The Expression Of GRIM-19 In Cervical Cancer And Its Reaction To Ionizing Radiation

Cervical cancer used to be the leading cause of cancer deaths for women in the World. However, in the past 40 years, the mobidity and the mortality rate of cervical cancer have decreased significantly,especially in developed country. This decline is largely the result of many women getting regular Pap tests which can find cervical precancer before it turns into cancer.Consistent infection of high risk-Human Papilloma Viruses (HR-HPV), such as HPV-16 and HPV-18 is one of the major cause of cervical cancer, E6 and E7 oncogenes of high-risk HPV play main roles in the development and the maintenance of cervical malignancy. However, more and more reports have shown that HPV infection alone is insufficient to spark off malignant changes, the abnormal expression of various oncogenes and tumor suppressor genes are also very important in the pathogenesis of cervical cancer.Cervical precancer is defined histologically as cervical intra-epithelial neoplasia (CIN), and graded as CINⅠ CINⅡ or CINⅢ The latter two grades are typically grouped together as high-grade CIN, which tends to develop into cancer if without treatment. However, the molecular basis of how normal cervical squamous epithelia transmutation into cervical intraepithelial neoplasia and invasive carcinoma has not been completely elucidated.Mutations/repression/loss of genes coding for transcription factors and apoptotic machinery have been implicated in tumor growth. Recently, it has been reported that viruses and other cellular factors bind to GRIM-19 and prevent its death-activating function. GRIM-19 is associated with apoptosis, growth inhibition, migration, invasion, and the epithelial-mesenchymal transition (EMT).The loss of GRIM-19 has been implicated in prostate cancer progression, primary renal cell carcinoma, oral squamous cell carcinoma, head and neck tumors, hepatocellular carcinoma and colorectal carcinomas. In most cases, tumor-derived GRIM-19 mRNA seems to harbor no mutations. We have reported that GRIM-19 can suppress tumor growth by down-regulating STAT3-induced gene expression and inducing p53 accumulation through a disruption of the E6/E6AP complex and an induction of E6AP auto-ubiquitination in cervical cancerous cells. In this study, we aim to investigate the expression of GRIM-19 in cervical tissues and exfoliated cells; we also want to make out the decrease of GRIM-19 in tumorigenesis, and to analysis the effects of irradiation on the expression of GRIM-19 and its regulation protein p53.This study is divided into two parts:Part I:To explore the silence of GRIM-19 in cervical cancer cells in promoting tumor growth in vivo and in vitro, and the effects of ionizing radiation on the expression of GRIM-19 and its regulation protein p53.Part II:To explore the expression of GRIM-19 in malignant cervical exfoliated cells and cervical intra-epithelial neoplasias, and its association with HR-HPV infection.The expression of GRIM-19 in cervical cancer and its reaction to ionizing radiationObjective:1. To explore the expression levels of GRIM-19 protein in cervical cancerous tissue;2. To explore the effects and mechanisms of silence of GRIM-19 on the growth of cervical tumor cell lines in vivo and in vitro.3. To explore the expression of GRIM-19 in cervical cancerous cells after ionizing radiation.Methods:HeLa (human cervical cancer cell line), SiHa cell lines (human cervical cancer cell line), HaCaT (human normal keratin cells) and clinical tissures were used to analysis the expression of GRIM-19. All cervical tissues from individual biopsy were collected between February 2010 and July 2013. Clinical stages were determined by a certified gynecologist according to the International Federation of Gynecology Obstetrics (FIGO 2014) staging system. The 30 cases of cervical carcinoma examined in these studies without metastases belonged to type Ⅰb for 10 patients, Ⅱa for 5 patients, ⅡB for 5 patients, III 10 patients; 10 patients with adenocarcinoma and 20 cases with squamous carcinoma. Additional 20 normal cervical tissues, from women who underwent hysterectomy for reasons other than neoplasias of either cervix or endometrium, were collected and used as controls in this study. HeLa and SiHa were used to extract proteins before and after irradiation with 300cGy; the samples considered for study entry included 20 slides of squamous cancerous cell (SCC) from patients with cervical cancer before and after radiotherapy with 40Gy. All Pap tests were collected in PreservCyte solution and processed using ThinPrep methodology (Hologic, Marlborough, MA). All of the slides were screened by at least two certified cytotechnologists. HeLa cells (stable transfected with GRIM-19 siRNA or control siRNA) were implanted subcutaneously on the dorsal flank of 6-week-old female athymic nude mice (each group containing 8 mice) to explore the tumor growth. The study including:① GRIM-19 expression was explored in cervical cancer cells and cancerous tissues by immunohistochemistry;② Tumor growth and its mechanisms were explored in cervical cancer with silence the expression of GRIM-19 by MTT and tumor-bearing nude mice expreriment in in vivo and in vitro;③ The effects of irradiation on the expression of GRIM-19 in cervical cell lines and cervical exfoliated cells in cervical cancer patients.Results:GRIM-19 Expression in cervical tissues:GRIM-19 was recognized as a potential tumor suppressor in recent studies, we first tested the expression status of GRIM-19 in primary cervical cancers. Thirty paraffin-embedded cervical squamous carcinoma and 20 normal cervical tissues were assayed for GRIM-19 expression using immunohistochemical analyses with a GRIM-19-specific monoclonal antibody. As shown in Figure 1, GRIM-19 was highly expressed in normal cervical squamous and glandular epithelial tissues; however, its expression was extremely reduced in cervical squamous cancers and adenocarcinomas (Fig.lA-B). The scores for the protein levels of GRIM-19 by immunohistochemical analyses was shown in the FiglB.The down-regulated of GRIM-19 was also been found in the cervical cancerous tissues by western blotting (Fig1C).Deceased expression of GRIM-19 promotes Cell Proliferation in HeLa:To test the biological relevance of GRIM-19 with cell growth, we firstly examined the GRIM-19 levels in the cervical cells. Compared to normal human keratinocyte line HaCaT, the levels of GRIM-19 was reduced in cervical cancer cells (HeLa and SiHa). After suppression the expression of GRIM-19 by siRNA, proliferation of HeLa/G19siRNAcells, as indicated by MTT, was markedly accelerated on day 4 and day 5 (p<0.05, Fig.3C) compared to the control cell line.Deceased expression of GRIM-19 promotes tumor growth through suppressing p53 and increasing Ki-67 expression in vivo. To evaluate the role of GRIM-19 in the tumor growth in vivo, we transplanted the HeLa/pcon and HeLa/G19 siRNA cells into athymic nude mice.Cells (1×107)were transplanted sub-cutaneously into the right flank. (n=8 per group) and tumor growth was monitored for 42 days (Fig.3 A-B). On day 42, animals were sacrificed and tumors were collected. Tumor sizes in HeLa/G19siRNA group were significantly increased when compared with the HeLa/pcon group (P< 0.05). Some representative mice bearing tumors and final tumor sizes are shown in fig.3A. We next examined the expression of p53 and GRIM-19 in tumors derived from mice using RT-PCR or Western blot analyses. As expected GRIM-19 expression levels were diminished in HeLa/G19siRNAgroup (Fig. 3D), and reduction of p53 levels also occurred in this group (Fig.3D). The effect of GRIM-19 on tumor growth was further assessed by an immunohistochemcial analysis of the expression of proliferation-associated antigen Ki67. As expected, GRIM-19 silencing tumors had an extremely increased expression of Ki67, compared to the control tumors (Fig.3E-F).Increased expression of GRIM-19 in the cancer cells after irradiation. We then examined the expression levels of GRIM-19 in HeLa and SiHa cells. The cells were treated with 300c Gy for 1.5min and 24 hours later, they were tested the expression of GRIM-19 and p53, we found the up-regulated of GRIM-19 and p53. The exfoliated cells in the patients diagnosed as cervical squamous cancer were collected before and after radiotherapy with 40Gy. The results showed that GRIM-19 and p53 in SCC cells were increased after radiotherapy.Conclusion:GRIM-19 was highly expressed in normal cervical squamous and glandular epithelial tissues; however, its expression was extremely reduced in cervical squamous cancers and adenocarcinomas. Down regulation of gene expression and activity of GRIM-19 resulted in cancer cells proliferation and tumor growth by decrease of p53 levels and increase STAT3 Levels. One of the mechanism of irradiation on killing tumor cells were probably the increase of GRIM-19 and p53 accumulation to anti-cancerogenesis.The expression levels of GRIM-19 in cervical intraepithelial neoplasias and exfoliated cells and its association with HR-HPV infectionObjective:1. To investigate the expression characteristics and levels of GRIM-19 in cervical exfoliated cells;2. To investigate the correlation of GRIM-19 expression and HR-HPV infection.;3. To investigate the expression levels of GRIM-19 in cervical intraepithelial neoplasias.Methods:The patients were selected from the gynecologic cytology practice at Anhui Provincial Hospital affiliated with Anhui Medical University, Hefei, China. The samples considered for study entry included 40 slides of normal squamous cells,40 slides of atypical squamous cells of undetermined significance (ASCUS),40 slides of low-grade squamous intraepithelial lesion (LSIL),40 slides of high-grade squamous intraepithelial lesion (HSIL), and 20 slides of squamous cancerous cell (SCC) from patients with cervical cancer. The samples were considered eligible if the patients were at least 21 years or older. Cases were excluded if the patients were younger than 21 years, if the samples were obtained from women with who had experienced hysterectomy, or if there was insufficient residual fluid to extract cellular proteins. All Pap tests were collected in PreservCyte solution and processed using ThinPrep methodology (Hologic, Marlborough, MA). All of the slides were screened by at least two certified cytotechnologists.A total of 20 cases of normal cervical tissues,35 cases of CIN 1,30 cases of CINII, 30 cases of CINIII, and 20 cases of SCC were used for the study. Colposcopy-directed biopsies were fixed in 10% buffered formalin and stained with hematoxylin and eosin using routine methods. The biopsies were included if they were obtained within three months of the abnormal Pap smear. All Pap smear slides and histological biopsy slides were reviewed by at least one experienced gynecologic pathologist also bspecialty certified in cytopathology and were interpreted using established criteria. The Bethesda System was used for the cytological diagnosis, and the biopsies were reported using CIN terminology. We have made the following experiments:① To investigate the expression characteristics and levels of GRIM-19 in the cervical exfoliated cells by immunohistochemistry;② To investigate the expression levels of GRIM-19 in cervical exfoliated cells and cancer cell lines by Western Blotting;③ HR-HPV infection was tested by HC-2, and the association of GRIM-19 expression with HR-HPV were analyzed by SPSS;④ To investigate the expression levels of GRIM-19 in cervical intraepithelial neoplasias and squamous cell carcinoma by immunohistochemistry and its correlation。Results:The expression of GRIM-19 in cervical exfoliated cells by immunohistochemistry Cervical squamous epithelial tissue is generally composed of three layers:the surface layer (cornified cells), middle layer, and basal layer. The cervical exfoliated cells used for a Pap smear are mainly cells from the middle and surface layers. Thus, as we can observe normal middle and surface layer cells on Pap slides, we first analyzed the expression of GRIM-19 in the exfoliated cells. Interestingly, GRIM-19 was highly expressed in 94.2±2.8%(188.4±5.5/200) of the normal middle layer squamous cells (Fig.1 a and a’) and in 0±0.0%(0±0.0/200) of the normal surface layer squamous cells (Fig.1 b and b’).We then calculated 200 ASCUS cells,200 LSIL cells,200 HSIL cells, and 200 SCCs in the corresponding ASCUS, LSIL, HSIL, and SCC Pap slides to detect the expression of GRIM-19. The expression of GRIM-19 was decreased in the ASCUS cells, LSIL cells, HSIL, and SCCs (Fig 2) and was negative in 16.5% of the ASCUS cells,32.0% of the LSIL cells,60.5% of the HSIL cells, and 97.5% of the SCCs (Table 1). These results suggest that the expression of GRIM-19 was gradually down-regulated in the progression of malignant cell transformation. There was a highly significant correlation between GRIM-19 expression and the cytological diagnosis (P<0.001).The expression of GRIM-19 in cervical exfoliated cells by western blottingWe further explored the GRIM-19 levels in cervical exfoliated cells by western blotting. Because GRIM-19 was not expressed in the surface layer exfoliated cells, these cells were counted (as a percentage) on each slide. After the GRIM-19/GAPDH proteins were quantified, the final relative expression level of GRIM-19 was calculated by the middle layer exfoliated cells/(middle layer exfoliated cells+surface layer exfoliated cells) ×100%, and GRIM-19/GAPDH in the normal middle layer exfoliated cells was normalized as 1. We found decreased GRIM-19 expression in the LSIL, HSIL, and SCC. Compared to the normal middle layer squamous cells, the expression of GRIM-19 was dramatically reduced in the LSIL cells (P<0.05), HSIL cells (P<0.05), and SCCs (P O.001) (Fig.3A-C).The expression of GRIM-19 and its association with HR-HPV infection in cervical precancersAs consistent HR-HPV infection is closely associated with SCC, we then explored the correlation of HR-HPV infection with the expression of GRIM-19 in exfoliated cells. There was a highly significant correlation between the expression of GRIM-19 and HR-HPV infection (*2= 57.583, r=-0.268, P＜0.001) (Table 2).GRIM-19 expression in cervical intraepithelial neoplasias and SCCs by immunohistochemistryTo confirm the results found in exfoliated cells, we then investigated GRM-19 expression in CIN I, CIN II, CINIII and SCC (Fig.4 A-E),all slides from colscopy-directed biopsies. High cytoplasmic expression of GRIM-19 was found in 100%(20/20) of the middle and basal layer cells of the normal cervical epithelium. The negative expression of GRIM-19 was found in 20.0%(7/35) of CIN I,23.3% (7/30) of CIN II,56.7%(17/30) of CINIII, and 95%(19/20) of SCC. The staining scores showed that, compared to the normal tissue, the expression of GRIM-19 was significantly decreased in CIN II (P<0.05), CINⅢ(P<0.05), and SCC (P＜0.001) (Fig.4F). There was a highly significant correlation between GRIM-19 expression and the biopsy diagnosis (P<0.001) (Table 3).Conclusions:1. GRIM-19 was major expressed in middle layer squamous cells, while was not expressed in surface layer squamous cells of the normal cervical epithelial cells.2. A highly significant correlation between GRIM-19 expression and the cytological diagnosis:the expression of GRIM-19 was gradually down-regulated in the LSIL、 HSIL、SCC cells.3. A highly significant correlation between GRIM-19 expression and the biopsy diagnosis:the expression of GRIM-19 was gradually down-regulated in the CIN Ⅰ、CIN Ⅱ、CINⅢ、SCC tissues; the expression of GRIM-19 was significantly decreased in CIN II, CINIII, and SCC.4. Negative correlation between the expression of GRIM-19 and HR-HPV infection, one of key factors associated with epithelial cells malignant transformation by HR-HPV infection was the decrease of GRIM-19 expression.