| Objective This study intends to observe the expression changes of Slit2 and Robo1 in breast cancer tissues and cells,and analyze their correlation with the clinicopathological characteristics of breast cancer.Through further in vitro cell experiments,verify the effect of Slit2 overexpression on the biological behavior of breast cancer cells,and then explore the mechanism of Slit2 /Robo1 signaling pathway involved in the development and met`astasis of br`east cancer,to provide new ideas for the follow-up treatment of breast cancer.Methods1.Tissue experimentImmunohistochemical technique was used to observe the localization and expression of Slit2 and Robo1 in breast cancer tissue,adjacent normal tissue and breast fibroadenoma,and the expression changes were detected by image analysis technique.Statistical methods were used to analyze the correlation between Slit2,Robo1 expression and p`atient’s a`ge,tum`or size,lym`ph no`de metast`asis,histological grade,pathological stage,molecular pathological index and tissue type.At the same time,cascading correlation analysis was used to verify the correlation between the expression levels of Slit2 and Robo1 in breast cancer.2.Cell experiment(1)The localization,quantitative expression and m RNA expression of Slit2 and Robo1 in immortalized breast epithelial cell MCF10 A and breast cancer cell line MCF-7 were detected by immunofluorescence,Wester`n blot and real-`time q`uantitative PC.R.(2)T.he Slit2 overexpression cell model was constructed(1)CCK8 method was used to detect the proliferation of breast cancer MCF-7 cells in Slit2 overexpression group,no-load group and blank control group for 48 hours,and the effect of Slit2 on breast cancer cell proliferation was analyzed.(2)Cell scratch test was used to detect the wound healing of breast cancer cells in Slit2 overexpression group,no-load group and blank control group for 24 hours and 48 hours,and the effect of Slit2 on the migration ability of breast cancer cells was analyzed.(3)The number of breast cancer cells passing through matrix glue in 24 hours in Slit2 overexpression group,no-load group and blank control group was counted by Transwell method,and the effect of Slit2 on the invasive ability of breast cancer cells was analyzed.(4)The number of colony formation of breast cancer MCF-7 cells in Slit2 overexpression group,no-load group and blank control group was detected by cell plate cloning test,and the effect of Slit2 on the colony formation ability of breast cancer cells was analyzed.(5)Flow cytometry was used to detect the cell cycle of breast cancer MCF-7cells in Slit2 overexpression group,no-load group and blank control group,and the effect of Slit2 on breast cancer cell cycle was analyzed.Results1.Tissue experiment(1)Slit2 was mainly expressed in the cytoplasm of cells,and its positive expression rates in breast fibroadenoma and normal breast tissue were 90.5%and 85.0% respectively,which were significantly higher than that in invasive breast cancer(44.2%),P<0.05.However,there was no significant difference between the positive expression rate of Slit2 in breast fibroadenoma and normal breast tissue(P>0.05).Slit2 was highly correlated with Robo1 expression in invasive breast cancer.Slit2 protein expression was positively correlated with ER,PR and Her2(P<0.05).Slit2 protein expression was not correlated with age,lymph node metastasis,tumor size,pathological stage and histological grade of invasive breast cancer patients(P>0.05).(2)Robo1 was m.ainly expressed in the nuc.leus and cytop.lasm o.f tumor cells.The pos`itive exp.ression rate of R.obo1 i.n fibroadenoma of breast and normal tissue of breast was 85.7% and 90.0%,respectively,which were significantly higher than that in invasive breast cancer(38.8%),P < 0.05.However,there was no sig.nifica`nt diffe.rence i.n the pos.itiv.e expres.sion rate betw`ee.n fibroadenoma and normal breast tissue(P > 0.05).In invasive breast cancer tissues,the expression of Robo1 protein was positively correlated with ER and PR(P < 0.05),but not with Her2 expression(P > 0.05).The expression of Robo1 protein was not correlated with age,lymph node metastasis,tumor size,pathological stage and histological grade of invasive breast cancer patients(P > 0.05).2.Cell experiment(1)Localization of Slit2 and Robo1 in immortalized mammary e.pithelial cell MC`F10A and br`east ca.ncer cell li.ne M`CF-.7 was detected by immunofluorescence technology.T`he results showed that Slit2 was mainly expressed in the cytoplasm of MCF10 A cells and M`CF-.7 cells,and Robo1 was mainly expressed in the nucleus and cytoplasm of MCF10 A cells and M`CF-.7cells.(2)The quantitative expression of Slit2 and Robo1 in immortalized breast epithelial cell MC.F10 A and breast cancer cell line M`CF-.7 was detected by Western blot.The results showed that the expression of Slit2 and Robo1 in MC.F10 A was higher than that in M`CF-.7 cells(P<0.05).(3)Real-time quantitative PCR was used to detect the m RNA expression of Slit2 and Robo1 in immortalized mammary epithelial cell MC.F10 A and breast cancer cell line M`CF-.7.The results showed that the m RNA expression of Slit2 in MC.F10 A cells was 2.63 times that of M`CF-.7.The m RNA expression of Robo1 in MC.F10 A cells was 2.92 times that of M`CF-.7cells,and the m RNA expression of Slit2 and Robo1 in MC.F10 A cells was significantly higher than that in M`CF-.7cells(P < 0.05).(4)The Slit2 overexpression cell model was constructed(1)The proliferation of breast cancer MCF-7 cells in 48 hours was detected by CCK8 method.The results showed that the proliferation of bre.ast cancer M.C.F-7 ce`lls in the Slit2 overexpression group was significantly lower than that in the empty load group and the blank control group(P<0.05).(2)Cell scratch test was used to detect the migration of breast cancer cells.The results showed that the migration ability of breast cancer M.C.F-7 ce`lls in the Slit2 overexpression group was significantly lower than that in the empty load group and the blank control group(P<0.05).(3)The number of breast cancer cells passing through the matrix glue in 24 hours was counted by Transwell method.The results showed that the number of M.C.F-7 ce`lls passing through the matrix glue in 24 hours in the Slit2 overexpression group was significantly lower than that in the empty load group and the blank control group(P<0.05).(4)The number of colony forming of breast cancer M.C.F-7 ce`lls was detected by c.ell plate cloning experiment.The results showed that the number of colony forming of breast cancer M.C.F-7 ce`lls in th.e Slit2 overe.xpression group w.as significantly lower tha.n that in the empty load group and the blank control group(P<0.05).(5)The cell cycle of breast cancer was detected by flow cytometry.The results showed that Slit2 overexpression had little effect on the cell cycle progression of breast cancer MCF-7(P>0.05).Conclusions1.The low expression of Slit2 and Robo1 protein in breast tissue is related to the occurrence,development and prognosis of breast cancer,suggesting that Slit2 /Robo1 signaling pathway may participate in the pathological process of breast cancer.2.The expression of Slit2 and Robo1 protein in MCF-7 cells was significantly lower than that in MCF10 A cells,suggesting that Slit2 and Robo1 may be potential inhibitors of breast cancer.3.The m RNA expression of Slit2 and Robo1 in MCF10 A cells was significantly higher than that in MCF-7 cells,suggesting that the occurrence of breast cancer may be related to the relative reduction of Slit2 and Robo1 gene expression.4.The proliferation,migration,invasion and colony formation of breast cancer MCF-7 cells in Slit2 overexpression group were significantly lower than those in no-load group and blank control group,suggesting that Slit2 may be a potential marker for inhibition of breast cancer proliferation,migration,invasion and cell colony formation. |
PDF Full Text Request