| Objective:The present study was designed to analyze the clinical significance of miR-372 via detecting its expression in breast cancer tissues and adjacent normal tissues.Furthermore,the effects of miR-372 on the occurrence,development and specific cellular biological behavior of breast cancer were further explored on the basis of cell and animal experiments,and the related mechanisms were also discussed.The purpose of the study is to elucidate the specific mechanism of miR-372 in the malignant process of breast cancer and provide the foundation for new molecular markers and therapeutic targets for breast cancer treatment.Methods(1)Samples of cancer tissues and adjacent normal tissues were collected from 24 breast cancer patients admitted to the People's Hospital of BeiHai city between July 2014 and July 2016.Expression of miR-372 in these tissues was determined by fluorescence quantitative PCR.Besides,the correlation was analyzed between miR-372 expression and clinicopathological characteristics of breast cancer patients.(2)Expression of miR-372 in normal human breast cell line MCF-10A and human breast cancer cell lines MCF-7,SK-BR-3,BT-474 and MDA-MB-231 was determined by fluorescence quantitative PCR.Cell line MDA-MB-231 cells were randomly divided into the following 2 groups:the scramble group(transfected with scramble),and the AS-miR-372 group(transfected with AS-miR-372),respectively.Expression of miR-372 in these cells was detected by fluorescence quantitative PCR.Proliferative activity of cells in each group was determined by MTT assay.The ability of colony-forming was detected by colony forming assay.Cell cycles and apoptosis were measured by flow cytometry.miR-372 target genes were predicted by bioinformatics software and validated by the luciferase reporter system.After inhibiting the expression of miR-372,mRNA and protein expression of LATS2 were detected by fluorescence quantitative PCR and Western blot respectively to further verify the miR-372 target gene.Furthermore,cells of the MDA-MB-231 cell line were randomly divided into the scramble group(transfected with scramble),the AS-miR-372 group(transfected with AS-miR-372),and the AS-miR-372+si-LATS2 group(co-transfected with AS-miR-372 and si-LATS2),respectively.Expression of LATS2 protein in each group was determined by Western blot,colony-forming ability was tested by colony forming assay,and cell proliferative activity was determined by MTT assay.(3)Cells of the MDA-MB-231 cell line were randomly divided into the scramble group(transfected with scramble),and the AS-miR-372 group(transfected with AS-miR-372),respectively.Expression of miR-372 in these cells was detected by fluorescence quantitative PCR.Cells of the two groups were inoculated subcutaneously to the right shoulder skin of nude mice to establish a breast cancer xenograft tumor model in nude mice.Tumor formation was observed,followed by the calculation of the volume and weight of the xenograft tumor.Results(1)The expression level of miR-372 in breast cancer tissues was significantly higher than that in normal breast tissues(P<0.05).The expression level of miR-372 was related to TNM staging and lymph node metastasis in breast cancer patients(P<0.05).However,there was no associated with the age,menopause,pathological type,tumor size,and the expression of c-erbB-2,PCNA,PR and ER(P>0.05).(2)The expression level of miR-372 in cell lines MCF-7,SK-BR-3,BT-474 and MDA-MB-231 was significantly increased compared with that of cell line MCF-10A(P<0.05).In cell line MDA-MB-231,the expression level of miR-372 in the AS-miR-372 group was significantly reduced compared with the scramble group,and the difference was statistically significant(P<0.05).Compared with the scramble group,there was decreased cell proliferation activity,reduced number of cell colony forming,increased number of cells arrested at G1 and M phases as well as decreased number at S phase,and increased cell apoptosis rate in the AS-miR-372 group,and the differences were statistically significant(P<0.05).LATS2 was predicted to be a target gene of miR-372,and miR-372 could bind to the 3'UTR region of LATS2 gene.In MDA-MB-231 cells,after inhibiting the expression of miR-372,the expression level of luciferase in cells transfected with wild type LATS2-WT expression vector was significantly higher than that of the scramble group(P<0.05).However,the expression level of luciferase in cells transfected with mutant type LATS2-MUT expression vector was not significantly changed versus the scramble group(P>0.05).Furthermore,the expression levels of LAST2 mRNA and protein were significantly increased following the inhibition of miR-372 expression(P<0.05).Compared with the scramble group,the expression level of LATS2 protein was significantly increased in the AS-miR-372 group(P<0.05),and that of the AS-miR-372+si-LATS2 group was significantly decreased(P<0.05).Meanwhile,the number of cell colony forming was significantly decreased in the AS-miR-372 group(P<0.05),while that of the AS-miR-372+si-LATS2 group remained unchanged(P>0.05).Additionally,the cell proliferation activity of the AS-miR-372 group was decreased at 24h,48h and 72h after transfection(P<0.05).But the proliferation activity of the AS-miR-3 72+si-LATS2 group remained unchanged at each time point(P>0.05).(3)Compared with the scramble group,the expression level of miR-372 was significantly reduced in cells of the AS-miR-372 group(P<0.05).Besides,the volume and weight of the transplanted tumor were significantly decreased in nude mice of the AS-miR-372 group(P<0.05).Conclusions(1)The expression of miR-372 is significantly increased in breast cancer cell lines and primary breast cancer tissues.(2)The decreased expression of miR-372 can inhibit the proliferation and growth of breast cancer cells,arrest cells at G1/M phase,and promote cell apoptosis.The mechanism is related to the reduced expression of LATS2 mediated by miR-372.(3)miR-372 can promote the growth of breast cancer cell xenograft tumor in nude mice. |
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