Patients
Cervical samples from 8 patients diagnosed with abnormal uterine bleeding (AUB) or uterine myomatosis (UM) and adnexal tumor (AT) were obtained by hysterectomy procedures. The patients diagnosed with AUB were classified according to the International Federation of Gynecology and Obstetrics (FIGO). The information concerning FIGO classification is included in Table 1. Additionally, all samples, were negative to pathological analysis to cervical cancer (NCC). Importantly 4 NCC samples (Lane P5 - P8) were positive for hrHPV by PCR, but negative for cervical intrapithelial neoplasia (CIN) by histopathological analysis (Table 1). In addition, the sample of each patient was divided into two parts; the first one for obtaining DNA and the remaining part was used to obtain total proteins extracts.
Table 1
Features of patients with NCC.
| P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 |
Age (years) | 49 | 36 | 40 | 38 | 51 | 50 | 39 | 47 |
IMC | 16.44 | 27.38 | 26.17 | 37.8 | 31.24 | 26.14 | 30.84 | 37.67 |
Marital status | Single | Married | Married | Divorced | Married | Divorced | Married | Single |
Place of residence | Mexico City | Mexico City | Michoacan | Mexico state | Mexico City | Mexico City | Michoacan | Mexico City |
Employed | Not | Not | Not | Not | Not | Yes | Not | Yes |
Diagnostic | AUB L | AUB L | AUB L | AUB L | AUB L | AUB L | AUB L | UM and AT |
Surgery | TAH | TAH | TAH | TAH | TAH | TVH | TAH | TAH |
CIN | NEG | NEG | NEG | NEG | NEG | NEG | NEG | NEG |
HPV genotype | NEG | NEG | NEG | NEG | 31 | 31, 66 | 18, 31 | 59 |
NCC, negative to pathological analysis to cervical cancer; IMC, body mass index; HPV, human papillomaviruses; UM, uterin myomatosis; AT, anexial tumoration; AUB L, abnormal uterine bleeding leiomyoma; TAH, total abdominal hysterectomy; TVH, total vaginal hysterectomy; CIN, cervical intraepithelial neoplasia; NEG, negative. |
Immunoblot Analysis
Immunoblot analysis
Immunoblotting assays were performed using total proteins extracts from NCC tissue. All samples were obtained with RSB buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% NP40) supplemented with complete protease inhibitor cocktail. Immunoblotting assays were performed as described elsewhere[13]. Briefly, samples were resuspended in 2X Laemmli-buffer with β-mercaptoethanol, heated to 95ºC for 8 min, and loaded onto a 15% SDS-polyacrylamide gel. Proteins were transferred to a PVDF membrane by damp blotting at 40 mA for 3 h. The PVDF membrane was blocked with 5% milk in TBS-T (10X TBS-T: 198 mM Tris, 1400 mM NaCl, 0.01% Tween 20, pH 7.6) for 1 h. Then the PVDF membrane was incubated overnight at 4ºC with the primary anti-H3 antibody (E.960.2, Thermo Fisher Scientific) or HRP conjugated-GAPDH antibody (14C10, Cell Signaling Technology). Anti-H3 antibody was used at a 1:500 dilution and GAPDH (used as loading control) at a 1:1000 dilution. The membrane was subsequently washed three times in TBS-T followed by incubation with HRP-conjugated secondary antibody (Jackson ImmunoResearch) for 1 h at room temperature. Then, the membrane was washed three times in TBS-T. Signals were detected using by chemiluminescence C-Digit (LI-COR).
Genotyping Hpv
DNA extraction from the patient’s tissue was performed following manufactures instructions (Invitrogen Thermo Fisher Scientific). After this, genotyping determination of HPV was carried out through Anyplex II HPV28 detection kit (Seegene) following the manufactures instructions. Briefly, in 20 µl PCR reaction 1x of HPV28 primer mix A (14 high-risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) or B (5 high risk HPV types: 26, 53, 69, 73, 82 and 9 low risk types: 6, 11, 40, 42, 43, 44, 54, 61, 70), 1x Anyplex master mix, and approximately 50 ng of DNA were added. The thermocycling conditions was as follow: an initial step at 50°C for 4 minutes and a denaturation step at 95°C for 15 minutes. The samples underwent 30 cycles at 95°C for 30 seconds, 60°C for 60 seconds and 72°C for 30 seconds. The PCR reaction were done in a CFX96 Real Time PCR System (Bio-Rad) as recommended by the manufacturer.