Notch2 Knockdown On The Proliferation,Apoptosis And Invasion Of Colorectal Cancer And Related Mechanisms

BackgroundColorectal cancer is one of the most common malignant tumors in the digestive tract.It is worth noting that the incidence rate has gradually increased in recent years.At present,colorectal cancer is mainly treated by surgery,but the recurrence rate after surgery is still high and the effect of late treatment is poor,so the quality of life and prognosis of patients are very low.This not only brings treatment problems to doctors,but also brings great pressure to patients and their families.Therefore,it is very important to find new therapeutic targets and find more iconic predictors.Notch signaling pathway is an evolutionary conserved signaling pathway.The participation of Notch pathway in the occurrence and development of tumors plays a key role that cannot be ignored.Many studies have shown that Notch’s gene mutation makes it over-activated or loses its function,thereby affecting the progression of tumors.At the same time,some researchers have concluded that not every receptor of the Notch family has the same function in the occurrence and development of tumors.The receptors can regulate each other to promote or antagonize the malignant progression of tumors.At present,most of the literature has proved that in colorectal cancer,the excessive activation caused by the mutation of Notch 1 can promote tumor proliferation,migration and invasion,and inhibit tumor apoptosis,making colorectal cancer seriously poor prognosis.However,the similar biological function and specific mechanism of Notch2 in colorectal cancer are not very clear,so further research is needed.Smad4 is one of the important members of the Smad protein family.It can bind to other phosphorylated proteins in the family or participate in some complex molecular signaling pathways,thereby regulating tumor-related mechanisms and affecting tumor progression.Studies have shown that Smad protein can participate in the mutual regulation of Notch pathway receptors,thereby affecting cell epithelial-mesenchymal transition(EMT)or stem cell differentiation.However,whether there is a similar relationship between Smad protein and Notch receptor in colorectal cancer is not yet known.ObjectiveThis study aims to explore the relationship between Notch2 and colorectal cancer grading and staging,and observe the effects of Notch2 on the biological behaviors of colorectal cancer such as proliferation,apoptosis,migration and invasion.Explore the relevant mechanism of Notchl regulation by the changes of Notch2 in colorectal cancer.Provide new ideas for clinical discovery of new therapeutic targets or predictors.Method1.The influence of Notch2 on the biological behavior of colorectal cancer(1)The biological information database analyzes the effect of Notch2 on the staging and apoptosis of colorectal cancer①Use the Oncomine biological information database to search the relationship between Notch2 and the stage of colorectal cancer.②Use GEPIA biological information database to search the correlation between Notch2 and related apoptosis protein caspase3.2.The influence of Notch2 changes on the biological behavior of colorectal cancer(2)Histological analysis of the relationship between the expression of Notch2 and colorectal cancer①71 cases of tissues obtained through clinical colorectal cancer surgery(39 cases of high-medium differentiation,32 cases of poorly differentiated)were stained with Hematoxylin Eosin(HE)and immunohistochemistry,and then scored by H-Score Observe the relationship between the expression of Notch2 and the grade and stage of colorectal cancer.②Analyze the relationship between the expression of Notch2 and other clinicopathological parameters in 71 tissues obtained from colorectal cancer surgery.③Using immunohistochemistry to verify the relationship between Notch2 and proliferation-related protein ki67 and apoptosis-related protein active-caspase3 through H-Score score from the perspective of histology.(3)Establish a cell line that knocks down Notch2 expression①Immunofluorescence test detects the sub-localization of Notch2 in colorectal cancer cell lines.②Using lentivirus to establish colorectal cancer knockdown Notch2 cell lines(HCT15-shCtrl and HCT15-shNotch2,HT29-shCtrl and HT29-shNotch2).The knockdown efficiency was detected by Western blot and RT-PCR experiments.(4)The effect of Notch2 changes on the biological behavior of colorectal cancer cells1)The effect of Notch2 changes on the proliferation of colorectal cancer cells①The effects of Notch2 on colorectal cancer cell viability were verified in vitro by CCK-8 using two knockdown cell lines.②Using two knockdown cell lines to verify the effect of Notch2 on the proliferation of colorectal cancer cells through clonal integration experiments.2)The effect of Notch2 changes on the apoptosis of colorectal cancer cells①Using two knockdown cell lines to detect the related apoptosis proteins BCL-2,BAX,cleaved-caspase3 and cleaved-PARP through Western.blot experiment through in vitro experiments to verify the effect of Notch2 on the apoptotic ability of colorectal cancer cells.②Two knockdown cell lines were used to verify the effect of Notch2 on the apoptosis of colorectal cancer cells by flow cytometry in vitro.3)The effect of Notch2 changes on the migration and invasion of colorectal cancer cells①Using two knockdown cell lines to verify the effect of Notch2 on the migration ability of colorectal cancer cells in vitro through scratch test and Transwell cell test.②Using two knockdown cell lines to verify the effect of Notch2 on the invasion ability of colorectal cancer cells in vitro through Transwell chamber experiment.(5)In vivo experiments to verify the effect of Notch2 changes on the biological behavior of colorectal cancer①Establish a mouse transplanted tumor model that knocks down Notch2,calculate the tumor volume and draw a growth curve.②Hematoxylin Eosin(Hematoxylin Eosin,HE)and immunohistochemical staining to detect the expression of Notch2,ki67,active-caspase3,and MMP-9,to verify the effect of Notch2 changes on proliferation in colorectal cancer.,Apoptosis and migration,invasion ability.2.In colorectal cancer,the relationship between Notch2 and Smad4 and Notchl(1)Western.blot experiment was used to detect the expression of Notchl,Notch2,and Smad4 in 4 colorectal cancer cell lines(HCT15,DLD1,S.W620,HT29.).(2)Two cell lines knocked down Notch2 were used to detect the expression changes of Smad4,Jaggedl and.Notch.1 by the Western.blot.experimental method.(3)Use knockdown Notch2 cell lines HCT15-shCtrl and HCT15-shNotch2 to knock down the expression of Smad4 again in a transient manner,and detect the changes in the expression levels of Jaggedl and Notchl by Western blot.Result1.The influence of Notch2 on the biological behavior of colorectal cancer(1)The biological information database analyzes the effect of Notch2 on the staging and apoptosis of colorectal cancer①According to the Oncomine biological information website,in colorectal cancer,the expression of Notch2 gradually decreases with the increase of stage(P<0.05).②The GEPIA biological information website shows that in colorectal cancer,the expression of Notch2 is positively correlated with the apoptotic protein caspase3(p-value=1.7e-05).(2)Histological analysis of the relationship between the expression of Notch2 and colorectal cancer①Immunohistochemical experiments show that the expression of Notch2 has nothing to do with the gender,age and age of the patient(P>0.05),but as the grade of the tumor decreases,the expression of Notch2 gradually decreases(P<0.001).②Analyzing the relationship between the expression of Notch2 and pathological parameters,it is concluded that the expression of Notch2 has nothing to do with the gender and age of the patient.On the basis of the relationship with the grade,the expression of Notch2 in patients with lymph node metastasis is lower than that in patients without lymph node metastasis.③After immunohistochemistry experiment,H-Score analysis showed that the expression of proliferation-related protein ki67 was low when Notch2 was high expression(P<0.001),while the expression of apoptosis-related protein active-caspase3 was high(P<0.001).(3)Establish a cell line that knocks down Notch2 expression①The immunofluorescence experiment shows that Notch2 is mostly expressed in the cytoplasm and nucleus in colorectal cancer cells.②After lentivirus transfection,the expression of Notch2 was successfully knocked down by Western blot and RT-PCR detection(P<0.05,P<0.01),and the transfection efficiency reached more than 90%.(4)The effect of Notch2 changes on the biological behavior of colorectal cancer cells1)The effect of Notch2 changes on the proliferation of colorectal cancer cells①CCK-8 experiment concluded that Notch2 knockdown can promote the cell viability of colorectal cancer cells(P<0.05,P<0.01).②Cloning integration experiments show that knockdown of Notch2 can promote the proliferation of colorectal cancer cells(P<0.05,P<0.01).2)The effect of Notch2 changes on the apoptosis of colorectal cancer cells①Western blot experiment detected apoptosis proteins BCL-2,BAX,cleaved-caspase3 and cleaved-PARP,and it was concluded that knockdown of Notch2 can improve the anti-apoptotic ability of tumor cells(P<0.05,P<0.01).②Flow cytometry concluded that the knockdown of Notch2 can improve the anti-apoptotic ability of tumor cells.3)The effect of Notch2 changes on the migration and invasion of colorectal cancer cells①Scratch experiment and Transwell cell experiment show that knockdown of Notch2 can promote tumor cell migration.②Transwell experiment shows that Notch2 knockdown can promote tumor cell invasion ability.(5)In vivo experiments to verify the effect of Notch2 changes on the biological behavior of colorectal cancer①The experimental model of transplanted tumor in mice was successfully established,and the tumor volume was measured and the growth curve was drawn.It is concluded that Notch2 knockdown can promote the proliferation of colorectal cancer(P<0.05).②According to immunohistochemical experiments,knockdown of Notch2 can promote the proliferation and migration of colorectal cancer,and inhibit the apoptotic ability of colorectal cancer(P<0.05,P<0.01).2.In colorectal cancer,the relationship between Notch2 and Smad4 and Notchl(1)Western blot experiments showed that the expression of Notchl and Notch2 in colorectal cancer cells was opposite(P<0.05,P<0.01).(2)Using two cell lines that knocked down Notch2,Western blot experiments showed that the expression of Smad4,Jagged.1 and Notch.1 increased after Notch2 knockdown(P<0.05,P<0.01).(3)After knocking down Smad4 in HCT15-shCtrl and HCT15-shNotch2 cells,Western blot showed that the expression of Jagged.1 and Notch.1 decreased(P<0.05,P<0.01).Conclusion1.Knockdown of Notch2 in colorectal cancer can promote tumor proliferation,migration and invasion,and inhibit tumor apoptosis;2.The expression of Notch2 and Notch1 in colorectal cancer is opposite,the decrease of Notch2 expression may induce the expression of Smad4 and Notchl.