Construction Of MiR-486-3p Specific Overexpression System And The Study Of Effects And Mechanism Of MiR-486-3p On Cancer

miRNA is a type of non-coding RNA conserved between 18-24 bases.It plays an important role in various biological processes,and participates in development,differentiation and stem maintenance.The abnormal expression of miRNA is closely related to the occurrence and development of many human diseases such as cancer.This study found that the expression of miR-486-3p in cancer tissues was significantly down-regulated relative to adjacent tissues.Subsequently,the correlation analysis of miR-486-3p expression data in TCGA database and breast cancer patients' survival rates revealed that miR-486-3p was highly expressed The survival rate of patients is significantly higher than that of patients with low expression,suggesting that miR-486-3p plays an important role in cancer.Therefore,this study will carry out the role and mechanism analysis of miR-486-3p in cancer.With the establishment of the miRNA storage platform and the development of bioinformatics,the special structure of miRNA complementary pairs has been discovered,and the miR-486 in this study can just be processed into complementary miRNA pairs,namely miR-486-5p and miR-486-3p mature body complementary pairing,this study found that miRNA-486 complementary pair special structure led to the use of traditional miRNA mimics and pri-miRNA overexpression methods can not achieve specific overexpression of miR-486-3p,for this reason,in sh RNA On the basis of the lentiviral expression system,a lentiviral system for miR-486-3p-specific overexpression was established in this study,and this system is also suitable for the specific overexpression of a single miRNA complemented by other miRNAs.Using the established specific over-expression lentiviral system of miR-486-3p,this study analyzed the effect of miR-486-3p on the biological characteristics of cancer,and found that miR-486-3p can significantly inhibit cancer cell proliferation by regulating the cancer cell cycle And clone formation,and tumor-bearing mouse epidermal tumor experiments confirmed that miR-486-3p also has the effect of inhibiting cancer cell proliferation in vivo.Secondly,overexpression of miR-486-3p can significantly induce cancer cell apoptosis and increase the sensitivity of cancer cells to chemotherapy.In addition,it was found that miR-486-3p can inhibit the invasion and migration of cancer cells by affecting the morphology of cancer cells.In order to further explore the molecular mechanism of miR-486-3p affecting cancer biological characteristics,this study used RNA-Seq technology to screen genes related to cell morphological changes TBCA,HMGA1,ARPC1 B and Rac3 as potential target genes of miR-486-3p Based on reporter gene activity and Western blot verification,this study confirmed for the first time that miR-486-3p targets TBCA,HMGA1,ARPC1 B,and Rac3 genes.Subsequently,we investigated whether the target gene participates in the process of miR-486-3p regulating cancer biological characteristics.The results show that down-regulating TBCA,HMGA1,ARPC1 B,and RAC3,respectively,affect the cell cycle of cancer cells,and down-regulating TBCA,HMGA1,and ARPC1 B can affect Morphology to inhibit cancer cell invasion and migration,but down-regulation of RAC3 gene has no effect on cell morphology and cancer cell invasion and migration.Finally,the tail vein injection of cancer cells stably transfected with firefly luciferase was used to prepare a mouse metastasis model.In combination with small animal live imaging,this study confirmed that overexpression of miR-486-3p and down-regulation of its target genes TBCA,HMGA1,and ARPC1 B were all detected.Can inhibit cancer cell metastasis in vivo.Using GO and KEGG enrichment to analyze RNA-Seq data,this study found and verified that overexpression of miR-486-3p can change the MAPK signaling pathway.Changes in the MAPK signaling pathway can affect cancer cell proliferation,apoptosis,invasion and migration,etc.Biological characteristics.And this study confirmed that miR-486-3p can at least affect the biological characteristics of cancer by targeting down-regulating its target genes to affect the phosphorylation level of ERK.Cancer stem cells are the source of cancer recurrence and metastasis.In order to further clarify the role of miR-486-3p in cancer metastasis,this study constructed a simple and efficient cancer stem cell screening and enrichment platform to simulate miR-486 by simulating the cancer stem cell microenvironment.The role of-3p in cancer stem cells.The results showed that overexpression of miR-486-3p and knockdown of its target genes TBCA,HMGA1 and ARPC1 B significantly inhibited the growth of cancer stem cell mass and metastasis in vivo.Mi R-486-3p can at least inhibit the self-proliferation and metastasis of cancer stem cells by targeting downregulating its target genes TBCA,HMGA1,and ARPC1 B to regulate the MAPK signaling pathway.In summary,this study successfully constructed a lentiviral system that specifically overexpresses miR-486-3p.Using the constructed system,it was confirmed that miR-486-3p can at least down-regulate its target genes TBCA,HMGA1,ARPC1 B,and RAC3 to regulate the MAPK signal pathway in cancer occurrence and development,cancer stem cell self-replication and proliferation,and metastasis in vivo.This study provides a new target for molecular treatment of cancer,and provides a theoretical basis for miR-486-3p for clinical cancer treatment.