Exploration of protein PCP4 as a potential tumor marker in uterine leiomyoma

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Objective: Evaluation of the possible role of Purkinje cell protein 4 (PCP4) as a potential tumor marker of uterine leiomyoma by measurement of antibody titer against this protein in blood serum and the applicability of this technique for evaluation of proteins as potential vaccine antigens.

Materials and methods: cDNA fragment clone was derived from PCP4 gene in leiomyoma nodule. After that the soluble PCP4-GFP chimera based on E. coli was constructed, and the recombinant protein was purified. This product was used to evaluate the upper limit of titer to detect antibodies in blood serum in patients using indirect enzyme immunoassay. Serum samples (24) were collected from donors among the patients undergoing treatment at the Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, who gave informed consent to participate in the study. Four groups of patients were formed to study the immunological activity of sera against the 6His-PCP4hs-GFP protein. Group 1 was the control group and was composed of men. Group 2 consisted of women without diagnosed fibroids who had five or more successful pregnancies in history. Group 3 included women with fibroids, who also had five or more successful pregnancies in history. Group 4 included women with recurrent myomas.

Results: The pRSET-EmGFP expression vector containing the PCP4 gene and the reporter gene of the green fluorescent protein EmGFP was derived from the cDNA of the myoma nodule with a driver mutation in the MED12 gene. Human PCP4 in preparative amounts was obtained using this construct embedded in the E. coli genome, that was sufficient for analysis of its reaction with antibodies from blood serum in different groups of patients. Blood sera in the control group of men showed high immunological reactivity against the 6His-PCP4hs-GFP protein, whereas the difference between the groups of women was insignificant. Minimum difference between sera was found in groups 2 and 4. Moreover, the reaction of sera in women with recurrent fibroids was higher compared with women without fibroids, who had five or more successful pregnancies. Comparison of women in groups 3 and 4 showed statistically significant differences in the optical density values obtained in dilution of sera 1:1600, 1:3200 and 1:6400. At the same time the reaction of sera of women with recurrent fibroids was higher compared with sera of women with fibroids, who had five or more successful pregnancies.

Based on the level of activity, sera were divided into 3 categories. It was found that reaction of sera of men against the 6His-PCP4hs-GFP antigen was most pronounced. The proportion of high activity in sera was 60%. In the group of women without fibroids, who had five or more successful pregnancies, the proportion of low activity in sera was 44%, that is the maximum value of indicator compared with the other groups. The group of women with fibroids, who had five or more successful pregnancies, is characterized by the moderate level of reactivity with the 6His-PCP4hs-GFP antigen. This category accounts for 75% of sera, that is the highest value of indicator among all groups. Finally, the group of women with recurrent fibroids is characterized by equal number of sera with high and moderate activity, that shows a greater tendency for production of antibodies against PCP4/PEP19 in this group of patients compared with multiparous women.

Conclusion: For the first time, human PCP4 was in preparative amounts sufficient for analysis of its reaction with antibodies from blood serum in different groups of patients. Testing showed that most samples in all groups had significant titers of antibodies against PCP4. The activity of these antibodies varies widely both in sera of men and women. Antibodies against PCP4 are most common in women with recurrent fibroids compared with multiparous women, especially without leiomyomas. Thus, it is unlikely that PCP4 immunization сarries the risk of any pathology. The titers of antibodies to PCP4 in the general group of multiparous women were significantly lower compared with men and patients with recurrent fibroids. Therefore, using PCP4 as the basis to develop a preventive vaccine against leiomyomas is not advisable.

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Sobre autores

M. Kuznetsova

Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia

Autor responsável pela correspondência
Email: mkarja@mail.ru
ORCID ID: 0000-0003-3790-0427

PhD, Senior Researcher at the Institute of Reproductive Genetics

Rússia, Moscow

A. Shevelev

Vavilov Institute of General Genetics, Russian Academy of Sciences

Email: shevel_a@hotmail.com
ORCID ID: 0000-0003-3564-7405

Chief Researcher

Rússia, Moscow

N. Pozdnyakova

Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia

Email: natpo2002@mail.ru
ORCID ID: 0000-0002-5765-3016

PhD, Senior Researcher at the Laboratory of Radionuclide and Radiation Technologies in Experimental Oncology

Rússia, Moscow

D. Samoilova

Avtsyn Research Institute of Human Morphology, Petrovsky National Research Centre of Surgery

Email: dashasam@mail.ru
ORCID ID: 0000-0001-5639-0835

Researcher at the Central Pathoanatomical Laboratory

Rússia, Moscow

V. Karyagina

Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia

Email: mkarja@mail.ru

PhD, Senior Researcher at the Laboratory of Molecular Genetic Methods, Institute of Translation Medicine

Rússia, Moscow

N. Tonoyan

Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia

Email: mkarja@mail.ru
ORCID ID: 0000-0002-1631-1829
Código SPIN: 8547-9399
Scopus Author ID: 57213609878

PhD, Doctor

Rússia, Moscow

Е. Trubnikova

Vavilov Institute of General Genetics, Russian Academy of Sciences; Kursk State University

Email: tr_e@list.ru
ORCID ID: 0000-0001-5025-9406

Dr. Bio. Sci., Associate Professor, Chief Researcher at the Laboratory of Genetics, Leading Researcher

Rússia, Moscow; Kursk

D. Zelensky

Kursk State University

Email: dmitriizelenskii@mail.ru

PhD student

Rússia, Kursk

P. Vishnyakova

Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of Russia; Patrice Lumumba Peoples’ Friendship University of Russia

Email: mkarja@mail.ru
ORCID ID: 0000-0001-8650-8240
Scopus Author ID: 57190971385

PhD, Head of the Laboratory of Regenerative Medicine, Head of the Laboratory of Molecular Pathophysiology, Research Institute of Molecular and Cellular Medicine

Rússia, Moscow; Moscow

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2. Fig. 1. Map of the pRSET-PCP4hs-EmGFP construct based on the pRSET-EmGFP vector (Thermo Fisher Scientific; USA), designed to produce the fusion trifunctional protein 6His-PCP4hs-GFP (A), and the sequence of the fusion gene in the region of PCP4/PEP19 (B)

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3. Fig. 2. Evaluation of the level of 6His-PCP4hs-GFP protein accumulation in the biomass of the E. coli NiCo21 strain carrying the pRSET-PCP4hs-EmGFP construct (2) and the non-recombinant NiCo21 strain grown under similar conditions (3). The lanes of 12.5% ​​denaturing PAGE were loaded with the total biomass obtained from 100 μl of the culture, denatured in Laemmli sample loading buffer (see Materials and Methods). The gel was stained with 1% Coomassie R-250. The location of the target protein with an estimated mass of 37.8 kDa is indicated by an arrow. Lane 1 was loaded with a pre-stained molecular mass standard from BioRad (cat. Nº 1610374S)

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4. Fig. 3. Evaluation of the purity of the 6His-PCP4hs-GFP protein purified from the lysate of the recombinant E. coli NiCo21 strain (pRSET-PCP4hs-EmGFP) culture using metal affinity chromatography on the HisPur™ Ni-NTA Resin sorbent (Thermo Fisher Scientific, USA). Electropherogram of proteins separated in 12.5% ​​denaturing PAGE after staining with Coomassie R-250. The following are applied to the gel lanes: (1) pre-stained molecular weight standard (BioRad, USA): marker masses of 250, 150, 100, 75, 50, 37, 25, 20, 15 and 10 kDa; (2) purified 6His-PCP4hs-GFP protein obtained as a result of elution from the sorbent; (3) fraction obtained by washing the sorbent with a buffer containing 100 mM imidazole; (4) fraction of soluble proteins of the E. coli NiCo21 (pRSET-PCP4hs-EmGFP) lysate that did not bind to the sorbent. The volumes of the fractions applied to lanes 2, 3 and 4 are normalized to the total volume of each fraction.

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5. Fig. 4. Study of the specificity of binding of antibodies from the used panel of sera to the recombinant protein 6His-PCP4hs-GFP. Electropherogram of proteins separated in 12.5% ​​denaturing PAAG after incubation with patient sera at a dilution of 1:1000 and a conjugate of protein G with peroxidase 1.1 mg/ml (Hytest, Russia) at a working dilution of 1:10,000 in PBS with the addition of 0.03% Tween-20. Peroxidase activity was visualized in a solution containing 20 μg/ml 3,3-diaminobenzidine and 0.01% H2O2. The following are applied to the lanes of the gel: (1) pre-stained molecular weight standard (BioRad, USA): marker weights 250, 150, 100, 75, 50, 37, 25, 20, 15 and 10 kDa; (2) purified 6His-PCP4hs-GFP protein obtained by elution from the sorbent. The panels are stained with sera from the following patients: A - staining without adding patients' serum (control of conjugate specificity); B - No. 5, male, ELISA titer 1:400; B - No. 3, male, ELISA titer 1:6400; D - No. 20, multiparous woman with myoma, ELISA titer 1:800

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