SSR4 promote gastric cancer progression by regulating mitochondrial oxidative phosphorylation via NDUFB11 and ATP6AP1

SSR4 promote gastric cancer progression by regulating mitochondrial oxidative phosphorylation via NDUFB11 and ATP6AP1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SSR4 promote gastric cancer progression by regulating mitochondrial oxidative phosphorylation via NDUFB11 and ATP6AP1 Lei Liu, Aoshuang Li, Baixue Liao, Kaiwen Wu, Ruiling Fan, Binjun Zhu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4890970/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Gastric cancer (GC) is one of the most common cancer worldwide. Cancer cell metastasis is a major factor leading to poor prognosis. Previous proteomic data suggested that SSR4 might be closely associated with the occurrence and development of GC. However, the role and molecular mechanism of SSR4 in GC is not yet clear. The present study found that the expression level of SSR4 was increased in GC tissue and serum from GC patients. In addition, SSR4 could promote the malignant biological behavior of GC cells in vitro and in vivo. The mechanism may be that SSR4 regulates the expression of NDUFB11 and ATP6AP1, and then enhanced the function of mitochondrial respiratory chain complex I (CI) and mitochondrial respiratory chain complex V (CV), which promoted the mitochondrial oxidative phosphorylation and thus promoted GC progression. These findings expand the understanding of the role of SSR4 and provide a new target for the treatment of GC. Biological sciences/Genetics/Cancer genetics Biological sciences/Cell biology/Mechanisms of disease SSR4 gastric cancer oxidative phosphorylation NDUFB11 ATP6AP1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Gastric cancer (GC) is a significant health issue, ranking fifth for incidence and fourth for mortality globally [ 1 ] . The incidence is highly correlated with regions, with the highest age-standardized incidence rates observed in East Asia [ 1 ] . The 5-year overall survival (OS) rate for patients with early detection and treatment of GC is usually higher (> 60%). However, for patients with local and distant metastasis, the 5-year OS rates decrease to 30% and 5%, respectively [ 2 ] . Unfortunately, the early clinical symptoms of GC were characterized by stealthiness and atypicality, with over 60% of patients already having local or distant metastases at the time of diagnosis [ 3 ] . The invasive and metastatic capabilities of GC cells are crucial factors contributing to mortality. In the case of early-stage GC, surgical resection is a viable option, whereas advanced-stage GC often necessitates a combination of surgery and chemotherapy. In severe instances, certain patients may opt for chemotherapy or biotherapy as their primary treatment modalities [ 4 – 6 ] . Therefore, exploring the process of GC cell metastasis can assist doctors in better understanding the developmental patterns of GC, preventing further spread of cancer cells, and ultimately improving the 5-year OS rate for patients. Currently, there is limited research on the gene SSR4, and studies regarding its association with cancer are also scarce. The translocation-associated protein complex (TRAP), also called signal sequence receptor (SSR), includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2, TRAPγ/SSR3 and TRAPδ/SSR4 [ 7 , 8 ] . SSR4, together with three other subunits, maintains the stability of the TRAP complex and plays a biological role [ 9 ] . Research has indicated that TRAP regulates N-glycosylation, while N-glycosylation primarily occurs in the rough endoplasmic reticulum. Additionally, the study suggested that SSR4 possessed chaperone-like properties, which could assist in N-glycosylation during endoplasmic reticulum stress [ 10 ] . The TRAP complex facilitates the translocation of proteins across the endoplasmic reticulum membrane and associates with the oligosaccharyl transferase (OST) complex to maintain proper glycosylation of nascent polypeptides [ 11 ] . Therefore, SSR4 was also associated with this congenital disorder of glycosylation. Through biochemical studies of fibroblasts in congenital disorders of glycosylation (CDG), it was found that the stability of TRAP complex was disrupted, SSR3 protein was completely lost, and SSR1 and SSR4 were partially lost [ 11 ] . SSR4 exerted a certain influence on insulin synthesis, deficient protein expression of SSR4, diminishes the protein levels of other TRAP subunits, concomitant with deficient steady-state levels of proinsulin and insulin [ 12 ] . There was a report showing that SSR4 was identified as significantly up-regulated in inflamed areas of the gingival connective tissue compared to non-inflamed tissue [ 13 ] . Cell metabolism could utilize carbohydrates (such as glucose via glycolysis, and pyruvate oxidation), fats (such as fatty acid β-oxidation), and proteins (such as amino acid oxidation and transamination) to synthesize the required energy. Molecules derived from these catabolic processes could enter the tricarboxylic acid cycle and serve as substrates for oxidative phosphorylation (OXPHOS), generating ATP. Under aerobic conditions, the primary source of energy for normal cells was the transfer of metabolites through the respiratory complexes (CI-CIV) on the inner mitochondrial membrane, ultimately synthesizing ATP via ATP synthase [ 14 ] . At present, there is limited research on the relationship between SSR4 and cancer. A study showed that SSR4 was a prognostic biomarker associated with immune cell infiltration in colon adenocarcinoma (COAD), and COAD patients with high SSR4 expression in tumor-infiltrating lymphocytes (TILs) had better overall survival [ 15 ] . In previous studies, increased expression of SSR4 has been observed in some cancers, such as colorectal cancer [ 15 ] and lung cancer [ 16 ] . However, there is currently no basic research exploring the relationship between SSR4 and GC. Results The expression level of SSR4 was increased in GC patients First, the expression of SSR4 mRNA in normal gastric tissues (n = 211) and GC tissues (n = 408) was analyzed using the GEPIA database. The results revealed that the mRNA expression level of SSR4 in GC tissues was increased than that in normal tissues (Fig. 1 A). Then, the concentration of SSR4 protein was detected in serum level. The specimens were collected from 66 Healthy volunteers and 86 GC patients. ELISA was performed to detect the concentration of SSR4 protein in serum. The data showed that the concentration of SSR4 in the serum of GC patients was higher than that in the normal group (Fig. 1 B). For the tissue microarray analysis, there were 96 cases of GC tissue remained after excluding the tissues with deletions. Due to the lack of patients classified as Grade I in this tissue array, we analyzed tissues from Grade II and Grade III patients. The results showed that SSR4 protein expression in Grade III was higher than that in Grade II (Fig. 1 C). Chi-square test and continuity-corrected Chi-square test showed that the expression level of SSR4 protein was significantly different between Grade II and Grade III group (p-value = 0.003). However, there were no significant differences among other clinical characteristics (Table 1 ). These results indicated that the expression level of SSR4 was increased in GC tissue and serum of GC patients, and SSR4 may be related to the differentiation degree of GC. Table 1 Correlation between the expression level of SSR4 and clinicopathological characteristics in gastric cancer Category n Evaluate Results p Positive Negative Gender 0.066 Male 75 52 23 Female 21 10 11 Age 0.200 ≤ 64 48 28 20 >64 48 34 14 Tumor size(cm) 0.420 ≤ 5 54 33 21 >5 42 29 13 Grades 0.001 ** Ⅱ 27 24 3 Ⅲ 69 37 32 Tumor growth status 0.437 T1-T2 10 8 2 T3-T4 84 52 32 Lymph node metastases 0.696 0 19 13 6 ≥ 1 77 49 28 Metastasis 0.948 M0 83 53 30 M1 13 9 4 Stages 0.880 1–2 32 21 11 3–4 64 41 23 SSR4 promoted the malignant biological behavior of GC cells in vitro First, the expression level of SSR4 was detected in human gastric mucosal epithelial cells GES-1 and five GC cell lines by qPCR (Fig. 2 A) and western blotting (Fig. 2 B). The expression level of SSR4 in these five GC cell lines was higher than that in normal gastric mucosal cells. SGC7901 and BGC823 cells were transfected with siRNA to interfere the expression of SSR4,, and siRNA-2 with higher interference efficiency was selected for subsequently study (Fig. 2 C). The CCK-8 cell proliferation assay continuously measured and recorded data for 5 days. The results showed that the proliferation rate of GC cells was decreased after SSR4 knockdown (Fig. 2 D). Apoptosis assay revealed an increased proportion of apoptotic cells in the siSSR4 group (Fig. 2 E), indicating that the decreased expression of SSR4 could promote apoptosis of GC cells. Cell cycle assay showed an increased proportion of G1 phase and a decreased proportion of S phase in the siSSR4 group, with no significant difference in the proportion in G2 phase (Fig. 2 F). This result indicates that when the expression level of SSR4 is reduced, cell division is arrested in the G1 phase.Subsequently, colony formation assays were conducted, and the results indicated that GC cell proliferation was inhibited in the siSSR4 group (Fig. 2 G). To evaluate the effect of SSR4 on cell migration ability, the transwell assays was performed. The result revealed that the migrated GC cells were reduced in siSSR4 group (Fig. 2 H). The wound healing assay showed that within the same time frame, there was a larger wound area in the siSSR4 group, which indicated that inhibit of SSR4 could reduce the migration ability in GC cells (Fig. 2 I-J). Thus, these results indicate that inhibition of SSR4 could reduce proliferation and migration abilities in GC cells. The effects of SSR4 on mitochondrial oxidative phosphorylation To predict the function of SSR4 in GC, SSR4 involved signal pathway was analyzed firstly. The signal pathway enrichment analysis was performed both in WiKi pathway database and in KEGG pathway database. The results revealed that there was a high correlation between SSR4 and the oxidative phosphorylation (OXPHOS) pathway (Fig. 3 A, B). The top ten gene related to OXPHOS pathway were listed, which include NDUFB11, NDUFA1, COX6B1, ATP6AP1, NDUFA2, etc (Fig. 3 C). The heatmap analysis was conducted to analyze the gene with high correlation to SSR4 in GC of TCGA database. Consistently, the top ten gene related to OXPHOS pathway were all included in the top 50 gene which positively correlated with SSR4 (Fig. 3 D). These results indicated that SSR4 may closely related to OXPHOS pathway in GC. Further, the expression level of these key gene in OXPHOS pathway was detected after knockdown of SSR4, and the expression level of NDUFB11 and ATP6AP1 was significantly reduced, which were selected for subsequent study. NDUFB11 is a crucial subunit of mitochondrial Complex I, associated with OXPHOS mechanism [ 17 ] . ATP6AP1, as an auxiliary subunit of the H + -ATPase complex, participates in H + transportation [ 18 ] . Both of NDUFB11 and ATP6AP1 were play important role in mitochondrial oxidative phosphorylation. We measured the ATP content in GC cells, as ATP is a product of mitochondrial OXPHOS. The results revealed that inhibition the expression of SSR4 led to a decrease of ATP content in GC cells (Fig. 4 A). Within living cells, ROS is primarily originated from mitochondria [ 19 ] and participated in regulating cellular processes such as apoptosis [ 20 ] , autophagy [ 21 ] , stem cell differentiation [ 22 ] , cellular [ 23 ] , and tissue inflammation [ 24 ] . With normal physiological conditions, ROS emission (production minus clearance) accounts for 2% of the total oxygen consumed by mitochondria [ 25 ] . Some studies have shown that the production of ROS in mitochondria fluctuates between 0.25% and 11% of total oxygen consumption, depending on the animal species and respiratory rate [ 26 ] . Both excessively high and low levels of ROS may have detrimental effects on cells. When ROS levels too low, cells cannot participate in appropriate cellular metabolism through the regulation of numerous biochemical reactions [ 27 ] . Conversely, when ROS levels are too high, cellular signal regulation becomes uncontrolled or disrupted [ 27 ] . Consequently, cancer cells accumulate high levels of ROS to promote their metabolic activities and growth [ 28 ] . We also assessed the levels of ROS in the control and siSSR4 cells, and the results showed a significant decrease in ROS levels in the siSSR4 group (Fig. 4 B). Mitochondrial membrane potential serves as a valuable indicator of cell health and functional status [ 29 ] . When the membrane potential in mitochondria is high, JC-1 exists in polymeric form, emitting red fluorescence at 595 nm; whereas, when the membrane potential is low, JC-1 exists in monomeric form, emitting green fluorescence at 530 nm [ 30 ] . The presence of red fluorescence indicates higher mitochondrial activity and stronger OXPHOS, whereas an increase in green fluorescence suggests decreased mitochondrial activity and weaker OXPHOS, which may lead to increased apoptosis [ 30 , 31 ] . The experimental results showed increased green fluorescence and decreased red fluorescence after SSR4 knockdown (Fig. 4 C). This indicates that decreased SSR4 expression results in reduced mitochondrial membrane potential, decreased mitochondrial activity, and inhibition of OXPHOS. Isocitrate dehydrogenases (IDH) are homodimers involved in various cellular metabolic processes. Both IDH2 and IDH1 catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate, converting NADP to NADPH + in the process. IDH1 is localized in the cytoplasm and peroxisomes, while IDH2 is localized in the mitochondria, serving as a crucial enzyme in the tricarboxylic acid cycle [ 32 ] . Therefore, IDH2 can be regarded as a marker enzyme for mitochondria, used to observe the status and quantity of mitochondria and understand mitochondrial biogenesis [ 33 ] . The experimental findings revealed a decrease in IDH2 expression when SSR4 expression was reduced (Fig. 4 D, E). In summary, inhibition of SSR4 could reduce the production of mitochondrial ATP and ROS, decrease mitochondrial membrane potential, restrain mitochondrial biogenesis, and ultimately inhibit mitochondrial OXPHOS. SSR4 affects mitochondrial OXPHOS through NDUFB11 and ATP6AP1 After SSR4 knockdown, western blotting revealed a decrease in the protein expression of NDUFB11 and ATP6AP1 (Fig. 5 A). Cell immunofluorescence showed that the fluorescence intensity of NDUFB11 and ATP6AP1 was decreased when SSR4 expression was reduced (Fig. 5 B, C). The result suggested that NDUFB11 and ATP6AP1 are the key molecules in the SSR4-mediated regulation of mitochondrial OXPHOS in GC cells. SSR4 regulate GC cells proliferation through NDUFB11 and ATP6AP1 in vivo To obtain cell lines with stable knockdown expression of SSR4, BGC823 cells were transfect with shSSR4 lentiviral. Western blotting was used to validate the knockdown effect, and cell lines efficient knockdown were selected for nude mouse subcutaneous tumor experiments (Fig. 6 A). Each BALB/c nude mouse was injected with the corresponding cell suspension on the outer side of the hind legs, with the left side injected with control cells and the right side injected with shSSR4 cells. After subcutaneous tumor formation, tumor lengths (L) and widths (W) were measured every 3 days, and tumor volume was calculated according to the formula 0.5 × L×W 2 . Tumor growth curves were plotted (Fig. 6 B). The curves showed that the tumor growth rate in the shSSR4 group was significantly slower than that in the control group. After 4 weeks, tumors were collected, photographed, and weighed. Analysis of tumor showed that the tumor volume and weight in the control group were greater than those in the shSSR4 group (Fig. 6 C). To explore whether SSR4 affects mitochondrial biogenesis in vivo, tumor tissues were labeled with IDH2. The fluorescence intensity of IDH2 in the shSSR4 group was decreased by comparing with the control group (Fig. 6 D), which indicated that SSR4 knockdown inhibited mitochondrial biogenesis in vivo. The result of immunofluorescence experiments on tumor tissues showed that the expression level of NDUFB11 and ATP6AP1 were also decreased in shSSR4 group (Fig. 6 E, F). These results indicated that inhibition of SSR4 could attenuate GC cells proliferation in vivo, which may through regulating NDUFB11 and ATP6AP1 expression. SSR4 regulate GC cells metastasis in vivo To explore the effect of SSR4 on the metastatic ability of GC cells in vivo, control and shSSR4 cells were intravenously injected into the tail vein (n = 6). The body weight of the nude mice was measured every 3 days, and the data were recorded to obtain the weight change curves. The curves showed no significant difference in body weight between the shSSR4 group and the control group (Fig. 7 A). Four weeks later, lung tissue was collected for weighing and photographing. It was observed that there were more visible metastatic nodules on the lung surface in the control group, accompanied by a reduction in lung volume and changes in lung morphology, while the lungs of the shSSR4 group appeared smoother (Fig. 7 B). The HE staining of lung tissues showed fewer metastatic nodules in the shSSR4 group by compared with control group (Fig. 7 C). Liver tissues were photographed and the weights were recorded and analyzed. However, the results showed no visible metastatic nodules on the liver surface in both the control and shSSR4 groups, with no statistical difference in liver weight (Fig. 7 D). The HE staining also revealed no apparent metastatic nodules in the liver (Fig. 7 E). These results suggested that inhibition of SSR4 could attenuate the metastasis ability of GC cells in vivo. Discussion Due to the high mortality and poor prognosis of GC patients, early detection and timely treatment could improve patient survival rates and quality of life [ 34 ] . Therefore, we attempted to screen for a molecule highly correlated with GC and explore its role in the occurrence and development of GC, as well as provide a new target for the treatment of GC. SSR4, as one of the components of the signal recognition particle (SRP) complex, participates in the protein translocation process of the endoplasmic reticulum [ 35 ] . There has been very little research on SSR4 and cancer both domestically and internationally. In previous studies, increased expression of SSR4 has been observed in some cancers, such as colorectal cancer [ 15 ] and lung cancer [ 16 ] . SSR4 serves as a prognostic biomarker and is associated with immune cell infiltration in colon adenocarcinoma [ 15 ] . According to the Warburg effect, it is known that under aerobic conditions, tumor tissues have a capacity to metabolize glucose into lactate that is ten times higher than normal tissues, demonstrating that the growth metabolism of cancer cells tends to favor aerobic glycolysis [ 36 ] . The proposition of the Warburg effect has led some researchers to mistakenly believe that the increased aerobic glycolysis in cancer cells is related to a decrease in mitochondrial OXPHOS (or respiratory impairment). Studies have shown that hypoxic tumor cells excrete lactate, which is subsequently recycled into pyruvate by stroma or tumor cells in aerobic environments for OXPHOS [ 37 , 38 ] . In fact, many cancer cells exhibit both the retention of OXPHOS and the Warburg effect, which collectively promote cancer cell proliferation and metabolism, as observed in urothelial bladder carcinoma [ 39 ] , melanoma [ 40 ] , breast cancer [ 41 ] , hepatocellular carcinoma [ 42 ] , etc. Furthermore, some studies indicate an upregulation of OXPHOS in certain cancers, including lymphoma, leukemia, pancreatic ductal adenocarcinoma, endometrial carcinoma, etc. [ 43 , 44 ] . In this study, we firstly reported the overexpression of SSR4 in GC tissue and serum. Subsequently, downregulation of SSR4 expression revealed its promotive role in the malignant biological behavior of GC, including the promotion of GC cell proliferation and migration ability. To further elucidate the mechanism of SSR4 action, we utilized different databases for analysis and determined that the pathway of SSR4 action is mitochondrial OXPHOS, with key molecules being NDUFB11 and ATP6AP1. Subsequent experiments validated that SSR4 can promote the production of ATP and ROS, stabilize mitochondrial membrane potential, thereby facilitating the process of OXPHOS in GC cells. It also promoted mitochondrial biogenesis. Furthermore, we further confirmed the effect of SSR4 on the OXPHOS pathway proteins NDUFB11 and ATP6AP1, showing that SSR4 can promote the expression of these two proteins. NDUFB11 is a protein subunit of mitochondrial Complex I, also known as NADH dehydrogenase (ubiquinone) subunit B11. It plays a crucial role in mitochondrial energy production, particularly in OXPHOS and electron transfer processes [ 45 , 46 ] . Impairment of NDUFB11 expression can lead to decreased activity of Complex I, thereby affecting mitochondrial OXPHOS [ 47 ] . Complex I represent the entry point of electrons from NADH into the mitochondrial respiratory chain (MRC), which pumps protons across the membrane through Complexes I-IV, driving H + pumping into the intermembrane space to generate the mitochondrial membrane potential used for ATP synthesis by the H-ATP (CV) of the OXPHOS system [ 48 ] . Additionally, Complex I is a major site of ROS generation, which can regulate numerous signaling pathways [ 49 , 50 ] , and elevated ROS levels generally exist in cancer cells to promote their growth and metabolism [ 28 ] . When SSR4 expression is reduced, it leads to decreased expression of NDUFB11, affecting Complex I function, resulting in decreased ROS production; affecting OXPHOS, leading to reduced ATP production; impacting the mitochondrial electron transport chain, causing a decrease in mitochondrial membrane potential, as indicated by an increase in green fluorescence in the JC-1 assay. ATP6AP1 is an auxiliary subunit of the V-ATPase complex, and it is a type I transmembrane protein associated with the membrane domain of V-ATPase [ 51 ] . Research suggests that V-ATPase is involved in various physiological processes, including endocytosis, exocytosis, intracellular membrane transport, membrane fusion, and intercellular fusion [ 52 , 53 ] . V-ATPase is believed to primarily contribute to tumor growth and metastasis by enhancing H + secretion, enabling tumor cells to survive in hypoxic and acidic tumor microenvironments [ 54 ] . Many studies currently suggest that ATP6AP1 primarily affects lysosomal transport function and exocytosis [ 51 , 55 ] , and there are also articles indicating its correlation with OXPHOS [ 56 , 57 ] . When SSR4 expression is reduced, it affects the expression of ATP6AP1, leading to a decrease in H + transport capacity, resulting in a reduction in mitochondrial membrane potential and ATP production. In the subcutaneous xenograft tumor model of nude mice, it was found that SSR4 promoted the proliferation of GC cells in vivo. Immunofluorescence experiments with IDH2 labeling indicated that SSR4 promoted mitochondrial biogenesis in vivo. The expression of NDUFB11 and ATP6AP1 decreased with the silencing of SSR4. In the nude mouse lung metastasis model, it was observed that there was no statistically significant difference in body weight between the shCtl group and shSSR4 group over time after tail vein injection. This may be because the tumors had not yet developed to the late stage, where significant weight loss due to cancer cachexia was observed [ 58 ] . Upon observation of lung tissue, it was found that the lung surface in the shSSR4 group was smoother with fewer metastatic foci, while the lung surface in the shCtl group appeared rougher with more metastatic foci, accompanied by a decrease in lung volume and deformation, which may be related to tumor tissue traction. Generally, in the process of lung metastasis in nude mice, lung volume and weight increase as the tumor grows [ 59 ] . However, in this study, lung tissue volume and weight decreased, possibly due to the formation of more metastatic foci and necrotic cavities in the later stages, resulting in a decrease in lung volume [ 60 ] . Histological examination of lung tissue sections stained with HE revealed fewer metastatic foci in the shSSR4 group. The liver surface of the nude mice appeared smooth with no visible metastatic foci, and there was no significant statistical difference in liver weight between the two groups. Cancer cells injected into the tail vein first pass through venous blood flow to the lungs, where they form metastatic foci. Liver metastasis, on the other hand, occurs later as cancer cells need to circulate through the bloodstream to the digestive system and then via the hepatic portal vein to the liver [ 61 ] . Therefore, HE staining of liver tissue did not reveal significant metastatic foci in this experiment. Conclusion SSR4 was identified as an oncogene that promoted the proliferation and migration of GC cells, thereby facilitating the progression of GC. SSR4 facilitated the process of mitochondrial biogenesis in GC cells. SSR4 though promoting the expression of NDUFB11, enhanced the function of mitochondrial complex I, enhancing OXPHOS, and increasing the production of ROS and ATP to promote the growth and metabolism of GC cells. Additionally, by upregulating ATP6AP1 expression, enhanced the transport function of H + across cellular and mitochondrial membranes, boosting OXPHOS, and generating more ATP to promote the growth and metabolism of GC cells. In vivo, SSR4 promoted the proliferation and metastatic ability of GC cells. SSR4 and its related molecules NDUFB11 and ATP6AP1 on the OXPHOS pathway can provide new ideas and references for the treatment of GC. Methods Cell culture The human GC cell lines were purchased from ATCC. BGC-823 and SGC-7901 GC cells were culturedin 1640 medium with 10% FBS (Gibco, USA) and 1% Penicillin–Streptomycin (Biosharp, China). All cell lines were incubated at 37°C in a humidified chamber containing 5% CO 2 . Enzyme-linked immunosorbent assay (ELISA) A total of 66 serum samples from healthy individuals and 86 serum samples from GC patients were collected, and basic demographic data of the patients were showed in Supplementary Material 1. Informed consent was obtained from all patients and their families for the collection of serum samples. This study was approved by the Ethics Committee of the Third People's Hospital of Chengdu. The levels of SSR4 were measured using commercial ELISA kits (MyBioSource, USA) according to the manufacturer’s protocol. Immunohistochemistry of GC samples Briefly, the paraffin-embedded tissues were deparaffinized in xylene and rehydrated in alcohol and were then incubated in H 2 O 2 for 10 min to block endogenous peroxidase activity. The samples were then incubated with normal goat serum (Zhong Shan Bio, China) for 30 min at room temperature. Subsequently, a rabbit monoclonal antibody against SSR4 (1:200 dilution) (Proteintech, USA.) was applied to the tissue array (Shanghai OUTDO BIOTECH Co., Ltd., China) and incubated overnight at 4°C. The secondary antibody (goat anti rabbit/mouse) was added to cover the tissue and incubated at 37℃ for 1 h. The peroxidase activity was visualized with the addition of DAB solution, and the samples were then counterstained with hematoxylin. All tissues or spots were scored by two independent observers in a blinded manner. Based on the proportion of positive cells in each specimen, the staining area ratio of cells was assessed and scored as follows: 0% staining scored 0, 75% scored 4. The staining intensity scores were assigned as follows: no signal, 0 points; weak signal, 1 point; moderate signal, 2 points; strong signal, 3 points. The histological score for SSR4 protein expression in each site was calculated using the following formula: Histological score = ratio score × intensity score. The total score range was determined to be 0–12 and categorized as negative (score = 0–1) or positive (score = 2–12) for further statistical analysis [ 62 ] . RNA extraction and real-time quantitative PCR Total RNA was extracted from tissues or cells using TRIzol reagent. Reverse transcription reagent (Vazyme, China) and PCR amplification reagent (Vazyme, China) were used for the reverse transcription and the qRT-PCR reactions to analyze mRNA expression. GAPDH was regarded as internal standard control to normalize the data. Experiment was repeated three times. The following primer sequences were used: GAPDH, 5'- GGA TTT GGT CGT ATT GGG − 3' (forward) and 5'- GGA AGA TGG TGA TGG GAT T -3' (reverse); and SSR4, 5'- CCG UCU UCA UUG UGG AGA UTT − 3' (forward) and 5'- AUC UCC ACA AUG AAG ACG GTT − 3' (reverse). Western blotting Cell pellets were collected at indicatedtime and washed twice with PBS. Cells were lysated in RIPA (Beyotime, China). After 15 min of centrifugation at 10,000 g, the supernatant was quantified using bicinchoninic acid (BCA) method (Thermo Fisher, USA). After quantification, sample was boiled at 100℃ with SDS buffer for 5 min. Protein extractions were separated using 12.5% SDS/PAGE gels. After incubated with primary and secondary antibodies (1:10000, Proteintech, USA), membrane signals were exposed by chemiluminescence system (Bio-Rad, USA). Antibodies used were as follows: Anti-SSR4 (1:1000, Proteintech, USA), Anti-GAPDH (1:1000, Proteintech, USA), Anti-IDH2 (1:1000, Proteintech, USA), Anti-NDUFB11 (1:1000, Proteintech, USA), Anti-ATP6AP1 (1:1000, Proteintech, USA). Transfection of siRNA and Lentiviral transduction of shRNA The SGC7901 and BGC823 cells were plated in six-well plates and cultured in RPMI1640 supplemented with 10% FBS. At 90% confluence, the cells were transfected with siRNA-SSR4 or siRNA-NC(GenePharma, China). The expression level of SSR4 was evaluated by Western blotting. The shRNA was used to down-regulate the expression of human SSR4 by lentivirus infection. The empty vectorwas served as control (GenePharma, China). The lentivirus were infected into the BGC823 cell lines according to the manufacturer’s protocol (GenePharma, China). The expression level of SSR4 was evaluated by Western blotting. Cell proliferation Assay The GC cells were plated into 96-well plates (5 × 10 3 cells/well), Cell proliferation were measured using the Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Japan) according to the manufacturer’s instructions. The absorbance was measured by an EnSpire Multimode Plate Reader (PerkinElmer, CA) at 450 nm. The experiment was performed three times independently. Flow cytometry Cells were seeded into a six-well plate in 10% FBS media. After 2 days, the cells were collected and washed with PBS. For cell apoptosis assay, cells were suspended in 100 µl of Annexin V binding buffer before staining with 5 µl of Annexin Ⅴ FITC and 5 µl of propidium iodide solution (Solarbio, China). After incubating in the dark at room temperature for 15 min, 400 µl PBS was added to each sample. Samples were analyzed by flow cytometry using a Bio-Rad ZE5 flow cytometer, and the data were processed using CytExpert software. For cell cycle assay, cells were seeded into a six-well plate in 10% FBS media. After 2 days, the cells were collected and prepared for cell cycle analysis by flow cytometry according to the Cell Cycle Phase Determination Kit (Beyotime, China). The data were processed using the Modfit software. Colony formation assay Cells were planted into 6-well plates with 1000 cells per well. The corresponding experimental treatment was carried out for each well. After 12 days, the cells colonywere fixed with 4% paraformaldehyde for 20 min and stained with 10% crystal violet. The formation of the clone was observed after washing with PBS. Cell migration Cell migration experiment was conducted utilizing a 24-well Transwell (8-m pore size; Corning, USA). The upper chamber has 2 × 10 5 cells suspended in 200 µl of 5% serum media; The lower chamber contains 600 µl of medium supplemented with 30% serum media. After 24 h, cotton swabs were used to remove the cells on the upper surface of the membrane. The lower surface cells were subsequently fixed with paraformaldehyde and stained with crystal violet. Finally, cells were enumerated and collected in five microscopic fields. Wound healing assay SGC7901 and BGC823 cells were seeded in 6-well plates, and wounds were generated at the center of the plates using 100 µl pipette tips. Floating cells were rinsed with PBS (Gibco, USA) and the remaining cells were grown in the serum-free RPMI-1640 medium. Cell migration was assessed by photographing the wound closure after 12 h. The wound closure area was measured using the ImageJ software. Three independent experiments were performed. Bioinformatic analysis The SSR4 mRNA expression in 408 GC tissues and 211 normal tissues was analyzed using the GEPIA ( http://gepia.cancer-pku.cn/ ) database. The function of SSR4 was predicted in TCGA database through LinkedOmics ( https://www.linkedomics.org/ ). Briefly, signal pathway enrichment analysis was performed in WiKi pathway database and KEGG pathway database, and a heatmap analysis was conducted to determine molecules with high correlation to SSR4. Mitochondrial ATP assay The cells were seeded into a 6-well plate (2 × 10 5 cells per well). After 3 days, 200 µl of lysis buffer was added to each well to lyse the cells. The lysates were then centrifuged at 4ºC and 12000 g for 5 min, and the supernatant was collected. A standard curve was prepared, and an ATP working solution (Beyotime, China) was prepared. Firstly, 100 µl of ATP detection working solution was added to each well of a 96-well plate. The plate was then left at room temperature for 3–5 min to consume all background ATP. Subsequently, 20 µl of sample or standard was added to the detection wells, gently mixed, with at least a 2 s interval between mixings, and the ATP levels were measured using a luminometer. Reactive oxygen species (ROS) assay Cells were seeded at a density of 1 × 10 4 cells/well in a 96-well plate and allowed to adhere. After adhesion, the cells were removed from the plate. DCFH-DA (Beyotime, China) was diluted in serum-free DMEM at a ratio of 1:1000, and 100 µl of the diluted DCFH-DA solution was added to each well. The plate was then incubated in a cell culture incubator for 20 min with gentle shaking every 5 min to ensure thorough contact between DCFH-DA probe and cells. After the incubation, the excess DCFH-DA probe was washed away with serum-free medium. The fluorescence intensity was measured using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm to detect the OD values of each group. Mitochondrial membrane potential assay. In a 12-well plate, sterile coverslips were placed, and cells were seeded at a density of 1 × 10 5 cells per well and allowed to adhere and grow to 80% confluency. The 12-well plate was then removed. An appropriate volume of JC-1 working solution (Beyotime, China) was prepared at a standard concentration of 1 ml per well, thoroughly mixed, and added to the wells. The plate was then placed in a cell culture incubator and incubated for 20 min. After washing with PBS, the coverslips with cells were removed and mounted using an anti-fade mounting reagent. Observation was performed under a laser confocal microscope with an excitation wavelength of 485 nm and an emission wavelength of 590 nm. Note: JC-1 is an ideal fluorescent probe widely used for detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix, producing red fluorescence; when the mitochondrial membrane potential is low, JC-1 remains in its monomeric form, emitting green fluorescence. Therefore, changes in fluorescence color can indicate alterations in mitochondrial membrane potential. Immunofluorescence Cells were seeded on coverslips (precoated with poly-L-lysine). Cells were fixed with 4% (v/v) paraformaldehyde. Cells were blocked using 5% bovine serum albumin (BSA) in PBS, then incubated for 1 h with primary antibodies, followed by three washes in PBS. Coverslips were incubated for 1 h with secondary antibodies and counterstained with 4′,6-diamidino-2 phenylindole (DAPI) for 5 min, then washed three times in PBS and once in water. Coverslips were observed by a laser confocal microscope. Animal experiments Six-week-old BALB/c nude mice were obtained from Charles River (China) and kept under specific pathogen-free conditions. The Ethics Committee on Animal Care of West China Hospital, Sichuan University authorized all animal experiments (approval number: 20231212001). For subcutaneous xenograft experiments, shSSR4 and control cells (1 × 10 7 cells per animal) were transplanted subcutaneously into the outer side of the hind legs of nude mice and tumor volumes were assessed every 3 days. Four weeks later, we collected tumors from mice and weighed them. For the lung metastasis assay, shSSR4 and control cells (5 × 10 6 cells per animal) cells were injected into the mice through tail veins. The weight of nude mice were recorded every 3 days. After four weeks, the lung and liver were fixed and embedded in paraffin for HE and IHC staining assays. Statistical analysis Statistics were analyzed using GraphPad Prism 8 (GraphPad Software, USA). The data were analyzed using unpaired t-tests, chi-square tests, and continuity-corrected chi-square tests between the two groups. All tests were two-tailed, and a significance level of p < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; NS, not significant).each experiment has a minimum of three replications. Declarations Supplementary Materials This file includes: Supplementary Material 1 Author contributions Conceptualization: Aoshuang Li, Xiaobin Sun. Methodology: Baixue Liao, Kaiwen Wu. Investigation: Ruiling Fan, Binjun Zhu. Visualization: Aoshuang Li. Supervision: Lei Liu, Xiaobin Sun. Writing—original draft: Aoshuang Li. Writing—review & editing: Aoshuang Li, Lei Liu, Xiaobin Sun. Funding Statement This work was supported by grants from The Third People's Hospital of Chengdu Scientific Research Project (2023PI19) Data and materials availability All data are available in the main text or the supplementary materials. Conflict of interest The authors declare no conflict of interest. Ethics statement The Ethics Committee on Animal Care of West China Hospital，Sichuan University authorized all animal experiments (approval number: 20231212001). Consent to participate The studies involving human participants were reviewed and approved by The Third People’s Hospital of Chengdu hospital. The patients/participants provided their written informed consent to participate in this study. References Sung H, Ferlay J, Siegel R L, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J]. CA Cancer J Clin, 2021, 71(3): 209–249. Yuan L, Xu Z Y, Ruan S M, et al. Long non-coding RNAs towards precision medicine in gastric cancer: early diagnosis, treatment, and drug resistance[J]. Mol Cancer, 2020, 19(1): 96. Thrift A P, El-Serag H B. Burden of Gastric Cancer[J]. Clin Gastroenterol Hepatol, 2020, 18(3): 534–542. Wang F H, Zhang X T, Li Y F, et al. 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Fernández-Ramos D, Lopitz-Otsoa F, Delacruz-Villar L, et al. Arachidyl amido cholanoic acid improves liver glucose and lipid homeostasis in nonalcoholic steatohepatitis via AMPK and mTOR regulation[J]. World J Gastroenterol, 2020, 26(34): 5101–5117. Lichtenstein D A, Crispin A W, Sendamarai A K, et al. A recurring mutation in the respiratory complex 1 protein NDUFB11 is responsible for a novel form of X-linked sideroblastic anemia[J]. Blood, 2016, 128(15): 1913–1917. Fernandez-Vizarra E, Zeviani M. Mitochondrial disorders of the OXPHOS system[J]. FEBS Lett, 2021, 595(8): 1062–1106. Mimaki M, Wang X, Mckenzie M, et al. Understanding mitochondrial complex I assembly in health and disease[J]. Biochim Biophys Acta, 2012, 1817(6): 851–62. Morán M, Rivera H, Sánchez-Aragó M, et al. Mitochondrial bioenergetics and dynamics interplay in complex I-deficient fibroblasts[J]. Biochim Biophys Acta, 2010, 1802(5): 443–53. Yang D Q, Feng S, Chen W, et al. V-ATPase subunit ATP6AP1 (Ac45) regulates osteoclast differentiation, extracellular acidification, lysosomal trafficking, and protease exocytosis in osteoclast-mediated bone resorption[J]. J Bone Miner Res, 2012, 27(8): 1695–707. Marshansky V, Futai M. The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function[J]. Curr Opin Cell Biol, 2008, 20(4): 415–26. Wada Y, Sun-Wada G H, Tabata H, et al. Vacuolar-type proton ATPase as regulator of membrane dynamics in multicellular organisms[J]. J Bioenerg Biomembr, 2008, 40(1): 53–7. Collins M P, Forgac M. Regulation and function of V-ATPases in physiology and disease[J]. Biochim Biophys Acta Biomembr, 2020, 1862(12): 183341. Wang F, Yang Y, Klionsky D J, et al. Mutations in V-ATPase in follicular lymphoma activate autophagic flux creating a targetable dependency[J]. Autophagy, 2023, 19(2): 716–719. Pottie L, Van Gool W, Vanhooydonck M, et al. Loss of zebrafish atp6v1e1b, encoding a subunit of vacuolar ATPase, recapitulates human ARCL type 2C syndrome and identifies multiple pathobiological signatures[J]. PLoS Genet, 2021, 17(6): e1009603. Teplova V V, Tonshin A A, Grigoriev P A, et al. Bafilomycin A1 is a potassium ionophore that impairs mitochondrial functions[J]. J Bioenerg Biomembr, 2007, 39(4): 321–9. Nishikawa H, Goto M, Fukunishi S, et al. Cancer Cachexia: Its Mechanism and Clinical Significance[J]. Int J Mol Sci, 2021, 22(16). Zhuo W, Liu Y, Li S, et al. Long Noncoding RNA GMAN, Up-regulated in Gastric Cancer Tissues, Is Associated With Metastasis in Patients and Promotes Translation of Ephrin A1 by Competitively Binding GMAN-AS[J]. Gastroenterology, 2019, 156(3): 676–691.e11. Stacchiotti S, Collini P, Messina A, et al. High-grade soft-tissue sarcomas: tumor response assessment–pilot study to assess the correlation between radiologic and pathologic response by using RECIST and Choi criteria[J]. Radiology, 2009, 251(2): 447–56. Schwab R S, Sweet W H, Et Al. Carcinoma of ascending colon, with metastases to brain, liver lung and lymph nodes[J]. N Engl J Med, 1949, 241(2): 73–5. Ren G, Tian Q, An Y, et al. Coronin 3 promotes gastric cancer metastasis via the up-regulation of MMP-9 and cathepsin K[J]. Mol Cancer, 2012, 11: 67. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4890970","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":339085624,"identity":"2804bd95-c332-4de8-b2c0-2249ef119f72","order_by":0,"name":"Lei 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patients.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4890970/v1/892bebd9bd0d73c3e74738fd.jpg"},{"id":66119117,"identity":"0f92ca9d-20e2-488d-b90c-ad528bddd27c","added_by":"auto","created_at":"2024-10-08 01:14:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":698575,"visible":true,"origin":"","legend":"\u003cp\u003eSSR4 promotes proliferation and migration of gastric cancer cells.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4890970/v1/d46ae31b0cc5301da84d386c.jpg"},{"id":66119115,"identity":"7b22d8a5-ee46-45fa-b094-48c81880ebfe","added_by":"auto","created_at":"2024-10-08 01:14:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":396590,"visible":true,"origin":"","legend":"\u003cp\u003eThe prediction of SSR4 involved signal pathway in the public database.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4890970/v1/86d1b12ff6cff2a17c2c5ee3.jpg"},{"id":66119116,"identity":"80d4a95b-821f-40d1-9716-0e007f12f63b","added_by":"auto","created_at":"2024-10-08 01:14:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":430282,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of SSR4 on mitochondrial oxidative phosphorylation.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4890970/v1/7ccffbe5b94390dae795a1ab.jpg"},{"id":66119120,"identity":"51d078ca-f2e9-42c2-92bd-43185f232c3b","added_by":"auto","created_at":"2024-10-08 01:14:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":427009,"visible":true,"origin":"","legend":"\u003cp\u003eSSR4 regulated mitochondrial oxidative phosphorylation in GC cells though NDUFB11 and ATP6AP1.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4890970/v1/2497ba4241b9cadc7340dfbe.jpg"},{"id":66119357,"identity":"c8720056-1b58-402b-bfdc-3ea116dd11c0","added_by":"auto","created_at":"2024-10-08 01:22:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":449590,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of SSR4 on GC cells proliferation in vivo.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4890970/v1/0781c2e03da5a0e757b5d371.jpg"},{"id":66119358,"identity":"d1b9dcda-7fec-4317-aa7e-2ef86717c6f4","added_by":"auto","created_at":"2024-10-08 01:22:47","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":483615,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of SSR4 on GC cells metastasis in vivo.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4890970/v1/bd30690523ccb3e0485daba4.jpg"},{"id":67228047,"identity":"6caf8e59-5980-4acc-b2c4-8bcce1923bf9","added_by":"auto","created_at":"2024-10-22 15:44:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3564412,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4890970/v1/d2a905f4-6424-478c-8429-cf2bf9172e67.pdf"},{"id":66119113,"identity":"3d6106f5-5bc8-44b9-a483-6af418323a0d","added_by":"auto","created_at":"2024-10-08 01:14:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4890970/v1/463ed2d7403818820cea4dde.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"SSR4 promote gastric cancer progression by regulating mitochondrial oxidative phosphorylation via NDUFB11 and ATP6AP1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer (GC) is a significant health issue, ranking fifth for incidence and fourth for mortality globally\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The incidence is highly correlated with regions, with the highest age-standardized incidence rates observed in East Asia\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The 5-year overall survival (OS) rate for patients with early detection and treatment of GC is usually higher (\u0026gt;\u0026thinsp;60%). However, for patients with local and distant metastasis, the 5-year OS rates decrease to 30% and 5%, respectively\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Unfortunately, the early clinical symptoms of GC were characterized by stealthiness and atypicality, with over 60% of patients already having local or distant metastases at the time of diagnosis\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The invasive and metastatic capabilities of GC cells are crucial factors contributing to mortality. In the case of early-stage GC, surgical resection is a viable option, whereas advanced-stage GC often necessitates a combination of surgery and chemotherapy. In severe instances, certain patients may opt for chemotherapy or biotherapy as their primary treatment modalities\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Therefore, exploring the process of GC cell metastasis can assist doctors in better understanding the developmental patterns of GC, preventing further spread of cancer cells, and ultimately improving the 5-year OS rate for patients.\u003c/p\u003e \u003cp\u003eCurrently, there is limited research on the gene SSR4, and studies regarding its association with cancer are also scarce. The translocation-associated protein complex (TRAP), also called signal sequence receptor (SSR), includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2, TRAPγ/SSR3 and TRAPδ/SSR4\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. SSR4, together with three other subunits, maintains the stability of the TRAP complex and plays a biological role\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Research has indicated that TRAP regulates N-glycosylation, while N-glycosylation primarily occurs in the rough endoplasmic reticulum. Additionally, the study suggested that SSR4 possessed chaperone-like properties, which could assist in N-glycosylation during endoplasmic reticulum stress\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The TRAP complex facilitates the translocation of proteins across the endoplasmic reticulum membrane and associates with the oligosaccharyl transferase (OST) complex to maintain proper glycosylation of nascent polypeptides\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Therefore, SSR4 was also associated with this congenital disorder of glycosylation. Through biochemical studies of fibroblasts in congenital disorders of glycosylation (CDG), it was found that the stability of TRAP complex was disrupted, SSR3 protein was completely lost, and SSR1 and SSR4 were partially lost\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. SSR4 exerted a certain influence on insulin synthesis, deficient protein expression of SSR4, diminishes the protein levels of other TRAP subunits, concomitant with deficient steady-state levels of proinsulin and insulin\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. There was a report showing that SSR4 was identified as significantly up-regulated in inflamed areas of the gingival connective tissue compared to non-inflamed tissue\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCell metabolism could utilize carbohydrates (such as glucose via glycolysis, and pyruvate oxidation), fats (such as fatty acid β-oxidation), and proteins (such as amino acid oxidation and transamination) to synthesize the required energy. Molecules derived from these catabolic processes could enter the tricarboxylic acid cycle and serve as substrates for oxidative phosphorylation (OXPHOS), generating ATP. Under aerobic conditions, the primary source of energy for normal cells was the transfer of metabolites through the respiratory complexes (CI-CIV) on the inner mitochondrial membrane, ultimately synthesizing ATP via ATP synthase\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt present, there is limited research on the relationship between SSR4 and cancer. A study showed that SSR4 was a prognostic biomarker associated with immune cell infiltration in colon adenocarcinoma (COAD), and COAD patients with high SSR4 expression in tumor-infiltrating lymphocytes (TILs) had better overall survival\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In previous studies, increased expression of SSR4 has been observed in some cancers, such as colorectal cancer\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e and lung cancer\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. However, there is currently no basic research exploring the relationship between SSR4 and GC.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe expression level of SSR4 was increased in GC patients\u003c/p\u003e \u003cp\u003eFirst, the expression of SSR4 mRNA in normal gastric tissues (n\u0026thinsp;=\u0026thinsp;211) and GC tissues (n\u0026thinsp;=\u0026thinsp;408) was analyzed using the GEPIA database. The results revealed that the mRNA expression level of SSR4 in GC tissues was increased than that in normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThen, the concentration of SSR4 protein was detected in serum level. The specimens were collected from 66 Healthy volunteers and 86 GC patients. ELISA was performed to detect the concentration of SSR4 protein in serum. The data showed that the concentration of SSR4 in the serum of GC patients was higher than that in the normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFor the tissue microarray analysis, there were 96 cases of GC tissue remained after excluding the tissues with deletions. Due to the lack of patients classified as Grade I in this tissue array, we analyzed tissues from Grade II and Grade III patients. The results showed that SSR4 protein expression in Grade III was higher than that in Grade II (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Chi-square test and continuity-corrected Chi-square test showed that the expression level of SSR4 protein was significantly different between Grade II and Grade III group (p-value\u0026thinsp;=\u0026thinsp;0.003). However, there were no significant differences among other clinical characteristics (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results indicated that the expression level of SSR4 was increased in GC tissue and serum of GC patients, and SSR4 may be related to the differentiation degree of GC.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between the expression level of SSR4 and clinicopathological characteristics in gastric cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eEvaluate Results\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.066\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size(cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.420\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrades\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅡ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅢ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor growth status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.437\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1-T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3-T4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node metastases\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.696\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetastasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.948\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStages\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.880\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSSR4 promoted the malignant biological behavior of GC cells in vitro\u003c/p\u003e \u003cp\u003eFirst, the expression level of SSR4 was detected in human gastric mucosal epithelial cells GES-1 and five GC cell lines by qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The expression level of SSR4 in these five GC cell lines was higher than that in normal gastric mucosal cells. SGC7901 and BGC823 cells were transfected with siRNA to interfere the expression of SSR4,, and siRNA-2 with higher interference efficiency was selected for subsequently study (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The CCK-8 cell proliferation assay continuously measured and recorded data for 5 days. The results showed that the proliferation rate of GC cells was decreased after SSR4 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Apoptosis assay revealed an increased proportion of apoptotic cells in the siSSR4 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), indicating that the decreased expression of SSR4 could promote apoptosis of GC cells. Cell cycle assay showed an increased proportion of G1 phase and a decreased proportion of S phase in the siSSR4 group, with no significant difference in the proportion in G2 phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). This result indicates that when the expression level of SSR4 is reduced, cell division is arrested in the G1 phase.Subsequently, colony formation assays were conducted, and the results indicated that GC cell proliferation was inhibited in the siSSR4 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eTo evaluate the effect of SSR4 on cell migration ability, the transwell assays was performed. The result revealed that the migrated GC cells were reduced in siSSR4 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). The wound healing assay showed that within the same time frame, there was a larger wound area in the siSSR4 group, which indicated that inhibit of SSR4 could reduce the migration ability in GC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-J). Thus, these results indicate that inhibition of SSR4 could reduce proliferation and migration abilities in GC cells.\u003c/p\u003e \u003cp\u003eThe effects of SSR4 on mitochondrial oxidative phosphorylation\u003c/p\u003e \u003cp\u003eTo predict the function of SSR4 in GC, SSR4 involved signal pathway was analyzed firstly. The signal pathway enrichment analysis was performed both in WiKi pathway database and in KEGG pathway database. The results revealed that there was a high correlation between SSR4 and the oxidative phosphorylation (OXPHOS) pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). The top ten gene related to OXPHOS pathway were listed, which include NDUFB11, NDUFA1, COX6B1, ATP6AP1, NDUFA2, etc (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The heatmap analysis was conducted to analyze the gene with high correlation to SSR4 in GC of TCGA database. Consistently, the top ten gene related to OXPHOS pathway were all included in the top 50 gene which positively correlated with SSR4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results indicated that SSR4 may closely related to OXPHOS pathway in GC. Further, the expression level of these key gene in OXPHOS pathway was detected after knockdown of SSR4, and the expression level of NDUFB11 and ATP6AP1 was significantly reduced, which were selected for subsequent study. NDUFB11 is a crucial subunit of mitochondrial Complex I, associated with OXPHOS mechanism\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. ATP6AP1, as an auxiliary subunit of the H\u003csup\u003e+\u003c/sup\u003e-ATPase complex, participates in H\u003csup\u003e+\u003c/sup\u003e transportation\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Both of NDUFB11 and ATP6AP1 were play important role in mitochondrial oxidative phosphorylation.\u003c/p\u003e \u003cp\u003eWe measured the ATP content in GC cells, as ATP is a product of mitochondrial OXPHOS. The results revealed that inhibition the expression of SSR4 led to a decrease of ATP content in GC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Within living cells, ROS is primarily originated from mitochondria\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e and participated in regulating cellular processes such as apoptosis\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, autophagy\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, stem cell differentiation\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, cellular\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, and tissue inflammation\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. With normal physiological conditions, ROS emission (production minus clearance) accounts for 2% of the total oxygen consumed by mitochondria\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Some studies have shown that the production of ROS in mitochondria fluctuates between 0.25% and 11% of total oxygen consumption, depending on the animal species and respiratory rate\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Both excessively high and low levels of ROS may have detrimental effects on cells. When ROS levels too low, cells cannot participate in appropriate cellular metabolism through the regulation of numerous biochemical reactions\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Conversely, when ROS levels are too high, cellular signal regulation becomes uncontrolled or disrupted\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Consequently, cancer cells accumulate high levels of ROS to promote their metabolic activities and growth\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. We also assessed the levels of ROS in the control and siSSR4 cells, and the results showed a significant decrease in ROS levels in the siSSR4 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eMitochondrial membrane potential serves as a valuable indicator of cell health and functional status\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. When the membrane potential in mitochondria is high, JC-1 exists in polymeric form, emitting red fluorescence at 595 nm; whereas, when the membrane potential is low, JC-1 exists in monomeric form, emitting green fluorescence at 530 nm\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. The presence of red fluorescence indicates higher mitochondrial activity and stronger OXPHOS, whereas an increase in green fluorescence suggests decreased mitochondrial activity and weaker OXPHOS, which may lead to increased apoptosis\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. The experimental results showed increased green fluorescence and decreased red fluorescence after SSR4 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This indicates that decreased SSR4 expression results in reduced mitochondrial membrane potential, decreased mitochondrial activity, and inhibition of OXPHOS.\u003c/p\u003e \u003cp\u003eIsocitrate dehydrogenases (IDH) are homodimers involved in various cellular metabolic processes. Both IDH2 and IDH1 catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate, converting NADP to NADPH\u003csup\u003e+\u003c/sup\u003e in the process. IDH1 is localized in the cytoplasm and peroxisomes, while IDH2 is localized in the mitochondria, serving as a crucial enzyme in the tricarboxylic acid cycle\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Therefore, IDH2 can be regarded as a marker enzyme for mitochondria, used to observe the status and quantity of mitochondria and understand mitochondrial biogenesis\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. The experimental findings revealed a decrease in IDH2 expression when SSR4 expression was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E).\u003c/p\u003e \u003cp\u003eIn summary, inhibition of SSR4 could reduce the production of mitochondrial ATP and ROS, decrease mitochondrial membrane potential, restrain mitochondrial biogenesis, and ultimately inhibit mitochondrial OXPHOS.\u003c/p\u003e \u003cp\u003eSSR4 affects mitochondrial OXPHOS through NDUFB11 and ATP6AP1\u003c/p\u003e \u003cp\u003eAfter SSR4 knockdown, western blotting revealed a decrease in the protein expression of NDUFB11 and ATP6AP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Cell immunofluorescence showed that the fluorescence intensity of NDUFB11 and ATP6AP1 was decreased when SSR4 expression was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). The result suggested that NDUFB11 and ATP6AP1 are the key molecules in the SSR4-mediated regulation of mitochondrial OXPHOS in GC cells.\u003c/p\u003e \u003cp\u003eSSR4 regulate GC cells proliferation through NDUFB11 and ATP6AP1 in vivo\u003c/p\u003e \u003cp\u003eTo obtain cell lines with stable knockdown expression of SSR4, BGC823 cells were transfect with shSSR4 lentiviral. Western blotting was used to validate the knockdown effect, and cell lines efficient knockdown were selected for nude mouse subcutaneous tumor experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Each BALB/c nude mouse was injected with the corresponding cell suspension on the outer side of the hind legs, with the left side injected with control cells and the right side injected with shSSR4 cells. After subcutaneous tumor formation, tumor lengths (L) and widths (W) were measured every 3 days, and tumor volume was calculated according to the formula 0.5 \u0026times; L\u0026times;W\u003csup\u003e2\u003c/sup\u003e. Tumor growth curves were plotted (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The curves showed that the tumor growth rate in the shSSR4 group was significantly slower than that in the control group. After 4 weeks, tumors were collected, photographed, and weighed. Analysis of tumor showed that the tumor volume and weight in the control group were greater than those in the shSSR4 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). To explore whether SSR4 affects mitochondrial biogenesis in vivo, tumor tissues were labeled with IDH2. The fluorescence intensity of IDH2 in the shSSR4 group was decreased by comparing with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), which indicated that SSR4 knockdown inhibited mitochondrial biogenesis in vivo. The result of immunofluorescence experiments on tumor tissues showed that the expression level of NDUFB11 and ATP6AP1 were also decreased in shSSR4 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). These results indicated that inhibition of SSR4 could attenuate GC cells proliferation in vivo, which may through regulating NDUFB11 and ATP6AP1 expression.\u003c/p\u003e \u003cp\u003eSSR4 regulate GC cells metastasis in vivo\u003c/p\u003e \u003cp\u003eTo explore the effect of SSR4 on the metastatic ability of GC cells in vivo, control and shSSR4 cells were intravenously injected into the tail vein (n\u0026thinsp;=\u0026thinsp;6). The body weight of the nude mice was measured every 3 days, and the data were recorded to obtain the weight change curves. The curves showed no significant difference in body weight between the shSSR4 group and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Four weeks later, lung tissue was collected for weighing and photographing. It was observed that there were more visible metastatic nodules on the lung surface in the control group, accompanied by a reduction in lung volume and changes in lung morphology, while the lungs of the shSSR4 group appeared smoother (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The HE staining of lung tissues showed fewer metastatic nodules in the shSSR4 group by compared with control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Liver tissues were photographed and the weights were recorded and analyzed. However, the results showed no visible metastatic nodules on the liver surface in both the control and shSSR4 groups, with no statistical difference in liver weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The HE staining also revealed no apparent metastatic nodules in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). These results suggested that inhibition of SSR4 could attenuate the metastasis ability of GC cells in vivo.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to the high mortality and poor prognosis of GC patients, early detection and timely treatment could improve patient survival rates and quality of life\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Therefore, we attempted to screen for a molecule highly correlated with GC and explore its role in the occurrence and development of GC, as well as provide a new target for the treatment of GC.\u003c/p\u003e \u003cp\u003eSSR4, as one of the components of the signal recognition particle (SRP) complex, participates in the protein translocation process of the endoplasmic reticulum\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. There has been very little research on SSR4 and cancer both domestically and internationally. In previous studies, increased expression of SSR4 has been observed in some cancers, such as colorectal cancer\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e and lung cancer\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. SSR4 serves as a prognostic biomarker and is associated with immune cell infiltration in colon adenocarcinoma\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccording to the Warburg effect, it is known that under aerobic conditions, tumor tissues have a capacity to metabolize glucose into lactate that is ten times higher than normal tissues, demonstrating that the growth metabolism of cancer cells tends to favor aerobic glycolysis\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The proposition of the Warburg effect has led some researchers to mistakenly believe that the increased aerobic glycolysis in cancer cells is related to a decrease in mitochondrial OXPHOS (or respiratory impairment). Studies have shown that hypoxic tumor cells excrete lactate, which is subsequently recycled into pyruvate by stroma or tumor cells in aerobic environments for OXPHOS \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. In fact, many cancer cells exhibit both the retention of OXPHOS and the Warburg effect, which collectively promote cancer cell proliferation and metabolism, as observed in urothelial bladder carcinoma\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, melanoma\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, breast cancer\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e, hepatocellular carcinoma\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e, etc. Furthermore, some studies indicate an upregulation of OXPHOS in certain cancers, including lymphoma, leukemia, pancreatic ductal adenocarcinoma, endometrial carcinoma, etc.\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we firstly reported the overexpression of SSR4 in GC tissue and serum. Subsequently, downregulation of SSR4 expression revealed its promotive role in the malignant biological behavior of GC, including the promotion of GC cell proliferation and migration ability. To further elucidate the mechanism of SSR4 action, we utilized different databases for analysis and determined that the pathway of SSR4 action is mitochondrial OXPHOS, with key molecules being NDUFB11 and ATP6AP1. Subsequent experiments validated that SSR4 can promote the production of ATP and ROS, stabilize mitochondrial membrane potential, thereby facilitating the process of OXPHOS in GC cells. It also promoted mitochondrial biogenesis. Furthermore, we further confirmed the effect of SSR4 on the OXPHOS pathway proteins NDUFB11 and ATP6AP1, showing that SSR4 can promote the expression of these two proteins.\u003c/p\u003e \u003cp\u003eNDUFB11 is a protein subunit of mitochondrial Complex I, also known as NADH dehydrogenase (ubiquinone) subunit B11. It plays a crucial role in mitochondrial energy production, particularly in OXPHOS and electron transfer processes\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Impairment of NDUFB11 expression can lead to decreased activity of Complex I, thereby affecting mitochondrial OXPHOS \u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Complex I represent the entry point of electrons from NADH into the mitochondrial respiratory chain (MRC), which pumps protons across the membrane through Complexes I-IV, driving H\u003csup\u003e+\u003c/sup\u003e pumping into the intermembrane space to generate the mitochondrial membrane potential used for ATP synthesis by the H-ATP (CV) of the OXPHOS system\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Additionally, Complex I is a major site of ROS generation, which can regulate numerous signaling pathways\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e, and elevated ROS levels generally exist in cancer cells to promote their growth and metabolism\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. When SSR4 expression is reduced, it leads to decreased expression of NDUFB11, affecting Complex I function, resulting in decreased ROS production; affecting OXPHOS, leading to reduced ATP production; impacting the mitochondrial electron transport chain, causing a decrease in mitochondrial membrane potential, as indicated by an increase in green fluorescence in the JC-1 assay.\u003c/p\u003e \u003cp\u003eATP6AP1 is an auxiliary subunit of the V-ATPase complex, and it is a type I transmembrane protein associated with the membrane domain of V-ATPase\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Research suggests that V-ATPase is involved in various physiological processes, including endocytosis, exocytosis, intracellular membrane transport, membrane fusion, and intercellular fusion\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. V-ATPase is believed to primarily contribute to tumor growth and metastasis by enhancing H\u003csup\u003e+\u003c/sup\u003e secretion, enabling tumor cells to survive in hypoxic and acidic tumor microenvironments\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. Many studies currently suggest that ATP6AP1 primarily affects lysosomal transport function and exocytosis\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e, and there are also articles indicating its correlation with OXPHOS \u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. When SSR4 expression is reduced, it affects the expression of ATP6AP1, leading to a decrease in H\u003csup\u003e+\u003c/sup\u003e transport capacity, resulting in a reduction in mitochondrial membrane potential and ATP production.\u003c/p\u003e \u003cp\u003eIn the subcutaneous xenograft tumor model of nude mice, it was found that SSR4 promoted the proliferation of GC cells in vivo. Immunofluorescence experiments with IDH2 labeling indicated that SSR4 promoted mitochondrial biogenesis in vivo. The expression of NDUFB11 and ATP6AP1 decreased with the silencing of SSR4.\u003c/p\u003e \u003cp\u003eIn the nude mouse lung metastasis model, it was observed that there was no statistically significant difference in body weight between the shCtl group and shSSR4 group over time after tail vein injection. This may be because the tumors had not yet developed to the late stage, where significant weight loss due to cancer cachexia was observed\u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. Upon observation of lung tissue, it was found that the lung surface in the shSSR4 group was smoother with fewer metastatic foci, while the lung surface in the shCtl group appeared rougher with more metastatic foci, accompanied by a decrease in lung volume and deformation, which may be related to tumor tissue traction. Generally, in the process of lung metastasis in nude mice, lung volume and weight increase as the tumor grows\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. However, in this study, lung tissue volume and weight decreased, possibly due to the formation of more metastatic foci and necrotic cavities in the later stages, resulting in a decrease in lung volume\u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. Histological examination of lung tissue sections stained with HE revealed fewer metastatic foci in the shSSR4 group. The liver surface of the nude mice appeared smooth with no visible metastatic foci, and there was no significant statistical difference in liver weight between the two groups. Cancer cells injected into the tail vein first pass through venous blood flow to the lungs, where they form metastatic foci. Liver metastasis, on the other hand, occurs later as cancer cells need to circulate through the bloodstream to the digestive system and then via the hepatic portal vein to the liver\u003csup\u003e[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e. Therefore, HE staining of liver tissue did not reveal significant metastatic foci in this experiment.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eSSR4 was identified as an oncogene that promoted the proliferation and migration of GC cells, thereby facilitating the progression of GC. SSR4 facilitated the process of mitochondrial biogenesis in GC cells. SSR4 though promoting the expression of NDUFB11, enhanced the function of mitochondrial complex I, enhancing OXPHOS, and increasing the production of ROS and ATP to promote the growth and metabolism of GC cells. Additionally, by upregulating ATP6AP1 expression, enhanced the transport function of H\u003csup\u003e+\u003c/sup\u003e across cellular and mitochondrial membranes, boosting OXPHOS, and generating more ATP to promote the growth and metabolism of GC cells. In vivo, SSR4 promoted the proliferation and metastatic ability of GC cells. SSR4 and its related molecules NDUFB11 and ATP6AP1 on the OXPHOS pathway can provide new ideas and references for the treatment of GC.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003eCell culture\u003c/p\u003e\u003cp\u003eThe human GC cell lines were purchased from ATCC. BGC-823 and SGC-7901 GC cells were culturedin 1640 medium with 10% FBS (Gibco, USA) and 1% Penicillin–Streptomycin (Biosharp, China). All cell lines were incubated at 37°C in a humidified chamber containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/p\u003e\u003cp\u003eA total of 66 serum samples from healthy individuals and 86 serum samples from GC patients were collected, and basic demographic data of the patients were showed in Supplementary Material 1. Informed consent was obtained from all patients and their families for the collection of serum samples. This study was approved by the Ethics Committee of the Third People's Hospital of Chengdu. The levels of SSR4 were measured using commercial ELISA kits (MyBioSource, USA) according to the manufacturer’s protocol.\u003c/p\u003e\u003cp\u003eImmunohistochemistry of GC samples\u003c/p\u003e\u003cp\u003eBriefly, the paraffin-embedded tissues were deparaffinized in xylene and rehydrated in alcohol and were then incubated in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 min to block endogenous peroxidase activity. The samples were then incubated with normal goat serum (Zhong Shan Bio, China) for 30 min at room temperature. Subsequently, a rabbit monoclonal antibody against SSR4 (1:200 dilution) (Proteintech, USA.) was applied to the tissue array (Shanghai OUTDO BIOTECH Co., Ltd., China) and incubated overnight at 4°C. The secondary antibody (goat anti rabbit/mouse) was added to cover the tissue and incubated at 37℃ for 1 h. The peroxidase activity was visualized with the addition of DAB solution, and the samples were then counterstained with hematoxylin.\u003c/p\u003e\u003cp\u003eAll tissues or spots were scored by two independent observers in a blinded manner. Based on the proportion of positive cells in each specimen, the staining area ratio of cells was assessed and scored as follows: 0% staining scored 0, \u0026lt; 25% staining scored 1, 25–50% staining scored 2, 50–75% staining scored 3, and staining \u0026gt; 75% scored 4. The staining intensity scores were assigned as follows: no signal, 0 points; weak signal, 1 point; moderate signal, 2 points; strong signal, 3 points. The histological score for SSR4 protein expression in each site was calculated using the following formula: Histological score = ratio score × intensity score. The total score range was determined to be 0–12 and categorized as negative (score = 0–1) or positive (score = 2–12) for further statistical analysis\u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRNA extraction and real-time quantitative PCR\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from tissues or cells using TRIzol reagent. Reverse transcription reagent (Vazyme, China) and PCR amplification reagent (Vazyme, China) were used for the reverse transcription and the qRT-PCR reactions to analyze mRNA expression. GAPDH was regarded as internal standard control to normalize the data. Experiment was repeated three times.\u003c/p\u003e\u003cp\u003eThe following primer sequences were used: GAPDH, 5'- GGA TTT GGT CGT ATT GGG − 3' (forward) and 5'- GGA AGA TGG TGA TGG GAT T -3' (reverse); and SSR4, 5'- CCG UCU UCA UUG UGG AGA UTT − 3' (forward) and 5'- AUC UCC ACA AUG AAG ACG GTT − 3' (reverse).\u003c/p\u003e\u003cp\u003eWestern blotting\u003c/p\u003e\u003cp\u003eCell pellets were collected at indicatedtime and washed twice with PBS. Cells were lysated in RIPA (Beyotime, China). After 15 min of centrifugation at 10,000 g, the supernatant was quantified using bicinchoninic acid (BCA) method (Thermo Fisher, USA). After quantification, sample was boiled at 100℃ with SDS buffer for 5 min. Protein extractions were separated using 12.5% SDS/PAGE gels. After incubated with primary and secondary antibodies (1:10000, Proteintech, USA), membrane signals were exposed by chemiluminescence system (Bio-Rad, USA). Antibodies used were as follows: Anti-SSR4 (1:1000, Proteintech, USA), Anti-GAPDH (1:1000, Proteintech, USA), Anti-IDH2 (1:1000, Proteintech, USA), Anti-NDUFB11 (1:1000, Proteintech, USA), Anti-ATP6AP1 (1:1000, Proteintech, USA).\u003c/p\u003e\u003cp\u003eTransfection of siRNA and Lentiviral transduction of shRNA\u003c/p\u003e\u003cp\u003eThe SGC7901 and BGC823 cells were plated in six-well plates and cultured in RPMI1640 supplemented with 10% FBS. At 90% confluence, the cells were transfected with siRNA-SSR4 or siRNA-NC(GenePharma, China). The expression level of SSR4 was evaluated by Western blotting.\u003c/p\u003e\u003cp\u003eThe shRNA was used to down-regulate the expression of human SSR4 by lentivirus infection. The empty vectorwas served as control (GenePharma, China). The lentivirus were infected into the BGC823 cell lines according to the manufacturer’s protocol (GenePharma, China). The expression level of SSR4 was evaluated by Western blotting.\u003c/p\u003e\u003cp\u003eCell proliferation Assay\u003c/p\u003e\u003cp\u003eThe GC cells were plated into 96-well plates (5 × 10\u003csup\u003e3\u003c/sup\u003e cells/well), Cell proliferation were measured using the Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Japan) according to the manufacturer’s instructions. The absorbance was measured by an EnSpire Multimode Plate Reader (PerkinElmer, CA) at 450 nm. The experiment was performed three times independently.\u003c/p\u003e\u003cp\u003eFlow cytometry\u003c/p\u003e\u003cp\u003eCells were seeded into a six-well plate in 10% FBS media. After 2 days, the cells were collected and washed with PBS. For cell apoptosis assay, cells were suspended in 100 µl of Annexin V binding buffer before staining with 5 µl of Annexin Ⅴ FITC and 5 µl of propidium iodide solution (Solarbio, China). After incubating in the dark at room temperature for 15 min, 400 µl PBS was added to each sample. Samples were analyzed by flow cytometry using a Bio-Rad ZE5 flow cytometer, and the data were processed using CytExpert software.\u003c/p\u003e\u003cp\u003eFor cell cycle assay, cells were seeded into a six-well plate in 10% FBS media. After 2 days, the cells were collected and prepared for cell cycle analysis by flow cytometry according to the Cell Cycle Phase Determination Kit (Beyotime, China). The data were processed using the Modfit software.\u003c/p\u003e\u003cp\u003eColony formation assay\u003c/p\u003e\u003cp\u003eCells were planted into 6-well plates with 1000 cells per well. The corresponding experimental treatment was carried out for each well. After 12 days, the cells colonywere fixed with 4% paraformaldehyde for 20 min and stained with 10% crystal violet. The formation of the clone was observed after washing with PBS.\u003c/p\u003e\u003cp\u003eCell migration\u003c/p\u003e\u003cp\u003eCell migration experiment was conducted utilizing a 24-well Transwell (8-m pore size; Corning, USA). The upper chamber has 2 × 10\u003csup\u003e5\u003c/sup\u003e cells suspended in 200 µl of 5% serum media; The lower chamber contains 600 µl of medium supplemented with 30% serum media. After 24 h, cotton swabs were used to remove the cells on the upper surface of the membrane. The lower surface cells were subsequently fixed with paraformaldehyde and stained with crystal violet. Finally, cells were enumerated and collected in five microscopic fields.\u003c/p\u003e\u003cp\u003eWound healing assay\u003c/p\u003e\u003cp\u003eSGC7901 and BGC823 cells were seeded in 6-well plates, and wounds were generated at the center of the plates using 100 µl pipette tips. Floating cells were rinsed with PBS (Gibco, USA) and the remaining cells were grown in the serum-free RPMI-1640 medium. Cell migration was assessed by photographing the wound closure after 12 h. The wound closure area was measured using the ImageJ software. Three independent experiments were performed.\u003c/p\u003e\u003cp\u003eBioinformatic analysis\u003c/p\u003e\u003cp\u003eThe SSR4 mRNA expression in 408 GC tissues and 211 normal tissues was analyzed using the GEPIA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database. The function of SSR4 was predicted in TCGA database through LinkedOmics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.linkedomics.org/\u003c/span\u003e\u003cspan address=\"https://www.linkedomics.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Briefly, signal pathway enrichment analysis was performed in WiKi pathway database and KEGG pathway database, and a heatmap analysis was conducted to determine molecules with high correlation to SSR4.\u003c/p\u003e\u003cp\u003eMitochondrial ATP assay\u003c/p\u003e\u003cp\u003eThe cells were seeded into a 6-well plate (2 × 10\u003csup\u003e5\u003c/sup\u003e cells per well). After 3 days, 200 µl of lysis buffer was added to each well to lyse the cells. The lysates were then centrifuged at 4ºC and 12000 g for 5 min, and the supernatant was collected. A standard curve was prepared, and an ATP working solution (Beyotime, China) was prepared. Firstly, 100 µl of ATP detection working solution was added to each well of a 96-well plate. The plate was then left at room temperature for 3–5 min to consume all background ATP. Subsequently, 20 µl of sample or standard was added to the detection wells, gently mixed, with at least a 2 s interval between mixings, and the ATP levels were measured using a luminometer.\u003c/p\u003e\u003cp\u003eReactive oxygen species (ROS) assay\u003c/p\u003e\u003cp\u003eCells were seeded at a density of 1 × 10\u003csup\u003e4\u003c/sup\u003e cells/well in a 96-well plate and allowed to adhere. After adhesion, the cells were removed from the plate. DCFH-DA (Beyotime, China) was diluted in serum-free DMEM at a ratio of 1:1000, and 100 µl of the diluted DCFH-DA solution was added to each well. The plate was then incubated in a cell culture incubator for 20 min with gentle shaking every 5 min to ensure thorough contact between DCFH-DA probe and cells. After the incubation, the excess DCFH-DA probe was washed away with serum-free medium. The fluorescence intensity was measured using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm to detect the OD values of each group.\u003c/p\u003e\u003cp\u003eMitochondrial membrane potential assay.\u003c/p\u003e\u003cp\u003eIn a 12-well plate, sterile coverslips were placed, and cells were seeded at a density of 1 × 10\u003csup\u003e5\u003c/sup\u003e cells per well and allowed to adhere and grow to 80% confluency. The 12-well plate was then removed. An appropriate volume of JC-1 working solution (Beyotime, China) was prepared at a standard concentration of 1 ml per well, thoroughly mixed, and added to the wells. The plate was then placed in a cell culture incubator and incubated for 20 min. After washing with PBS, the coverslips with cells were removed and mounted using an anti-fade mounting reagent. Observation was performed under a laser confocal microscope with an excitation wavelength of 485 nm and an emission wavelength of 590 nm. Note: JC-1 is an ideal fluorescent probe widely used for detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix, producing red fluorescence; when the mitochondrial membrane potential is low, JC-1 remains in its monomeric form, emitting green fluorescence. Therefore, changes in fluorescence color can indicate alterations in mitochondrial membrane potential.\u003c/p\u003e\u003cp\u003eImmunofluorescence\u003c/p\u003e\u003cp\u003eCells were seeded on coverslips (precoated with poly-L-lysine). Cells were fixed with 4% (v/v) paraformaldehyde. Cells were blocked using 5% bovine serum albumin (BSA) in PBS, then incubated for 1 h with primary antibodies, followed by three washes in PBS. Coverslips were incubated for 1 h with secondary antibodies and counterstained with 4′,6-diamidino-2 phenylindole (DAPI) for 5 min, then washed three times in PBS and once in water. Coverslips were observed by a laser confocal microscope.\u003c/p\u003e\u003cp\u003eAnimal experiments\u003c/p\u003e\u003cp\u003eSix-week-old BALB/c nude mice were obtained from Charles River (China) and kept under specific pathogen-free conditions. The Ethics Committee on Animal Care of West China Hospital, Sichuan University authorized all animal experiments (approval number: 20231212001). For subcutaneous xenograft experiments, shSSR4 and control cells (1 × 10\u003csup\u003e7\u003c/sup\u003e cells per animal) were transplanted subcutaneously into the outer side of the hind legs of nude mice and tumor volumes were assessed every 3 days. Four weeks later, we collected tumors from mice and weighed them. For the lung metastasis assay, shSSR4 and control cells (5 × 10\u003csup\u003e6\u003c/sup\u003e cells per animal) cells were injected into the mice through tail veins. The weight of nude mice were recorded every 3 days. After four weeks, the lung and liver were fixed and embedded in paraffin for HE and IHC staining assays.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistics were analyzed using GraphPad Prism 8 (GraphPad Software, USA). The data were analyzed using unpaired t-tests, chi-square tests, and continuity-corrected chi-square tests between the two groups. All tests were two-tailed, and a significance level of p \u0026lt; 0.05 was considered statistically significant (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, and ****P \u0026lt; 0.0001; NS, not significant).each experiment has a minimum of three replications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eSupplementary Materials\u003c/p\u003e\n\u003cp\u003eThis file includes:\u003c/p\u003e\n\u003cp\u003eSupplementary Material 1\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eConceptualization: Aoshuang Li, Xiaobin Sun. Methodology: Baixue Liao, Kaiwen Wu. Investigation: Ruiling Fan, Binjun Zhu. Visualization: Aoshuang Li. Supervision: Lei Liu, Xiaobin Sun. Writing\u0026mdash;original draft: Aoshuang Li. Writing\u0026mdash;review \u0026amp; editing: Aoshuang Li, Lei Liu, Xiaobin Sun.\u003c/p\u003e\n\u003cp\u003eFunding Statement\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from The Third People\u0026apos;s Hospital of Chengdu Scientific Research Project (2023PI19)\u003c/p\u003e\n\u003cp\u003eData and materials availability\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the supplementary materials.\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eEthics statement\u003c/p\u003e\n\u003cp\u003eThe Ethics Committee on Animal Care of West China Hospital，Sichuan University authorized all animal experiments (approval number: 20231212001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe studies involving human participants were reviewed and approved by The Third People\u0026rsquo;s Hospital of Chengdu hospital. The patients/participants provided their written informed consent to participate in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel R L, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J]. CA Cancer J Clin, 2021, 71(3): 209\u0026ndash;249.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan L, Xu Z Y, Ruan S M, et al. Long non-coding RNAs towards precision medicine in gastric cancer: early diagnosis, treatment, and drug resistance[J]. Mol Cancer, 2020, 19(1): 96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThrift A P, El-Serag H B. Burden of Gastric Cancer[J]. Clin Gastroenterol Hepatol, 2020, 18(3): 534\u0026ndash;542.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang F H, Zhang X T, Li Y F, et al. 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Mol Cancer, 2012, 11: 67.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SSR4, gastric cancer, oxidative phosphorylation, NDUFB11, ATP6AP1","lastPublishedDoi":"10.21203/rs.3.rs-4890970/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4890970/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGastric cancer (GC) is one of the most common cancer worldwide. Cancer cell metastasis is a major factor leading to poor prognosis. Previous proteomic data suggested that SSR4 might be closely associated with the occurrence and development of GC. However, the role and molecular mechanism of SSR4 in GC is not yet clear. The present study found that the expression level of SSR4 was increased in GC tissue and serum from GC patients. In addition, SSR4 could promote the malignant biological behavior of GC cells in vitro and in vivo. The mechanism may be that SSR4 regulates the expression of NDUFB11 and ATP6AP1, and then enhanced the function of mitochondrial respiratory chain complex I (CI) and mitochondrial respiratory chain complex V (CV), which promoted the mitochondrial oxidative phosphorylation and thus promoted GC progression. These findings expand the understanding of the role of SSR4 and provide a new target for the treatment of GC.\u003c/p\u003e","manuscriptTitle":"SSR4 promote gastric cancer progression by regulating mitochondrial oxidative phosphorylation via NDUFB11 and ATP6AP1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-08 01:14:42","doi":"10.21203/rs.3.rs-4890970/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cf35ced4-8eed-4c80-a9f8-07792531a5df","owner":[],"postedDate":"October 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35915798,"name":"Biological sciences/Genetics/Cancer genetics"},{"id":35915799,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2024-10-22T15:36:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-08 01:14:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4890970","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4890970","identity":"rs-4890970","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}