| Objective: RRS1(Regulator of ribosome synthesis 1)is a new ribosomal synthesis regulatory protein discovered by Shimadzu in 1999.It is involved in the maturation of 25 srRNA and assembly of large ribosomal subunits in 60 s in ribosomal biosynthesis.In recent years,studies have found that RRS1 is overexpressed in some human tumors,which is related to the proliferation of colorectal cancer,liver cancer and breast cancer,but its role in breast cancer metastasis is still unclear.In this study,the effect of human RRS1/ murine Rrs1 on breast cancer metastasis was investigated through in vitro breast cancer cell function experiment and in vivo mouse breast cancer model.Methods:(1)Real-time quantitative PCR(qPCR)and Western Blotting(WB)were used to detect the expression of RRS1 in human breast cancer MDA-MB-231 cells,mouse breast cancer 4T1 cells,and normal breast epithelial cells HMEC.(2)MDA-MB-231 cells were transfected with lentiviral ShRNA-RRS1 to knock down the expression of RRS1 gene,while 4T1 cells were transfected with small interfering sirna-rrs1 to knock down the expression of Rrs1 gene,and plasmid transfection was used to over-express Rrs1.At the end of transfection,the transfection efficiency was observed by fluorescence microscope,and RRS1 knockdown efficiency,Rrs1 knockout and overexpression efficiency were further detected by qPCR and WesternBlotting.(3)CCK8 assay was used to detect the proliferation activity of RRS1 in MDA-MB-231 cells and Rrs1 in 4T1 cells after knockdown and overexpression.;The change of migration capacity of MDA-MB-231 and 4T1 cells after transfection was detected by scratch test and Transwell transfer test.Transwell invasion assay was used to detect the changes in invasion capacity of the two cells before and after transfection.(4)LUC-4T1 cells were cultured and transplanted tumor model was established in BALB/C mice with the fourth breast pad.After successful tumor bearing,mice were randomly divided into three groups: control group(transfection reagent injection),sirna-ctrl transfection group,and sirna-rrs1 transfection group.Every two days,each tumor bearing mouse was injected with siRNAs and transfection reagent for one month.Tumor growth and metastasis in tumor-bearing mice were detected using in vivo imaging system of small animals.After the mice were killed,the tumor was exfoliated and photographed to extract tumor RNA and protein.The lungs of the mice were removed and fixed with Bouin's fixative for 24 hours.The number of tumor nodules was counted.Results:(1)The expression level of RRS1 mRNA and protein in MDA-MB-231 and 4T1 cells was higher than that in HMEC cells(P <0.01).(2)The fluorescence efficiency of MDA-MB-231 and 4T1 cells was observed to be more than 70% under a fluorescence microscope.After ShRNA-RRS1 transfection,the expression of RRS1 in MDA-MB-231 cells was significantly lower than that in the negative control group.After transfection of siRNA-Rrs1,the expression of Rrs1 in 4T1 cells was 0.58 ± 0.095.The expression of Rrs1 in 4T1 cells after transfection of Rrs1 plasmid was 1.62 ± 0.133.(3)After transfection of MDA-MB-231 cells with ShRNA-RRS1,the proliferation activity decreased(P<0.01),and the invasion and migration ability was significantly lower than that of the control group(P<0.05).After transfection of 4T1 cells with siRNA-Rrs1,the proliferation activity decreased(P<0.01).Invasion and migration capacity decreased(P<0.05).The cell proliferation activity of Rrs1 plasmid overexpression group was significantly higher than that of other groups(P<0.01).Invasion and migration capacity increased(P<0.05).(4)Mouse tumor-bearing model was successfully established.After one month of treatment,tumor metastasis occurred in the three groups of mice to different degrees.However,tumor cell metastasis was significantly inhibited in the mice injected with si RNA-Rrs1,and distal lung metastasis occurred in the other two groups of mice.Moreover,the tumor volume collected from the si RNA-Rrs1 treatment group was significantly smaller than that of the other two groups,and the number of pulmonary nodules was significantly less than that of the other two groups.Conclusions: Overexpression of Rrs1 at cellular level promotes the proliferation,migration and invasion of 4T1 cells,while knockdown of Rrs1 gene expression inhibits the proliferation,migration and invasion of 4T1 cells;knockdown of RRS1 expression also inhibits the proliferation,migration and invasion of MDA-MB-231 cells.The tumor growth and metastasis of BALB/C mice were inhibited by injection of siRNA-Rrs1 in vivo.The experimental results indicated that RRS1/ RrS1 overexpression may promote the invasion and metastasis of breast cancer... |
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