Investigating Effects And Mechanisms Of RCC2 In Breast Cancer

Background:RCC2(Ragulator of chromosome condensation 2),also termed TD60,locus on 1p36.13.Its coding protein belongs to CPC(chromosomal passenger complex),which is involved in cell mitosis and spindle assembly,and is associated with cell proliferation and migration.The important pathological features of tumors are abnormal cell proliferation and cell metastasis.There are reports that RCC2 plays an important role in tumors.It has been shown that RCC2 is associated with the recurrence of melanoma,colorectal cancer and basal cell carcinoma,the expression of RCC2 was significantly raised in gastric cancer,and RCC2 can promote the transfer of lung adenocarcinoma.All of these evidence that RCC2 plays an important role in tumors.Breast cancer is the most common malignant tumor in women and its incidence is 30%in female malignant tumors.The biological behavior of tumor cells,such as proliferation、migration and apoptosis influence the development and prognosis of breast cancer,and the development of breast cancer is associated with abnormal signal pathway of oncogenes and supressor genes,but the exact molecular mechanism has not been fully elaborated.Therefore,to explore the cellular and mechanism of breast cancer,to find effective prognostic is of great importance on diagnosis of breast cancer.RCC2 is associated with a variety of cancers,this finding let us infer RCC2 may be associated with breast cancer.But there has not been about RCC2 effect on breast cancer in vitro and in vivo.Estrogen receptor positive breast cancer is the most common type of breast cancer.So,we explore the relationship of RCC2 and regulation-mechanism of breast cancer at the same time studying whether estrogen plays an important role in breast cancer through RCC2.In this topic,we explore the pathogenesis mechanism of RCC2 in breast cancer and its relationship with estrogen through clinical specimens、cell models and animal models,clarify RCC2 how to participate in the development of breast cancer and its mechanism,thus providing a new idea to explore the pathogenesis of breast cancer.Methods:1.Collect breast.tissues(n=32,contained estrogen receptor+(ER+)breast cancer tissues 16 cases,estrogen receptor-(ER-)breast cancer tissues 9 cases and breast fibroma tissues 7 cases),extract total protein,detect the protein level of RCC2 in breast tissues by western blot.2.Use breast cancer cell line MCF-7 as cell model,inhibit or promote the expression of RCC2 in MCF-7,detecting whether the change of RCC2 can effect cell proliferation n cell migration and cell apoptosis.3.After inhibiting the expression of RCC2,using PAHS-131ZA Human Breast Cancer RT2 ProfilerTM PCR Array to observe the 84 genes in breast cancer signal pathway whether have obvious changes compared with control.Validate the results in mRNA and protein level,screen RCC2 downstream genes.4.Create virus inhibiting the expression of RCC2,stable transfection in breast cancer cell line MCF-7.Choose 4-5 weeks BALB/C female nude mice,subcutaneous injection MCF-7 cells that can steady inhibiting the expression of RCC2,establish the xenotransplanted tumor models.Measure tumor volume,create tumor growth curve.Using magnetic resonance imaging to detect ADC(Apparent Diffusion Coefficient)and weighing tumors,to observe whether the change of RCC2 can influence the growth of nude mice subcutaneous tumors.5.Culture MCF-7 cells using defferent concentrate of estrogen,detect the protein expression of RCC2.Using optimal estrogen concentration to culture MCF-7 cells,dectect the mRNA and protein expression of RCC2 downstream genes:TWIST1(Twist Family BHLH Transcription Factor 1)and IGF1(Insulin-Like Growth Factor 1)that screens from PCR Array chip.Observe co-working of RCC2 and estrogen to the effect of MCF-7 cell prolferation and apoptosis.Results:1.The expressions of RCC2 among ER+breast cancer tissues(n=16),ER-breast cancer tissues(n=9)and breast fibroma tissues(n=7)have statistically significant(F=9.497,P=0.001).Then in post hoc we found no matter compared with ER+breast cancer tissues(0.795 ± 0.342)or ER-breast cancer tissues(0.569 ± 0.153),the expression of RCC2 in breast fibroma tissues(0.284±0.085)were lower(P<0.001,P=0.039),and the expression of RCC2 in ER+breast cancer tissues were higher than ER-breast cancer tissues(P=0.048).2.Small interference RNA inhibit the expression of RCC2 in MCF-7 cell successfully in mRNA and protein levels(P=0.019,P=0.017),expression plasmid successfully make MCF-7 cell RCC2 quantity increased(P<0.001).Inhibiting the expression of RCC2 can make the MCF-7 cell migraion ability decreased significantlly(P4day=0.004),cell apoptosis rate increased significantly(P=0.037),but there have no obvious change in cell proliferation(F处理=0.003,P=0.957).Overexpression of RCC2 can make the MCF-7 cell migraion ability increased significantlly(P4day=0.009),cell apoptosis rate decreased significantly(P=0.037),but there have no obvious change in cell proliferation(F处理=2.304,P=0.149).3.After inhibiting the expression of RCC2 in MCF-7 cells,PAHS-131ZA Human Breast Cancer RT2 ProfilerTM PCR Array chip screen out three genes that change ≥2 times compared with control group.Among them,IGF1 decrease 2.6 times,SLIT2(Slit Guigance Ligand 2)increase 2.23 times,and TWIST1 decrease 2.53 times.Real-time PCR shows,after inhibiting the expression of RCC2,the mRNA level of TWIST1 and IGF1 decreased significantly(P=0.019,P=0.013),which conform with the result of PCR Array.But there has no obvious change In the mRNA of SLIT2(P=0.832).Western-blot shows,after inhibiting the expression of RCC2,the protein level of TWISTI and IGF 1 decreased significantly(P=0.036,P=0.007).4.Subcutaneous injection MCF-7 cells that stable inhibit the expression of RCC2,establish xenotransplanted tumor models.Compared with control group,inhibiting the expression of RCC2 made nude mice tumor growth slow,the volumes of tumors are significantly decreased(P=0.002),the weights of tumors are decreased(P=0.001),the ADC values of tumors are increased(P=0.044).These show that inhibiting the expression of RCC2 in xenotransplanted tumor models can delay tumor growth.5.Compared with control,10-4-10-12mol/L Estrogen can promote the expression of RCC2 in MCF-7 cells,and the expression of RCC2 is highest when the concentration of estrogen is 10-8mol/L.Estrogen also promote the expression of TWIST1 and IGF1 which were screened from breast cancer signal pathways use PCR Array in MCF-7 cells.Estrogen can promote cell proliferation(P<0.001),but when with the change of RCC2,there had no obvious effect on cell proliferation(P=0.200,P=0.101);in the presence of estrogen,inhibiting the expression of RCC2 can make cell apoptosis rate increased(P=0.020),Overexpression of RCC2 can make cell apoptosis rate decreased(P=0.019).Conclusions:1.RCC2 is highly expressed in breast cancer tissues,and in ER+ breast cancer tissues were higher than in ER-breast cancer tissues.2.RCC2 can promote MCF-7 cell migration and inhibit MCF-7 cell apoptosis.3.RCC2 positive regulation TWIST1 and IGF1 in human breast cancer signaling pathways.TWIST1 can induce cell migration,invation and EMT,IGF1 plays an important role in cell proliferation,differentiation and inhibiting apoptosis.4.In xenotransplanted tumor models,inhibiting the expression of RCC2 can delay the tumor growth of nude mice.5.Estrogen positive regulate the expression of IGF1 and TWIST1 through RCC2 to promote tumor growth.The above results suggest that RCC2 is breast cancer oncogene,estrogen stimulate the expression of RCC2 and its downstream genes TWIST1 and IGF1 to inhibit apoptosis and promote cell metastasis.