| Background:Breast cancer is one of the major malignant tumors that endanger the health of women.Although our country is a low incidence area,with the development of the national economy and the improvement of living standards,the incidence of breast cancer has also been increasing year by year.In recent years,mortality in urban breast cancer patients rises fastest.Lymphoid metastasis is the main and most common metastasis pathway of breast cancer.Early lymphangiogenesis of breast cancer may play a crucial role in lymphatic metastasis by local lymphatic metastasis to peripheral lymph nodes,Is to determine the most important factor in breast cancer staging and treatment options.The newly discovered vascular endothelial growth factor-C(VEGF-C),vascular endothelial growth factor-D(VEGF-D)and its receptor VEGFR-3 growth factor receptor-3 play an important role in lymphangiogenesis and metastasis.The VEGF-C and D/VEGFR-3 signaling pathways have provided favorable environment for lymphangiogenesis and lymphatic metastasis in breast cancer.And with the deepening of the research,more and more signaling pathways are found to be involved in lymphangiogenesis,with complicated cross-talk between different signaling pathways.Phosphatidylinositol-3-kinase/protein kinase B,PI3K/AKT is one of the more important signal pathways.PI3K/AKT signaling pathway plays an important regulatory role in cell proliferation,differentiation and apoptosis,tumorigenesis,metastatic resistance and other processes,which cross with a variety of signal regulatory molecules,forming a complex network of regulatory pathways.AKT,a key downstream molecule of the PI3K signaling pathway,is mainly involved in the biological regulation of PI3K initiating process.The function of AKT also plays an important role in pathological processes such as tumor angiogenesis,lymphangiogenesis and invasion and metastasis in addition to the regulation of cell growth and proliferation and the initiation of apoptosis program.In the PI3K/AKT signaling pathway,AKT functions by phosphorylation of Thr308 and Ser473 residues.Studies have shown that in invasive ductal carcinoma of the breast,a variety of cytokines can act on the membrane receptor in breast cancer cell membrane activation of tyrosine kinases at the cell membrane,and then transduce extracellular signals into the cell to promote AKT phosphorylation,Phosphorylated AKT(p-AKT)can promote downstream VEGF-C protein expression and lymphangiogenesis,AKT protein plays an important role in tumor angiogenesis and lymphangiogenesis.P-AKT is a key molecular target in the PI3K signaling pathway.It is upstream of the PI3K signaling pathway and can accelerate the activation of many downstream biological molecules,thereby accelerating the development of breast cancer.It can also make the traditional treatment of breast cancer drug resistance;can be used as a candidate target for breast cancer gene therapy.The activity of PI3K/AKT signaling pathway is negatively regulated by tumor suppressor gene lipid phosphatases PTEN and SHIP2,a clear tumor suppressor gene that blocks the PI3K/AKT signaling pathway to regulate a variety of cellular processes.SHIP gene can inhibit cell proliferation and promote apoptosis,resulting in PI3K/AKT pathway inactivation,SHIP partial deletion may activate PI3K/AKT signaling pathway.MicroRNAs(miRNAs,miRs)have obvious tissue-specific features,especially in malignant tumors.The findings show that miRNAs have different expression patterns and even some miRNAs are expressed in whole body cells.However,there are obvious differences in the expression levels of microRNAs in different tissues or different developmental stages.Related studies have shown that microRNAs play a key regulatory role in the development of tumors.These microRNAs play a specific role in the role of oncogene/oncogene or tumor suppressor genes.In view of this specific characteristic,detection of specific of miRNAs contribute to the diagnosis of tumors,and can predict the prognosis of patients,but also can assess the clinical efficacy,to provide a theoretical support for the development of a reasonable treatment plan.In breast cancer,miR-139-5p,miR-33b,miR-27b,miR-204 and other miRNAs and their role is getting more and more attention.The key miRNAs related to the progress of breast cancer have become hot spots in recent years.MiR-3941 has been identified as a regulator of the progression of breast cancer by mediating immunoglobulin-binding protein 1(IGBP1)expression.The interaction between ABCA6 in ABC(ATP-binding cassette)subfamily A and miR-3941 was also found to be involved in the development of colorectal cancer.In view of AKT phosphorylation and miR-3941 specific role in breast cancer is not clear,further study is needed,in order to further clarify AKT phosphorylation and miR-3941 in breast cancer and its possible molecular mechanism,this research has carried on the research of the following two parts,the first part is the study of the role of AKT phosphorylation in lymphatic metastasis of breast cancer.The second part is about the mechanism of microRNA-3941 regulating IGF-1 in breast cancer cells in order to further analyze the phosphorylation of AKT and miR-3941 in breast cancer and its possible signal pathways,providing possible molecular biomarkers for the early diagnosis,curative effect evaluation and prognosis prediction of breast cancer,and provide a new direction for exploring new gene targeting therapy.Chapter One The role of AKT phosphorylation in breast cancer lymphatic metastasisResearch purposes:Detecting breast cancer specimens lymphangiogenesis(LMVD),regulatory factors VEGF-C,VEGFR-3,Survivin,Ang2,PI3K,AKT,p-AKT(Thr308),p-AKT(Ser473)and tumor suppression factors SHIP,PTEN expression levels;To analyze the relationship between AKT phosphorylation and lymphatic vessel density,lymphangiogenic growth factor and lymph node metastasis in breast cancer.To investigate the role of AKT phosphorylation in the PI3K/AKT pathway,VEGF-C,D/VEGFR-3 signaling pathway and its mechanism of action in the process of lymphatic metastasis of breast cancer.To elucidate the interaction between these factors in the regulation of lymphangiogenesis the specific molecular mechanism,in order to provide a basis for the inhibition of lymphatic metastasis of breast cancer and an effective target.Research methods:(1)Sixty cases of breast cancer tissue samples were collected and embedded in paraffin,sectioned and immunohistochemically detected for VEGF-C,VEGFR-3,LMVD,Survivin,Ang2,PI3K,AKT and P-AKT(Thr308)?P-AKT(Ser473)and tumor suppressor SHIP,PTEN protein expression levels.(2)Twenty fresh specimens of invasive ductal carcinoma of the breast were selected;Western-blot was used to detect the expression of VEGF-C,VEGFR-3,P-AKT(Thr308),P-AKT(Ser473)and tumor suppressor SHIP,PTEN protein.(3)Twenty fresh specimens of breast invasive ductal carcinoma were selected and the expression of VEGF-C,VEGFR-3,PI3K,AKT,PTEN,SHIP,Survivinv and Ang2 mRNA were detected by RT-PCR.To analyze the relationship between AKT phosphorylation and lymphatic vessel density,lymphangiogenesis factor and lymph node metastasis in breast cancer.Research Results:1 Analysis of immunohistochemistry results1.1 The expression and correlation of PI3K and AKT and VEGFR-3 in breast cancer tissuesIn 60 cases of breast cancer,the positive rate of PI3K was 66.67%(40/60),the positive rate of AKT was 61.67%(37/60)and the positive rate of VEGFR-3 was 86.67%(52/60);The positive expression rate(78.13%,25/32)of PI3K in group with lymph node metastasis was significantly higher than that in group with non-lymph node metastasis by 53.57%(15/28),the difference was statistically significant(X2 =4.051,P=0.044);The positive rate(71.88%,23/32)of AKT in lymph node metastasis group was significantly higher than that in non-lymph node metastasis group(50.00%,14/28),the difference was statistically significant(X2 = 4.029,P=0.045).The positive rate(96.89%,31/32)of VEGFR-3 in lymph node metastasis group was significantly higher than that in non-lymph node metastasis group(75.00%,21/28),the difference was statistically significant(X2 = 4.436,P = 0.035).The expression of PI3K,AKT and VEGFR-3 in patients with lymph node metastasis was significantly higher than that in patients without lymph node metastasis.The expressions of PI3K,AKT and VEGFR-3 were closely related to the progression of breast cancer.The results of clinical examination showed that the positive rate of VEGFR-3 in PI3K-positive group was 95.00%(38/40),while the positive rate of VEGFR-3 in PI3K-negative group was 70.00%(14/20);The positive rate of VEGFR-3 expression was 94.59%(35/37)in patients with AKT positive expression,while the positive rate of VEGFR-3 expression in patients with AKT negative expression was 73.91%(17/23).Spearman correlation analysis showed that there was a significant positive correlation between the expression of PI3K and VEGFR-3 in breast cancer(r=0.369,P=0.004).There was also a significant positive correlation between the expression of AKT and the expression of VEGFR-3 in breast cancer(r=0.415,P=0.001).It indicate that P13K,AKT,VEGFR-3 play an important role in the pathogenesis of breast cancer.1.2 LMVD count,expression and correlation of Survivin and Ang2 in breast cancerAmong 60 breast cancer cases,the LMVD count was(16.39 ± 3.16)/HP.The LMVD count(18.84 ± 4.12)/HP in lymph node metastasis group was significantly higher than that in non-lymph node metastasis group(14.45 ± 3.71)/HP,the difference was statistically significant(t = 4.312,P = 0.000).The positive rate of Survivin in breast cancer was 85.00%(51/60),while the positive rate of Ang2 in breast cancer was 73.33%(44/60).The positive rate(96.86%,31/32)of Survivin in patients with lymph node metastasis was significantly higher than that in patients with non-lymph node metastasis(71.43%,20/28).There was significant difference between them(?2 = 5.72,P = 0.020).Compared with the positive rate(57.14%,16/28)of Ang2 in patients without lymph node metastasis,the positive expression rate of Ang2 in patients with lymph node metastasis was significantly high(87.50%,28/32),the difference was statistically significant(?2 = 7.04,P = 0.008).There was no significant difference in the expression of Survivin and Ang2 between breast cancer and tumor size,age and histopathological stage(P>0.05).Clinical data analysis showed that the positive expression rate of Ang2 in breast cancer wih survivin positive expression group was 74.51%(38/51),the positive expression rate of Ang2 in breast cancer wih survivin negative expression group was 66.7%(6/9),The results of Spearman correlation analysis showed that there was a positive correlation between Survivin expression and Ang2 expression in breast cancer(r=0.325,P=0.012).1.3 breast cancer tissue p-AKT,PTEN,SHIP,VEGF-C expression analysisThe positive rates of p-AKT308 and p-AKT473 in breast cancer tissues were respectively 70.00%(42/60)and 65.00%(39/60).The positive rate of p-AKT308 in non-lymph node metastasis was 46.43%(13/28).The positive rate(90.63%,29/32)of p-AKT308 in lymph node metastasis group was significantly higher than that in non-lymph node metastasis group.The difference was statistically significant(x2=13.891,P=0.002).The positive rate(90.63%,29/32)of p-AKT 473 in lymph node metastasis group was significantly higher than that in non-lymph node metastasis group(35.71%,10/28),the difference was statistically significant(x2 = 19.792,P=0.000).P-AKT 308 expression and P-AKT 473 expression in breast cancer tissues were not related to tumor size,age and TNM stage(P>0.05).The positive rates of VEGF-C,PTEN and SHIP in breast cancer tissues were 81.67%(49/60),56.67%(34/60)and 61.67(37/60)respectively in 60 specimens.The positive rate(100.00%,32/32)of VEGF-C in lymph node metastasis group was significantly higher than that in non-lymph node metastasis group(60.71%,17/28),the difference was statistically significant(x2=20.863,P=0.000).The positive expression rate(34.38%,11/32)of PTEN in lymph node metastasis group was significantly lower than that in non-lymph node metastasis group(82.14%,23/28)(X2 = 13.877,P = 0.000).Compared with positive expression rate(89.29%,25/28)of SHIP in group without lymph node metastasis,SHIP positive expression rate in lymph node metastasis group was significantly decreased by 37.50%(12/32),the difference was statistically significant(X2 =16.942,P=0.000).The positive rate of VEGF-C in p-AKT308 positive expression group in breast cancer was 92.86%(39/42),while the positive expression rate of VEGF-C in p-AKT308 negative expression group in breast cancer was 55.56%(10/18).The positive rate of VEGF-C expression was 94.87%(37/39)in patients with positive p-AKT473 expression,and 57.14%(12/21)in patients with negative p-AKT473 expression.Spearman correlation analysis,p-AKT308 and VEGF-C expression was positively correlated(r=0.5677,P = 0.000).There was a positive correlation between the expression of p-AKT473 and VEGF-C(r = 0.455,P = 0.000).2 Western-blot test results analysisIn the 20 fresh specimens,12 patients with invasive ductal carcinoma of the breast had lymph node metastasis,8 patients without lymph node metastasis.There were positive expression of VEGF-C,VEGFR-3,AKT,p-AKT308 and p-AKT473 in patients with lymph node metastasis.There were 3 cases of PTEN without expression and 4 cases of SHIP without expression.In the cases without lymph node metastasis,VEGF-C was not expressed in 1 case,VEGFR-3 was not expressed in 2 cases,p-AKT308 was not expressed in 2 cases,and p-AKT473 was not expressed in 3 cases.AKT,PTEN and SHIP were all expressed.Compared with the expression of VEGF-C,VEGFR-3,AKT,P-AKT(Thr308)and P-AKT(Ser473)in breast cancer tissues without lymph node metastasis,VEGF-C,VEGFR-3,AKT,P-AKT(Thr308)and P-AKT(Ser473)were significantly increased,the results were statistically significant(P<0.05).However,compared with PTEN and SHIP protein expression in breast cancer without lymph node metastasis,the expression of PTEN and SHIP protein in breast cancer tissues with lymph node metastasis was significantly decreased,and the results were statistically different(P<0.05).The relative expression levels of P-AKT(Thr308)and P-AKT(Ser473)in 20 specimens of invasive ductal carcinoma of the human breast were 0.20 ± 0.13 and 0.25±0.19,respectively,and the LMVD count was 16.39±5.49.By linear correlation analysis,P-AKT308 expression was positively correlated with LMVD(r=0.922,P<0.01).The positive expression of P-AKT(Ser473)was positively correlated with LMVD(r=0.946,P<0.01).3 RT-PCR test results analysisThe expression of VEGF-C,VEGFR-3,Survivin,Ang2 and PI3K,AKT mRNA were all found in 20 cases of breast invasive ductal carcinoma with lymph node metastasis.The expression of PTEN was not observed in 3 cases.Four patients did not express SHIP.In breast cancer tissues without lymph node metastasis,AKT was not expressed in one case,VEGF-C was not expressed in three cases,VEGFR-3 was not expressed in two cases,and the others were all expressed.Compared with the expression of VEGF-C,Ang2,PI3K and AKT mRNA in breast cancer tissues without lymph node metastasis,the expression of VEGF-C,Ang2,PI3K and AKT mRNA in breast cancer with lymph node metastasis was significantly increased.The results were statistically significant(P<0.05).Compared with the expression of Survivin,PTEN and SHIP mRNA in breast cancer tissues without lymph node metastasis,the expression levels of Survivin,PTEN and SHIP mRNA in breast cancer tissues with lymph node metastasis were significantly decreased(P<0.05).VEGFR-3 mRNA expression in breast cancer with lymph node metastasis was significantly higher than that in non-lymph node metastasis(P>0.05).Conclusions:(1)The expression of VEGF-C,VEGFR-3 protein and gene and lymphangiogenesis of lymphangiogenesis signal pathway VEGF-C/VEGFR-3 exist in breast cancer tissues,VEGF-C,VEGFR-3 positive expression rate and lymphatic vessel count in patients with lymph node metastasis were significantly higher than those without lymph node metastasis.(2)The PI3K/AKT signaling pathway-associated proteins PI3K,AKT,phosphorylated P-AKT(Thr308)and P-AKT(Ser473)were highly expressed in breast cancer patients.The expressions of PI3K,AKT,phosphorylated P-AKT(Thr308)and P-AKT(Ser473)in patients with lymph node metastasis were significantly higher than those without lymph node metastasis.It is suggested that there is a relationship between PI3K/AKT signaling pathway and lymph node metastasis in breast cancer.(3)The tumor suppressor PTEN and SHIP were involved in lymphangiogenesis and lymph node metastasis of breast cancer.The expression of PTEN and SHIP in patients without lymph node metastasis was higher than that in patients with lymph node metastasis.(4)The phosphorylation of AKT in breast cancer tissues was positively correlated with VEGF-C expression,VEGFR-3 expression and lymphatic vessel count,but negatively correlated with PTEN expression and SHIP expression,indicating that phosphorylation of AKT contributes to promote the expression of VEGF-C and VEGFR-3,PTEN,SHIP play a negative regulatory role,induced lymphangiogenesis,and further promote the lymph node metastasis of breast cancer.(5)AKT phosphorylation promoted the secretion of cytokines through the PI3K/AKT signal transduction pathway in breast cancer and also interacted with the VEGF-C and D/VEGFR-3 signaling molecules to promote the expression of lymphangiogenesis,which may be the mechanism of lymph node metastasis in breast cancer.Chapter Two The Study of the mechanism of microRNA-3941 regulating IGF-1 in breast cancer cellsResearch purposes:To detect the expression of miR-3941 and IGF-1 in breast cancer tissues and cells,further study the effect of miR-3941 overexpression and inhibition on the proliferation,invasion and metastasis of cancer cells,The relationship between miR-3941 and IGF-1 is to clarify the key role of miR-3941 in the development of breast cancer and possible regulatory mechanisms.Research Methods:Sixty breast cancer patients were selected as the research object to collect the breast cancer tissues and matched normal tissues.The human breast cancer cell line MDA-MB-231 and mammary epithelial cell line MCF-10 were selected at the same time,MiR-3941 mimic,miR-3941 inhibitor,Scramble RNA and IGF-1 specific small interfering RNA(siRNA)were transiently transfected by cationic liposomes,small molecule inhibitor LY294002 blocked PI3K/AKT signaling pathway,MTT assay for cell proliferation,clone assay for assessing cell proliferation,Transwell cell migration assay and in vitro invasion assay for cell migration and invasion,the target relationship between IGF-1 and miR-3941 was evaluated using a dual-luciferase reporter assay.The expression of IGF1,E-cadherin,N-cadherin and vimentin in breast cancer tissues and cells were detected by Western Blot.The expression of miR-3941 and IGF1 mRNA in breast cancer tissues was detected by RT-PCR.Results:1.Reverse expression of miR-3941 and IGF-1 in breast cancer tissues and cellsThe expression of miR-3941 in breast cancer tissues was lower than that in adjacent normal breast tissues(P<0.01).In vitro cell analysis showed that miR-3941 in MDA-MB-231 cells was also expressed at lower levels(P<0.01)compared to normal mammary epithelial MCF-10 cells.In addition,IGF-1 was highly expressed in breast cancer tissues and MDA-MB-231 cells(P<0.01)as compared to the expression in adjacent normal tissues and normal cells.It suggested the reverse expression of miR-3941 and IGF-1 in breast cancer tissues and cells.2 overexpression of miR-3941 inhibits breast cancer cell viabilityMDA-MB-231 cells were transfected with miR-3941 mimics and miR-3941 inhibitors.We found that the expression of miR-3941 was significantly up-regulated in miR-3941 mimics and significantly downregulated in miR-3941 inhibitor group(P<0.05),indicating that miR-3941 was successfully overexpressed and induced in breast cancer MDA-MB-231 cells.In addition,MTT assay showed that miR-3941 overexpression resulted in a significant decrease in cell viability compared with control and disruption groups,whereas the opposite effect was observed after miR-3941 inhibition(P<0.05).In addition,the result of colony detection was consistent with that of MTT assay,indicating that the number of colonies significantly decreased after miR-3941 was overexpressed and significantly increased after miR-3941 was inhibited(P<0.05).It showed that miR-3941 overexpression significantly inhibited the proliferation of breast cancer cells.3 Overexpression of miR-3941 may significantly inhibit the migration and invasion of cancer cells through the regulation of EMT-related proteinsTranswell analysis was used to evaluate the effect of miR-3941 imbalance on cell migration and invasion.Compared with the control group,miR-3941 overexpression significantly decreased the number of migrated or infiltrated cells,and significantly increased after miR-3941 inhibition(P<0.05).In addition,expression of E-cadherin was significantly up-regulated,while expression of N-cadherin and vimentin was down-regulated after miR-3941 was overexpressed(P<0.05).The opposite trend in the expression of these proteins was observed after miR-3941 inhibition(P<0.05).It is suggested that miR-3941 overexpression may inhibit cell migration and invasion by up-regulating E-cadherin and down-regulating N-cadherin and vimentin.4 IGF-1 is the target of miR-3941The relative luciferase activity of cells bearing IGF-1 3'-UTR-WT was significantly lower(P<0.05)after miR-3941 was overexpressed compared to control cells.Mutations in the predicted binding site of IGF-1 3'-UTR by miR-3941 restored a decrease in luciferase activity(P<0.05).In addition,the expression of IGF-1 in miR-3941 mimic group was significantly lower than that in miR-3941 inhibitor group(P<0.05).IGF-1 is a direct target of miR-3941 and miR-3941 negatively regulates IGF-1 expression.5 miR-3941 overexpression regulates migration,invasion and EMT-related proteins by targeting IGF-1Compared with the control group,the expression of IGF-1 was significantly decreased after si-IGF-1 transfection(P<0.01),indicating that IGF-1 was successfully knocked down.In addition,we found that inhibition of miR-3941 overexpression on cell migration and invasion was reversed(P<0.05)when miR-3941 mimics and si-IGF-1 co-cotransfected cells.In addition,an increase in E-cadherin expression and a decrease in N-cadherin and vimentin expression induced by miR-3941 overexpression were significantly reversed when cells were co-transfected with miR-3941 mimics and si-IGF-1(P<0.05).In addition,the effect of miR-3941 inhibition on cell migration,cell invasion and EMT-related proteins was also reversed(p<0.05)when co-transfected with miR-3941 inhibitor and si-IGF-1.It showed that miR-3941 overexpression regulates migration,invasion and EMT-related proteins in breast cancer cells by targeting IGF-1.6 IGF-1 regulates breast cancer MDA-MB-231 cell migration through the PI3K/AKT signaling pathwayAt the concentration of IGF-1,the migration ability of MDA-MB-231 cells was increased,compared with the control group,the difference was statistically significant(P<0.05);when the PI3K/AKT signaling pathway was blocked by LY294002,migration of MDA-MB-231 cells was inhibited,and also inhibited the migration of cells under the intervention of IGF-1,compared with the control group,the difference was statistically significant(P<0.05).The expression of phosphorylated AKT(Thr308/Ser473)was significantly increased after 30 minutes of intervention with IGF-1,and there was a statistically significant difference from the control group(P<0.05).It was shown that IGF-1 can enhance the migration ability of breast cancer cells,PI3K/AKT signaling pathway may be the pathway of IGF-1 enhancement,and the key molecule AKT phosphorylation may be the core target.Conclusions:(1)In breast cancer tissues and breast cancer MDA-MB-231 cells,the expression of miR-3941 was down-regulated and IGF-1 expression was up-regulated.MiR-3941 and IGF-1 were involved in the development of breast cancer.(2)MiR-3941 overexpression may inhibit the migration and invasion of breast cancer cells by up-regulating E-cadherin and down-regulating N-cadherin and vimentin.(3)IGF-1 is a direct target of miR-3941,and miR-3941 negatively regulates the expression of IGF-1.Overexpression of miR-3941 can regulate the migration,invasion and EMT-related proteins of breast cancer cells by targeting IGF-1.(4)MiR-3941 is down-regulated in breast cancer cells.Down-regulation of miR-3941 may promote the proliferation,migration,and invasion of breast cancer cells through targeted regulation of IGF-1,which may be achieved through the PI3K/AKT signaling pathway.Phosphorylation of AKT is a key target,and miR-3941 and IGF-1 may serve as diagnostic markers or potential targets for the treatment of breast cancer. |
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