Skip to main content
NCBI home page
3 Biotech logoLink to 3 Biotech
. 2020 May 11;10(6):246. doi: 10.1007/s13205-020-02237-x

Expressions and clinical significances of STAT3 and Grim19 in epithelial ovarian cancer

Yiru Wang 1,2, Ying Yan 1,2, Mengying Yang 1,2, Zhijun Yang 1,2,✉
PMCID: PMC7214566  PMID: 32411570

Abstract

This study aimed to explore the expressions of signal transducer and activator of transcription 3 (STAT3) and a gene associated with retinoid-interferon induced mortality (Grim19) in epithelial ovarian cancer (EOC), and to determine their correlations with tumor progression and metastasis as well as the related mechanism. Ovarian tissue specimens resected through operation in our hospital were collected, and the correlations of Grim19 and STAT3 expressions with clinicopathological indexes were detected via immunohistochemistry (IHC) and Western blotting. Their positions in cells were observed through immunofluorescence. IHC assay results showed that STAT3 had the lowest expression level in the normal ovary, followed by those in benign ovarian tumor and borderline ovarian tumor (BOT), but it had high expression in EOC; The expression level of Grim19 was the lowest in EOC, followed by those in BOT and benign ovarian tumor successively, while it was highly expressed in the normal ovary; The expressions of STAT3 and Grim19 presented negative correlations in all kinds of ovarian tissues (p < 0.05). The expression level of STAT3 in EOC had no obvious correlations with FIGO staging or WHO classification (p > 0.05). The expression level of Grim19 in EOC in stage FIGO III–IV was higher than that in stage FIGO I–II (p < 0.05), Grim19 expression was not obviously associated with WHO classification (p > 0.05). The expressions of Grim19 and STAT3 in lymphatic metastasis lesion had significantly positive correlations with the primary lesion (p < 0.05). The Western blotting assay results were identical with the IHC results. The immunofluorescence demonstrated that STAT3 and Grim19 were mainly localized in the cytoplasm and they were colocalized in mitochondria. In conclusion, STAT3 presents high expression in EOC tissues while Grim19 is expressed in EOC tissues at a low level, which may be related to its interaction with STAT3 as well as progression, metastasis and poor prognosis of ovarian cancer.

Keywords: Grim19, Metastasis, Ovarian cancer, Prognosis, Progression, STAT3

Introduction

As one of the three major gynecologic malignant tumors, ovarian cancer (OC) is the primary death cause for gynecologic malignant tumors among women (Torre et al. 2018), where epithelial ovarian cancer (EOC) is the most common, accounting for over 95% of ovarian malignant tumors, and its clinical manifestations and therapeutic effects may vary from subtype to subtype (Lheureux et al. 2019a). In the past 20 years, the overall 5-year survival rate of malignant tumors belonging to different systems is elevated, but the 5-year survival rate of OC is not obviously elevated (< 47%) due to concealed onset, fast progression and frequent recurrence of OC (Lheureux et al. 2019b). Distant metastasis and chemotherapy resistance are difficulties faced in the clinical treatment of EOC (Christie and Bowtell 2017).

Signal transducer and activator of transcription 3 (STAT3), one of the member of STAT protein family, can be activated by Janus protein tyrosine kinase (JAK). Regulating target genes after binding to DNA, STAT3 plays a significant role in physiological activities such as cell proliferation, differentiation and angiogenesis (Verhoeven et al. 2020). The activation of STAT3 is rapid and transient under normal physiological conditions. The cellular anti-apoptosis ability will be enhanced under persistent activation of STAT3, thus resulting in inactivation of the normal cellular apoptotic function and genesis of a tumor, moreover, this will exert important effects on proliferation and invasion of tumor cells and induction of immunosuppression (Huang et al. 2020; Loh et al. 2019; Yu et al. 2014).

The gene associated with retinoid-interferon induced mortality (Grim19), also known as type I NADH dehydrogenase 1 alpha subcomplex13 (NDUFA13), is a new-type tumor suppressor protein jointly induced by all-trans retinoic acid (ATRA) and interferon β. It is reported that Grim19 specifically acts upon STAT3 to inhibit its anti-apoptosis ability and it presents a low expression trend in various malignant tumors such as prostatic cancer and cutaneous squamous cell carcinoma (Lin et al. 2019; Wu et al. 2017). Hu et al. deems that moderate down-regulation of Grim19 in diseases promotes the generation of mitochondria electron leakage, increases H2O2 in cytoplasm, up-regulates the expression of PRX2 and accelerates the dimerization of STAT3, thus activates anti-apoptotic signals (Hu et al. 2017). STAT3 plays an important role in tumor progression and tumor microenvironment, which has become one of new targets of tumor-targeted therapy (Li et al. 2018), but the effect of Grim19 on tumors through its specific action on STAT3 remains to be further investigated.

In the present study, the expression levels and intracellular localizing of STAT3 and Grim19 in EOC were detected, and their correlations with clinicopathological indexes were also explored.

Materials and methods

Data source

All tissue sections were collected from ovarian specimens resected through operative treatment in Guangxi Medical University Affiliated Tumor Hospital from January 2013 to October 2018, where 145 were collected from EOC with patients aging from 25 to 78 years old (average age: 51.6 years old) (Table 1); 23 from metastatic lymph glands obtained through excision in the same period of OC in an advanced stage; 25 from the epithelial borderline ovarian tumor (BOT) with patients aging from 17 to 52 years old (average age: 34.6 years old); 81 from benign epithelial ovarian tumor with patients aging from 13 to 83 years old (average age: 41.4 years old); 83 from normal ovary experiencing adnexectomy as required by the treatment with patients aging from 43 to 67 years old (average age: 50.8 years old). All of the obtained tissue specimens were excised for the first time after definite pathological diagnosis with complete data. Tissues were embedded with paraffin following methanol polymer fixation, gradient ethanol dehydration and xylene hyalinization, 4 μm of paraffin sections were continuously cut from each specimen. Fresh tissue specimens: ovarian specimens excised in Guangxi Medical University Affiliated Tumor Hospital from September 2018 to February 2019 were collected, where six specimens were from EOC, five from benign epithelial ovarian tumor and six from the normal ovary, all tissue specimens had the definite pathological diagnosis and complete data, and they were rapidly taken after excision in middle of an operation and frozen in liquid nitrogen. The acquisition and use of all tissue specimens obtained informed consent from patients and approved by Ethics Committee of Guangxi Medical University Affiliated Tumor Hospital.

Table 1.

WHO histological classification and FIGO classification of epithelial ovarian cancer

FIGO staging WHO histological classification of epithelial ovarian cancer (case) Total
Low-grade serous ovarian cancer High-grade serous ovarian cancer Mucinous ovarian cancer Endometrioid ovarian cancer Ovarian clear cell carcinoma
I 0 3 1 0 2 6
II 4 13 1 3 1 22
III 6 72 4 5 5 92
IV 3 20 2 0 0 25
Total 13 108 8 8 8 145
Open in a new tab

Immunohistochemistry (IHC)

Paraffin sections were taken and placed in a 60 ℃ incubator for 1 h. Antigen retrieval was implemented using sodium citrate antigen retrieval solution (Solarbio, Beijing, China) through microwave method after dewaxing and rehydration with xylene and gradient ethanol, endogenous peroxidase was removed using 3% H2O2, nonspecific binding site was confined for 1 h using confining liquid (CST, Boston, USA) at room temperature, and STAT3 (Catalog no. ab68153; Dilution 1:500; Abcam, Cambridge, UK) and Grim19 antibodies (Catalog no. ab186848; Dilution 1:500; Abcam, Cambridge, UK) were incubated at 4 °C and stayed overnight; On the next day, the sections were rinsed using PBS after returning to room temperature to incubate second antibodies (CST, Boston, USA), followed by developing using DAB (CST, Boston, USA) for 10 min, rinsing using PBS and cell nucleus staining using hematoxylin (Solarbio, Beijing, China). After blue restoration in clear water, the sections were dehydrated and confined using neutral resin, then images (400×) were acquired via a digital imaging system (Motic-BA600Mot; Motic, Beijing, China). The IHC positive results were quantitatively determined through the image processing software Image J 1.51 (National Institutes of Health, Bethesda, MA, USA). On the precondition that there were no false positive or false negative results, the gray levels (maximum gray level of Image J was Gmax = 256) in five visual fields of the same size with the strongest coloring from each specimen were taken, their area fractions were calculated for quantitative analysis.

Immunofluorescence assay

Frozen tissues were taken out from liquid nitrogen and placed in freezing microtome for rewarming, the sections were continuously cut after optimal cutting temperature (OCT) embedding. The immunofluorescent staining was implemented in darkroom together with antibody incubation and IHC assay. The next day, after sections were rewarmed, they were divided into DAPI staining group and Mito-tracker staining group and placed at room temperature for second antibody incubation with fluorescence, the sections were confined after being rinsed using PBS (glycerin: PBS = 1:1), and then they were rapidly placed under a laser confocal scanning microscope (FV1000; Olympus, Tokyo, Japan) to observe the intracellular localizing conditions of target proteins.

Western blotting

The frozen tissues were taken from liquid nitrogen, RIPA lysate and protease inhibitor (Solarbio, Beijing, China) were mixed according to the proportion of 100:1, and the mixed solution was added into the frozen tissues, the specimens were put under pyrolysis on ice for 30 min after being fully grinded in a homogenizer, followed by centrifugation at 12,000 rpm for 30 min, the supernatant was taken, and protein concentrations were determined using fresh BCA working solution (Thermo Fisher Scientific, Waltham, MA, USA), the protein working solution was configured according to loading quantity of protein and measured protein concentrations, and the protein solution was placed in 99 °C water bath for 10 min to ensure full protein degeneration. 10% SDS–polyacrylamide was prepared according to molecular weights of proteins for gel electrophoresis (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membrane (Millipore, Boston, MA, USA) for 2 h confining, then it was incubated using primary antibodies of STAT3 and Grim19 (Dilution 1:500; Abcam, Cambridge, UK), respectively, GAPDH was used for internal reference, and they stayed overnight at 4 ℃. The next day, PVDF membrane was incubated using the second antibodies at room temperature for 2 h, after washing with TBST, membrane was visualized in a gel imaging system (Protein simple, Santa Clara, CA, USA).

Databases

The interactive relationship between target proteins was initially defined using the search tool String for the retrieval of interacting genes/proteins. The relationships of STAT3 and Grim19 with OC were determined via HPA (Human Protein Atlas) database (Knut and Alice Wallenberg Foundation, Sweden).

Statistical analysis

All data were analyzed using SPSS 24.0 software (SPSS Inc, Chicago, IL, USA). Analysis of variance was used for comparing multiple sample averages. If variances were proved homogeneous through homogeneity test of variances, LSD test would be used. If heterogeneity of variances existed, Dunnett’s test would be chosen. Independent-sample t test was used for two sample averages. Spearman correlation test was used for correlation between two samples. The interaction between two samples was analyzed via a simple linear regression model. A value of p < 0.05 was considered to be statistically significant.

Results

String database and HPA database

The interaction between the proteins was inquired from String database, and as verified by the experiment and reported by related literatures, STAT3 and Grim19 presented a strong interaction. As a constituent part of mitochondria complex I, Grim19 participates in activities of STAT3, the two proteins bind to each other and their specific interaction influences their functions.

STAT3 and Grim19 expressions in human ovarian tumor were observed by inquiring HPA database. As a cancer-related gene, STAT3 is commonly expressed in all kinds of cancers, but the specificity of Grim19 is low in all detected cancers. Based on the survey of 373 OC patients and semi-quantitative expression levels of STAT3 and Grim19, STAT3 and Grim19 proteins were detected in OC tissues of most patients, and the content of Grim19 was correlated with the prognosis of OC patients (Fig. 1a, b).

Fig. 1.

Fig. 1

Open in a new tab

The interactive relationship between Grim19 and STAT3 proteins and their correlations with OC inquired in String database and HPA database. a The String database indicates the correlation between STAT3 and Grim19. (1) According to experimental verification and literature reports, STAT3 interacts with Grim19. (2) The edge confidence of the interaction between STAT3 and Grim19 is the strongest. (3) STAT3 binds to Grim19; b HPA database shows semi-quantitative results of Grim19 and survival analysis of 373 OC patients. The expression level of Grim19 is obviously correlated with the survival time of patients, p < 0.05

Expressions of STAT3 and Grim19 proteins in different tissues via IHC assay

The IHC results showed that STAT3 protein was commonly expressed in OC tissues and mainly located in the cytoplasm. A small quantity of STAT3 protein was distributed in the cell nucleus and expressed on the wall of the capillary vessel, being brown or chocolate brown. Most Grim19 proteins were expressed in cytoplasm in granular form, and positive expressed on the wall of the capillary vessel (Fig. 2a).

Fig. 2.

Fig. 2

Open in a new tab

Expression levels of STAT3 and Grim19 in ovarian tissues and their correlation via IHC assay. a Distributions of STAT3 and Grim19 in EOC via IHC assay (×400). The two proteins are mainly distributed in cytoplasm, being brown or chocolate brown, expressed on the wall of capillary vessel, and red arrow points at capillary vessel; b expressions and distributions of STAT3 and Grim19 in ovarian tissues via IHC assay (×400), the row indicates HE staining, IHC-STAT3 and IHC-Grim19 successively according to different staining methods, and the column denotes different histological types, being (from left to right); c, d statistical analysis of quantitative results of expression levels of STAT3 and Grim19 proteins measured through IHC assay. The histogram shows (from left to right) normal ovary, benign ovarian tumor, BOT and EOC. The comparative differences between every two groups are statistically significant, p = 0.000; e, f correlation between the expression level of STAT3 and that of Grim19 via correlation analysis and regression statistical analysis, where e denotes P–P plot of residual error and approximately normal distribution graph, f displays scatter diagram and linear regression graph, independent variable is Grim19 and dependent variable is STAT3

The quantitative results showed that the expression of STAT3 presented obvious decline trend in EOC, epithelial BOT, benign epithelial ovarian tumor and normal ovarian tissues, and the expression differences in various tissues were of statistical significance (p = 0.000); The expression of Grim19 presented obvious progressive increase trend in EOC, epithelial BOT, benign epithelial ovarian tumor and normal ovarian tissues, and the expression differences in various tissues were statistically significant (p = 0.000) (Table 2, Fig. 2b–d).

Table 2.

Comparison of STAT3 and Grim19 protein expression in different ovarian tissues

Group N STAT3 F p Grim19 F p
Normal ovary 83 31.40 ± 1.25b,c,d 152.48 0.000 37.55 ± 1.02b,c,d 184.074 0.000
Benign ovarian tumor 81 40.58 ± 1.52a,c,d 22.84 ± 0.67a,c,d
Borderline ovarian tumor 25 52.07 ± 3.02a,b,d 19.47 ± 1.12a,b,d
Epithelial ovarian cancer 145 68.08 ± 1.20a,b,c 14.17 ± 0.56a,b,c
Open in a new tab

Compared with normal ovarian group, ap = 0.000; compared with benign ovarian tumor, bp = 0.000; compared with borderline ovarian tumor, cp = 0.000; compared with epithelial ovarian cancer, dp = 0.000

The Spearman correlation test of expression levels of STAT3 and Grim19 in all specimens was performed, and the correlation coefficient between the two proteins was determined as rs = − 0.550 (p = 0.000), indicating that STAT3 presented a negative correlation with Grim19 in ovarian diseases, namely the rising expression of STAT3 was correlated with low expression of Grim19. The simple linear regression model was used to analyze the interaction between the two proteins in ovarian tissues. By drawing the scatter diagram, we found that STAT3 had a linear correlation with Grim19. By drawing standardized residual scatter diagram and P–P plot, we further observed the residual error, homogeneity of variance and approximately normal distribution of quantitative results of the samples, the regression equation was: STAT3 protein content = 72.57 − 0.93 × Grim19 protein content, and the effect of Grim19 protein content in ovarian tissues on STAT3 was statistically significant (F = 122.987, p = 0.000) (Fig. 2e, f).

Correlation of expressions of STAT3 and Grim19 with clinicopathological indexes

According to WHO classification, EOC could be divided into five types. The five phenotypes had no statistical differences in the expression levels of STAT3 and Grim19 (p > 0.05). According to FIGO staging, EOC could be divided into four stages, and the patients were divided into two groups (early-stage I–II and advanced stage III–IV). The expression of STAT3 in early-stage I–II was comparable to that in advanced stage III–IV, and the expression levels in different FIGO stages were not statistically significant (p > 0.05); The expression of Grim19 in advanced stage III–IV was higher than that in early-stage I–II, and the difference was statistically significant (p < 0.05).

According to initial treatments, the patients were divided into accepting primary cytoreductive surgery group and accepting the interval cytoreductive surgery group after conventional chemotherapy using platinum drugs. The expression of STAT3 had no obvious correlation with initial treatment method (p > 0.05); The expression level of Grim19 in patients accepting NACT + IDS treatment was higher than accepting PDS followed by chemotherapy, and the difference was statistically significant (p < 0.05) (Table 3, Fig. 3a, b).

Table 3.

Expressions of Grim19 and STAT3 in epithelial ovarian cancer and their relationships with clinical pathological parameters

Clinical pathological index N STAT3 Grim19
Expression level t/F p Expression level t/F p
FIGO staging
 I–II 28 66.24 ± 3.70 − 0.713 0.477 11.49 ± 1.12 − 2.514 0.017
 III–IV 117 68.48 ± 1.23 14.73 ± 0.63
WHO histological type
 Serous carcinoma 121 67.11 ± 1.41 0.236 0.790 13.55 ± 0.67 0.111 0.895
 Other types (mucinous carcinoma, Endometrioid carcinoma, clear cell carcinoma) 24 69.83 ± 2.90 14.61 ± 1.27
Initial treatment method
 PDS + chemotherapy 72 67.78 ± 1.83 − 0.227 0.820 12.85 ± 0.76 − 2.167 0.032
 NACT + IDS 73 68.34 ± 1.60 15.22 ± 0.79
Metastatic
 Primary lesion 23 69.75 ± 2.53 0.058 0.954 15.05 ± 1.12 − 0.225 0.823
 Metastatic lymph node 23 69.94 ± 2.19 14.66 ± 1.27
Open in a new tab

Fig. 3.

Fig. 3

Open in a new tab

Correlations of STAT3 and Grim19 with different clinicopathological indexes via IHC assay (×400). a The expression levels of STAT3 and Grim19 in different FIGO stages of EOC via IHC assay. Positive expression is indicated by brown or chocolate brown. The row denotes different staining methods, being HE staining, IHC-STAT3 and IHC-Grim19 in succession. The column displays (from left to right) stage FIGO I–II and stage FIGO III–IV; b the expression levels of STAT3 and Grim19 under different WHO histological types in EOC via IHC assay. The row denotes different staining methods, being HE staining, IHC-STAT3 and IHC-Grim19 in succession. The column displays different WHO histological types, being (from 1 to 5) low-grade serous ovarian cancer (LGSOC), high-grade serous ovarian cancer (HGSOC), endometrioid ovarian cancer, ovarian clear cell carcinoma (OCCC) and mucinous ovarian cancer (MOC)

A total of 23 high-grade serous ovarian cancer (HGSOC) specimens in an advanced stage and metastatic lymph gland specimens resected in the same period were collected. The expression levels of STAT3 and Grim19 had no statistical differences between the primary lesion and metastatic lymph node (p > 0.05). According to simple linear regression and Spearman correlation test, the expression levels of STAT3 and Grim19 had a positive correlation and linear regression between the primary lesion and metastatic lymph node, and the difference was statistically significant (p < 0.05) (F = 24.427, p = 0.0005) (Fig. 4a–e).

Fig. 4.

Fig. 4

Open in a new tab

Expression levels of STAT3 and Grim19 in the primary lesion of EOC and lymphatic metastatic lesion via IHC assay. a Expression levels of STAT3 and Grim19 in primary lesion and lymphatic metastatic lesion of EOC (×400). Positive expression is indicated by brown or chocolate brown. The row represents IHC-STAT3 and IHC-Grim19, and the column (from left to right) displays primary lesion and metastatic lymph node successively. b, c Correlations of quantitative results of STAT3 in primary lesion and metastatic lymph node through correlation analysis and regression statistical analysis, b is P–P plot of residual error and approximately normal distribution, c displays scatter diagram and linear regression graph, the independent variable is expression level of STAT3 in primary lesion and the dependent variable is expression level of STAT3 in Metastatic lymph node (R2 = 0.350, y = 34.18 + 0.51x); d, e correlations of quantitative results of Grim19 in primary lesion and metastatic lymph node through correlation analysis and regression statistical analysis, d is P–P plot of residual error and approximately normal distribution, e displays scatter diagram and linear regression graph, independent variable is expression level of Grim19 in primary lesion and dependent variable is expression level of Grim19 in metastatic lymph node (R2 = 0.538, y = 2.16 + 0.83x)

Expression levels of STAT3 and Grim19 in different tissues via Western blotting assay

To further verify the expression levels of STAT3 and Grim19 in ovarian tissues, Western blotting was used to detect the expressions of STAT3 and Grim19 in EOC, benign epithelial ovarian tumor and normal ovarian tissues. The expression level of STAT3 was gradually elevated with the progress of diseases, which were identical with the IHC results, and the differences were statistically significant (p < 0.05). The expression level of Grim19 presented an overall decline trend with the progression of diseases, which were also identical with the IHC results, and the differences were statistically significant (p < 0.05) (Fig. 5a–c).

Fig. 5.

Fig. 5

Open in a new tab

Expressions and Localizations of STAT3 and Grim19 in EOC. a–c Expression levels of STAT3 and Grim19 proteins in ovarian tissues via Western blotting assay. a Expression levels of STAT3 and Grim19 in different ovarian tissues via Western blotting assay, The row indicates different proteins, being Grim19, STAT3 and GAPDH; the column represents different ovarian tissue types, being (from left to right) OC, benign ovarian tumor and normal ovary; b, c histograms: statistical analysis of expression levels of STAT3 and Grim19 in different ovarian tissues via Western blotting assay, and the comparative differences between every two groups are statistically significant. d Cellular localization of STAT3 and Grim19 in EOC through immunofluorescence assay (×400): the first row displays DAPI, STAT3, Grim19 and DAPI + STAT3 + Grim19 from left to right, and the second row represents Mito Tracker, STAT3, Grim19 and Mito Tracker + STAT3 + Grim19 from left to right

Localization of STAT3 and Grim19 in EOC via immunofluorescent assay

To define subcellular localization of STAT3 and Grim19 in EOC, five specimens from EOC were performed by immunofluorescence assay and divided into two groups: the DAPI group, which DAPI was used to localize cell nucleus, Alexa Fluor488 to mark STAT3 protein and Cy3 to mark Grim19, and the Mito Tracker group, which Mito Tracker was used to mark mitochondria, Alexa Fluor488 to mark STAT3 protein and Alexa Fluor405 to mark Grim19, the subcellular localization of specimens were observed under an electron microscope. We observed that STAT3 and Grim19 were co-localized in cytoplasm, and a small quantity of STAT3 protein was expressed in the cell nucleus. Grim19 was mainly distributed in the cytoplasm of EOC and presented obvious co-localization phenomenon with mitochondria, Grim19 and STAT3 were co-localized in mitochondria (Fig. 5d).

Discussion

Among the members of STAT protein family, it is proved that STAT3 and STAT5 play vital roles in the progression of cancers (Han et al. 2019). STAT3 is closely associated with inflammatory reaction, angiogenesis, metabolism, invasion and metastasis in tumors. It has been found that STAT3 is excessively activated in various cancers like breast cancer and lung cancer, and it is correlated with poor clinical prognosis (Banerjee and Resat 2016; Caetano et al. 2018). STAT3 may be a promising target of tumor therapy.

As the accessory subunit of mitochondria complex I, Grim19 exists in FeS cluster with low electrochemical potential and it presents low expression in many tumors (Ni et al. 2018). Moderate down-regulation of Grim19 will promote dimerization of STAT3 so as to repress tumor cell apoptosis. STAT3 can interact with Grim19 upon mitochondria, and it has become a potential therapeutic target because of regulating effect and functions of mitoSTAT3 in cellular activities (Rincon and Pereira 2018). This study aims at investigating the expressions and localization of STAT3 and Grim19 in EOC and exploring their correlations with tumor progression and metastasis as well as the related mechanisms.

In this study, according to String database and HPA database, STAT3 and Grim19 present a strong interaction and commonly expressed on EOC. And the content of Grim19 is correlated with the prognosis of OC patients. Further detection results show that STAT3 and Grim19 mainly locate in the cytoplasm and expressed strongly on the wall of capillary vessel. This study demonstrates that STAT3 and Grim19 have obvious differences and interact with each other in the progression of ovarian diseases, and the expression of STAT3 is negatively correlated with that of Grim19. The overall trends of expression levels of STAT3 and Grim19 detected via Western blotting are identical with the IHC results, further confirming their roles in EOC. This suggests that STAT3 and Grim19 have a significant effect on both the tumor cell and tumor microenvironment. Recent studies have confirmed that down-regulating STAT3 expression level can effectively inhibit tumor progression and angiogenesis (Chang et al. 2017; Zhang et al. 2020). The expression level of STAT3 in EOC is higher than those in other ovarian diseases in the control group, meaning that STAT3 is continuously expressed in OC, so as to exert its anti-apoptosis effect, while apoptosis repression in tumor cells is closely related to cancer recurrence and resistance of platinum drugs (Peng et al. 2018). On the contrary to STAT3, the expression level of Grim19 is reduced with the progression of the disease and it is obviously reduced in EOC. Under the interaction with STAT3, its specific inhibiting effect on anti-apoptotic protein STAT3 will be degraded.

The expression level of Grim19 is different with the progression of EOC, the expression level of Grim19 in EOC in the advanced stage is slightly higher than that in early stage, which is also closely related to high expression of STAT3. The standard therapy of EOC is usually PDS+ chemotherapy. A hot discussion prevails in recent years, comparing the curative effect and prognosis of NACT + IDS treatment and traditional standard treatment (Ghirardi et al. 2020; Himoto et al. 2019). This study points out that among the primary treatment methods of EOC, the expression level of Grim19 in patients accepting NACT + IDS treatment is significantly higher than those accepting PDS+ chemotherapy, and its inhibiting effect on anti-apoptosis is enhanced, indicating the sensitivity of Grim19 to chemotherapeutics is high. In the meantime, the expression levels of STAT3 and Grim19 in the primary lesion of ECO in an advanced stage and metastatic lesion are obviously correlated, indicating that they may have an important effect on lymphatic metastasis of EOC.

Mitochondria play a crucial role in the development of cancer (Pustylnikov et al. 2018). Grim19 acts as the accessory subunit of mitochondrial complex I to participate in various cellular activities. The immunofluorescent staining results show that STAT3 and Grim19 are mainly distributed in the cytoplasm in EOC, and they are co-localized in mitochondria, participating in the metabolic regulation effect of mitochondria (Tammineni et al. 2013).

STAT3 is an important role in apoptosis, its abnormal increasing can exert adverse effects in the progression and prognosis of malignant ovarian diseases (Wen et al. 2018). According to our study, the high expression of STAT3 in EOC indicates that it is persistently activated to exert the apoptosis-inhibiting effect while repressing targets of JAK/STAT3 signaling pathway in different stages is a possible therapeutic target of OC (Johnson et al. 2018; Li et al. 2020). As an important subunit of mitochondria complex I, Grim19 plays a significant role in cellular activities. In EOC, the specific negative correlation between STAT3 and Grim19 has provided a direction for target therapy of OC, thus becoming a potential treatment spot. In the meantime, partial elevating expression of Grim19 in EOC of advanced stage after NACT + IDS treatment hints its sensitivity to chemotherapeutics, and this provides a direction for further exploring the effects of chemotherapeutics in OC (Ni et al. 2018).

Taken together, STAT3 interacts with Grim19 in EOC. The anti-apoptosis ability can be fully exerted by moderately down-regulating the expression level of Grim19 and increasing that of STAT3 in EOC. The two proteins are associated with progression, metastasis and poor prognosis of OC, which may become potential therapeutic targets in OC.

Author contributions

ZY designed, analyzed the experiments and wrote the manuscript. YW performed the experiments and co-wrote the manuscript. YY and MY analyzed some experiment data and co-wrote the manuscript.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical statements

The protocol of all procedures had been approved by the Ethics Committee of Guangxi Medical University Affiliated Tumor Hospital.

References

  1. Banerjee K, Resat H. Constitutive activation of STAT3 in breast cancer cells: a review. Int J Cancer. 2016;138:2570–2578. doi: 10.1002/ijc.29923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Caetano MS, Hassane M, Van HT, Bugarin E, Cumpian AM, McDowell CL, Cavazos CG, Zhang H, Deng S, Diao L, Wang J, Evans SE, Behrens C, Wistuba II, Fuqua SAW, Lin H, Stabile LP, Watowich SS, Kadara H, Moghaddam SJ. Sex specific function of epithelial STAT3 signaling in pathogenesis of K-ras mutant lung cancer. Nat Commun. 2018;9:4589. doi: 10.1038/s41467-018-07042-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang N, Ahn SH, Kong DS, Lee HW, Nam DH. The role of STAT3 in glioblastoma progression through dual influences on tumor cells and the immune microenvironment. Mol Cell Endocrinol. 2017;451:53–65. doi: 10.1016/j.mce.2017.01.004. [DOI] [PubMed] [Google Scholar]
  4. Christie EL, Bowtell DDL (2017) Acquired chemotherapy resistance in ovarian cancer. Ann Oncol, 28: viii13-viii15 [DOI] [PubMed]
  5. Ghirardi V, Moruzzi MC, Bizzarri N, Vargiu V, D'Indinosante M, Garganese G, Pasciuto T, Loverro M, Scambia G, Fagotti A. Minimal residual disease at primary debulking surgery versus complete tumor resection at interval debulking surgery in advanced epithelial ovarian cancer: a survival analysis. Gynecol Oncol. 2020;157:209–213. doi: 10.1016/j.ygyno.2020.01.010. [DOI] [PubMed] [Google Scholar]
  6. Han Y, Wu J, Yang W, Wang D, Zhang T, Cheng M. New STAT3-FOXL2 pathway and its function in cancer cells. BMC Mol Cell Biol. 2019;20:17. doi: 10.1186/s12860-019-0206-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Himoto Y, Cybulska P, Shitano F, Sala E, Zheng J, Capanu M, Nougaret S, Nikolovski I, Vargas HA, Wang W, Mueller JJ, Chi DS, Lakhman Y. Does the method of primary treatment affect the pattern of first recurrence in high-grade serous ovarian cancer? Gynecol Oncol. 2019;155:192–200. doi: 10.1016/j.ygyno.2019.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hu H, Nan J, Sun Y, Zhu D, Xiao C, Wang Y, Zhu L, Wu Y, Zhao J, Wu R, Chen J, Yu H, Hu X, Zhu W, Wang J. Electron leak from NDUFA13 within mitochondrial complex I attenuates ischemia-reperfusion injury via dimerized STAT3. Proc Natl Acad Sci USA. 2017;114:11908–11913. doi: 10.1073/pnas.1704723114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Huang Q, Zhong Y, Dong H, Zheng Q, Shi S, Zhu K, Qu X, Hu W, Zhang X, Wang Y. Revisiting signal transducer and activator of transcription 3 (STAT3) as an anticancer target and its inhibitor discovery: where are we and where should we go? Eur J Med Chem. 2020;187:111922. doi: 10.1016/j.ejmech.2019.111922. [DOI] [PubMed] [Google Scholar]
  10. Johnson DE, O'Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Rev Clin Oncol. 2018;15:234–248. doi: 10.1038/nrclinonc.2018.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lheureux S, Braunstein M, Oza AM. Epithelial ovarian cancer: evolution of management in the era of precision medicine. CA Cancer J Clin. 2019;69:280–304. doi: 10.3322/caac.21559. [DOI] [PubMed] [Google Scholar]
  12. Lheureux S, Gourley C, Vergote I, Oza AM. Epithelial ovarian cancer. Lancet. 2019;393:1240–1253. doi: 10.1016/S0140-6736(18)32552-2. [DOI] [PubMed] [Google Scholar]
  13. Li F, Qi J, Qin C, Fu Z, Ren W. Taurolidine promotes cell apoptosis by enhancing GRIM19 expression in liver cancer. Oncol Rep. 2018;40:3743–3751. doi: 10.3892/or.2018.6711. [DOI] [PubMed] [Google Scholar]
  14. Li H, Qian Y, Wang X, Pi R, Zhao X, Wei X. Targeted activation of Stat3 in combination with paclitaxel results in increased apoptosis in epithelial ovarian cancer cells and a reduced tumour burden. Cell Prolif. 2020;53:e12719. doi: 10.1111/cpr.12719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lin H, Lin T, Lin J, Yang M, Shen Z, Liu H, Zou Z, Zheng Z. Inhibition of miR-423-5p suppressed prostate cancer through targeting GRIM-19. Gene. 2019;688:93–97. doi: 10.1016/j.gene.2018.11.021. [DOI] [PubMed] [Google Scholar]
  16. Loh CY, Arya A, Naema AF, Wong WF, Sethi G, Looi CY. Signal transducer and activator of transcription (STATs) proteins in cancer and inflammation: functions and therapeutic implication. Front Oncol. 2019;9:48. doi: 10.3389/fonc.2019.00048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ni F, Yan CY, Zhou S, Hui PY, Du YH, Zheng L, Yu J, Hu XJ, Zhang ZG. Repression of GRIM19 expression potentiates cisplatin chemoresistance in advanced bladder cancer cells via disrupting ubiquitination-mediated Bcl-xL degradation. Cancer Chemother Pharmacol. 2018;82:593–605. doi: 10.1007/s00280-018-3651-3. [DOI] [PubMed] [Google Scholar]
  18. Peng Z, Zhang C, Zhou W, Wu C, Zhang Y. The STAT3/NFIL3 signaling axis-mediated chemotherapy resistance is reversed by Raddeanin A via inducing apoptosis in choriocarcinoma cells. J Cell Physiol. 2018;233:5370–5382. doi: 10.1002/jcp.26362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pustylnikov S, Costabile F, Beghi S, Facciabene A. Targeting mitochondria in cancer: current concepts and immunotherapy approaches. Transl Res. 2018;202:35–51. doi: 10.1016/j.trsl.2018.07.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rincon M, Pereira FV. A new perspective: mitochondrial Stat3 as a regulator for lymphocyte function. Int J Mol Sci. 2018;19:E1656. doi: 10.3390/ijms19061656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tammineni P, Anugula C, Mohammed F, Anjaneyulu M, Larner AC, Sepuri NB. The import of the transcription factor STAT3 into mitochondria depends on GRIM-19, a component of the electron transport chain. J Biol Chem. 2013;288:4723–4732. doi: 10.1074/jbc.M112.378984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Torre LA, Trabert B, DeSantis CE, Miller KD, Samimi G, Runowicz CD, Gaudet MM, Jemal A, Siegel RL. Ovarian cancer statistics, 2018. CA Cancer J Clin. 2018;68:284–296. doi: 10.3322/caac.21456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Verhoeven Y, Tilborghs S, Jacobs J, De Waele J, Quatannens D, Deben C, Prenen H, Pauwels P, Trinh XB, Wouters A, Smits ELJ, Lardon F, van Dam PA. The potential and controversy of targeting STAT family members in cancer. Semin Cancer Biol. 2020;60:41–56. doi: 10.1016/j.semcancer.2019.10.002. [DOI] [PubMed] [Google Scholar]
  24. Wen W, Lowe G, Roberts CM, Finlay J, Han ES, Glackin CA, Dellinger TH. Pterostilbene suppresses ovarian cancer growth via induction of apoptosis and blockade of cell cycle progression involving inhibition of the STAT3 pathway. Int J Mol Sci. 2018;19:E1983. doi: 10.3390/ijms19071983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wu N, Hui H, Cui L, Yang F. GRIM-19 represses the proliferation and invasion of cutaneous squamous cell carcinoma cells associated with downregulation of STAT3 signaling. Biomed Pharmacother. 2017;95:1169–1176. doi: 10.1016/j.biopha.2017.09.055. [DOI] [PubMed] [Google Scholar]
  26. Yu H, Lee H, Herrmann A, Buettner R, Jove R. Revisiting STAT3 signalling in cancer: new and unexpected biological functions. Nat Rev Cancer. 2014;14:736–746. doi: 10.1038/nrc3818. [DOI] [PubMed] [Google Scholar]
  27. Zhang ZH, Li MY, Wang Z, Zuo HX, Wang JY, Xing Y, Jin C, Xu G, Piao L, Piao H, Ma J, Jin X. Convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer. Phytomedicine. 2020;68:153172. doi: 10.1016/j.phymed.2020.153172. [DOI] [PubMed] [Google Scholar]

Articles from 3 Biotech are provided here courtesy of Springer

RESOURCES