Regulation Of RAB22A By MiR-193b Inhibits Breast Cancer Growth And Metastasis Mediated By Exosomes

PURPOSE and BACKGROUND:Breast cancer is one of the main types of cancer affecting the health of females worldwide.Despite improvements in therapeutic approaches,cancer patients succumb to the disease due to metastasis itself,rather than the primary tumor from which metastases arise,emphasizing the need for the better understanding of the biological bases that contribute to disease progression.Recent studies have shown that mi R-193 b plays an important inhibitory role in the proliferation and metastasis of various tumor cells and is closely related to clinicopathological features and prognosis of patients.RAB22 A,a member of the proto-oncogene RAS family,plays an important role in the formation,trafficking and metabolism of exosomes,and is associated with the occurrence and development of multiple human cancers.The specific mechanism of action in breast cancer remains unclear.As a "new pathway" for intercellular communication,exosomes are microvesicles that are produced by cells and secreted into the extracellular microenvironment,between tumor cells themselves or interaction with other cells,such as lymphocytes or antigens recognition cell,relying on special transmitter components contained therein to transmit special biosignals,thereby affecting the proliferation,migration and invasion ability of the tumor cell.In this study,we will examine the expressionlevels of mi R-193 b and RAB22 A in human mammary gland epithelial cells,breast tumor cells and human breast cancer tissues,and identify the relationship between their expression;explore the decrease of RAB22 A expression affecting tumor cell proliferation,colony formation,infiltration and migration abilities;studying the key role of mi R-193 b in breast cancer cells in the regulation of RAB22A-mediated exocytogenesis in cancer cell growth and metastasis,which may be important for cancer treatment.METHODS:We collected 242 groups of breast cancer samples at the clinic,and breast cancer cell lines MCF-10 A,MCF-7,MDA-MB-231,SK-BR-3,MDA-MB-453,MDA-MB-468,ZR-75 and T47 D,RT-q PCR and immunoblotting used to detected the expression levels of RAB22 A and mi R-193 b and observed relationship between these two genes.Kaplan-Meier survival curves were used to analyze the influencive ability of RAB22 A m RNA expression levels to patient prognosis.To establish a cell model with low expression of RAB22 A gene: MCF-7and SK-BR-3 cell overexpressing mi R-193 b,and the expression levels of mi R-193 b and RAB22 A in the experimental and control cells were detected by RT-q PCR.Transfection cells with either pc DNA3.1-RAB22 A or pc DNA3.1was performed in cells overexpressing mi R-193 b,and the growth rate of transfected cells was observed to confirm that RAB22 A is a target gene ofmi R-193b;Lentiviruses HBLV-RAB22 A sh RNA-Puro(sh RAB22A)were used to infect MCF-7 and SK-BR-3 cells,and HBLV-Scramble-Puro control(SC)was used for negative control.RAB22 A has been knocked down was confirmed by RT-q PCR and immunoblotting.Exosome extraction and enzymatic treatment: Cell culture supernatants were collected,110,000 g ultra-high speed centrifugation twice for 70 minutes at 4 ° C,resuspended sediment in PBS solution,and exosomes were extracted.Analyze.Exosomes extracted from SK-BR-3 cells are inactivated by proteases and RNases.Exosomes uptake assay: exosomes labeled with Di O probe were co-cultured with SK-BR-3 cells for 24 hours,washed with PBS,fixed with 4%paraformaldehyde for 15 minutes,and stained with DAPI at room temperature.minute.Placed under a fluorescence confocal microscope and photographed.Co-culture experiment of exosomes and cells: MCF-7 and SK-BR-3 cells were added with exosomes to be tested to a final concentration of 20 ?g/ml,placed at 37 ° C,5% CO2 constant temperature and humidity culture for 2 days,cells were collected for subsequent experimental studies.Cell proliferation assay: The effect of low expression of RAB22 A gene or co-culture with exosomes on the growth rate of MCF-7 or SK-BR-3 cells was observed.A colony formation assay was used to evaluate the effect of knockdown of the RAB22 A gene on the proliferative capacity of MCF-7 orSK-BR-3 cells.Transwell cell migration and infiltration experiments were used to evaluate the effect of knockdown of the RAB22 A gene or removal of exosomes on SK-BR-3 cell metastatic ability.RESULTS:1.Expression of mi R-193 b and RAB22 A genes in human breast cancer cells and tissues.In this study,we demonstrate that the upregulation of RAB22 A is associated with breast cancer progression and lymph node metastasis.We identified a signature of RAB22 A and mi R-193 b that exhibited a negative association in metastatic as opposed to the surrounding normal cells.2.RAB22 A was identified as the target gene of mi R-193 b.After transfecting pre-mi R-193 b mimic,the expression of mi R-193 b was increased to8-fold and 10-fold compared to the negative control group in MCF-7 and SK-BR-3 cells,while RAB22 A protein content was increased.Reduced to 40%and 30%.3.RAB22 A is the regulatory target gene of mi R-193 b.In the SK-BR-3cells transfected with both mi R-193 b and RAB22 A gene,the ability of proliferation was restored from the third day.However,overexpression of RAB22 A alone significantly promoting proliferation than the negative control group.4.RAB22 A gene regulates proliferation and metastasis of breast cancer.After knocking down RAB22 A in MCF-7 and SK-BR-3 cells,the expression level of RAB22 A decreased to ~30% and ~35% of control cells,and the protein content of RAB22 A decreased to ~50% and ~20%.Low expression of RAB22 A gene lost promoting effect on SK-BR-3 cell proliferation after the third day culturing,which is more pronounced with increasing number of culturing days,the same effect can also be observed in MCF-7 cells.In addition,after knocking down the RAB22 A gene,the number of clones formed was ~20% and~30% lower to the negative control group.The low expression of RAB22 A gene can significantly deprived the migration and distant invasion abilities in SK-BR-3 cells.5.Low expression of RAB22 A decreased the level of exosomes in SK-BR-3 cells.Western blot analysis also revealed the reduced expression of the exocrine marker proteins after RAB22 A expression decreased.6.RAB22 A was found to regulate exosomes-mediated breast cancer cell proliferation and migration,these biological characteristics were diminished in the breast cancer cells in which the RAB22 A gene was knocked down or in the cells in which the exosomes were dissolved by proteinase K/RNase treatment.CONCLUSION:1.The up-regulation of RAB22 A gene expression is positively correlated with breast tumorigenesis and lymph node metastasis,which can be used as a marker of clinical prognosis of breast cancer.2.Mi-193 b inhibits the proliferation of breast cancer cells by targeting RAB22 A gene expression.3.RAB22 A gene promotes breast cancer cells proliferation,migration and invasion,which may be related to its regulation of exosome synthesis and secretion.