The Serum Expression Level Of MiR-1247-5p In Patients With Primary Liver Cancer And It Regulats The Proliferation And Invasion Of HepG2 By Targeting Smad2

Objective In order to explore the role of miR-1247-5p in the serum of Primary liver cancer patients, we detect the serum miR-1247-5p expression level from patients,thereby to study the role of micro RNA regulation of diagnostic value in Primary liver cancer. Explore miR-1247-5p effects on Smad2 which is a key protein in TGF-? signaling pathway. Discussing the influence of miR-1247-5p and its targeted protein Smad2 on HepG2 cells proliferation and migration ability, and find new molecular mechanisms and treatment methods for primary liver cancer.Method 1.Collection of Primary liver cancer patients serum,and healthy check-up people serum samples were collected at the same time, totolly 82 cases from General Hospital of Ningxia Medical University from April, 2014 to February, 2015. Real-time PCR was used to test the miR-1247-5p and mi R-21 expression levels in the two groups.2.Using the prediction softwares mi RBase and targetscan to find the potential target genes of miR-1247-5p, the potential target gene Smad2 was selected. Based on chemical technology to synthetic stem-loop structure RNA of Smad2 3’-UTR which is complementary to miR-1247-5p, and cloned it into the linearized Report plasmid, the point mutation sites are also cloned. Then sequenced, the positive recombinants were transfected into HEK-293 T cells; use the relative luciferase activity test, and Western blot test to validate the targeted regulatory relationship between miR-1247-5p and Smad2 3’-UTR.3.HepG2 cells were transfected with the miR-1247-5p mimic, miR-1247-5p inhibitor, Samd2 over expression vector and Smad2 RNAi vector. The HepG2 cells without transfecting any vector were used as normal blank,cell proliferation was detected by using CCK-8 assay kit detecting 0 h,24 h, 48 h, 72 h. Besides, HepG2 cell migration changes were measured by healing assays at 0h、24h、48h.Results1.According to the Real-Time PCR results by using the relative quantitative method calculates.Compared with healthy controls, mi R-21 expression significantly raised three times, miR-1247-5p expression significantly descend eight times in the patients with primary liver cancer( 0.01<P<0.05).Through the ROC curve analysis, miR-1247-5p has certain diagnostic value to the HCC.2. By the predicted soft, we identified two Smad2 target sites. Both of the recombinant plasmid of Report-Smad2-3’UTR were constructed successfully, dual luciferase reporter showed that miR-1247-5p mimics can suppress Smad2 site3390 recombinant vector luciferase activity, miR-1247-5p inhibitors can incresase Smad2 site3390 recombinant vector luciferase activity(P<0.05), miR-1247-5p has no influence on Smad2 site4420. The results of Western blot confirmed that the over-expression of miR-1247-5p suppressed the expression level of Smad2 protein significantly(P<0.05); suppression of miR-1247-5p expression increased the expression level of Smad2 protein significantly(P<0.05).3.CCK-8 results showed that transfection with over-expression Smad2 vector promoted HepG2 cells proliferation, miR-1247-5p mimics inhibited the proliferation of HepG2 cells,and co-transfecting miR-1247-5p mimics and Smad2 RNAi inhibited the proliferation of HepG2 cells. Howerver,when co-transfection miR-1247-5p mimics and over-expression Smad2 vector, the cell proliferation increased, which showed that the restoration of Smad 2-expression significantlyameliorated the miR-1247-5p-induced suppression of cell proliferation. The results of wound healing assays showed that miR-1247-5p mimics can reduce the migration of HepG2 cells after 48 h, but transfection with over-expression Smad2 vector can accelerate the migration process of HepG2 cells.Conclusion 1.Compared with healthy control group, patients with primary liver cancer have significant changes in serum miR-1247-5p expression. Serum miR-1247-5p has certain reference significance in the clinical diagnosis of primary liver cancer, and is expected to become the HCC molecular diagnostic potential targets.2.miR-1247-5p can targetedly regulated Samd2 expression, and this kind of regulation effect is related to the target site3390 of Samd2. miR-1247-5p can targetedly regulated Samd2 expression to influence the proliferation and invasion of HepG2.3.miR-1247-5p has the potential to be small molecule substrate for therapeutic targets development on Smad2.