The Regulate Effect Of MiR-96 On The Proliferation By Permethrin Induced In Human Breast Cancer MCF-7 Cells

Pseudoestrogen chemicals are abundant in the environment.According to the report,they can interfere with the endocrine system,reproductive system toxicity,resulting in metabolic disorders,birth defects,developmental disorders and other diseases.Pyrethroids,as its representative,are widely used in household and agricultural deinsectization,In addition to disrupting endocrine metabolism,it also induces the proliferation of certain cells and induces the occurrence of tumors.The United States environmental protection agency classifies it as a"potential carcinogen".Therefore,in order to further understand and elucidate its specific mechanism of inducing cell proliferation.In this paper,the human MCF-7 cell line was selected to establish a model of Permethrin exposure MCF-7 cells,to detect the changes at the cellular,physiological,biochemical and molecular levels,to speculate the biological pathways that these compounds induce cell proliferation,and to elucidate the possible biological mechanisms of their estrogen effect.The results were as follows:1.CCK-8 method was used to detect the proliferation effect of permethrin on MCF-7 cells.The results showed that permethrin had time-dependent and dose-dependent effects on cell proliferation.According to the results of CCK-8detection,the screening time of 72 hour and concentration of 10^-4mol/L had the strongest effect on cell proliferation,Under these conditions,the changes in cell morphology were observed by fluorescence staining and light microscopy.Compared with the control,it was found that the cell morphology was uniform,the cell adhesion was strong,and the amount of DNA in apoptotic bodies in the nucleus was significantly reduced.In addition,and by CCK-8 cell proliferation detection method,the optimal concentration of estradiol to promote the proliferation of MCF-7 cells at72 hour treatment was screened to be 10^-7mol/L.2.When the concentration of permethrin was 10^-4mol/L,the concentration of estradiol was 10^-7mol/L,and the treatment time was 72 hours,Western Blot also found that permethrin,like estrogen,significantly up-regulated the protein expression levels of estrogen receptor（ER-α）and Signal transducer and activator of transcription3（STAT3）,which was significantly higher than that of the blank group.The fluorescence quantitative PCR method was used to detect miR-96,and the results showed that permethrin promoted the differential up-regulation of miR-96 expression,which was about 1.5 times that of the control group.Combined with literature reports,STAT3 is a miR-96 activated transcription factor.It was speculated that STAT3 might be the cause of miR-96 up-regulation caused by permethrin.3.The effect of transfected miR-96 on cell proliferation was detected by fluorescence quantitative PCR and CCK-8.Mimics and inhibitors of miR-96 were transfected with normally cultured MCF-7 cells.The results showed that the expression level of miR-96 in mimics transfected cells was significantly increased and the cell proliferation ability was significantly enhanced,while the expression level of miR-96 in transfected inhibitor cells was significantly down-regulated and the cell viability was significantly decreased.It was revealed that miR-96 was one of the causes of MCF-7 cell proliferation.The scratch assay showed the cell migration ability of the permethrin-treated group and the mimics group exposed to permethrin transfection was significantly enhanced,while the migration of inhibitor exposed to permethrin transfection was significantly inhibited.Combined with the above experiments,it was speculated that the up-regulation of miR-96 might be one of the causes of promoting permethrin cells.Combined with the above experiments,it was speculated that permethrin up-regulated miR-96 to promote the proliferation and migration of MCF-7 cells.4.Bioinformatics was used to predict the target genes of miR-96.Results the GO and KEGG methods showed that these target genes were mainly involved in the regulation of cell proliferation pathways and cancer cell pathways.The expression of HOXA5 was detected by Western Blot.Results showed that the expression of HOXA5 protein was significantly down-regulated in the miR-96mimics group treated with permethrin and exposed to permethrin,and significantly up-regulated in the miR-96inhibitor group compared with the permethrin group and the mimics group,but lower than the control group.Permethrin inhibited the expression of HOXA5 in MCF-7 cells by up-regulating miR-96.According to the above studies,combined with the results of scratch experiments,it was concluded that miR-96 and its target gene HOXA5 may mediate the effects of permethrin on the proliferation and migration of MCF-7 cells.In summary,we reasoned that permethrin,As a pseudoestrogen,it can up-regulate the key gene of the estrogen receptor pathway in MCF-7 cells,the estrogen receptor ER-α、STAT3 and miR-96,STAT3 may activate the transcription of miR-96,and up-regulation of miR-96 targeting HOXA5 can cause cell proliferation and migration.The specific mechanism needs to be further studied...