NKAIN1, as an oncogene, promotes the proliferation and metastasis of breast cancer, affecting its prognosis

Na, K‐ATPase interaction (NKAIN) is a transmembrane protein family, which can interact with Na, K‐ATPase β1 subunit. NKAIN1 plays an important role in alcohol‐dependent diseases such as endometrial and prostate cancers. However, the relationship between NKAIN1 and human breast cancer has not been studied. Hence, this study aimed to explore the relationship between NKAIN1 expression and breast cancer. Data used in this study were mainly from the Cancer Genome Atlas, including differential expression analysis, Kaplan–Meier survival analysis, receiver operating characteristic curve analysis, multiple Cox regression analysis, co‐expression gene analysis, and gene set enrichment analysis. Analyses were performed using reverse transcription‐quantitative polymerase chain reaction, western blot analysis, and immunohistochemistry on 46 collected samples. The knockdown or overexpression of NKAIN1 in vitro in MCF‐7 and MDA‐MB‐231 cell lines altered the proliferation and migration abilities of tumor cells. In vivo experiments further confirmed that NKAIN1 knockdown effectively inhibited the proliferation and migration of cancer cells. Therefore, our study identified NKAIN1 as an oncogene that is highly expressed in breast cancer tissues. The findings highlight the potential of NKAIN1 as a molecular biomarker of breast cancer.

function. 6The prognosis of patients has improved with the development of molecular targeted therapies, including human epidermal growth factor receptor-2 (Her2), 7 poly ADP ribose polymerase, 8 cyclin-dependent kinase, 9 and immune checkpoint inhibitors. 10However, the current therapeutic outcomes remain unsatisfactory.
Recently, the diagnosis and treatment of breast cancer has been contentious, but it is still essential to identify and treat breast cancer before the onset of metastasis. 11Therefore, further studies are required to identify potential prognostic molecular biomarkers and novel therapeutic targets.
Based on the existing research on breast cancer by our group, we have screened several breast cancer genes that are co-regulated by DNA methylation and microRNAs (miRNAs) based on messenger RNA (mRNA) expression profiles (unpublished articles).Among these, the evolutionarily conserved membrane protein Na, K-ATPase interaction (NKAIN)1 has been of particular interest.
NKAIN is a new transmembrane protein family, which can interact with Na, K-ATPase β1 Subunit and includes four evolutionarily conserved membrane proteins (NKAIN1, 2, 3, and 4). 12NKAIN proteins do not contain characteristic functional domains except for the highly conserved transmembrane domains.Remarkable amino acid conservation in the first two transmembrane domains suggests that these proteins may play a role in the membrane bilayer.Research on NKAIN1 has been conducted for alcohol-dependent diseases. 13,14wever, knowledge of its relationship with human tumors is limited.
NKAIN1 can be optimized as a practical gene to predict the prognosis of prostate cancer and response rate to immunotherapy. 15In addition, NKAIN1 expression levels are known to increase in esophageal adenocarcinoma and positively correlate with its poor prognosis. 16However, the association between NKAIN1 and human breast cancer remains unclear.
Hence, this study aimed to verify NKAIN1 overexpression as a cancer-promoting gene in breast cancer using bioinformatics, 17 including method such as differential expression analysis, Kaplan-Meier survival analysis, receiver operating characteristic (ROC) curve analysis, multiple Cox regression analysis, coexpression gene analysis, and gene set enrichment analysis (GSEA).The 46 samples included in the study were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis.Moreover, NKAIN1 knockdown effectively inhibited the proliferation and migration of cancer cells in MCF-7 and MDA-MB-231 cells and was conducive to improving prognosis.Thus, NKAIN1 may serve as a potential molecular biomarker for breast cancer.

| Data source and online analysis tool
Gene expression data with clinical information (Workflow Type: HTSeq-TPM and HTSeq-FPKM) were acquired from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). 18mples with insufficient clinical information were excluded from the

| Differentially expression
We performed a logistic regression analysis to determine the correlation between NKAIN1 mRNA expression and clinical characteristics in breast cancer.We compared the differential NKAIN1 expression levels between tumors and corresponding normal tissues in 23 human tumor types using the Wilcoxon rank-sum test.In the breast cancer cohort, we analyzed the differential NKAIN1 expression levels between the tumor tissues and adjacent normal tissues.The expression levels in paired samples and breast cancer subtypes were also explored.The results are presented as scatterplots.

| Prognostic value
Overall survival (OS) was defined as the time from randomness to death due to any cause.Relapse-free survival (RFS) was defined as the time from randomness to the first disease recurrence or death from any cause.Kaplan-Meier OS and RFS curves were obtained based on gene chip data from the Kaplan-Meier plotter database.
The log-rank test was used for statistical analysis.The forest plot of the multivariate Cox regression analysis was then processed to identify the prognostic factors for breast cancer.We calculated each potential predictor's p value, hazard ratio (HR), and 95% confidence interval (CI).Prognostic factors (HR > 1 and p < 0.05) were risk factors for breast cancer prognosis.Meanwhile, those with HR < 1 and p < 0.05 were regarded as protective factors.The ROC curve was generated based on data acquired from the TCGA-breast cancer data set, to assess the accuracy of predicting the outcome of patients with breast cancer and normal controls.We developed a nomogram to predict the probability of 1-, 3-, and 5-year OS by integrating NKAIN1 and other prognostic factors in patients with breast cancer.

| Co-expression networks and GSEA
To predict the potential biological mechanism of NKAIN1 in breast cancer, we used the LinkFinder module on the LinkedOmics website to study the co-expression network of NKAIN1 in the TCGA-breast cancer data set.In the LinkInterpreter module of the same website, the GSEA method was used to identify terms significantly related to NKAIN1 co-expression genes.Gene Ontology (GO) biological process (GO-BP), GO cellular component (GO-CC), GO molecular function (GO-MF), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed.The Pearson correlation test was used as the statistical approach, and the false discovery rate (FDR) < 0.05 was defined as significantly related or enriched.

| Immune infiltrates analysis
The TIMER web server is a comprehensive resource for the systematic analysis of immune infiltrates across diverse cancer types.TIMER uses a deconvolution algorithm to deduce the abundance of tumor-infiltrating immune cells (TIICs) from gene expression profiling.The correlation between the copy number variation (CNV) of NKAIN1 and TIICs was studied.The relationship between the level of immune infiltration and NKAIN1 expression in breast cancer was also explored.

| Cell culture and treatment
Human breast cancer cells, MCF-7 and MDA-MB-231 (preserved in our laboratory), were cultured at 37°C with 5% CO 2 in Dulbecco's modified Eagle medium (DMEM; Cat#C11995500BT; GIBCO) supplemented with 10% fetal bovine serum (Cat# 04-001-1ACS; BI) and 1% antibiotics (100 U/mL penicillin and streptomycin).After reaching 90% confluence, the cells were initially washed with phosphatebuffered saline (PBS), followed by the addition of proper trypsin and incubation at 37°C for 1 min.After digestion, serum-containing medium was added to the cells, to terminate the reaction, and the cells were blown to single cells.An appropriate amount of culture medium was left, fresh culture medium was added, and cell morphology was microscopically analyzed.The cells were then placed in an incubator to continue the culture and passaged every 3 days.

| RT-qPCR
Total RNA was isolated from tissues or cultured cells using the TRIzol reagent (Tiangen Biotech) and reverse-transcribed into cDNA using a PrimeScript RT kit (Takara Bio).qPCR was performed using a C1000 Thermal Cycler CFX96 Real-Time System (Bio-Rad; Hercules) with SuperReal PreMix Plus (SYBR Green).

| Western blot analysis
Briefly, MCF-10A, MCF-7, MDA-MB-231, and breast cancer tissues (approximately 30 mg) were homogenized with Radio-Immunoprecipitation Assay buffer (Cat#R0010; Beyotime Institute of Biotechnology) for 30 min on ice and sonicated.Total protein was centrifuged at 12,000g for 15 min and the supernatant was quantified using a bicinchoninic acid assay kit (Cat#PC0020; Solarbio).The lysates were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes.After blocking with 5% fat-free dry milk, the membranes were incubated using anti-β-tubulin (Cat#10094-1-AP; Proteintech) and anti-NKAIN1 (Cat#Ag24499; Proteintech).Following primary incubation, the membranes were washed three times with Tris Buffered Saline with Tween 20 (TBST) at room temperature and incubated with Goat Anti-Rabbit immunoglobulin G (IgG) (H + L) horseradish peroxidase (1:5000, Cat# S0001; Affinity) or Goat Anti-Mouse IgG (H + L) horseradish peroxidase (1:5000, Cat# S0002; Affinity) for 1 h at room temperature.After washing three times with TBST, protein bands were visualized using an enhanced chemiluminescence kit (Cat# KF003; Affinity) and a chemiluminescence system (Amersham Imager 600).ImageJ software (version 1.51; National Institute of Health) was used to quantify the bands of each protein; β-actin was used as the loading control.

| Immunohistochemistry
Immunohistochemical staining was performed to determine NKAIN1 expression in breast cancer and adjacent tissues.Briefly, antigens were unmasked in ethylenediaminetetraacetic acid (C1034; Solarbio) using a microwave, and immunostaining was performed using the avidin-biotinylated enzyme complex method with antibodies against NKAIN1 and Ki67 or equivalent concentrations of polyclonal nonimmune IgG controls.After incubation with the appropriate biotin-conjugated secondary antibody, and subsequently with streptavidin solution, color was developed using diaminobenzidine (ZLI-9018; Zhongshanjinqiao) as a chromogen.The sections were counterstained with hematoxylin and dehydrated using increasing concentrations of ethanol and xylene.

| Lentivirus transfection
MCF-10A, MCF-7, and MDA-MB-231 cells in good condition were digested and resuspended.An appropriate number of cells were inoculated in a 24-well plate.The cells were incubated at 37°C overnight.The cells were 30%-50% fused the next day.NKAIN1 knockdown lentivirus, overexpressed lentiviruses, and their corresponding negative control viruses, were mixed and diluted proportionally with DMEM to a total volume of approximately 500 µL.The original medium from the 24-hole plate was replaced with freshly prepared medium, and cultivated in a 37°C incubator.
After 24 h, the virus-containing culture medium was replaced with fresh culture medium, and cultivated at 37°C.Transfection efficiency was determined using an inverted fluorescence microscope at 48-96 h.expressed, and their corresponding negative control groups were digested and resuspended, and 200 cells were prepared in 96 well plate μL cell suspension was cultured in 37°C incubator for 24, 48, 72, and 96 h respectively.Cell counting kit-8 (CCK-8) solution (10 µL)   was added to each well and the culture plates were incubated for 4 h.
Absorbance at 450 nm was measured using a microplate reader.The same method was used for the MCF-10A and MDA-MB-231 cells.

| Cell cloning experiment
MCF-7 cells from the NKAIN1 knockdown, overexpression, and their corresponding negative control groups in the logarithmic growth period were used to prepare cell suspensions and inoculated into sixwell plates containing culture medium for 2-3 weeks.A culture terminates when visible clones are present in the well.The supernatant was discarded, the cells were fixed with 4% paraformaldehyde, and stained with gamma isotope mapping system staining solution for 10-30 min and he number of clones formed was calculated.The same method was used for the MDA-MB-231 cells.

| Transwell
In the NKAIN1 knockdown group, the overexpressed group, and their corresponding negative control group, MCF-7 cells were serumstarved for 12 h, digested, centrifuged, the culture medium was discarded after terminating digestion, and the cells were resuspended in serum-free medium.100 μL of the cell suspension was added to the Tranwell Wells.To 24-well plates, 600 μL serum-containing medium was added and cultured for 24 h.The transwell chamber was removed, the culture solution was discarded in the hole, washed twice with calcium-free PBS, fixed with methanol for 30 min, and air dried.After staining with 0.1% crystal violet for 20 min, the upper layer of nonmigrated cells was gently wiped off using a cotton swab and washed with PBS three times.The cells were observed in five visual fields under a ×400 magnification microscope and counted.
The same method was used for the MDA-MB-231 cells.ShCTRL or shNKAIN1 cells (5 × 106) were injected in situ into the fourth pair of mouse breast pads (five per group).After 7 days, the width and length of the tumors were measured twice weekly.Tumor volume was calculated as follows: Tumor volume = (length × width 2)/2.After 21 days, the mice were euthanized, and tumor weights were measured.

| Statistical analysis
PRISM version 9 (GraphPad Software Inc.) was used for data analysis.
Student's t test or one-way analysis of variance (ANOVA) was used to determine significant differences between the groups.Two-way ANOVA was used to determine significant differences between treatments in different cell cohorts or at different time points.p < 0.05 was considered statistically significant.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns represents no significance.

| Baseline characteristics of patients and logistic regression analysis
The 1083 included patients were divided into two groups based on NKAIN1 expression level.Detailed baseline characteristics, including T stage, N stage, M stage, pathological stage, age, histological type, progesterone receptor (PR) status, estrogen receptor (ER) status, Her2 status, prediction analysis of microarray 50-genepanel (PAM50) molecular subdivisions, menopausal status, and radiation therapy are listed in Table 1.Logistic analysis of the correlation between NKAIN1 expression and the clinical characteristics of breast cancer is shown in Table 2.

| NKAIN1 expression is upregulated in breast cancer tissues
Pan-cancer differential expression analysis showed that NKAIN1 expressions in breast cancer, cholangiocarcinoma, esophageal carcinoma, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, liver hepatocellular carcinoma, prostate adenocarcinoma, and uterine corpus endometrial carcinoma were significantly higher than those in the corresponding adjacent normal tissues.Moreover, NKAIN1 expression was significantly lower in rectal adenocarcinoma, colon adenocarcinoma, and Head and Neck squamous cell carcinoma than those in normal tissues (Figure 1A).To assess NKAIN1 expression levels exclusively in patients with breast cancer, we analyzed the expression status data from the TCGA-breast cancer data set.The results indicated that NKAIN1 expression was significantly higher in breast cancer tissues than those in the adjacent normal tissues (Figure 1B).These results were consistent in paired samples (Figure 1C).Subgroup analysis revealed significantly higher NKAIN1 expression in the luminal type than that in other breast cancer types (Figure 1D).expression (Figure 2A).However, on further grouping based on whether the hormone receptor was positive, the difference in OS was no longer significant (Figure 2B,C).In contrast, high NKAIN1 expression implied a worse prognosis in RFS (Figure 2D).On grouping the cohort according to hormone receptor positivity, the difference in RFS was even more remarkable (Figure 2E,F).Multivariate Cox analysis indicated that old age, high N stage, and high M stage were independent risk factors for OS in patients with breast cancer, while high NKAIN1 expression level was an independent protective factor for OS (Figure 3A).The ROC curve showed that NKAIN1 levels could accurately predict normal and breast cancer outcomes (Figure 3B).By integrating NKAIN1 with other clinical prognostic factors, we constructed a nomogram to predict the probability of 1-, 3-, and 5-year OS (Figure 3C).

| The interaction between NKAIN1 and co-expressed genes may play a role in the occurrence and development of breast cancer
Linked Omics is a publicly available portal which includes multiomics data from all 32 TCGA cancer types and 10 clinical proteomic tumor analysis consortium cancer cohorts. 20On the LinkedOmics platform, 20155 entries linked to NKAIN1 were analyzed.Significant (FDR < 0.05) positively (red dots) and negatively (green dots) correlated genes are shown in volcano plots (Figure 4A).All linked genes are listed in Supplementary Table 1.The top 20 positively and negatively linked genes were selected and ranked in heat maps (Figure 4B,C).
These results suggest the potential biological functions of NKAIN1.

| Relationship between NKAIN1 expression and TIICs
Differences in the 16 types of TIICs between the NKAIN1 low-and NKAIN1 high-expression groups were compared.The results indicated that the low-expression group had higher number of activated T cells, B cells, CD8+ T cells, cytotoxic cells, dendritic cells, macrophages, neutrophils, Th1 cells, Th17 cells, and regulatory T (Treg) cells.Natural killer (NK) cells, mast cells, and Th2 cells were more significantly activated in the NKAIN1 high-expression group, while other TIICs showed no significant intergroup differences (Figure 5A).The TIMER database was used to investigate whether NKAIN1 expression in breast cancer is related to the level of immune invasion.The results suggested that CNV in NKAIN1 was significantly related to the infiltration levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells (Figure 5B).
This suggests that NKAIN1 levels affect the number and infiltration of tumor immune cells.

| NKAIN1 was overexpressed in breast cancer samples and cells
We performed qPCR analysis using RNA from 46 pairs of breast cancer and paired paracancerous tissues.The results revealed that NKAIN1 expression in cancerous tissues was significantly higher than that in paracancerous tissues (Figure 6A).Further, immunohistochemical staining of samples from these 46 cases revealed that NKANI1 was significantly overexpressed in breast cancer tissues compared to that in paracancerous tissues, regardless of the subtype (Figure 6B).In addition, western blot analysis of proteins extracted from these samples revealed significantly higher NKAIN1 protein expression in cancerous tissues than that in paracancerous tissues (Figure 6C).Next, we extracted RNA and proteins from the three cell lines, MCF-10A, MCF-7, and MDA-MB-231.The results for qPCR and western blot analysis revealed significantly higher NKAIN1 expression in cancer cell lines than that in normal epithelial cells (Figure S1a,b).bioinformatics analysis results, we screened the top forward and reverse-ranked genes in Figure 4A,B, and used mouse tumors to extract mRNA for qPCR experiments.The results showed that NKAIN1 knockdown reduced the expression levels of NKAIN1, RIMS4, FISP1, and KCNK15 (Figure S2a), while increasing the expression levels of SMCO4, RASAL1, and PADI2 (Figure S2b).This finding is consistent with the results of our analysis.Furthermore, immunohistochemical analysis revealed decreased expressions of KI67 and NKAIN1 in tumors of the shNKAIN1 group (Figure S2c).Breast cancer includes different subtypes exhibiting different clinical outcomes. 21Understanding this heterogeneity is key to developing targeted interventions for cancer prevention and treatment. 22idence from past and existing research, highlighting the progress in screening, diagnosis, and treatment strategies in breast cancer management, is critical for improving clinical outcomes. 23This study identified NKAIN1 as a biomarker of breast cancer prognosis.Data mining revealed significantly higher NKAIN1 expression in breast cancer tissues than that in adjacent tissues.This conclusion holds true for the paired samples.Subgroup analysis showed significantly higher NKAIN1 expression in luminal A and B-type breast cancers than those in Her2-positive and basal-like-type breast cancers.
Baseline data also showed that the proportion of hormone receptor positivity was significantly higher in the high-expression group than that in the low-expression group.Patients with luminaltype breast cancer have a significantly better prognosis than those of other types, which is a strong protective factor. 24Logistic regression analysis 25 implied that high NKAIN1 expression significantly correlates with the luminal breast cancer type.This may explain superior OS and RFS in the high NKAIN1 expression group compared to that in the low-expression group.Cox regression analysis 26 also identified NKAIN1 overexpression as a prognostic protective factor.Interestingly, when the cohort was subdivided according to hormone receptor status, the difference in OS was no longer significant.In contrast, based on the RFS results, high NKAIN1 expression implied a worse prognosis.Notably, NKAIN1 overexpression was also significantly related to advanced N stage, which might explain these seemingly contradictory results.
We further explored co-expressed genes associated with NKAIN1 expression.Through data retrieval, some of the coexpressed genes were found to be related to neurological diseases, consistent with previous reports. 27Among these, Growth Regulating Estrogen Receptor Binding 1 (GREB1) was a distinct significantly coexpressed gene. 28GREB1 is a protein-encoding gene.Diseases associated with GREB1 include Mayer-Rokitansky-Kuster-Hauser syndrome and ovarian sex cord-stromal tumors.Among the related pathways are ESR-mediated signaling and the validated nuclear ER alpha network. 29GREB1 expression is necessary for breast cancer cell proliferation; GREB1 regulates PI3K/Akt signaling to control hormone-sensitive breast cancer proliferation. 30Based on these results, we speculated that NKAIN1 might be related to the upregulation of GREB1, thereby promoting the growth and invasion of breast cancer cells.This explains the high expression of NKAIN1, particularly in hormone receptor-positive breast cancers.
Animal experiments were approved by the Medical Ethics Committee of Qilu Hospital (Qingdao) of Shandong University (ethical code: KYLL-K5-2022118).Naked mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd.The MDA-MB-231 breast cancer cell line containing fluorescently labeled lentivirus with stably knocked-down NKAIN1 and a negative control were screened, and a tumorigenic model of nude mice was constructed by in situ injection.

T A B L E 1
Clinical characteristics of the patients with breast cancer based on data from TCGA.

F I G U R E 1
Na, K-ATPase interaction (NKAIN)1 is overexpressed in breast cancer tissues.(A) Differential expression analysis of NKAIN1 in a human pan-cancer cohort.NKAIN1 expression in malignant tumors was significantly higher than that in adjacent normal tissues; (B) NKAIN1 expression in breast cancer tissues and adjacent normal tissues; (C) NKAIN1 expression in paired breast cancer tissues and adjacent normal tissues; (D) NKAIN1 expression in different types of breast cancer.

F I G U R E 2
High Na, K-ATPase interaction (NKAIN) expression is an independent protective factor for overall survival (OS) in patients with breast cancer.Kaplan-Meier curves for OS (A-C) and recurrence-free survival (RFS) (D-F) based on gene chip data from the Kaplan-Meier Plotter website.Kaplan-Meier curve of OS for (A) all cohorts; (B) hormone receptor-positive patients with breast cancer; (C) hormone receptornegative patients with breast cancer.Kaplan-Meier curve of RFS for (D) all cohorts; (E) hormone receptor-positive patients with breast cancer; (F) hormone receptor-negative patients with breast cancer.HR, hazard ratio.

F I G U R E 3
High Na, K-ATPase interaction (NKAIN) expression is associated with good breast cancer prognosis.(A) Forest plot for multivariate Cox analysis of NKAIN1 and clinical parameters in breast cancer.(B) Receiver operating characteristic curve for NKAIN1 in predicting breast cancer outcomes based on the TCGA-breast cancer data set; (C) Nomogram for predicting the probability of 1-, 3-, and 5-year overall survival in patients with breast cancer.AUC, area under the curve; CI, confidence interval; HR, hazard ratio; TPR, true positive rate.

F I G U R E 4
The Na, K-ATPase interaction (NKAIN) co-expression genes and gene set enrichment analysis (GSEA) in breast cancer.(A) Volcano plot of the highly positively (red plots) and negatively (green plots) linked NKAIN1 genes in breast cancer.(B) Heat map of the top 20 positively linked genes.(C) Heat maps of the top 20 negatively linked genes.Bar charts of the enriched terms in (D) Gene Ontology (GO) biological processes (GO_BP), (E) GO cellular components (GO_CC), (F) GO molecular function (GO_MF), and (G) KEGG pathway analysis using GSEA.

3. 7 | 3 . 8 |
Changes in NKAIN1 levels affect the proliferation and migration of breast cancer cellsWe transduced short hairpin RNAs (shRNAs) targeting NKAIN1 in the breast cancer cell lines, MDA-MB-231 and MCF-7, to knockout NKAIN1 expression.After 48 h of transduction, shNKAIN1 was found to significantly inhibit NKAIN1 expression in MDA-MB-231 and MCF-7 cells (Figure6D and S1c).We then performed CCK-8 cell proliferation and clone formation assays using scrambled shRNAtransduced MDA-MB-231 and MCF-7 cells (shCTRL) and shNKAIN1 transduced cancer cells.The CCK-8 results revealed that shNKAIN1 cell proliferation was significantly inhibited after 48 h of culture, compared to that of the shCTRL group, (Figure6E).Clone formation experiments revealed significantly reduced number of clones formed by shNKAIN1 cells compared to those in the shCTRL group (Figure6F).We investigated the role of NKAIN1 in the invasiveness of breast cancer cells.Transwell assays showed that NKAIN1 knockout effectively inhibited the migration of MDA-MB-231 and MCF-7 cells (Figure6G).Therefore, knocking down NKAIN1 expression in MCF-7 and MDA-MB-231 cells significantly decreased the proliferation, cloning, and migration abilities of breast cancer cells.We transduced overexpression plasmids targeting NKAIN1 in the breast cancer cell lines MDA-MB-231 and MCF-7 to overexpress NKAIN1.After 24 h of transduction, the overexpression group (overNKAIN1) showed significantly increased NKAIN1 expression in MDA1-MB-231 and MCF-7 cells, relative to the corresponding overCTRL group (Figure7A).We performed CCK-8 cell proliferation and colony formation assays on overCTRL and overNKAIN1 cells.The CCK-8 results showed that compared to the overCTRL group, overNKAIN1 cell proliferation was significantly accelerated after 72 h of culture (Figure7B).In addition, compared to the overCTRL group, the overNKAIN1 group showed nonsignificantly enhanced cell proliferation in MCF-10A cells (FigureS1d).The clone formation assay showed that overNKAIN1 cells formed a significantly higher number of clones than those by CTRL-overexpressing cells (Figure7C).We investigated the effect of NKAIN1 overexpression on the invasive ability of breast cancer cells.Transwell assays showed that NKAIN1 overexpression enhanced the migration ability of MDA-MB-231 and MCF-7 cells (Figure7D).In conclusion, enhanced NKAIN1 expression significantly accelerated the proliferation, cloning, and migration abilities of breast cancer cells.Depletion of NKAIN1 inhibits the growth of breast cancer cells in vivo To further investigate the role of NKAIN1 in breast cancer, MDA-MB-231 cells stably transfected with shCTRL or shNKAIN1 lentiviral vectors were injected in situ in nude mice (five nude mice per group).Measurement of the tumor volume twice weekly revealed significantly lower tumor volume in the shNKAIN1 group than that in the shCTRL control group on Day 14 (Figure 7E).On Day 21, mice were euthanized, and tumor size and weight were measured.Tumors in the shNKAIN1 group had significantly lower tumor size and weight than those in the shCTRL group (Figure 7F,G).To further validate the F I G U R E 5 Na, K-ATPase interaction (NKAIN) affects the number and infiltration level of tumor immune cells.(A) Relationship between NKAIN1 expression levels and immune infiltration in breast cancer.(B) NKAIN1 copy number variation influenced the infiltration level of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells.F I G U R E 6 Silencing Na, K-ATPase interaction (NKAIN) inhibited cell proliferation and migration.(A) Analysis of NKAIN1 RNA expression in 46 pairs of breast cancer and paired paracancerous tissues.(B) NKAIN1 expression in 12 pairs of breast cancer and paracancerous tissues (Luminal A, Luminal B, Her overexpression and triple negative, three pairs for each subtype) using immunohistochemistry, ×100.(C) NKAIN1 protein expression in six pairs of breast cancer and paracancerous tissues.(D) Results of western blot after NKAIN1 knockdown in MCF-7 and MDA-MB-231 cells.(E) Change in the proliferation ability of breast cancer cells after NKAIN1 knockdown mediated by lentivirus compared with the control group in MCF-7 and MDA-MB-231 cells.(F) Changes in cell cloning ability after NKAIN1 knockdown in MCF-7 and MDA-MB-231 cells compared with the shCTRL group.(G) Changes in the migration ability of breast cancer cells in the shCTRL and shNKAIN1 groups in MCF-7 and MDA-MB-231 cells.*p < 0.05.

Thus,
NKAIN1 knockdown effectively inhibited the growth of cancer cells in vivo.In summary, NKAIN1 promoted the proliferation and invasion of breast cancer cells both in vivo and in vitro.Simultaneously, NKAIN1 knockout significantly inhibited breast cancer development.F I G U R E 7 The levels of Na, K-ATPase interaction (NKAIN) alter breast cancer cell growth both in vivo and in vitro.(A) Western blot analysis after NKAIN1 overexpression in MCF-7 and MDA-MB-231 cells.(B) Change in the proliferation ability of breast cancer cells after NKAIN1 overexpression compared with that in the control group in MCF-7 and MDA-MB-231 cells.(C) Change in the clonogenic ability of breast cancer cells NKAIN1 overexpression compared with that in the shCTRL group in MCF-7 and MDA-MB-231 cells.(D) Changes in migration ability after NKAIN1 overexpression compared with those in the control group in MCF-7 and MDA-MB-231 cells.(E) Tumor volumes in shCTRL and shNKAIN1 groups on the indicated days.(F) Representative images and (G) tumor weights in shCTRL and shNKAIN1 groups on Day 21.Five mice were used for each set of experiments.*p < 0.05.
GO and KEGG enrichment analyses showed that T cell activation, NK cell activation, lymphocyte-mediated immunity, and other factors negatively correlated with NKAIN1 expression.Signaling pathways such as NK cell-mediated cytotoxicity and tumor necrosis factor (TNF) signaling negatively correlated with NKAIN1 expression.NK cells were initially identified because of their antitumor ability.They play essential roles in the tumor microenvironment, preventing cancer development and/or progression, and in the recognition of human tumor cells. 31TNF, a critical cytokine, can activate several intracellular signaling pathways, including apoptosis, cell survival, inflammation, and immunity.Presence of TNF-α-induced selective apoptotic cell death in ER-α-positive MCF-7 human breast cancer cells. 32In this study, we found that different NKAIN1 expression levels affect the levels of immune infiltration.High NKAIN1 expression inversely correlated with the infiltration levels of T cells, B cells, CD8 T cells, cytotoxic cells, dendritic cells, macrophages, neutrophils, Th1 cells, Th17 cells, and Treg cells.These findings suggest that NKAIN1 expression inhibits the tumor immune response in humans.In conclusion, using bioinformatics analysis, we confirmed higher NKAIN1 expression in cancers than that in adjacent noncancerous tissues.Lentivirus transfection successfully reduced NKAIN1 levels and significantly inhibited cancer cell proliferation and migration.We supplemented the cell phenotype experiment with the overexpression of NKAIN1, and the results showed that NKAIN1 overexpression significantly promoted the proliferation and metastasis of cancer cells.In addition, in vivo experiments revealed that NKAIN1 depletion inhibits the growth of cancer cells.In summary, our study preliminarily confirmed the promising role of NKAIN1 as a molecular biomarker in determining the occurrence and development of breast cancer.However, the underlying molecular mechanism and cellular pathways remain unclear and warrant further study.
Logistic analysis of correlation between NKAIN1 expression and clinical characteristics in breast cancer.
19breviations: ER, estrogen receptor; HER2, human epidermal growth factor receptor-2; PAM50, prediction analysis of microarray 50-genepanel; PR, progesterone receptor;3.3 | High NKAIN1 expression indirectly affects prognosis in patients with breast cancerThe Kaplan-Meier plotter is a web-based survival analysis tool used to assess the correlation among gene expressions (mRNA, miRNA, and protein) and survival in various tumor types based on databases, including Gene Expression Omnibus, European Genome-phenome Archive, and TCGA.19Analysis of gene chip data from the Kaplan-Meier plotter revealed significantly better OS in patients with NKAIN1 overexpression than in those with low NKAIN1 T A B L E 2 Abbreviations: NKAIN1, Na, K-ATPase interaction 1; PAM50, prediction analysis of microarray 50-genepanel.