Study Of The Function And Mechanism Of JMJD2A In The Pathogenesis And Progression Of Human Breast Cancer

Human breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females. According to the GLOBOCAN estimates, breast cancer accounted for 23% of the total cancer cases and 14% of the cancer deaths. Prognosis of patients with advanced stage breast cancer is poor because of the progression and metastasis of the tumor, even if various therapies were employed for most cases.Multiple factors, including genetic background, hormone disorder, and environmental impact are involved in breast cancer complex pathogenesis. Polygenic variation causes breast cancer. The expression of genes is mutative during the pathogenesis and progression of human breast cancer. Alteration of these genes contributes to breast cancer biological behaviors and clinical phenotypes. Gene expression level and gene mutation/rearrangement affect its regulating function. Activation of oncogenes or inactivation of tumor-suppressor genes are frequently reported to trigger breast cancer. Further study and explanation on the molecular mechanism in the pathogenesis and progression are significantly meaningful in order to understand and treat breast cancer.JMJD2A, about 130 KD, belongs to the JMJD2 protein family, containing JmjN, JmjC, two tandem PHD zinc finger motifs and Tudor domains. JmjC is considered as main functional area in the JMJD2 protein family, which can catalysis histone demethylation to break the balance of histone methylation and demethylation. PHD zinc finger motif and Tudor domain can take part in the protein-protein interaction, binding transcription factors leading to the chromatin reconstruction. Histone deacetylase (HDAC) is the most common and important in the chromatin reconstruction complexes.JMJD2A is widely expressed in human cancer tissues and cell lines, such as lung cancer, bladder cancer and prostate cancer. High endogenous expression of JMJD2A mRNA was found in several human cell lines, including human T-cell lymphotropic virus 1-infected cell lines, the HT1376 bladder carcinoma cell line, the U2OS osteosarcoma cell line and the prostate cancer cell line.JMJD2A can combine with histone deacetylases, and the histone deacetylation is considered to repress gene transcription. Thus, JMJD2A may accelerate histone deacetylation to repress gene transcription. JMJD2A can bind pRb, E2F and HDAC structurally and functionally to form complex to repress the expression of downstream genes. E2F-HDAC complex plays an important role in this process to inhibit the expression of downstream genes, in which tumor suppressor gene ARHI is critical.JMJD2A is widely expressed in human tissues, and correlate with pathogenesis and progression of various cancers. However, there is no literature reporting whether the expression of JMJD2A in human breast cancer is abnormal and whether the expression of JMJD2A correlates with pathogenesis and progression of breast cancer. If JMJD2A correlates with breast cancer, what pathway does JMD2A involve in to regulate the pathogenesis and progression of breast cancer? This study is devoted to research on the function and mechanism of JMJD2A in the pathogenesis and progression of human breast cancer from the three aspects.Part I The expression of JMJD2A in human breast cancer and correlation with ARHI, p53, ER, PR and CerbB-2Background:JMJD2A is widely expressed in human cancer tissues and cell lines, but there is no literature reporting the correlation between JMJD2A and breast cancer. This study is devoted to detect the expression of JMJD2A in human breast cancer. Further, the correlation between JMJD2A and ARHI, p53, ER, PR and CerbB-2 was investigated.Methods:104 human breast cancer samples and 60 fibroadenoma samples were collected. HE staining and histological examination were used to validate the cilincal diagnosis. Immunohistochemical examination, western blot and quantitative real-time PCR were performed to detect the expression of JMJD2A in breast cancer tissues and benign lesion tissues respectively. Immunohistochemical examination and quantitative real-time PCR were performed to detect the expression of ARHI, p53, ER, PR and CerbB-2 in the breast cancer, and SPSS statistic software was used to analysis their correlation with JMJD2A.Results:The clinical diagnosis of these samples is correct. According to immunohistochemical analysis,104 infiltrating duct carcinoma tissue samples were made up of 22 strongly positive results (21.2%),34 moderately positive results (32.7%),30 weakly positive results (28.8%) and 18 negative results (16.7%). While 2 strongly positive results (3.3%),5 moderately positive results (8.3%),6 weakly positive results (10.0%) and 47 negative results (78.3%) composed 60 benign lesion tissue samples. Western blot showed that, the expression of JMJD2A protein in infiltrating duct carcinoma tissue was notablely higher than benign lesion tissue. The mean optical density of infiltrating duct carcinoma tissue was 1.094±0.151, while that of benign lesion tissue was 0.563±0.105. Quantitative real-time PCR showed that, there was significant difference in the levels of JMJD2A mRNA between infiltrating duct carcinoma tissue (0.621±0.117) and fibroadenoma tissue (0.286±0.105). Spearman and Pearson correlation analysis suggested that, expression of JMJD2A was negatively correlated with ARHI, and positively correlated with p53 and ER.Conclusion:It was confirmed that the expression of JMJD2A in human breast cancer was higher than benign lesion tissue from in situ, protein level and mRNA level. Moreover, according to the statistic software analysis, JMJD2A is negatively correlated with tumor suppressor gene ARHI, and positively correlated with p53 and ER in in infiltrating duct carcinoma.Part Ⅱ Study on the correlation between JMJD2A and highly invasive human breast cancer cell line MDA-MB-231Background:Part Ⅰ has proved that JMJD2A is highly expressed in human breast cancer tissue, but the correlation between JMJD2A and breast cancer is still unaccounted, which needs further experiments to investigate. It is simple, specific and effective to use small interfering RNA (siRNA) to silence target gene. Recently, siRNA is widely used. In this study, siRNA was used to silencing JMJD2A and then biological behavior of cancer cells was observed.Methods:MDA-MB-231 cells in siRNA group were transfected with chemically synthesised JMJD2A-specific siRNA, at the same time negative control group was transfected with negative control siRNA and blank control group just used culture media. Quantitative real-time PCR and western blot were performed to detect expression of JMJD2A at mRNA level and protein level in three groups. Flow cytometric anlysis and MTT assay were used to evaluate cell cycle and proliferation in three groups. The abilities of invasion and migration in three groups were evaluated by cell migration and invasion assay with Transwell chambers.Results:The transfection efficiency was about 72.3%. According to the results of quantitative real-time PCR, the mRNA expression of siRNA group (0.386±0.108) were significantly lower than that in blank control group (0.998±0.170) and negative control group (0.997±0.150), respectively. Western blot analysis showed that, the levels of JMJD2A protein expression in the siRNA group (0.093±0.051) were significantly lower than that in blank control group (0.203±0.042) and negative control group (0.210±0.050), respectively. Flow cytometric anlysis showed that, the percentage of cells in G0/G1 phase in siRNA group was 44.3±1.6%, higher than that in blank control group (30.3±2.7%) and negative control group (34.2±2.3%). The percentage of cells in S phase in siRNA group was 43.4±2.3%, lower than that in blank control group (58.4±2.1%) and negative control group (52.8±2.2%). The percentage of cells in G2/M phase in siRNA group was 12.1±2.2%, and in blank control group was 11.0±1.2%, in negative control group was 13.3±1.8%. Proliferation index was calculated, which showed that, proliferation index in siRNA group (55.6±2.1%) was lower than that in blank control group (69.6±2.1%) and negative control group (65.9±2.2%). MTT assay showed the average actual absorbance of siRNA group (1.711±0.087) was significantly lower than blank control group (2.136±0.135) and negative control group (2.089±0.115). In the migration assay, transmembrane cell number in siRNA group was 67 ±10.2, lower than blank control group (173±17.7) and negative control group (168±16.4). In the invasion assay, transmembrane cell number in siRNA group was 175±14.4, lower than blank control group (327±20.8) and negative control group (311±15.3).Conclusion:The in vitro cell experiments proved that transfection with JMJD2A-spefic siRNA in highly invasive human breast cancer cell line MDA-MB-231 could down-regulate JMJD2A mRNA and protein expression to silence it. The transfection with JMJD2A-spefic siRNA in highly invasive human breast cancer cell line MDA-MB-231 was successful. Furthermore, silencing JMJD2A can cause cell cycle change and inhibition of proliferation, migration and invasion of MDA-MB-231. This study suggested that silencing JMJD2A gene can inhibit malignant biological behavior of cancer cells. In human breast cancer, high expression of JMJD2A is not a coincidence, but correlated with the biological behavior of cancer cells.Part III Study on the correlation between JMJD2A and lowly metastatic human breast cancer cell line MCF-7Background:Previous two parts confirmed that JMJD2A was highly expressed in human breast cancer and JMJD2A was correlated with the biological behavior of cancer cells. Transfection with JMJD2A-spefic siRNA in highly invasive human breast cancer cell line MDA-MB-231 could silence JMJD2A gene, down-regulate JMJD2A protein, change cell cycle and inhibit proliferation, migration and invasion of MDA-MB-231. Transfection was effective. However, it is flawed to merely reveal the correlation between JMJD2A and highly invasive cell line MDA-MB-231. Other breast cancer cell line can’t be represented. Thus, experiments in other cell line are still required to complete the research on the correlation between JMJD2A and breast cancer. Lowly metastatic human breast cancer cell line MCF-7 combined with highly invasive MDA-MB-231 could mimic the expression patterns of their in vivo counterparts. Therefore MCF-7 cell line is an effective supplement to Part II.Methods:In this study, we chose lowly metastatic human breast cancer cell line MCF-7. MCF-7 cells in siRNA group were transfected with chemically synthesised JMJD2A-specific siRNA, at the same time negative control group was transfected with negative control siRNA and blank control group just used culture media. Quantitative real-time PCR and western blot were performed to detect expression of JMJD2A at mRNA level and protein level in three groups. Flow cytometric anlysis and WST-8 assay were used to evaluate cell cycle and proliferation in three groups. The abilities of invasion and migration in three groups were evaluated by cell migration and invasion assay with Transwell chambers.Results:The transfection efficiency was about 70.4%. According to the results of quantitative real-time PCR, the mRNA expression of siRNA group (0.55±0.03) were significantly lower than that in blank control group (0.98±0.02) and negative control group (0.94±0.03), respectively. Western blot analysis showed that, the levels of JMJD2A protein expression in the siRNA group (0.083±0.031) were significantly lower than that in blank control group (0.223±0.053) and negative control group (0.208±0.047), respectively. Flow cytometric anlysis showed that, the percentage of cells in G0/G1 phase in siRNA group was 53.80±1.80%, higher than that in blank control group (44.24±1.86%) and negative control group (46.37±1.29%). The percentage of cells in S phase in siRNA group was 36.55±1.52%, lower than that in blank control group (47.06±1.26%) and negative control group (44.72±1.86%). The percentage of cells in G2/M phase in siRNA group was 9.64±1.26%, and in blank control group was 8.70±0.63%, in negative control group was 8.58±1.35%. Proliferation index was calculated, which showed that, proliferation index in siRNA group (46.19±1.80%) was lower than that in blank control group (55.76±1.86%) and negative control group (53.63±1.29%). WST-8 assay showed the average actual absorbance of siRNA group (1.45±0.05) was significantly lower than blank control group (1.73±0.02) and negative control group (1.69±0.03). In the migration assay, transmembrane cell number in siRNA group was 57±11.3, lower than blank control group(114±19.5) and negative control group (109±8.7). In the invasion assay, transmembrane cell number in siRNA group was 133±17.4, lower than blank control group (197±20.6) and negative control group (181±16.3).Conclusion:In vitro cell experiments in this part proved that transfection with JMJD2A-spefic siRNA in lowly metastatic human breast cancer cell line MCF-7 could also down-regulate JMJD2A mRNA and protein expression to silence it. Moreover, silencing JMJD2A can cause cell cycle change and inhibition of proliferation, migration and invasion of MCF-7. Based on the conclusion of Part I and Part Ⅱ, this study proved that transfection with JMJD2A-specific siRNA in both MCF-7 and MDA-MB-231 cell lines could silence JMJD2A gene and inhibit malignant biological behavior of cancer cells such as proliferation, migration and invasion. The correlation between JMJD2A and the pathogenesis and progression of breast cancer is a common.Part IV Study on the mechanism of JMJD2A in regulating tumor suppressor gene ARHIBackground:Previous three parts have proved that JMJD2A is highly expressed in human breast cancer tissue and correlated with the pathogenesis and progression of breast cancer. The negative correlation between JMJD2A and ARHI was revealed in Part I. Tumor suppressor gene ARHI is suppressed in malignant tumors such as breast cancer and ovarial cancer. JMJD2A could interact with HDAC1, HDAC3 and pRb. Researches showed that E2Fs were negative correlated with ARHI. And HDACs were reported involved in ARHI negatively regulation. We conjectured that ARHI was the target gene JMJD2A regulated, and JMJD2A regulated the expression of ARHI. However, the regulation mechanism needs further exploration.Methods:Western blot was performed to detect expression of JMJD2A and ARHI in human breast cancer and paired tumor-adjacent non-cancerous breast tissues. Immunohistochemical examination was performed for JMJD2A and ARHI in serial sections of breast cancer and benign lesion. Quantitative real-time PCR and western blot were performed to detect expression of JMJD2A and ARHI at mRNA level and protein level after transfection with JMJD2A-specific siRNA in MCF-7 and MDA-MB-231 cell line. Co-immunoprecipitation assay was used to determine proteins binding to JMJD2A. Chromatin immnoprecipitation assay was used to determine whether JMJD2A binds to ARHI promoter.Results:Western blot showed that, JMJD2A highly expressed and ARHI lowly expressed in human breast cancer, while JMJD2A lowlier expressed and ARHI more highly expressed in the paired tumor-adjacent non-cancerous breast tissues than breast cancer. Immunohistochemical examination showed that ARHI was positive when JMJD2A was negative in breast cancer and benign lesion, and ARHI was negative when JMJD2A was positive in breast cancer. Expression of ARHI increased at mRNA level and protein level after silencing JMJD2A in two cell lines. JMJD2A could specifically co-immunoprecipitated E2Fs and HDACs. JMJD2A could immunoprecipitate ARHI promoter at A1 and A2 sites.Conclusion:We confirmed that the expression of JMJD2A was negatively correlated with ARHI, and silencing JMJD2A gene caused the expression of tumor suppressor gene ARHI increased. JMJD2A really can specifically bind to HDACs and E2Fs to form complex. JMJD2A can specifically bind to the ARHI promoter to negatively regulate ARHI. The mechanism was revealed that JMJD2A negatively regulate ARHI to promote the pathogenesis and progression of breast cancer.