The Effects Of Nicotine On Ovarian Cancer Cell Line HO-8910 By Upregulating CD147

Ovarian cancer is one of the three major malignant gynecological tumors,which is the most lethal gynecological malignancy.Due to its deep anatomical location, no obvious early symptoms and rapid morbidity, 70% of patients are diagnosed at late phase, and their survival rate for five years is lower than 30%. Although the recent surgery and chemotherapy has a significant improvement in survival rate of patients with ovarian cancer, treatment failure and disease progression are still unavoidable.With the increase of global female smokers, more and more research organizations consider that smoking is a potential risk factor for ovarian cancer.Nicotine is the main component of cigarettes. There are many studies have shown that nicotine in many systems can promotethe growth and metastasis of tumor, through the induction of cell cycle progression, epithelial mesenchymal transition(EMT), migration, invasion, angiogenesis and evade apoptosis. However, whether smoking is directly related to ovarian cancer remains controversial, One study has demonstrated smoking is closely associated with mucinous ovarian cancer, but its relationship with serous ovarian cancer remains unclear.Serous ovarian cancer(SOC) constitutes the most common and lethal histological type of ovarian cancer,therefore, even a small increase in smoking-related risk of invasive serous ovarian cancer could have important implications for the worldwide incidence of ovarian cancer.CDl47, widely distributed in the human body, not only participates in a variety of physiological functions, but also is closely related to proliferation, infiltration and metastasis of tumor. CDl47 has been proven be expressed in a variety of human tumor cells.By stimulating fibroblast secrete matrix metalloproteinases(MMPs) and degrading extracellular matrix, CD147 plays an important role in many cancer progressions, in the MMPs family MMP2 and MMP9 is most closely related with tumor invasion and metastasis. The expression level is closely related to the malignant degree of tumor, Therefore, CDl47 expression is associated with the poor prognosis of patients with ovarian cancer, which is a potential prediction of ovarian epithelial carcinoma and invasive biomarkers.Research purposes:Discuss the effects of nicotine on the proliferation and migration of ovarian cancer HO-8910 cells,and the effects and molecular mechanisms of CD147 in the process, to provide theoretical and experimental basis for clarifying the effects and molecular mechanisms of CD147 on nicotine inducing cell proliferation and migration.Research methods:1. In vitro cultivation of ovarian cancer cell line HO-8910, treated with different concentrations of nicotine, after a period of time, to detect the proliferation of ovarian cancer cells by MTT(methyl thiazolyl tetrazolium) colorimetric method, and to detect the migration of ovarian cancer by wound-healing migration assay, by Western Blot detecting CD147, MMP2, MMP9 protein expression level.2. Use siRNA CD147 first knock down the expression of CD147 level, and then treat with 1uM nicotine, determined by MTT, Western Blot, wound-healing migration assay to detect the proliferation of ovarian cancer migration and CD147, MMP2, MMP9 expression.3. Use the expression of ERK inhibitor U0126 inhibit p-ERK first, and then treated with 1uM nicotine, using Western Blot to detection the influence of the expression of CD147, MMP2 and MMP9.Research results:1. The appropriate concentration and time of nicotine affecting HO-8910 cellsThe survival rate of HO-8910 cells determined by MTT shows: nicotine within a certain range may promote the proliferation of ovarian cancer cell line HO-8910 as the concentration increases, the maximum proliferation appears with 1uM, if continue to increase in nicotine concentration, cell growth significantly suppressed.Nicotine on the proliferation of ovarian cancer cell line HO-8910 also increased with time, the time dependence.2. Nicotine has influence in CD147, MMP2 and MMP9 expressions of HO-8910 cellsUsing Western Blot test protein expression levels of CD147, MMP2, MMP9 of HO-8910 cells with different nicotine concentrations(0, 0.1, 1, 10 uM) processed by 48 hours, results show that with the increasing of concentration, CD147, MMP2, MMP9 protein level is higher, the maximum appears with 1uM.By 1uM nicotine effect respectively in HO-8910 cells for 0 hours, 24 hours, 48 hours, 72 hours, the results show that the longer duration of action, CD147, MMP2, MMP9 protein expression level of HO-8910 cellsis higher, there is time dependence.3.Nicotine has an effect on the activity of ERKUsing Western Blot test ERK phosphorylation levels of HO-8910 cells with different nicotine concentrations(0, 0.1, 1, 10 uM) processing for 48 hours, results show that with the increasing concentration, p-ERK level also gradually increased, the maximum also appears with 1uM.4.Nicotine promotes the ERK activity, proliferation and migration of HO-8910 cells by CD147After transfected with siRNA CD147, HO-8910 cells processed with nicotine, with a determined by MTT method and Western Blot, wound-healing migration assay,the results show: The silence of CD147 can lead decreases to CD147, MMP2, MMP9 expression level and ERK phosphorylation level, and cell proliferation and migration ability is lower at the same time.5.ERK inhibitors have negative effect on nicotine raising CD147, MMP2, MMP9After pretreat with nicotine and U0126 to HO-8910 cells, using Western Blot, the results show that inhibition of ERK signaling pathway can lead to decreases of CD147, MMP2, MMP9 expression levels.Conclusion:1.Nicotine can selectively increase the expression of CD147 in human ovarian cancer cell line HO-8910, then increase the expression of MMP2 and MMP9, and then cause cells enhance the ability of proliferation and migration.2.The effect of nicotine that promotes the proliferation and migration can be inhibited by siRNA CD147, prompting CD147 plays a key role in proliferation and migration effect with nicotine.3.In the process of nicotine effect, ERK signaling pathway activated, and ERK inhibitor U0126 can inhibit the increase of nicotine mediated CD147, MMP2, MMP9, prompting that ERK signaling pathway in the process of nicotine mediated proliferation and migration is indispensable.