Effect Of MicroRNA-133a-5p On The Migration And Invasion Of QBC939 Cholangiocarcinoma Cells By Regulating LASP-1 Protein

Objective: Cholangiocarcinoma(CCA)is a malignant tumor of the digestive system and is difficult to be diagnosed in the early stage.The typical symptoms usually appear in the progression period of CCA.At this time,surgery cannot guarantee a cure for CCA patients.In addition,CCA is insensitive to traditional treatment including radiotherapy and chemotherapy,these situations made patients with CCA have difficulty obtaining a good prognosis.Micro RNA(micro RNA,miRNA)can regulate the physiological and pathological processes of the human body through the induction and modification of the translation process after transcription.Current evidences show that the unregulated expression of miRNA may directly or indirectly give rise to the occurrence and progression of CCA.We studied the expression of Micro RNA-133(miR-133)in CCA cell line QBC939,analyzed the role of miR-133 in the migration and invasion of QBC939 cells,and explored the possible molecular mechanisms.Methods: Seeding and culturing the QBC939 cells in DMEM,qRT-PCR(Quantitative real time PCR)was used to detect the expression of the three subtypes of miR-133(miR-133a-5p,miR-133a-3p,miR-133b)in QBC939 cell,to select the suitable subtype for subsequent experiments.QBC939 cells were divided into three groups by transfection with different reagents: Interference group(transfection with miR-133a-5p inhibitor),MiR-133a-5p mimics group(transfection with miR-133a-5p mimic)and Blank control group.After cultivation,the expression of miR-133-5p in the three groups of QBC939 cells were detected using qRT-PCR.Transwell assay was used to detect the migration ability and invasion ability of each group;Western blot was used to detect the expression of LASP-1(LIM、SH3-1)protein in each group,and one-way analysis of variance was used to analyze the results above.Results: qRT-PCR detected the relative expression of the three miR-133 subtypes:miR-133a-5p(1.07±0.06),miR-133a-3p(0.97±0.11),miR-133b(0.73±0.07).The result showed that the relative expression of miR-133a-5p is the highest(P<0.05),so we chose miR-133a-5p to continue the next experiment.qRT-PCR was used to detect the relative expression of miR-133a-5p in three groups of QBC939 cells: Interference group(0.70±0.08),MiR-133a-5p mimics group(1.43±0.34),Blank control group(0.90±0.17),the result showed that the relative expression of miR-133a-5p in the interference group was the lowest(P<0.05).Transwell assay was applied to evaluate the migration invasion ability of the QBC939 cells of the three groups.Number of migration: Interference group(72.0±11.0),MiR-133a-5p mimics group(110.3±4.2),Blank control group(106.0±10.4);Invasion number: Interference group(20.0±3.0),MiR-133a-5p mimics group(65.0±2.6),Blank control group(83.7±3.5),and the number of migration and invasion of QBC939 cells in the interference group was the least(P<0.05).The relative expression level of LIM in the three groups of QBC939 cells detected by Western blot were: Interference group(0.85±0.02),MiR-133a-5p mimics group(0.30±0.06),Blank control group(0.28±0.05),and the interference group expressed the highest level of LIM(P<0.05);the relative expression level of SH3-1 in the three groups of QBC939 cells detected by Western blot were: Interference group(0.71±0.01),MiR-133a-5p mimics group(0.98±0.03),Blank control group(0.99±0.01),and the interference group expressed the least SH3-1(P<0.05).Conclusion: 1.MiR-133a-5p can regulate the expression of LASP-1 protein in vitro,knockdown miR-133a-5p can upregulate LIM expression and downregulate SH3-1expression in QBC939 cells;2.Knockdown miR-133a-5p can inhibit the migration and invasion of QBC939 cells,which may be associated with the regulation of LASP-1protein expression by miR-133a-5p.In conclusion,miR-133a-5p can affect the malignant behaviour of QBC939 cells by regulating the expression of LASP-1 protein.This provides a broad prospect for early diagnosis and targeted therapy of CCA.