Expressions of PKCα, PKCε and PKCδ in cervical cancer and their relationships with chem

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Journal of Hainan Medical University 2019; 25(2): 43-48

Journal of Hainan Medical University

✉Corresponding author: Qing-Zhi Guo, Department of Obstetrics and Gynecology,

Affiliated Hospital of Binzhou Medical College, Shandong Province 256603. E-mail: gqz9266 @

Fund Project: Shandong Medical and Health Science and Technology Development Plan Project: Study on the Mechanism of Candesartan Regulating PKC Delta Regulating Chemotherapy Sensitivity of Cervical Cancer (Fund No. 2016WS0039).

1. Introduction

Cervical cancer is a common malignant tumor of the reproductive

system in gynecology, which seriously endangers women's reproductive health and life safety. At present, the incidence of cervical cancer has a global upward trend, and the incidence of cervical cancer in young women has increased significantly [1].

Chemotherapy means the systemic chemotherapy performed before the implementation of partial treatment, which is mainly to reduce tumor volume and eliminate invisible metastatic cells to further weaken the vitality of cancer cells, thereby lessening the disability caused by surgery and improving the effect of surgical radiotherapy [2,3]. Protein kinase C (PKC) is involved in various signaling pathways in vivo . At the same time, it has been found that different subtypes of PKC are abnormally expressed in cancer cells such as colon cancer, breast cancer and lung cancer [4]. At present, there are few reports on the expression of PKC subtypes in cervical cancer, so 38 cases of cervical cancer patients were studied for this paper, analyzing the expression of three subtypes of PKC , PKC and PKC in cervical cancer and their relationship with chemosensitivity.

Qing-Zhi Guo et al./ Journal of Hainan Medical University 2019; 25(2): 43-48

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2. Cases and methods

2.1. Case information

A total of 38 patients with cervical cancer admitted to our hospital from November 2015 to November 2016 were selected, aged 25-65 years, with an average of (45.42±3.87) years old. According to the diagnosis of the lesion diameter≧4 cm by three-dimensional color Doppler ultrasonography and confirmed by two or more senior physicians, 30 of the 38 cervical cancer patients had squamous cell carcinoma and 8 cases of adenocarcinoma. The clinical stages adopt the FIGO criteria[5], then it turned out that 13 cases were in stage IIA, 15 cases in stage II

B and 10 cases in stage III, in which 29 cases were effective and 9 cases were ineffective after chemotherapy. The pathological sections used were reviewed by senior pathologists and no chemotherapy contraindications were found. Inclusion criteria: (1) no radiotherapy or chemotherapy and surgery were operated before; (2) normal liver and kidney function, generally in good condition; (3) patients were informed and have signed the informed consent. Exclusion criteria: (1) combined with other serious medical diseases and malignant tumors; (2) clinical data was incomplete.

2.2 Sample collection

Cervical biopsy forceps were used to obtain cervical lesions before and after chemotherapy. The surgically removed tumor tissue and normal cervical tissue were obtained about 100 mg. The lesions were cleaned with saline and then stored in liquid nitrogen for storage. Three-dimensional color Doppler ultrasound was used to detect VI, FI, and VFI.

2.3 Methods

2.3.1 Treatment methods

Adopting the NACT program. All patients underwent systemic intravenous infusion chemotherapy with a paclitaxel plus carboplatin regimen. The specific steps are as below: paclitaxel (175 mg/m2); Carboplatin was taken based on AUC Calvert formula. AUC was taken as 5, one course of chemotherapy with 3-week intervals. Patients underwent clinical and CT examinations after chemotherapy, successfully pulling through the extensive hysterectomy and pelvic lymphadenectomy after three to four weeks of chemotherapy. After chemotherapy, the patient's blood routine, liver and kidney function were monitored, and the colony cell stimulating factor Leukocyte elevation therapy was adopted when myelosuppression occurred after chemotherapy.

Evaluation of NACT. Before, during and after chemotherapy, the changes in the size of cervical lesions after interventional therapy were evaluated to determine the clinical efficacy. The tumor volume was measured by gynecological examination and three-dimensional color Doppler ultrasound. Efficacy evaluation criteria for WHO solid tumor chemotherapy are following: Complete Remission (CR): visible target lesion completely disappears; Partial Remission (PR): the product of maximum diameter and vertical diameter of lesion are reduced by 50%; Stable Disease (SD): the product of lesion’s two diameters is less than 50% and enlargement is less than 25%; Progressive Disease (PD): the increase of lesions’ two diameters is over 20%, or new lesions is found. According to the efficacy, it is divided into effective group and ineffective group. Effective group: CR+PR; ineffective group: SD+PD.

2.3.2 Detection of PKCα, PKCε, PKCδ mRNA expression levels in tissues by RT-PCR

The cervical cancer tissue specimens before and after chemotherapy with frozen liquid nitrogen were extracted in accordance with the instructions of Trizol kit (Fermenas). RNA was reverse-transcribed into cDNA according to the reverse transcription kit and used as a template for RT- PCR to detect the expression levels of PKC , PKC , PKC mRNA. The RT-PCR reaction system was 25 L: SYBR Green I qPCR Master Mix 12.5 L, 1 L of upstream and downstream

primers, 2.5 L of cDNA template, ddH

2

O 8 L. The reaction procedure was: 95 ħ for 10 min, 95 ħ for 15 s, 48 C for 30 s and 72 ħ for 30 s, 40 cycles in total. Primer synthesis is shown in Table 1. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd. After the reaction, data was collected and the Ct value of the obtained data was analyzed. Using β-actin as an internal reference gene, the relative expression levels of PKC , PKC , and PKC were calculated by 2- Ct method.

2.3.3 Western blot analysis of PKCα, PKCε, PKCδprotein expression in tissues

About 0.1 g of cervical cancer tissue was frozen with the liquid nitrogen, and 1 mL of RIPA lysate containing 1% protease inhibitor phenylmethylsulfonyl fluoride (PMSF) was added to extract the total protein of the sample. The protein concentration of the extracted sample was detected by the BCA method, then the absorbance at

Table 1.

qRT-PCR primer sequences.