Study on the correlation between hTREC and HPV load and cervical CINI/II/III lesions and cervical cancer

Abstract Objective To investigate the correlation between hTREC and human papillomavirus (HPV) load and cervical intraepithelial neoplasia (CIN) grade II/III lesions and cervical cancer. Methods A total of 135 patients with cervical lesions of different degrees admitted to our hospital from January 2016 to February 2017 were selected, including CIN I/III 65 cases, grade III 39 cases, and cervical cancer 31 cases. The expression of hTERC gene was detected by fluorescence in situ hybridization (FISH) in three groups, and the HPV load was detected by second‐generation hybridization capture (HC II) method, and its relationship with cervical lesion grade was analyzed. Department. Results The positive expression rate of hTERC gene amplification was cervical cancer > CIN I/II lesion > CIN III lesion; the positive expression rate of HPV was cervical cancer > CIN I/II lesion > CIN III lesion. After treatment, the positive rate of hTERC gene amplification and HPV expression decreased significantly within 1 year (P < .05). Spearman's analysis showed that the degree of cervical lesion was positively correlated with hTREC and HPV load (P < .05). Conclusion hTREC and HPV are closely related to the occurrence and development of cervical precancerous lesions and cervical cancer. The abnormal amplification of hTERC gene increases with the grade of cervical lesions. Both of them can be used as auxiliary indicators for early screening, treatment, and prognosis of cervical cancer.

CIN III, and eventually, the cancer cells will break through the basement membrane and develop into invasive cervical cancer. Therefore, people should attach great importance to it. Many studies have shown that telomerase is strongly related to cell canceration. HTERC is the main part of telomerase, and its expansion will cause cervical cells to be very prone to atypical development, and then evolve into cervical cancer. 5 In this study, hTERC gene detection was performed by fluorescence in situ hybridization (FISH) in patients with different cervical CIN lesions, and the hybrid capture two generation test (HC2) was used to detect their HPV load, which were analyzed in order to provide evidence for the diagnosis and treatment of cervical cancer and precancerous lesions. Exclusion criteria: (a) Patients with other malignant tumors; (b) Patients whose cervical cancer were failed to control or relapse after previous treatment. According to the pathological diagnosis results of surgical resection specimens and biopsy specimens, all cases were divided into CIN I/II lesion group (65 cases), CINI III lesion group (39 cases), and cervical cancer group (31 cases). The general information of the three groups was not obvious Difference (P > .05).

| hTERC gene expression detection
All patients had intercourse banned 1-2 days before the test. We had the patient's cervix fully exposed using a speculum and slowly cleaned the cervical secretions with a dry cotton swab. Then, we rotated the special sampling brush five times clockwise at the cervical canal for 10 seconds to collect the detached cells once. After brushing, we put the sampling brush into a thin layer of liquid-based cytology examination preservation solution, and then extracted 10 mL, and centrifuge at 10,000 g for 10 minutes (centrifugation radius 15 cm). After removing the supernatant, we cultured the cells with 5 mL collagenase B at 37°C for half an hour and then added 5 mL deionized water and pipetted the suspended cells three times for at 37°C for half an hour. Afterward, we slowly added 2 mL of fixative solution for thorough mixing, and we centrifuged at 10,000 g for 10 minutes, removed the supernatant, and then added 5 mL of fixative solution for 10 minutes; then, we centrifuged again and fixed the cells with fixative solution, and lately, slowly blew the suspension cells, extracted the liquid from the tube, and prepared the tablet; predried after natural drying (preheated, rinsed with SSC solution, soaked with hydrochloric acid solution, fixation with CH 2 O solution, dehydration), we hybridized with the denatured probe overnight at 42°C. After washing the slide, we added 10 μL of DAPI counterstain and determined the gene expression level with a fluorescence microscope.

| HPV-DNA viral load detection
The HC2 method was used to detect the HPV viral load. A sampling brush was used to rotate the patient's cervix counterclockwise three times, and the sample was taken after 10 seconds. The sampling brush was placed in a test tube, and the double-stranded DNA was broken down into single strands by lysis. The HPV probe solution hybridizes the lysed specimen, captures the hybrids, and amplifies the signal, and then uses a chemiluminescence meter (Digene American Corporation) for interpretation.

| Treatment
All patients were given comprehensive radiotherapy, external wide field irradiation, the upper limit of the field was located between the level of the 4th or 5th lumbar vertebra, the lower limit was about 5 cm below the upper margin of the pubic symphysis, and the left and right boundary was 0.5 cm away from the lateral midline of the femoral head, before and after 6MV-X-ray irradiation, 200 cGy/1f, five times per week. When the dose DT reached 3000 cGy, the lead plate was used to block the middle of the pelvic cavity, and the irradiation continued. DT was added to 2500 cGy/13f for six times.

| Determination of positive hTERC gene expression
Observe the FISH results of cervical exfoliated cells under a 100× objective lens and use red and green filters to record TERC and

| Statistical processing
Data were processed using SPSS19.0 (SPSS Inc) software. Measurement data were expressed as mean ± standard deviation by using t test; count data were expressed as rate, using χ 2 test; The correlation was analyzed by Spearman. The difference was statistically significant at P < .05.

| The results of hTERC gene amplification in patients with different grades of cervical lesions
The positive expression rate of hTERC gene amplification is ranked from high to low as manifested as cervical cancer > CIN III lesions > CIN I/II lesions (P < .05), as shown in Table 1. From the positive rate of amplification, it can be concluded that the positive rate of hTERC will increase significantly with the severity of cervical lesions.
The results of hTERC gene amplification in different cervical lesions are shown in Figures 1-3.

| Results of HPV load in patients with different levels of cervical disease
The HPV positive rate is ranked from high to low: cervical cancer > CIN III lesions > CIN I/II lesions (P < .05). It can be seen that as the severity of cervical lesions increases, the HPV load is also increasing in Table 2.

| The correlation between the hTREC
Human papillomavirus load, and the degree of cervical lesions, Spearman's correlation analysis results: There was a positive correlation between the degree of cervical lesions and hTREC and HPV load (r = .436, 0.385, P < .05). As shown in Table 5.

| D ISCUSS I ON
Cervical cancer is one of the most common gynecological malignancies that ranks second in the incidence of gynecological malignancies. Besides, cervical cancer can be divided into two types, squamous cell carcinoma, and adenocarcinoma. The former is the most common, accounting for about three-quarters of cervical cancer. The latter is highly malignant, patients are prone to metastasis, the prognosis is extremely poor, and the mortality rate is high. 6,7 Middle-aged and elderly women are a high-incidence group, but their incidence tends to be younger in recent years, posing a serious threat to women's health, and has been raised to the height of a public health problem. 8  only induces the body to produce an anti-viral immune response, but also escapes the body's immune response, making the infection a persistent feature, which weakens the body's ability to remove viruses and greatly increases the prevalence of cervical cancer. 16 The results of this study showed that the positive rate of HPV was The results of this study show that after active treatment, HPV positive rates in different groups of patients within 1 year. All of them decreased significantly, thus preventing the precancerous lesions of the cervix.
Telomerase is a reverse transcriptase that maintains telomere length and plays an important role in the process of cell canceration. 18 Relevant research shows that telomerase is highly expressed in more than 90% of malignant tumors, so telomerase activity can be detected clinically as a judgment of benign and malignant tumors. 19 Although HPV detection technology is currently