Expression And Significance Of USP22 And HIF-1α In Cervical Cancer

Carcinoma of uterine cervix is one of the most frequent and high malignant degree of tumor, its morbidity and mortality have been high in our country. There are more than 130,000 new cases per year on average in our country, accounts for about 1/4 ~ 1/3 of the world’s incidence. With the progress of cervical cancer screening in recent years, the treatment and the detection rate of cervical cancer increased significantly, but the total incidence is not reduced, especially the younger trend are increasing obviously. This phenomenon is a serious menace to the reproductive health of women who are at childbearing age. The occurrence of cervical cancer development has a lot of factors, steps, such as HPV infection, the behavior factors, cervical erosion, etc. We mainly take the etiology and development as a basis for cervical cancer prevention and treatment. Such as HPV vaccination and prevention programs for regular screening, cooperate with surgery, drugs and gene therapy, etc Although the above measures are taken,cervical cancer is still ranking the second in gynecologic malignant tumors in China, and the 5-year survival rate is still not satisfied. The nosogenesis of cervical neoplasm has not been made sure, the treatment remains to be further improved, so look for cervical cancer‘s related indexes of molecular biology and the specific target in the development of cervical cancer is urgently needed.Ubiquitin specific peptidase 22(USP22) is one builder of the specificity of ubiquitin proteasomes, expresses highly among various kinds of malignant tumors. It is located in human chromosome 17, contains 1578 base pairs. The proteins encoded by it mainly play roles by deubiquitination in order to adjust modify a series of biochemical reactions within the cell, such as the growth and differentiation of cell,controlling cell cycle, activation of transcription factors, regulation of signal transduction pathways involved in tumor development. Protein encoded by USP22 express mainly in the nucleus, there is a zinc finger construction at its amino terminal which is common for ubiquitin enzyme. And the finger construction is the main foundation for USP22 to work with the substrate protein. There is a His box and a Cys box at the carboxyl terminal, the Cys box include Cysteine, histidine, aspartic acid/asparagine group, whose main function is to take down the ubiquitin from the aim protein. Tumor stem cells study consider that there is a small number of tumor cells has infinite proliferation, self-renewal and multi-directional differentiation potential, we call them cancer stem cells. They are causes for oncogenesis, inbreaking and metastasis. Someone said that USP22 is a new tag to be used as tumor stem cells, and it is expected to become the key point of controlling tumor. Studies remaindered that the mechanism of action maybe USP22 can remove the ubiquitin which will degrade histone H2 A, H2 B in order to control gene copy and expression, and will affect the cell cycle at the same time. Study after study has shown that the tumor cells lacking of USP22 protein has a slower proliferation, most of them are stagnating in the G1 phase. In recent years, USP22 has a high expression in colorectal cancer, liver cancer, bladder cancer, non-small cell lung cancer, oral squamous cell carcinomas, and esophageal squamous carcinoma are reported, but its tumor specific mechanism is still not very clear.Hypoxia-inducible factor HIF- 1 is the core transcription factors related to the tumor hypoxia microenvironment in present study. HIF- l is composed by α and βsubunits as a kind of heterologous dimers. α subunit is the Regulatory proteins for oxygen, determine the activity of HIF – 1. β subunit is the fundamental protein,connecting with the stability of HIF. When tumor cells increase quickly will make a hypoxic microenvironment, HIF-1α will concentrate and transfer to the nucleus, then combine with HIF- 1β to form an actived HIF – 1. The activated HIF – 1 will couple with the aim genes of hypoxia response element, to start the expression of aim genes.For example, working on the key enzyme of glycolytic to provide energy for the growth of tumor, or work on vascular endothelial growth factor VEGF, to promote tumor angiogenesis, to break a path for the development, invasion and metastasis of tumor.ObjectiveResearches about USP22 or the correlation between USP22 and HIF – 1α in cervical cancer have not been reported in our country. This study adopts the method of immunohistochemistry and RT-PCR to detect the protein and gene expression of USP22 and HIF – 1α, grouped by normal cervical tissues, CIN and carcinoma of cervix. Analysis of the difference between them in clinical classification, pathologic stage, lymph node metastasis, judge that is there a correlation between USP22 and HIF – 1α in the occurrence and development of cervical cancer. It may bring new hope for the early diagnosis, biological evaluation and molecular targeted therapy for cervical cancer.Materials and methods1. Materials: We got in 85 cases of cervical samples in the Third Affiliated Hospital of Zhengzhou University all the year of 2014, put these cases in three groups, the number of CIN is is 25, cervical carcinoma is 45 and the number of normal cervix is15.(from the patients with the operation of total hysterectomy due to hysteromyoma or adenomyosis, not except cervicitis). And the means of the ages is 40,49,50respectively;Going by the FIGO standard of cervical carcinoma,there are 20 cases of stage I samples,25 cases of stage II;At the same time,according to the cervical cancer pathology classification standard,there are 20 casesof G1 stage,14 cases of G2 stage,and 11 cases of G3 level.And there are 24 ones with lymph node metasis and 21 ones not.16 ones are older than 50, 29 ones are not. All the samples were had not received chemotherapy or radiotherapy before operations. Took part of the tissue in liquid nitrogen then stored at the temperature of 80 below zero,the other part was fixed in formaldehyde,embedded by paraffin and then make serial section at 4 μm,all the samples were made a definite diagnosis by two qualified pathologist.2. Methods : We use the methods of Immunohistochemistry and fluorogenic quantitative PCR to detect protein and m RNA expression level of USP22 and HIF-1αin carcinoma of cervix group, CIN and normal cervical tissues.Then, find the roles of both in development and incidence of cervical cancer and demonstrate the correlation between them. It May bring new hopes for an early diagnosis, biological evaluation, molecular targeted therapy for cervical cancer.3. Statistics:The Statistical anylysis was carried out by the statistical software of SPSS 16.0,and the quantitive datas were made in the form ofsx ±.The datas are analyzed by t test, K-W test,One-way analysis of variance,ect;and the qualitative datas are analyzed by Chi—Square Tests,Fisher’s exact probability method and so on.Pearson correlation method is used to analyze the relationship of USP22 and HIF-1α in cervical cancer.Size of a test is 0.05.Results1. The expression of USP22 and HIF-1αImmunohistochemical results shown: USP22 proteins are mainly expressed in the nucleus, nucleus and cytoplasm are where HIF-1α proteins appear, all the positive cells are dyed in claybank under the microscope.RT-PCR results shown: Observe the CT value of purpose gene after amplification, ΔCT = CT(purpose gene)- CT(internal), Δ CT value was on behalf of the cervical tissue purpose gene expression, 2-ΔΔ C T was on behalf of how many times of cervical tissue gene expression compared with normal cervix purposes gene expression.2. The expression of USP22 and HIF-1α in distinct cervical tissuesProtein and nucleic acid expression of USP22 in cervical cancer, CIN and normal cervix tissue were gradually reduced. The positive rate of protein expression was as follows: 57.8%, 32% and 0%, respectively, and the differences was statistically significant(2c =16.583, P < 0.05). The relative expression of m RNA were5.822±5.489、2.214±0.866 、1.047±0.339,severally, prompt the expression of USP22 in cervical cancer and CIN was 5.822 times and 5.822 times compared with normal cervix, the different expression were: 3.975±1.417、4.851±0.574、5.890±0.445,and it had significant differences(H=18.233, P<0.05).Protein and nucleic acid expression of HIF-1α in cervical cancer, CIN and normal cervix tissue were gradually reduced, too. The positive rate of protein expression were as follows: 82.2% 、 52% 、 20%, severally,the differences was statistically significant(2c =19.665, P < 0.05). The relative expression of m RNA were4.313±3.122、2.305±1.083、1.048±0.31519.665,severally, prompt the expression of HIF-1α in cervical cancer and CIN was4.313 times and 2.305 times compared with normal cervix, different expression were: 4.928±1.155、5.582±0.727、6.623±0.475,it had significant differences(H=18.302, P<0.05).3. The correlation between the age of cervical cancer cases and the expression of USP22 and HIF-1α.Protein expression of USP22 in cervical cancer has no correlation with age, the positive rates of protein expression were as follows: 56.25%(older than 50) 、58.62%( younger than 50),differences was not statistically significant(P=0.878). The relative expression of m RNA were3.764±1.361、4.091±1.458,difference was not statistically significant(P=0.466),too.Protein expression HIF-1α of in cervical cancer has no correlation with age, the positive rate of protein expression were as follows: 68.75%(older than 50)、89.66%( younger than 50),difference was not statistically significant(P=0.177). The relative expression of m RNA were4.558±1.06、5.132±1.71,difference was not statistically significant(P=0.112).4. The correlation between the clinical stages of cervical cancer and the expression of USP22 and HIF-1α.The expressions of USP22 and HIF-1α protein in cervical cancer are related to clinical stages. The positive rates of USP22 Protein in stage I,II were 50%、64%,difference was not statistically significant(P=0.345), The expression of m RNA were4.763±1.326、3.344±1.166, difference was statistically significant(P<0.05).The positive rates of HIF-1α Protein in stage I,II were 75%、88%, difference was not statistically significant(P=0.459). The expression of m RNA were5.573±1.096 、4.412±0.931, difference was statistically significant(P<0.05).5. The correlation between lympho node metastasis of cervical cancer and the expression of USP22 and HIF-1α.The expressions of USP22 and HIF-1α protein in cervical cancer are correlated to lympho node metastasis. The positive rate of USP22 and HIF-1α samples with lympho node metastasis is75%,95.83%, higher than that without(38.10%,66.67%)difference was statistically significant(P<0.05);The same consequence had been found from the relative expression of m RNA.6. The correlation between pathologic grades of cervical cancer and the expression of USP22 and HIF-1α.The expressions of USP22 and HIF-1α protein in cervical cancer are correlated to pathologic grades.The positive rates of USP22 Protein in G1 stage, G2 stage, G3 stage are40% 、57.14% 、 90.91%, difference was statistically significant(P<0. 05), The relative expression of m RNA were5.595±0.096、3.71±1.172、2.558±0.772, difference was statistically significant(P<0.05).The positive rates of HIF-1α Protein in G1 stage, G2 stage, G3 stage are60% 、92.86% 、 100%difference was statistically significant(P<0. 05). The relative expression of m RNA were5.595±0.996、4.841±1.082、3.826±0.477, difference was statistically significant(P<0.05).7. The correlation between the size of cervical cancer and the expression of USP22 and HIF-1α.The expressions of USP22 and HIF-1α protein in cervical cancer are correlated to the size. The positive rate of USP22 and HIF-1α samples with the size bigger than 4cm are74.07%,96.30%,higher than that smaller than 4cm(33.33%,61.11%) difference was statistically significant(P<0.05);The same consequence had been got from the relative expression of m RNA.8. The correlation between USP22 and HIF-1α in cervical cancerExpression of protein results suggest that USP22 positively related with HIF-1α, the same as nucleic acid expression.Conclusions1. In cervical cancer, the expression of USP22 and HIF- 1α increased gradually may promote the occurrence development, invasion and metastasis of cervical cancer.2. USP22 is positively related with HIF-1α, it means that there may be some kind of interaction to promote the occurrence and development of the tumor.3. Combining USP22 and HIF- 1α testing may be used as auxiliary indexes in the diagnosis of cervical cancer, or bring new hope for targets treatment.