| Background and objective:According to the latest statistics released by the International Agency for Research on Cancer(IARC)in December 2020: Breast cancer accounts for 11.7% of new cancer cases in 2020,surpassing lung cancer(11.4%)as the highest incidence type of malignant tumor in the world.Studies have found that long-chain non-codingRNA homeobox transcript antisenseRNA(HOX transcript antisenseRNA,HOTAIR)can silence some tumor suppressors in breast cancer,and the increase in HOTAIR expression means a worse prognosis.HOTAIR can also interfere with signaling molecules related to the development of breast cancer,but the mechanism is still not fully understood.The LncRNA-miRNA network is involved in many cellular processes and human diseases,including tumorigenesis.Studies have found that miR-203 can inhibit LncRNA HOTAIR in renal cell carcinoma,and affect the occurrence of renal cell carcinoma by regulating the epithelial-mesenchymal transition pathway.MiR-203 is located on chromosome 14q32.33 and is a short non-codingRNA.MiR-203 is associated with tumor resistance to cisplatin chemoth-erapy,tumor aggressiveness,tumor proliferation and metastasis,and can be used as a tumor biomarker.In addition,miR-203 can inhibit tumor cell migration and invasion through caveolin-1(CAV1)in pancreatic cancer cells.CAV1 is an important part of caveolae,which has rich functions such as mediating endocytosis,endocytosis,mechanical protection,cell metabolism and lipid homeostasis.CAV1 is related to many human diseases,including pulmonary hypertension,hypertriglyceridemia and cancer.Based on the above research status,this topic will deeply study the role of HOTAIR/MIR203/CAV1 in breast cancer.This experiment attempts to explore: in breast cancer cells,whether lncRNA HOTAIR can regulate MiR-203 and whether MiR-203 can target CAV1 to affect tumor proliferation,migration and invasion,with a view to preventing and treating breast cancer Provide new ideas and theoretical basis.Methods:1、To observe the effect of LncRNA HOTAIR on the biological function of breast cancer cell MDA-MB231.Firstly,HOTAIR homo pc DNA3.1 was transfected into MDA-MB231 cells,and the expression level of the HOTAIR was detected by RT-q PCR.Each siRNA was transfected and then screened out the most effective one of down-regulating HOTAIR.CCK was used to detect the proliferation ability of MDA-MB231 cells after HOTAIR overexpression and inhibition.Then the invasion ability of MDA-MB231 cells after overexpression and inhibition of HOTAIR were detected by Transwell invasion experiment.Finally,the migration ability of MDAMB231 cells after overexpression and inhibition of HOTAIR were detected by Transwell migration experiment and cell scratch experiment.2、To observe whether LncRNA HOTAIR affects the proliferation,invasion and migration ability of MDA-MB231 cells by regulating miR-203.Dual-luciferase reporter assay was used to detect whether HOTAIR binds to miR-203.RT-q PCR was used to detect the expression level of miR-203 after overexpression and inhibition of HOTAIR;miR-203 mimics and miR-203 inhibitor were transiently transfected into MDA-MB231 cells respectively,and the expression of miR-203 was detected by RT-q PCR.CCK,Transwell invasion test,Transwell migration test and cell scratch test were respectively used to detect the proliferation,invasion and migration ability of MDA-MB231 cell line after overexpression of miR-203,inhibition of miR-203,and inhibition of miR-203+ down-regulating HOTAIR.3、To observe whether miR-203 affects the proliferation,invasion and migration ability of MDA-MB231 cells through Caveolin-1.Dual-luciferase reporter assay was used to detect whether miR-203 is bound to CAV1.MiR-203 mimics and miR-203 inhibitor were transfected into MDA-MB231 cells,the expression of CAV1 was detected by Western Blot.Using RT-q PCR and Western Blot experimental methods to confirm whether CAV1 homo pc DNA3.1 has the effect of up-regulating CAV1;screen out the siRNA that most effectively down-regulate CAV1.The proliferation,invasion and migration ability of MDA-MB231 cell line after overexpression of CAV1,inhibition of CAV1,overexpression of CAV1 and overexpression of miR-203 were detected by CCK,Transwell invasion experiment,Transwell migration experiment and cell scratch experiment.Result:1、 After transfection of HOTAIR homo pc DNA3.1,the expression level of HOTAIR increased significantly.After transfection of NC,siRNA536,siRNA1162,siRNA1597 to MDA-MB231,siRNA536(HOTAIR-homo-536)was selected as the most effective siRNA in down-regulating the expression level of HOTAIR.Enhancing the expression of HOTAIR in MDA-MB231 can promote proliferation,invasion and migration ability of cancer cells;knocking down HOTAIR in MDAMB231 can inhibit the proliferation,invasion and migration ability of cancer cells.2、The results of Dual-luciferase reporter assay showed that there was a binding site between HOTAIR and miR-203.The RT-q PCR experiment found that the expression of miR-203 in breast cancer cell line(MDA-MB231)was negatively correlated with HOTAIR.After transfection of miR-203 mimics,the expression of miR-203 in MDA-MB231 cells increased.For miR-203 inhibitor,after transfection the expression of miR-203 in MDA-MB231 cells decreased.Up-regulation of MiR-203 can inhibit the proliferation,invasion and migration ability of MDA-MB231,while down-regulation of MiR-203 can promote the proliferation,invasion and migration of ability MDA-MB231.In addition,down-regulation of HOTAIR can reverse the effect of down-regulation of miR-203 on the proliferation,invasion and migration of MDA-MB231.3、 The results of Dual-luciferase reporter assay showed that there is a binding site between miR-203 and CAV1.Using WB to detect the expression of CAV1 in MDA-MB231,it was found that,compared with the control group,the expression of CAV1 decreased when miR-203 increased.Also the expression of CAV1 increased when miR-203 decreased.The expression level of CAV1 increased significantly after transfection of CAV1 homo pc DNA3.1.After transfecting the NC,siRNA439,siRNA548,siRNA710 to MDA-MB231,siRNA710(Caveolin1-homo-710)was picked as the most effective one in down-regulating the expression level of CAV1 in terms of protein and transcription level.Up-regulation of CAV1 can promote the proliferation,invasion and migration ability of MDA-MB231 cells,while down-regulation of CAV1 can inhibit it.In addition,overexpression of miR-203 reversed the effect of up-regulation of CAV1 on the proliferation,invasion and migration ability of MDA-MB231.Conclusion:1、HOTAIR may regulate the proliferation,invasion and migration of breast cancer cell line MDA-MB231.2、LncRNA HOTAIR and miR-203 can be combined with each other.LncRNA HOTAIR may negatively regulates the expression of miR-203,thereby affecting the proliferation,invasion and migration capabilities of MDA-MB231.3、 MiR-203 and CAV1 can be combined with each other.MiR-203 may negatively regulates the expression of CAV1,thereby affecting the proliferation,invasion and migration of MDA-MB231. |
