This study assessed the hypothesis that the protein levels of high mobility group box 1 (HMGB1) and hepsin can be used as markers for diagnosis and prognosis in cervical carcinoma. Seventy patients with cervical cancer who were hospitalized in Xuzhou Central Hospital from May 2008 to June 2010 and underwent surgical treatment were selected for the observation group. At the same time, 20 patients with cervical benign lesions who underwent tumor stripping or accessory resection were selected for the control group. Immunohistochemical (SP) detection methods were used to detect hepsin and HMGB1 protein levels in tissues. The positive rates of HMGB1 cells in normal, paracancerous and cancerous cervical tissues were 5.0% (1/20), 22.9% (16/70) and 95.7% (67/70) (F=24.581, P=0.001) respectively. The positive rates of hepsin in normal, paracancerous and cancerous cervical tissues were respectively 10% (2/20), 61% (43/70) and 90% (63/70) (F=11.538, P=0.001). The HMGB1 expression level was related to the degree of tumor differentiation, lymph node metastasis and TNM stage (P<0.05). While the level of hepsin expression was related to the degree of tumor differentiation, invasion depth, lymph node metastasis and TNM stage (P<0.05). Furthermore, a positive correlation between the levels of hepsin and HMGB1 was found (r=15.27, P<0.05). The overall 5-year survival rates of patients with high expression of HMGB1 (+++) and low expression of HMGB1 (+ to ++) were respectively 51.2 and 29.2% (HR=11.637, 95% CI=4.351–38.213; P=0.002). The overall 5-year survival rates of patients with high hepsin expression (+++) and low hepsin expression (+ to ++) were respectively 41.3 and 35.3% (HR=10.143, 95% CI=4.285–33.275; P=0.006). Based on our results, the higher the levels of expression of hepsin and HMGB1 in tissues the higher the degree of invasiveness of the cervical cancers, and the worse the prognoses for the patient.
Cervical cancer is the most common malignant tumor of the female reproductive system in China. It is diagnosed mostly at advanced stages when its prognosis is poor. The 5-year survival rate is only 60%. The survival rate of patients with distant metastasis is even lower. In recent years, with the progresses made in surgical treatments, radiotherapy and chemotherapy, the 5-year survival rate of patients has improved. However, the long-term survival rate is still not satisfactory. Therefore, newer approaches like gene therapy against tumor proliferation and invasion have gained widespread interest (
Seventy cervical cancer patients who initially received surgical treatments and had completely preserved clinical and pathological data, in the Department of Obstetrics and Gynecology, Xuzhou Central Hospital from May 2008 to June 2010, were selected to participate in the study. Inclusion criteria included: i) A definite pathological diagnosis; ii) clinical data were complete; iii) no radiotherapy or chemotherapy had been performed before operation; and iv) there were no brain, liver, kidney or other organ dysfunctions. The age of the patients ranged from 22 to 74 years with an average of 42.6±13.2 years. There were 59 cases of squamous cell carcinoma and 11 cases of adenocarcinoma. Classification according to the FIGO standards (
Paraffin-embedded tissues were collected and immunohistochemistry used the SP method. Mouse polyclonal hepsin antibody (dilution, 1:100; cat. no. sc-517056) and mouse monoclonal HMGB1 antibody (dilution, 1:150; cat. no. sc-135809) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used. SP immunohistochemical kits were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co. (Beijing, China). Experimental steps were conducted according to the kit's specifications. Phosphatase-buffered saline (PBS) was used instead of primary antibody as a negative control, and the positive control was an antibody known to be positive on the samples.
HMGB1 positive staining signal was brown and disseminated in small particles, all of which were located in the cytoplasm of the cells. The hepsin protein was distributed in the nucleus and the cytoplasm and consisted of brownish yellow or brown particles. For each slice, 10 consecutive views at ×400 magnification were examined, 200 cells were counted in each view, and all the positive cells were counted, in order to calculate the positivity rate. The percentage of positive cells were then calculated, and each sample was assigned points: 0–5% was noted as 0 points, 6–25% was noted as 1 point, 26–50% was 2 points, and more than 50% was noted as 3 points. Additionally, the staining intensity was also ranked by points: Unpigmented cells were marked 0 points, pale yellow cells were given 1 point, brownish-yellow cells were given 2 points, and brown cells were given 3 points. Multiplying the points of both scores resulted in definite comprehensive scores: A score of 0–1 meant tissue was the negative for HMGB1 or hepsin staining, a score of 2–3 meant the tissue was weakly positive (+), 4–6 meant tissue was moderately positive (++) and a score of 9 meant the tissue was strongly positive (+++).
The SPSS software package (SPSS, Inc., Chicago, IL, USA) was used for data analysis, measurement data were expressed by mean ± SD. The t-test (Fisher's exact test) was used in comparison between groups, χ2 test and exact tests were used to compare the rate of two samples, and for correlations the Pearson's test was adopted. The survival rate was calculated using the Kaplan-Meier survival curve and was analyzed by the log-rank test. A P<0.05 was considered to indicate a statistically significant difference.
HMGB1 was mainly expressed in the cytoplasm of cervical cancer cells, and was almost absent from normal tissues (only 5% of them had some expression). The positive rate for paracancerous tissues was 22.9% (16/70), and the differences between these two groups had statistical significance (P<0.05). The positive expression rate of HMGB1 in cervical cancer tissues was 95.7% (67/70), and when compared to the rates in the normal and paracancerous groups the differences were statistically significant (P<0.05).
The hepsin protein was mainly expressed in the cytoplasm of the cervical cancer cells, and there were only 2 cases of hepsin expression in 20 cases of normal tissues (positive rate of 10%). The positive expression rate in paracancerous tissues was 61% (43/70), and in the cervical carcinoma group was 90% (63/70), the differences among the three groups were statistically significant (P<0.05) (
In 70 cases of cervical cancer, the expression of HMGB1 was independent of age, histological type, the degree of differentiation, and TNM stage (P-values >0.05). Only the depth of tumor invasion, and presence of lymph node metastasis correlated with the expression of HMGB1 (P<0.05).
The expression of hepsin was not related to the tumor pathological type (P>0.05). But, it was related to the degree of differentiation, the depth of invasion, the presence of lymph node metastasis and the TNM stage (P-values <0.05) (
In 70 cases of cervical cancer tissues, the expressions of hepsin and HMGB1 were simultaneously positive 67.1% (47/70), and simultaneously negative 21.4% (15/70), here was a positive correlation between them (r=15.27, P<0.05) (
All subjects were followed-up for 5 years. On July 1, 2015, 66 patients completed their follow-up, while 3 patients with hepsin positive expression and 1 with HMGB1 positive expression failed to complete it. The rate of failure to complete the 5-year follow-up was 5.7%, and the data of patients without a complete follow-up were not used anymore. During the follow-up period, in the 66 cases of cervical cancer patients, 34 (51.5%, 34/66) died, and 32 (48.5%, 32/66) were alive, the 5-year survival rate was 39.3%, and the median survival time was 36.9 months. The log-rank test showed that expression of hepsin and HMGB1 protein (either strong or weak) were related to the patients' 5-year survival rate and median survival time (P<0.05). Comparing the risk of death (HR) in cervical cancer patients with the strongly positive expression of hepsin and HMGB1 protein with weakly and moderately positive expressions, the differences had statistical significance (P<0.05) (
Cervical cancer is one of the most common malignant tumors in females. The incidence rate increases year by year, with a high degree of malignancy and a high mortality rate. The typical features of malignant tumors are the invasiveness and high metastasis rates. It is thought that cell regulatory factors and other patient inherent factors play an important role in allowing for invasion and metastasis of the tumors. A large number of clinical trials have shown that patients with high expression of invasive biological factors have a worse prognosis. Therefore, it is very important to find new gene therapy targets for cervical cancer (
The relationship between HMGB1 protein expression and cervical cancer has been studied. HMGB1 is a cytokine discovered 30 years ago, it is believed to be important for the survival, proliferation, invasion and metastasis of malignant tumor cells, but the research on it is still in the exploratory stages (
At present, there is no unified theory on the role of HMGB1 in the development of cervical cancer. It was found that HMGB1 stimulates the endothelial progenitor cell migration and neovascularization using its receptor RAGE (
The hepsin gene was first found in the cDNA of hepatocellular carcinoma cells, and mainly locates on the human chromosome 19 (q11-q13.2) (
A large number of studies have confirmed that hepsin protein is highly expressed in various tumor tissues (
In conclusion, this study showed that the expression levels of hepsin and HMGB1 were related to the depth of invasion, the presence of lymph node metastasis of cervical cancer and the prognosis for patients. This suggests that both proteins may play a synergistic role in the genesis and development of cervical cancer. We believe that the combined detection of hepsin and HMGB1 expression is helpful in the diagnosis and prognosis of cervical cancer, and propose them as a pair of new markers for cervical cancer. Moreover, blocking both signal transduction pathways may become a successful strategy for inhibiting cervical cancer.
Expression of HMGB1 and hepsin in normal cervical tissues, paracancerous tissues and cervical cancer tissues. (A) HMGB1 protein expression was negative in normal cervical tissues. (B) HMGB1 protein expression in paracancerous tissues was pale yellow moderately positive (++). (C) HMGB1 protein expression in cervical carcinoma tissues was tan color strong positive (+++), and located in the cytoplasm. (D) Hepsin protein expression in normal cervix group was negative. (E) Hepsin protein expression was yellow positive (++) in paracancerous tissues. (F) Hepsin protein expressed in cervical carcinoma tissues was tan strong positive (+++), and located in the cytoplasm. HMGB1, high mobility group box 1.
Expression levels of HMGB1 and hepsin in different tissues (n, %).
Positive rate | |||
---|---|---|---|
Tissues | Cases | HMGB1 | Hepsin |
Normal | 20 | 5.0% (1/20) | 10% (2/20) |
Paracancerous | 70 | 22.9% (16/70) | 61% (43/70) |
Cervical cancer | 70 | 95.7% (67/70) | 90% (63/70) |
F-value | 24.581 | 11.538 | |
P-value | 0.001 | 0.001 |
HMGB1, high mobility group box 1.
Relationship of HMGB1 and hepsin with clinicopathological factors of cervical cancer.
HMGB1 | Hepsin | ||||
---|---|---|---|---|---|
Pathological factors | Cases | + | P-value | + | P-value |
Age (years) | |||||
<55 | 48 | 47 (97.9) | 0.257 | 44 (91.7) | 0.126 |
≥55 | 22 | 20 (90.9) | 18 (81.8) | ||
Pathological types | |||||
Localization | 34 | 31 (91.2) | 0.204 | 28 (82.4) | 0.067 |
Infiltrating | 36 | 36 (100) | 35 (97.2) | ||
Degree of differentiation | |||||
Poor | 37 | 36 (97.3) | 0.191 | 36 (97.3) | 0.044 |
High and middle | 33 | 30 (90.9) | 27 (81.8) | ||
Depth of invasion | |||||
T1+T2 | 14 | 11 (78.6) | 0.043 | 8 (57.1) | 0.013 |
T3+T4 | 56 | 56 (100) | 55 (98.2) | ||
Lymph node metastasis | |||||
Negative | 23 | 20 (87.0) | 0.048 | 18 (78.3) | 0.035 |
Positive | 47 | 47 (100) | 45 (95.7) | ||
TNM stage | |||||
I+II | 28 | 25 (89.3) | 0.087 | 24 (85.7) | 0.021 |
III+IV | 42 | 42 (100) | 39 (92.9) |
HMGB1, high mobility group box 1.
Correlation analysis of hepsin and HMGB1 in cervical cancer.
HMGB1 | ||||
---|---|---|---|---|
Hepsin | + | − | R-value | P-value |
+ | 47 | 7 | ||
− | 1 | 15 | 15.27 | 0.01 |
HMGB1, high mobility group box 1.
Survival analysis of cervical cancer patients with different expression intensity levels for hepsin and HMGB1 protein.
Category | Cases | 5-year survival rate (%) | Median survival time (month) | HR value | 95% CI | P-value |
---|---|---|---|---|---|---|
HMGB1 protein expression | ||||||
+ to ++ | 43 | 22 (51.2) | 54.1 | 11.637 | 4.351–38.213 | 0.002 |
+++ | 24 | 7 (29.2) | 24.8 | |||
Hepsin protein expression | ||||||
+ to ++ | 46 | 19 (41.3) | 41.6 | 10.142 | 4.285–33.275 | 0.006 |
+++ | 17 | 6 (35.3) | 27.3 |
HMGB1, high mobility group box 1; HR, hazard ratio; CI, confidence interval.