Effect Of Silencing SUZ12Expression On Repression Of Proliferation And Metastasis In Human Non-small Cell Lung Cancer And Its Mechanism

Background:Lung cancer is a malignant tumor which originated in bronchial mucosa or gland. Its incidence is in the first bit of male malignant tumor and second bit of female malignant tumor. It is one of the most common cause of death in malignant tumor. With the air pollution and the increase of smoking, lung cancer will pose a big threat to human health and life. Lung cancer in early stage has no obvious symptoms, especially in non-small cell lung cancer which accounted for about80%of lung cancer. Most patients diagnosed when already at middle-late stage, and had the focal shift in the distance, at last lost the opportunity of radical surgery. Although chemotherapy and radiation can shrink tumors to a certain extent and improve the quality of life, the susceptibility to radiation and chemotherapy of non-small cell lung cancer is so poor, while the side effects of radiation and chemotherapy drugs which lead to the low survival rate and poor prognosis of patients with lung cancer. The research has pointed out that the5-year survival rate for patients with NSCLC of A stage is15-23%, while only about6-7%of patients with NSCLC of B stage. The research has pointed out that the5-year survival rate for patients with NSCLC of A stage is15-23%, while only about6-7%of patients with NSCLC of B stage. The tolerance to chemotherapy and radiotherapy of patients with lung cancer and the growth, recurrence, metastasis of tumor are the main factors influencing the prognosis.As well as other tumor diseases, the occurrence of lung cancer is a complex process of accumulating genetic damage which involved in multiple genetic factors. The activation and expression of oncogene, the deficiency or inactivation of tumor suppressor genes and other genetic mutation is in the every period of the development, invasion, metastasis of tumor, while mutation can be genetic. The development, invasion, metastasis of tumor have extremely close relations with the abnormal of gene. The level of gene therapy offers hope for the radical cure of tumor which showed the good prospects for treatment. In recent years, with the deepening research on mechanism of tumor development and the development of molecular biology techniques, the gene therapy of tumor developed rapidly which has become the hot topic and new hope in the study of tumor therapy. Among them, a branch of gene therapy is that using antisense technology, ribozymes or RNAi technology specificity inhibit the cancer genes and their products, thereby inhibit the growth of tumors cells or restore its sensitivity to the radiation and chemotherapy. RNAi has quickly become a hot field in life science research since it has been found. RNAi use the21-23bp long double-stranded RNA to induce the degradation process of mRNA with homologous sequences. It can block the expression of the specific gene in vivo effectively and show a lack of specific gene phenotypes in cells. Compared with the antisense nucleic acid and nuclear enzyme technology, RNAi technology has the characteristic of high specificity, long time inhibition, high efficiency of the inhibition of gene expression and so on. It is a powerful tool for the regulation of gene expression. The emergence of RNAi technology offers a new approach for various diseases such as the prevention and cure of tumor and the disease caused by virus and the research of the new gene function. Therefore, looking for the right epigenetics interference target, using RNAi technology to inhibit the related genes and their products, thereby inhibiting the growth and metastasis of tumor cell is expected to be the new way for tumor treatment.SUZ12is one of the core subunits of PcG family, it realize its function of transcription and modification by influencing the activity of histone methylation transferase and silencing the gene of EED-EZH2compounds. As a kind of proto-oncogenes, it plays an important regulation role in promoting cell proliferation, inhibiting cell apoptosis and enhancing cell malignant. Several studies have confirmed that the abnormally high expression of SUZ12in prostate cancer, gastric cancer, breast tumor, nervous system neoplasms and other malignant tumor are closely related to the poor prognosis of tumor, The wild-type SUZ12expression plasmid was shifted into normal stomach and mammary gland epithelial cells which can promote cell malignant transformation, while down-regulated the expression of SUZ12can effectively inhibit the proliferation of tumor cells, it prompt that SUZ12may be involved in the formation of tumor.SUZ12was closely associated with the invasion and metastasis of a variety of malignant tumor. Some scholars have studied the relationship of SUZ12and the most common type infiltrating ductal carcinoma of breast cancer, it has been found that the expression of mRNA and protein level in SUZ12were high, The high expression of SUZ12has the obvious relevance to the metastasis of axillary lymph node and the positive conditions of estrogen receptor which suggest that SUZ12is related to the progression and metastasis of breast infiltrating ductal carcinoma. Li and others has found that the high expression of SUZ12in ovarian cancer tissue is associated with tumor progression. In addition, the down-regulated of SUZ12can reduce the invasive ability of MNK1and other gastric cancer cells.SUZ12is overexpression in parietal cell of immortalization and plays an important role in the development and transfer of gastric cancer cells.Lung cancer is one of the malignant tumor which has a serious hazard to human health and life. The malignant proliferation, invasion, metastasis of tumor cell in vivo is the main cause of the high mortality rate. The high expression of SUZ12in a variety of malignant tumor cells is closely related to the ability of proliferation and metastasis. Throughout the research abroad about PcG and lung cancer, although the relationship between the expression of EZH2and Bmi-1in lung cancer tissue and the clinical staging in patients with lung cancer, the depth of tumor invasion has gradually cleared, it is still lack of the systemic research about the expression of SUZ12in lung cancer cells and the relationship between SUZ12and cell proliferation and metastasis.In conclusion, this topic proposed to use human non-small cell lung cancer A549cell line as the experiment object and observe the ability of cell proliferation and transfer after silencing the expression of SUZ12by RNA interference and discuss its molecular mechanism preliminarily which will provide experimental foundation and theoretical basis for finding the new target and approach for lung cancer treatment.Objective:This topic proposed to use human non-small cell lung cancer A549cell line to detect the expression of SUZ12gene and observe the effects on cell proliferation and migration ability after silencing the expression of SUZ12and explore the relevant molecular mechanisms, which will provide the theoretical and experimental basis for SUZ12as a novel target for clinical treatment of lung cancer.Methods:1. Effect of silencing SUZ12expression on repression of proliferation in human non small lung cancer and its mechanism.(1) The different expression of SUZ12gene in L132, NHBE,16HBE and A549cell line was detected with western blot.(2) Lung cancer cell line was adopted, the pairs of SUZ12-siRNA were designed according to the SUZ12gene sequence and synthesized chemically. After SUZ12-siRNA transfection, the proliferation of A549cell and normal cell was examined by MTT. The ability of A549cell proliferation was measured by colony-forming unit assay. The tumor experiment in vitro was measured by nude mice. The cell cycle status and apoptosis of A549cell were detected by flow cytometry (FCM). The senescence of A549cell was examined by β-Galactosidase dyeing assay.(3)72hours after SUZ12-siRNA transfection, western blot assay was used to determine the change of level of protein of EZH2, EED, H3K27me3, CyclinD1, CDK2, CDK4, CDK6, P21, p27, P16INK4a, P14ARF, P53, Rb, pRb and E2F1. (4) SUZ12-siRNA and CDKN2A-siRNA transfection at the same time to silence the expression of SUZ12, P16INK4a and P14ABF in A549cell line, the proliferation of A549cell was examined by MTT. The ability of A549cell proliferation was measured by colony-forming unit assay. The cell cycle status of A549cell were detected by flow cytometry (FCM). The senescence of A549cell was examined by β-Galactosidase dyeing assay.2. Effect of silencing SUZ12expression on repression of metastasis in human non small lung cancer and its mechanism.(1) After SUZ12-siRNA transfection, the ability of cell invasion in vitro was tested by transwell chamber model.(2) After SUZ12-siRNA transfection, the ability of cell metastasis in vitro was tested by transwell chamber model.(3) Western blot assay was used to determine the change of level of protein of Snail, E-cadherin, N-cadherin, Vimentin, MMP-2and TIMP-1.Results:1. Effect of silencing SUZ12expression on repression of proliferation in human non small lung cancer and its mechanism.(1) Expression of SUZ12protein were overexpressed by (5.16±0.77),(2.44±0.29),(2.32±0.34) times in A549cell compared with NHBE,16HBE, L132cells(P<0.01).(2) siRNA transfection can silence the expression of SUZ12gene in A549cell effectively and western blot revealed that SUZ12-siRNA-003group has the best silencing effect. MTT revealed that the rate of A549cell proliferation decreased (52.03±5.31)%and the rate of normal cell proliferation has no difference compared to the control group. Colony-forming unit assay revealed that the clone number in untreated group, Ctrl-siRNA group and SUZ12-siRNA group were86.00±11.00,82.30±10.50and26.00±6.60respectively, the number and rate of colony formation in SUZ12-siRNA were largely decreased (p<0.05). Tumor formation can be found in every nude mice. Tumor tissue mean weights of the untreated group and Ctrl-siRNA group were342.97±28.88mg and310.03±59.32mg respectively.However, tumor tissue weight of SUZ12-siRNA group was 177.15±24.32mg. Tumor growth was in inhibited significantly (P<0.05) and the inhibition rate was48.35%. FCM revealed that the percentage of G1stage cells of untreated group, Ctrl-siRNA group and SUZ12-siRNA group were (57.08±2.30)%,(57.16±3.51)%and (78.29±2.88)%respectively; the percentage of S stage cells were (23.53±4.35)%,(26.45±1.38)%and(13.82±0.86)%respectively; the percentage of G2stage cells were (19.39±2.39)%,(16.39±3.61)%and (7.98±2.55)%respectively. These data showed that SUZ12-siRNA restrained the conversion of G0/G1stage. FCM revealed that the percentage of apoptotic cells of untreated group, Ctrl-siRNA group and SUZ12-siRNA group were (2.49±0.94)%,(2.78±0.64)%and (3.66±1.25)%respectively, β-Galactosidase dyeing assay revealed that SUZ12-siRNA group has the senescence obviously compared with the untreated group and Ctrl-siRNA group.(3) Western blot assay revealed that SUZ12-siRNA transfection down-regulated the level of expression of EZH2, EED, H3K27me3, down-regulated the level of expression of CyclinD1, CDK2, CDK4, CDK6and up-regulated the level of expression of P21and P27, down-regulated the level of expression of E2F1, pRb and up-regulated the level of expression of P16INK4a, P14ARF, P53and Rb in A549cell line compared with the untreated group and Ctrl-siRNA group.(4) Comparing with the SUZ12-siRNA group, the rate of cell proliferation increased (62.64±4.16)%in (SUZ12+CDKN2A)-siRNA group. The rate of clone formation increased (59.84±14.47)%in (SUZ12+CDKN2A)-siRNA group. The percentage of Gl stage cells decreased (13.72±5.29)%in (SUZ12+CDKN2A)-siRNA group.2. Effect of silencing SUZ12expression on repression of metastasis in human non small lung cancer and its mechanism.(1) Transwell assay revealed that the number of migration cells decreased (70.33±7.17)%.(2) Transwell assay revealed that the number of invasion cells decreased (72.86±6.72)%.(3) Western blot assay revealed that SUZ12-siRNA transfection down-regulated the level of expression of Snail, N-cadherin, Vimentin, MMP-2and up-regulated the level of expression of E-cadherin and TIMP-1Conclusion:1. The expression of SUZ12is high in A549cell lines and low in lung normal cell lines.2. The inhibition of proliferation in lung cancer cell line A549after silencing the expression of SUZ12gene may be via lifting the effect of gene silencing mediated by PRC2and reactivating INK4a/ARF signaling pathways to block cycle process and induce cell aging.3. The inhibition of invasion and migration in lung cancer cell line A549after silencing the expression of SUZ12gene may be by reducing the activity of cell proliferation and lifting its inhibition effect to calcium adhesive proteins to reverse the occurrence of EMT in tumor cells and to change the expression level of extracellular matrix metalloproteinases, but the specific molecular mechanism remains to be futher studied.4. The expression of SUZ12is closely related to the proliferation, invasion, migration and other biological behavior of lung cancer cell and it is expected to become an important target to inhibit the recurrence and metastasis of lung cancer.