In the present study, tumor tissues and the corresponding para-carcinoma normal tissues of 60 ovarian cancer patients admitted into the Department of Surgery in Dongying Hospital (Dongying, China) from June, 2012 to June, 2015, were selected. The patients were definitively diagnosed with ovarian cancer by clinical pathology and did not receive chemotherapy and surgical therapy. Patients were aged 24–78 years, and the median age was 52 years. This study was approved by the Clinical Ethics Committee of Dongying People's Hospital. At the same time, all the patients enrolled signed the informed consent form.

RNA extraction, reverse transcription, and RT-qPCR kit (all from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), HMGB1, BRCA1, P62, GAPDH antibodies (1:800; cat. nos. 10829-1-AP, 22362-1-AP, 18420-1-AP, 10494-1-AP) and HRP-labeled secondary antibodies (1:1000; cat. no. SA00001-2) (all from Proteintech Biotechnology Co., Ltd.; Wuhan Sanying Biotechnology, Wuhan, China); immunohistochemical staining kit SP-9001 (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd.; OriGene Technologies, Inc., Beijing, China), primer synthesis (Takara Biotechnology Co., Ltd., Dalian, China) were used in the present study.

Approximately 100 mg tumor and para-carcinoma normal tissues of patients frozen in liquid nitrogen were taken and the total RNA was extracted according to the instructions of the RNA extraction kit. An ultraviolet and visible spectrophotometer (Hitachi Ltd., Tokyo, Japan) was used to detect the absorbance value of the total RNA extracted at 260 and 280 nm, and the total RNA samples with the A260/A280 value within 1.8–2.0 were selected for the follow-up experiment.

The reverse transcription reaction was carried out according to the instructions of the reverse transcription kit, and the cDNA obtained served as the template. The mRNA expression of HMGB1, BRCA1 and P62 was detected according to the instructions of the PCR kit with GAPDH as the control gene. The primer sequences are shown in Table I. The reaction conditions were: 95°C for 10 min, 95°C for 15 sec, 60°C for 30 sec, 72°C for 30 sec, a total of 42 cycles for amplification; 72°C for 5 min. The C_q value was read, and the 2^−ΔCq method was used to calculate the relative expression level. The formula used was: ΔC_q (target gene) = C_q (target gene) - C_q (control gene).

After the tissues were obtained during surgery, they were fixed using 4% formalin, and then embedded in paraffin, followed by conventional sections. The operation was carried out according to the instructions of the SP-9001 immuno-histochemical kit. The paraffin sections were dewaxed and hydrated for pretreatment, and incubated at 3% H₂O₂ at room temperature for 10 min. Then they were heated and repaired by citric acid buffer and sealed using 10% goat serum. The primary antibody (dilution 1:100) was added for incubation at 4°C overnight. After the sections were washed with phosphate-buffered saline (PBS), the biotin-labeled secondary antibody was added for incubation at room temperature for 20 min, followed by washing using PBS, color development via diaminobenzidine, hematoxylin staining, dehydration, transparency via xylene, sealing via neutral resin, and microscopic image (TE2000-U; Nikon Instruments Europe BV, Amsterdam, The Netherlands).

Five fields (×400) were selected randomly in each section. The staining results were scored according to the staining intensity and the percentage of positive cells: The number of positive cells were scored as: <5%: 0 point; 5–25%: 1 point; 26–50%: 2 points; >50%: 3 points. The staining intensity was scored as: No staining: 0 point; light yellow: 1 point; brown yellow: 2 points; dark brown: 3 points. Both scores above were added: ≤3 points indicated a negative expression, while >3 points indicated a positive expression, and the statistical results were analyzed.

Tumor tissues of the patients were obtained during surgery, immediately digested using the digestive enzyme, filtered and centrifuged to collect cells. RPMI-1640 culture medium containing 10% fetal calf serum was added to prepare the cell suspension. Cell-collagen mixture was prepared in an ice bath, then inoculated onto a 24-well plate and cultured for 24 h. The cells were divided into the administration group (cisplatin) and blank group (no drug) and cultured for 24 h. Then neutral red solution was added and cells were fixed with 10% formaldehyde solution. The in vitro drug sensitivity was evaluated by calculating the number of cells in the administration group/the number of cells in blank group, with <50% indicating drug sensitivity, and ≥50% indicating cell drug resistance (10).

According to the expression status of HMGB1, BRCA1 and P62 in ovarian carcinoma tissue, 60 ovarian cancer patients were divided into the positive and negative expression groups. A Chi-square test was used to analyze the association of the expression of HMGB1, BRCA1 and P62 in ovarian carcinoma tissue with the drug resistance of patients to cisplatin.

SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for data processing. Measurement data were expressed as mean ± standard deviation. A t-test was used for intergroup comparison, the Chi-square test was used for intergroup comparisons of enumeration data. P<0.05 was considered to indicate a statistically significant difference.

The RT-qPCR detection results are shown in Fig. 1. Compared with those in para-carcinoma normal tissues, the mRNA expression of HMGB1, BRCA1 and P62 was significantly higher in ovarian carcinoma tissues, and the differences were statistically significant (P<0.01).

Immunohistochemistry detection results are shown in Fig. 2. The positive immunohistochemical staining of HMGB1, BRCA1 and P62 were all yellow brown, with HMGB1 protein mainly being identified in the cytoplasm, BRCA1 mainly in the cell nucleus, and partially in the cytoplasm; and P62 mainly being identified in the cytoplasm.

The scores of statistical staining are shown in Table II. The positive expression rates of HMGB1 in ovarian carcinoma and para-carcinoma normal tissues were 61.67% (37/60) and 13.33% (8/60), and the difference was statistically significant (P<0.01); the positive expression rates of BRCA1 in ovarian carcinoma and para-carcinoma normal tissues were 78.33% (47/60) and 8.33% (5/60), and the difference was statistically significant (P<0.01). The positive expression rates of P62 in ovarian carcinoma and para-carcinoma normal tissues were 71.67% (43/60) and 11.67% (7/60), and the difference was statistically significant (P<0.01).

The results of in vitro resin droplet experiment showed that 38 out of 60 ovarian cancer patients had drug resistance to cisplatin, while 22 cases were sensitive to cisplatin (Table III).

The results showed that the positive rates of the protein expression of HMGB1, BRCA1 and P62 in 38 patients in the drug resistance group were 84.21, 94.74 and 92.11%, respectively, while those in the sensitive group were 22.73, 50.00 and 36.36%, respectively. The results of the Chi-square test showed that the positive rates of the protein expression of HMGB1, BRCA1 and P62 in the drug resistance group were significantly higher than those in the sensitive group (P<0.01) (Table IV).