Relevance research between the expression of p16INK4a , Notch1, and hTERC genes: The development of HPV16-positive cervical cancer.

Abstract Background GLOBOCAN 2018 latest data show cervical cancer ranks fourth in morbidity and mortality among women. Many genes in cervical lesions differ in sensitivity and specificity. However, the diagnostic molecules for early cervical cancer are not very clear. This paper screens biomarkers for early molecular diagnosis of Mongolian patients with cervical cancer. Methods Immunohistochemical SP method was used to detect the expression of p16INK4a and Notch1 protein in paraffin sections of 226 Mongolian patients with HPV16‐positive cervical lesions after pathological examination, and 100 of them were randomly selected by fluorescence in situ hybridization to detect hTERC gene. The HPV16‐binding human cervical cancer SiHa cell line was used to silence the expression of HPV16 E6/E7 gene by RNA interference, and the expression of p16INK4a, Notch1, and hTERC genes and protein expression levels were detected by RT‐PCR and Western blot. Results The positive expression rates of p16INK4a, Notch1, and hTERC genes in HPV16‐positive cervical cancer, CIN‐III, CIN‐II, CIN‐I, uterine leiomyoma, and chronic cervicitis were significantly different (P < .05); the positive expression rates of the three genes were also significantly different in the same type of cervical lesions (P < .05); RNA interference can effectively inhibit HPV16 E6/E7, p16INK4a and Notch1 gene expression, but has no effect on hTERC gene expression. Conclusion The p16INK4a gene can be used as a biomarker for early screening of cervical cancer, and the hTERC gene can be used to confirm the clinical diagnosis of cervical cancer.


| INTRODUC TI ON
In the past 50 years, due to the extensive development of gynecological census, the incidence and mortality of cervical cancer have decreased significantly. However, it is still the third most common malignant tumor after breast and colorectal cancer and is one of the important causes of female death worldwide. 1

Cervical cancer (CC)
is the most common malignant tumor of the female reproductive system, which seriously endangers women's life and health. Cervical cancer and precancerous lesions are currently considered to be a persistent, progressive, multifactorial, and multi-step disease, and human papilloma virus (HPV) infection is the leading cause of cervical cancer development. 2 The positive rate of HPV infection in patients with cervical cancer is as high as 99%. [3][4][5] Human papilloma virus is a non-enveloped double-stranded circular DNA virus consisting of 7900 base pairs. The HPV gene structure basically includes three important regions: early region (E), late region (L), and long control region (LCR). The early region (E) encodes products E6 and E7, and their abnormal expression is a key event in the malignant development of infected cells, which is related to various alteration pathways of viruses and cells. 6 Clinically, HPV is classified into the low-risk type and high-risk type according to the virulence of HPV subtype or the risk of cancer. Among them, highrisk HPV infection has a closer relationship with the development of cervical cancer and its precancerous lesions. There are two states after HPV infection of the cervix, which are free and combined, and the persistent infection of HPV in the combined form is an important cause of cervical cancer development. 7 Wang et al 8 found that especially high-risk HPV16/18 is closely related to the occurrence of cervical cancer. HPV16 is recognized as the most important genotype for the development of squamous cell carcinoma and adenocarcinoma worldwide. 9 Therefore, this study used HPV16-infected cervical lesions and cervical cancer cells as the research object. By detecting the differential expression of p16 INK4a , Notch1, and hTERC genes, the relationship between these genes and the occurrence and development of Mongolian patients with cervical cancer was analyzed, and screen the best reference biomarkers for early diagnosis of cervical cancer in Mongolian population, and establish the relationship between these three genes and HPV16 infection. The Notch1 signaling pathway plays an important role in some key steps regulating cell differentiation, proliferation, and apoptosis. Notchl expression is increased in cervical intraepithelial neoplasia (CIN) and cervical cancer tissues. Laura et al believe that the carcinogenesis of the normal cervical epithelium may be related to the increased expression of Notchl protein, leading to the development of cervical cancer. 11 The hTERC gene has a certain inhibitory effect on apoptosis and is closely related to tumorigenesis. 12 In recent years, the National Institutes of Health research on cervical cancer showed that the majority of cervical epithelial cell carcinogenesis is accompanied by an increase in 3q copy number. The human telomerase RNA component (hTERC gene, located at 3q26.3) may be the most important gene involved. Meng-Lan O et al 13 found that the hTERC gene is activated in the early stages of cervical cancer. Therefore, it is possible to diagnose cervical cancer based on the activity of telomerase and predict the development of cervical cancer. In summary, p16 INK4a , Notch1, and hTERC genes are abnormally expressed in HPVinfected cervical diseases. [14][15][16] However, there are large differences in the reports of p16 INK4a -positive expression rates in cervical tissues in different articles. 17 We speculate that the expression levels of these three genes are possibly to be different in population expression. Studies have shown that the E6 and E7 proteins produced by HPV16 E6 E7 mRNA can inactivate cell growth inhibitory genes such as p53 and pRB. 18 The increased risk of cervical cancer is due to the overexpression of E6 and E7 oncoproteins. In this study, Mongolian cervical cancer patients was designed to detect gene expression of  (Table S1). All patients were not treated with physical therapy, biological therapy, drug therapy, chemotherapy, radiotherapy, or surgery. And all the sections were diagnosed with the same opinion obtained by two senior professors of pathology.

| Immunohistochemistry
Immunohistochemistry was performed using SP-9000 DAB staining solution (streptavidin-biotin method) kit manufactured by Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Abcam mouse anti-human p16 monoclonal antibody and rabbit monoclonal antibody Notch1 were used. The paraffin-embedded tissue sections with a thickness of 4 μm were used as specimens.
Immunohistochemical examination was performed on 226 patients with HPV16-positive tissue, and brown-yellow coloring was recorded as positive expression. Greenspan semi-quantitative method 19 : 0 is that the cells are basically not colored, 1 is a lighter coloration, 2 is a moderate coloring, and 3 is a deeper coloring.

| RT-PCR and Western blot
Total RNA from the above three groups of SiHa cells was prepared using TRIzol (Invitrogen), and OD values of 260 nm and 280 nm wavelength were measured by NanoDrop2000 ultra-micro spectrophotometer, and Abs260/Abs280 ratio and RNA concentration were recorded. cDNA was synthesized using a universal RT-PCR kit purchased from Beijing Dingguo Changsheng Biotechnology co., LTD. 3ug of total RNA was taken as the template, and cDNA was amplified using gene-specific primers ( Table 1). The amplified products were separated by 2% agarose gel electrophoresis.
Total protein was extracted from three groups of SiHa cells by using the total cell protein extraction kit (Solarbio), and the concentration was measured on a Nanodrop 2000 ultra-micro spectrophotometer (Gene Co., Ltd.). Take 0.5 mg of total protein and transfer to PVDF membrane (Millipore). Specific protein detection using Western blot kits and antibodies purchased from Proteintech, the PVDF membrane was placed in a Bio-Rad scanner for photography.

| Statistical analyses
Statistical analysis was performed using SPSS 21.0 statistical software. The chi-square test was performed. The difference was statistically significant at P < .05. The correlation test was performed using the Spearman rank correlation analysis.

| The correlation between p16 INK4a gene and the occurrence and development of cervical cancer is most significant
The expression of p16 INK4a Table 2 in various cervical lesions, it can also be seen that the sensitivity of its expression before cancer is low, and it has little significance for early diagnosis of cervical cancer. According to Table 2 and Figure 2B Figure 2C,D).
In conclusion, the p16 INK4a gene has the most significant correlation with the occurrence and development of cervical cancer, which can be considered as a biomarker for early diagnosis and screening of cervical cancer.

| The expression of p16 INK4a and Notch1 genes in HPV16-positive cervical cancer is associated with HPV infection
Interference with HPV16 E6/E7 siRNA on HPV16-positive SiHa cells, as shown in Figure 3A,B, the expression of the HPV16 E6/E7 gene in

SiHa cells was inhibited, and the expression levels of HPV16 E6/E7
protein and HPV16 E6/E7 mRNA in the siHPV16 E6/E7 group were significantly lower than those in the nontransfection and siNControl groups. HPV16 E6/E7 siRNA interference plays a significant role.
The expression level of the housekeeping gene (GAPDH gene) had no effect before and after interference, indicating that there was no effect on the normal growth of cells before and after interference. As shown in Figure 3C, the relative mRNA levels of p16 INK4a , Notch1, and hTERC genes were analyzed. The results showed that the mRNA changes of Notch1 gene and p16 INK4a gene were very similar to HPV16 E6/E7 gene and had significant correlation ( Figure 3D), while hTERC gene was no significant change in mRNA levels.

| D ISCUSS I ON
In Western blot was used to detect the total protein extracted from three groups of SiHa cells after interference, with beta-actin as the reference. Three groups of cells included nontransfection group, siNControl group: transfection negative control disordered siRNA sequence, siHPV16 E6/E7 group: transfection specific siRNA sequence; B, the expression of specific gene after HPV16 E6/E7 SiRNA interfered with HPV16-positive SiHa cells. The specific gene was amplified by RT-PCR, the mRNA was derived from the disturbed SiHa cells, and the PCR product was detected by 2% agarose gel electrophoresis; C, changes in specific gene mRNA levels. The total RNA of SiHa cells was extracted, and the relative mRNA levels of each specific gene were obtained by fluorescence quantitative PCR with reference to GAPDH. D, Comparison of the correlation coefficients between the p16INK4a, Notch1, and hTERC genes and the HPV16 E6/E7 gene. Correlation coefficients were obtained by Spearman rank correlation analysis with respect to the relative mRNA levels of the p16INK4a, Notch1, hTERC, and HPV16 E6/E7 genes a significant correlation with HPV16 infection (r = 0.753, P < .05),

AUTH O R CO NTR I B UTI O N S
All the authors have accepted responsibility for the entire content of this submitted manuscript and approved the submission.