In Vitro Study On The Invasion And Metastasis Effect Of Stanniocalcin 2 In Human Infiltrating Duct-type Breast Cancer

Research backgroundBreast cancer has become the most malignant tumor among women in the world,and it seriously threatens women’s life and health.In China,the incidence of breast cancer is increasing year by year and has become the most common cancer among Chinese women.Although breast cancer is moving toward "early detection,early diagnosis,and early treatment," as breast cancer screening technology and early diagnostic technology are continuously updated,and breast cancer comprehensive treatment methods are continuously optimized,there is still a great deal of progress.Some patients with breast cancer already have metastases of regional lymph nodes or distant metastases throughout the body at the time of diagnosis.In addition,breast cancer patients who have undergone regular treatment including surgery,chemotherapy,radiotherapy,endocrine therapy,molecular targeted therapy,and immunotherapy are at risk of high or low recurrence and distant metastases.It is also a serious problem that all breast cancer patients and our clinicians must face.Stanniocalcin is a generic term for secretory glycoproteins.A large number of studies have shown that stanniocalcin can participate in the body’s calcium and phosphorus regulation,metabolism,reproduction,stress response and development.There have been many reports that STC2 is associated with the proliferation or invasion and metastasis of a variety of tumors,including ovarian cancer,kidney cancer,and the like.But for different types of tumors,the role of STC2 may be completely different.Therefore,one of the main purposes of this article is to study the effect of STC2 on the invasion and metastasis of breast cancer.In addition,studies have found that STC2 may be involved in the regulation of HER2 and/or ER signaling pathways.To understand the relationship between the three,we must first study the effect of STC2 on the phenotype of breast cancer cells and its mechanism.Epithelial to mesenchymal transition(EMT)is a process in which cancer cells lose epithelial cell characteristics and acquire stromal cell characteristics and increase the ability to invade and metastasize.In recent years,EMT is gradually becoming a hot topic in tumor research.The EMT process is regulated by a sequence of proteins,including PKC,ZEBl,Slug,Snail,Twist,and Vimentin.Law et al.It has been reported that STC2 can promote EMT of hypoxic ovarian cancer cells.However,little is known about the correlation between STC2 and EMT in breast cancer cells.Yook et al.found that Wnt triggers the EMT process of breast cancer cells through its classical pathway,the Wnt/p-catenin pathway.In this paper,the expression of related proteins was detected by Western blot.PurposeIn summary,this study intends to complete the following three research objectives from in vitro experiments:(1)clinical data to clarify the relevance of STC2 and human breast cancer invasion,metastasis;(2)cell experiments to determine the STC2 invasion and metastasis of human breast cancer cells(3)To explore the mechanism of STC2 on the invasion and metastasis of human breast cancer cells.Methods1.Cell culture All cell lines were cultured in DMEM medium containing 10%fetal bovine serum.Both were placed in a 5%C02,37℃ cell culture incubator,and the cells were grown to 90%of the bottom area of the 25 cm2 cell culture flask for digestion and passage.The cells were grown to about 80%of the area of the six-well plate for experiments.2.Construction of STC2 Over-expression and Interfering shRNA Cell Lines After the STC2 fragment was obtained,the vector and the vector were digested by the restriction endonucleases EcoRI and BamHI and then ligated and transformed.Positive clones were picked from the STC2 fragment,and then subjected to plasmid extraction,restriction digestion,and sequencing verification.The constructed STC2 over-expressed plasmid(38 STC2)and the corresponding control plasmid,the empty vector(38 Vector),the STC2 interfering ShRNA plasmid(38 HM STC2i)and the corresponding control plasmid(38 HM Scr)were passed through the lentivirus.The packaging system infects the target cells and is screened by puromycin to obtain stable transformed cells.3.q-PCR reaction First,take 2 μl PCR product for agarose gel electrophoresis to see if there is a target band,and then the target product of the glue recovery.Based on the design of primers and multiple cloning sites of vector plasmids,the restriction enzymes Nhel and Notl multiple products and vector plasmids were used for processing.4.Observation of cell morphology by microscopy The cells were cultured using a small cover slip culture method(ie slides,coverslips)until the exponential proliferative phase,rinsed and fixed.Then it was dehydrated,dried and sealed.The morphology of the cells was observed with an optical microscope and images were collected.5.CCK-8 Proliferation Assay Each group of cells was inoculated into a 96-well plate,and 10 ul of CCK-8 solution was added to each well in the dark.After incubation for 1 hour,they were placed in a microplate reader and the absorbance of each cell culture well was measured at a wavelength of 450 nm.6.Transwell Migration and Invasion AssayMigration test:Take exponential growth cells for digestion,culture suspension cells in serum-free medium,count and adjust cell concentration to 2×105.In the lower chamber,600 ul of medium containing 10%serum was added and 200 μl of cell suspension was added to the upper chamber for 48 h.After the chamber was removed,the upper chamber liquid was aspirated and fixed with methanol for 30 minutes at room temperature.Stained for 30 min at room temperature with hematoxylin violet stain.After rinsing,dry the upper chamber liquid and wipe the cells on the bottom membrane of the upper chamber with a cotton swab.Remove the membrane(bottom side facing up)and slide onto the slide.Using a Leica DC 300F upright microscope,select up、down、left、right and center 5 fields for observation and photographing.Invasion test:Matrigel was thawed and diluted with serum-free medium to a concentration of 1 mg/ml.100 ul of diluted Matrigel was added to the upper chamber of the chamber and incubated at 37C for 4 hours to dry it into a gel.Follow-up test steps are the same as the migration test.7.Protein extraction Cells were harvested and appropriate amount of cell IP lysis buffer(containing protease inhibitors)was added and lysed on ice or at 4℃ for 30 min,then centrifuged at 12,000g for 30 min and the supernatant was removed.8.Western Blot The glass plate was assembled on a plastic frame.After confirming that there is no leakage,the separating glue mixture is injected into the glass plate.After the ethanol was injected into the upper layer of the separation gel,it was allowed to stand still for 1 hour at room temperature.After pouring anhydrous ethanol,it was washed three times with double distilled water.Prepare the concentrated glue and inject it between the glass plates.Insert the 15-hole comb into the concentrated gel and let stand for 1 hour at room temperature.The gel plate was mounted in an electrophoresis tank and filled with 1 × electrophoretic fluid.Remove the comb after loading.Connect the power supply for electrophoresis.After the end of the transfer membrane,the gel and PVDF membrane was placed in the filter paper and sponge sandwich,the membrane clip was inserted into the electrorotation tank and placed in an ice basin.After the end,the PVDF membrane was closed for 1 hour at room temperature.After overnight incubation in a 4℃ refrigerator in a primary antibody solution.Re-warm at room temperature for 30 minutes.After rinsing,add secondary antibody and incubate for 1 hour.The luminescent liquid was dropped on the front surface of the PVDF film,and an exposure program was started to perform image acquisition and preservation.9.Immunohistochemistry The tissue sections were successively subjected to xylene dewaxing,gradient alcohol hydration,endogenous peroxidase blocking,antigen retrieval,blocking,primary antibody blocking,secondary antibody incubation,DAB coloration,counterstaining,and organization.Dehydrated and transparent,sealing.Then score the results.10.Statistical analysis All data were statistically processed using SPSS 16.0(SPSS Inc.,Chicago,USA)statistical software.Count data were analyzed by variance and t-test,non-parametric test and correlation test were used for statistical analysis.p<0.05 was considered statistically significant.Results1.STC2 is associated with invasion and metastasis of human breast cancerThrough the collection and analysis of clinical data,it was found that the expression of STC2 in breast cancer tissues with lymph node metastasis,no distant metastasis,and patients with lymph nodes and distant metastases was higher than that of tissues without lymph node and distant metastatic breast cancer.Reduced.This shows that there is a correlation between STC2 and invasion and metastasis of human breast cancer,and may play a role in inhibiting the invasion and metastasis of human breast cancer.2.STC2 plays an important role in maintaining the morphology of breast cancer cellsThe morphology of the 38 STC2 cells became wavy,whereas the 38 HT STC2i cells became longer and morphologically more resembled mesenchymal fibroblasts than the control group..3.STC2 has no obvious effect on the proliferation of breast cancer cellsSTC2 was transfected into 38 and 38 HM cells of breast cancer.The growth rate of STC2 overexpressing cells was slightly increased,but the difference was not statistically significant.4.STC2 inhibits the migration and invasion of breast cancer cellsCompared with control cells,the number of 38 STC2 cells passing through the basement membrane of Transwell cells was smaller,while the number of 38 HM STC2i cells passing through the basement membrane of the cells was greater than that of the control group.At the same time,the results of cell invasion experiments were similar to those of cell migration experiments.The number of 38 STC2 cells that passed through the Matrigel cells at the bottom of the cell was less than that of the control 38 Vector cells,and the number of 38 HM STC2i cells crossed more than the control group..5.STC2 regulates breast cancer cell EMT through Wnt/β-catenin pathwayWnt expression was reduced in 38 STC2 cells compared to the control and increased in 38 HM STC2i cells.At the same time,the expression of β-catenin was consistent with the trend of Wnt.After 6,12 and 24 hours of XAV-939 treatment,the expression of Wnt/β-catenin decreased.It shows that STC2 may regulate breast cancer cell EMT through Wnt/β-catenin pathway.Conclusion:Clinical data and tissue experiments have shown that STC2 is associated with invasion and metastasis of human breast cancer and may play an inhibitory role.After shRNA interference reduced the expression of STC2 in 38 HM cells,38 HM cells exhibited fibroblast morphology with high metastasis characteristics,and increased cell invasion and metastasis capacity.Conversely,the invasiveness and metastasis of cells were decreased after overexpression of STC2 in 38 cells.In addition,a preliminary study on the influencing mechanism of stanniocalcin 2 on invasion and metastasis of human breast cancer cells found that STC2 may regulate breast cancer cell invasion by regulating EMT process through Wnt/β-catenin regulating pathway in breast cancer cells.Transfer ability.