference between the high and low MARCHF9 expression subgroups, suggesting that MARCHF9 may not signifcantly afect the overall survival of female COAD patients (Figure 3(a), P � 0.35).In contrast, male patients exhibited a notable diference in survival.Specifcally, male patients in the high-MARCHF9 group showed signifcantly poorer survival outcomes compared to those in the low-MARCHF9 group (Figure 3(b), P � 0.025).Tis gender-specifc divergence underscores the potential importance of considering gender-related diferences in the prognostic impact of MARCHF9 expression in COAD.
Further analyses delve into the relationship between MARCHF9 expression and survival in COAD cases stratifed by microsatellite instability (MSI) status.Intriguingly, higher MARCHF9 expression predicted worse survival in COAD patients with high MSI, suggesting that MARCHF9 may have a detrimental impact on prognosis in this specifc molecular subgroup (Figure 3 Moreover, we assessed the interaction between MARCHF9 expression and postoperative adjuvant chemotherapy in COAD patients.In those who received postoperative adjuvant chemotherapy, higher MARCHF9 expression was correlated with worse prognosis, suggesting that MARCHF9 may negatively impact survival in the context of chemotherapy (Figure 3(e), P � 0.009).Conversely, in COAD patients who did not receive postoperative adjuvant chemotherapy, MARCHF9 expression showed no statistical signifcance in predicting survival outcomes (Figure 3(f), P � 0.13).Tese fndings emphasize the potential relevance of MARCHF9 as a predictive biomarker for treatment response and highlight the need to consider treatment modalities when assessing its prognostic role in COAD.
In summary, these subgroup survival analyses provide valuable insights into the multifaceted prognostic role of MARCHF9 in COAD.Te gender-specifc diferences, MSIdependent efects, and interactions with adjuvant chemotherapy underscore the complexity of MARCHF9's impact on survival outcomes in COAD patients, highlighting the need for personalized approaches in prognostic assessment and therapeutic decision making.

Inhibition of COAD Cell Proliferation and Invasion through MARCHF9 Silencing.
To explore the functional implications of MARCHF9 in COAD, we next conducted in vitro experiments aimed at silencing MARCHF9 expression in SW480 and LoVo cell lines.Representative western blotting images demonstrate the successful knockdown of MARCHF9 in both SW480 and LoVo cell lines.Tis knockdown validation underscores the efectiveness of our experimental approach in modulating MARCHF9 expression (Figure 4(a)).
Next, we assessed the impact of MARCHF9 knockdown on COAD cell proliferation, revealing that MARCHF9 knockdown signifcantly reduced the growth rate of COAD cells compared to control cells (Figures 4(b) and 4(c)).Tese fndings indicate that MARCHF9 plays a crucial role in Genetics Research promoting the proliferation of COAD cells, highlighting its potential as a key regulator of tumor growth.Furthermore, we investigated the infuence of MARCHF9 knockdown on COAD cell invasion using Matrigel-Transwell assays.Accordingly, MARCHF9 knockdown led to a notable suppression of COAD cell invasion compared to control cells.Genetics Research

Genetics Research
Tis suggests that MARCHF9 may contribute to the invasive properties of COAD cells, further emphasizing its potential as a critical factor in cancer progression (Figures 4(d) and 4(e)).
Terefore, our in vitro experiments demonstrate that silencing MARCHF9 can efectively inhibit the proliferation and invasion of COAD cells.Tese fndings provide valuable mechanistic insights into the oncogenic role of MARCHF9 in COAD and highlight its potential as a promising therapeutic target for colorectal adenocarcinoma.

Discussion
Colorectal cancer (CRC) remains a signifcant global health burden, necessitating comprehensive investigations into the molecular mechanisms that drive its pathogenesis [16].Te present study focused on the oncogenic role of Membrane-Associated Ring-CH-Type Finger 9 (MARCHF9), a RING-Type E3 ubiquitin transferase, in CRC.Our fndings shed light on the clinical signifcance of MARCHF9, its functional impact on CRC progression, and its potential as a prognostic biomarker.
Our analysis of data from the Cancer Genome Atlas (TCGA) cohort revealed a substantial increase in MARCHF9 expression in CRC tissues compared to normal colorectal tissues.Tis upregulation aligns with fndings in other cancer types, suggesting that MARCHF9 may serve as an oncogenic factor in multiple malignancies.Interestingly, our study demonstrated that high MARCHF9 expression was associated with advanced clinical stages, distant metastases, and the presence of residual tumors in CRC patients.Tese clinicopathological associations suggest that MARCHF9 might play a pivotal role in promoting tumor progression and metastasis.Te association between MARCHF9 expression and advanced clinical stages is particularly intriguing.Tis fnding echoes prior studies that have implicated MARCHF9 in cancer progression.Survival analyses in our study demonstrated that high MARCHF9 expression was signifcantly associated with unfavorable overall and disease-specifc survival outcomes in CRC patients.Tis observation underscores MARCHF9's potential as a prognostic biomarker in CRC, aligning with emerging evidence from other cancer types.Notably, MARCHF9 has been implicated in the prognosis of lung adenocarcinoma, where it suppresses tumor progression by downregulating ICAM-1 [17].Low MARCHF9 expression has been linked to poor prognosis and adverse clinicopathological characteristics of lung adenocarcinoma.Te distinct pattern of MARCHF9's association with poor survival outcomes across various cancers suggests its broad applicability as a prognostic marker.Of note, in our multivariate analysis, the signifcance of the pathological stage (TNM) was compromised, which may be due to collinearity with other variables.Te multivariate model included several interrelated factors that could impact the results, causing the pathological stage to lose its statistical signifcance (P � 0.997), emphasizing the need for cautious interpretation of these fndings.
In vitro experiments in our study revealed that siRNAmediated knockdown of MARCHF9 in COAD cell lines (SW480 and LoVo) led to a signifcant reduction in cell proliferation and invasion.Tese fndings highlight the functional relevance of MARCHF9 in promoting CRC progression.While the exact mechanisms underlying MARCHF9's oncogenic efects in CRC warrant further investigation, it is conceivable that MARCHF9 may impact key pathways involved in cell proliferation, invasion, and metastasis.Te role of MARCHF9 in promoting cancer cell proliferation has been documented in other malignancies.In glioblastoma, MARCHF9 has been implicated in tumor immune microenvironment [18].Our fndings align with these reports and suggest that MARCHF9's pro-proliferative efects may extend to CRC.Moreover, our study demonstrated that MARCHF9 knockdown signifcantly inhibited the invasion of COAD cells.Te inhibition of COAD cell invasion upon MARCHF9 knockdown in our study suggests that MARCHF9 may similarly infuence the invasive behavior of CRC cells.
However, it is essential to acknowledge diferences in MARCHF9's role across cancer types.For instance, in lung adenocarcinoma, MARCHF9 overexpression has been correlated with favorable clinicopathological characteristics and can inhibit tumor invasion while showing little efect on cell proliferation [17].In glioblastoma, MARCHF9 has been reported to be involved in suppressive immune microenvironments [18].In summary, while MARCHF9's clinical signifcance is widely recognized, its functional roles and mechanisms of action may vary depending on the specifc cancer type.
Te clinical signifcance of MARCHF9 in CRC, as highlighted in our study, presents opportunities for its translation into clinical practice.MARCHF9's association with advanced disease stages, distant metastases, and poor prognosis suggests its potential utility as a prognostic biomarker.Patients with high MARCHF9 expression may beneft from more intensive monitoring and personalized treatment strategies.Moreover, our functional fndings suggest that MARCHF9 may serve as a promising therapeutic target in CRC.Strategies aimed at inhibiting MARCHF9 expression or activity could be explored to impede cancer cell proliferation and invasion.Given the heterogeneity of CRC, patient stratifcation based on MARCHF9 expression levels may help identify individuals who are most likely to respond to targeted therapies.
While our analysis was based on substantial datasets from TCGA and GEO, retrospective analyses inherently have limitations related to data quality and potential confounders.Further prospective studies and functional experiments are warranted to validate our fndings and elucidate the exact mechanisms underlying MARCHF9's oncogenic efects in CRC.In addition, future research endeavors should delve into elucidating the precise molecular mechanisms by which MARCHF9 infuences CRC progression.Investigating its downstream targets and interacting partners could provide valuable insights into potential therapeutic interventions.Additionally, the impact of MARCHF9 in preclinical models and its evaluation as a therapeutic target in clinical trials should be explored to assess its translational potential.

. Conclusions
In conclusion, our study illuminates the oncogenic role of MARCHF9 in colorectal cancer.High MARCHF9 expression is associated with advanced disease stages, metastasis, and adverse prognosis, while in vitro experiments demonstrate its functional impact on cancer cell proliferation and invasion.Tese fndings underscore MARCHF9's clinical relevance as a potential prognostic biomarker and therapeutic target in CRC.

Figure 1 :
Figure 1: Expression diference of MARCHF9 in colorectal cancers and pan-cancers.(a) Diferential mRNA levels of MARCHF9 between pan-cancer tissues and corresponding normal tissues.Analysis conducted using unpaired Student's t-test based on the Cancer Genome Atlas (TCGA) dataset.(b) Distinct mRNA levels of MARCHF9 in COAD and READ compared to normal colorectal tissues.Analysis performed using unpaired Student's t-test based on TCGA dataset.(c) Variations in mRNA levels of MARCHF9 in pan-cancers compared to their respective paired normal tissues.Analysis conducted using paired Student's t-test based on TCGA dataset.(d) Diferential mRNA levels of MARCHF9 in COAD and READ relative to their paired normal colorectal tissues.Analysis performed using paired Student's t-test based on TCGA dataset.* P < 0.05.

Figure 2 :
Figure 2: Kaplan-Meier survival curves to assess the prognostic role of MARCHF9.(a) Overall survival (OS) analysis and hazard ratio (HR) of COAD and READ cases in the TCGA cohort according to MARCHF9 expression levels.(b) Disease-specifc survival (DSS) analysis and hazard ratio (HR) of COAD and READ cases in the TCGA cohort according to MARCHF9 expression levels.(c) Overall survival analysis and hazard ratio (HR) of COAD cases in a combined cohort of TCGA and GEO datasets, stratifed by MARCHF9 expression levels.(d) Overall survival analysis and hazard ratio (HR) of READ cases in a combined cohort of TCGA and GEO datasets, stratifed by MARCHF9 expression levels.

Table 1 :
Clinical characteristics of colorectal cancer patients stratifed by MARCHF9 expression.

Table 2 :
Disease-specifc survival of colorectal cancer patients.