| Objective: Breast cancer is one of the most common malignant tumors in women,and its incidence has jumped to the top of female malignant tumors in the world.According to the expression of estrogen progesterone receptor and human epidermal growth factor 2(HER-2),breast cancer can be clinically divided into 4 subtypes,namely luminal type A,luminal type B,her-2 overexpression type and triple negative type.The clinical progress and treatment,disease outcome and prognosis of each type are different.Among them,triple negative breast cancer has attracted much attention due to the lack of corresponding,targeted treatment,rapid tumor progression,lymph node and distant metastasis.In recent years,many researchers have focused on the molecular mechanism of triple negative breast cancer and tried to find new therapeutic targets,but so far,there are still no effective therapeutic methods and therapeutic targets except chemotherapy.Local invasion and distant metastasis are the most important factors affecting the prognosis and therapeutic effect of tumor,and epithelial-mesenchymal transformation is the most closely related to these.Epithelial-mesenchymal transformation is the transformation of cancer cells from an epithelial phenotype to a mesenchymal phenotype.Morphologically,the cells change from polygonal,round to spindle shape.From a molecular perspective,the expression levels of epithelial markers(CK and E-cadherin)decreased,while the expression levels of mesenchymal markers(Vimentin and N-cadherin)increased.Compared with other types of breast cancer,the cancer cells of triple negative breast cancer are polygonal,or even long spindle,with obvious interstitial reaction,relatively active epithelial interstitial transformation,and prone to lymph nodes and distant metastasis.This is also an important cause of poor prognosis of triple negative breast cancer.In recent years,with the development of biotechnology,more and more evidences show that a variety of nc RNAs,including lncRNAs,play an important role in tumorigenesis.A new mechanism of RNA interaction(ce RNA)has become the focus of recent research,in which RNA competitively binds to the same type of mi RNA to influence its expression level.Lnc RNA is the molecular "sponge" of one kind of mi RNA,which contains the binding site of mi RNA and can adsorb mi RNA and affect the functional expression.Mi RNA is an endogenous,non-coding single stranded RNA that acts as a post-transcriptional regulator to down-regulate the translation of target m RNA.Lnc RNA is involved in the regulation of m RNA by binding to mi RNA.The action axis of lncRNA-mi RNA-m RNA plays a pivotal role in the occurrence and development of diseases.Therefore,this study will carry out relevant studies on the action axis of lncRNA-mi RNA-m RNA for breast cancer,especially triple negative breast cancer,in an attempt to find therapeutic targets for triple negative breast cancer,and provide new basis for diagnosis and treatment of triple negative breast cancer.Methods: Part I: In this study,lnc01094 was screened as differentially expressed lncRNA of breast cancer by searching GEPIA database and related literatures.q RT-PCR was used to detect the m RNA expression levels of breast cancer,adjacent tissues and different molecular types of breast cancer,and analyze the relationship between the clinicopathological factors.Meanwhile,q RT-PCR was used to detect the m RNA expression levels of different types of breast cancer cell lines and normal breast epithelial cell lines.By knocking down and overexpressing lnc01094 in MDA-MB-231 and HCC-1937 triple negative breast cancer cell lines,using MTT assay,The proliferation,migration and invasion abilities of MDA-MB-231 and HCC-1937 cell lines with different lnc01094 levels were detected by scratch repair assay and transwell assay,respectively.Western blot was used to detect epithelial and stromal cell markers of different lnc01094 levels to evaluate EMT changes in MDA-MB-231 cell lines and HCC-1937 cell lines.Part II: The mi RNAs that may have a targeted relationship with lnc01094 and the mrnas that may have a targeted relationship with mir-877-5p were screened and predicted by database and bioinformatics software.Dual luciferase assay was used to verify the targeting relationship between lnc01094 and mir-877-5p and mir-877-5p and Del-1.The m RNA expression levels of mir-877-5p and Del-1 in breast cancer and adjacent tissues were detected by q RT-PCR,and the correlation between lnc01094 and mir-877-5p and lnc01094 and Del-1 was analyzed.Meanwhile,the m RNA expression of mir-877-5p and Del-1 in different types of breast cancer cell lines and normal breast epithelial cells was detected by q RT-PCR.By regulating the expression levels of lnc01094 and mir-877-5p in MDA-MB-231 cell line and HCC-1937 cell line,q RT-PCR was used to detect the m RNA expression levels of mir-877-5p and Del-1.The effects of lnc01094,mir-877-5p and Del-1 expression on proliferation,migration and invasion of MDA-MB-231 and HCC-1937 cell lines were detected by MTT assay,cell scratch repair assay and transwell assay,respectively.Western blot was used to detect the effects of changes in expression levels of lnc01094,mir-877-5p and Del-1 on markers of epithelial and mesenchymal cells.Part III: Western blot was used to detect the protein expression level of Del-1 in breast cancer tissues and normal breast tissues,immunohistochemistry was used to detect the expression level of Del-1 in breast cancer tissues,and the relationship between clinicopathology and prognosis was analyzed.At the same time,the expression levels of E-cadherin,N-cadherin and Vimentin in breast cancer tissues were detected by immunohistochemistry,and the relationship between them and Del-1 was analyzed.The expression levels of CD34 and D2-40 were detected,and the density of microvessels and lymphatic vessels was counted.Results: Part I: The expression level of lnc01094 in invasive breast carcinoma(BRCA)tissues was increased by searching the GEPIA database.q RT-PCR further verified that the expression level of lnc01094 was increased in breast cancer tissues and cell lines,and the highest expression level was found in triple negative breast cancer.The high expression of lnc01094 was associated with high histological grade,high expression of Ki-67 and negative expression rate of ER,PR and HER-2.After lnc01094 was knocked out,MTT assay showed that the proliferation rate of MDA-MB-231 and HCC-1937 cell lines decreased,and scratch repair assay showed that the migration ability of MDA-MB-231 and HCC-1937 cell lines decreased.Transwell assay results showed that the invasion ability of MDA-MB-231 and HCC-1937 cell lines was decreased.Western blot results showed that after lnc01094 was knocked out,the expression of epithelial cell markers(E-cadherin)was increased and the expression of mesenchymal cell markers(N-cadherin and Vimentin)was decreased in MDA-MB-231 and HCC-1937 cell lines.lnc01094 inhibited mesenchymal transformation,which was contrary to the results of reverse functional validation experiments.Functional experiments showed that lnc01094 promoted the proliferation,migration and invasion of triple negative breast cancer cell lines,and promoted epithelial mesenchymal transformation.Part II: Dual luciferase assay verified the targeted binding relationship between lnc01094 and mir-877-5p,mir-877-5p and Del-1.q RT-PCR detection of mir-877-5p m RNA expression in breast cancer tissues showed that compared with para-cancer tissues,mir-877-5p m RNA expression was decreased and Del-1 m RNA expression was increased,and mir-877-5p was negatively correlated with the expression of lnc01094.Del-1 was positively correlated with lnc01094 expression.The same expression trend was also obtained in cell line validation,and it was found that compared with other cell lines,the m RNA expression level of mir-877-5p in all triple negative breast cancer cell lines was the lowest,while that of Del-1 was the highest.Subsequent cell rescue experiments verified that lnc01094 negatively regulated the expression of mir-877-5p in triple negative breast cancer cell line.In cell function experiments,simultaneous inhibition of the expression of triple negative breast cancer lnc01094 and mir-877-5p can reverse the changes of malignant phenotypes caused by mir-877-5p alone,including cell proliferation measured by MTT assay,cell migration measured by scratch repair assay,and cell invasion measured by transwell,western blot analysis of EMT.Meanwhile,we inhibited mir-877-5p while knocking down lnc01094.The m RNA expression level of Del-1 was detected by q RT-PCR,and the results showed that knocking down lnc01094 could down-regulate the expression of Del-1,while knocking down mir-877-5p could up-regulate the expression of Del-1.Knockdown of mir-877-5p can reverse del-1downregulation caused by knockdown of lnc01094.Cell function experiments showed that the up-regulation of del-1 expression while knocking down lnc01094 could reverse the changes of malignant phenotype of triple negative breast cancer cell lines caused by knocking down lnc01094 alone,including cell proliferation measured by MTT assay,cell migration measured by scratch repair assay,cell invasion measured by transwell,western blot analysis of EMT.Part III: Western blot detection of fresh breast cancer specimens and normal breast tissues showed that Del-1 expression was elevated in breast cancer tissues compared with normal breast tissues.We also carried out immunohistochemical staining on 200 breast cancer tissue samples,and the results showed that there were different degrees of Del-1expression in breast cancer tissues,among which the positive rate and positive intensity of triple negative breast cancer expression were higher.The positive expression rate of Del-1 was significantly correlated with the negative expression rate of ER,PR and HER-2,the high expression rate of Ki-67,the vascular tumor plug and histological grade,but not with tumor size and patient age.The expression of epithelial cell markers(E-cadherin)and mesenchymal cell markers(N-cadherin and Vimentin)were decreased in the Del-1 high expression group.In addition,the density of microvessels and lymphatic vessels was higher in the Del-1 high expression group.The disease-free survival and overall survival were shorter in the Del-1 high expression group.Conclusions: 1.Lnc01094 is up-regulated in breast cancer,and the expression level of triple negative breast cancer is higher than that of other types of breast cancer.Lnc01094 can promote the proliferation,migration,invasion and epithelial-mesenchymal transformation of MDA-MB-231 and HCC-1937 cells breast cancer cells.2.In the breast cancer cell line MDA-MB-231,lnc01094 up-regulated the expression of Del-1 by adsorption of mir-877-5p,promoted the proliferation,migration and invasion of breast cancer cells,and induced the occurrence of EMT.3.Del-1 is highly expressed in breast cancer,and is significantly correlated with immunohistochemical phenotype,high expression rate of Ki-67,vascular tumor thrombolus and histological grade.EMT and the density of microvessels and lymphatic vessels in Del-1 group is higher,DFS and OS is shorter.Del-1 expression was higher in triple negative breast cancer compared to other types of breast cancer. |
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