The Role And Mechanism Of MiR-141-3P In Malignant Biological Behavior Of Lung Adenocarcinoma

miR-141-3p is an important member of the miR-200 family and is located at chromosome 12p13.31,where expression levels differ in human cancers.In the early stage of the research team,the miR-141-3p gene was highly expressed in lung adenocarcinoma by miRNA PCR gene chip screening,but the regulation of miR-141-3p on the malignant biological behavior of lung adenocarcinoma cells,and related specific The mechanism is not fully understood.Moreover,studies have confirmed that miR-141-3p is involved in biological behaviors such as proliferation,metastasis,invasion and apoptosis in different tumor cells,but the literature on the biological characteristics of lung cancer cells is not fully elucidated in lung adenocarcinoma.The research has rarely been reported.At the same time,in order to explore the related mechanism of miR-141-3p,we screened the target genes UBAP1,MALAT1,KLF12,STAT4 and PTEN to study miR-by bioinformatics analysis and cancer genomic map(TCGA)dataset.The important regulatory mechanism of 141-3p in lung adenocarcinoma,therefore,this study intends to use Real-time PCR technology to verify the expression level of miR-141-3p in lung adenocarcinoma,using RT-qPCR technology,cell function experiments,Western blotting(western-blot)explored the malignant biological behavior of miR-141 in lung adenocarcinoma and its correlation with target genes,and analyzed the relationship between miR-141-3p expression and clinical pathological parameters.It provides new clues for the mechanism of lung adenocarcinoma and also provides a more effective target for the early diagnosis and treatment of lung adenocarcinoma.[Objective]1.Collect and screen specimens,expand the sample size,and further verify the expression level of miR-141-3p in lung adenocarcinoma by RT-qPCR.2.The cell line was selected and cultured,and the expression level of miR-141-3p was knocked down and overexpressed by RNA interference technology.The main regulation of miR-141-3p in lung adenocarcinoma cells was explored and analyzed.3.The expression of miR-141-3p and its correlation with target genes were initially explored and analyzed by RT-qPCR and western blot.4.To analyze the correlation between miR-141-3p and the target gene,and the relationship between miR-141-3p expression level and clinicopathological parameters.[Method]1.The Department of Thoracic and Cardiovascular Surgery,the Third Affiliated Hospital of Kunming Medical University,selected from 30 cases of lung adenocarcinoma and normal lung tissue from January 2017 to December 2018.The patients had no history of chemotherapy drugs.The preoperative clinical diagnosis and intraoperative frozen section diagnosis results were consistent.The normal tissue is more than 5 cm from the cancerous tissue,and the tissue block is less than 0.5 cm Tumor type and malignancy were determined by histopathological evaluation of hematoxylin and eosin(H&E)staining according to WHO criteria.All patients were selected to sign informed consent and approved by the hospital ethics committee.The criteria for tissue inclusion were as follows:1 surgically resected specimens were pathologically diagnosed as lung adenocarcinoma 2 without anti-tumor treatment before surgery 4 surgically removed,and.the margin was negative.After the specimens were collected,they were immersed in a cryotube containing RNAlater solution,placed in a 40 refrigerator,and then transferred to a-80? refrigerator for overnight use.2.The lung adenocarcinoma cell line was screened by RT-qPCR technique,and the expression level of miR-141-3p was knocked down or overexpressed by RNAi interference technology.Each cell line was set up with a blank group and a negative control(NC)group.In the experimental group,cell proliferation experiments,scratch experiments,cloning experiments,and invasion experiments were performed to observe whether there were differences in malignant biological behavior between the groups.3.RT-qPCR was used to screen for target genes with high correlation with miR-141-3p.4.Western-blot was used to determine the correlation between cells and the expression of target gene proteins after RNAi interference.5.The expression level of miR-141-3p in tissue samples,the expression level of screened target genes and the correlation between them were analyzed by RT-qPCR.6.SPSS 19.0 software package was used for statistical analysis.The experimental data of each group were expressed as mean±standard deviation(x±s).The comparison between groups was analyzed by one-way analysis of variance.The data obtained from RT-qPCR of tissue samples were expressed as median.The relationship between miRNA expression and its downstream target genes was compared by Spearman test.The expression levels of miRNA and mRNA were compared with age,sex,region,tumor size,TNM stage,lymph node metastasis,etc.The relationship between the clinicopathological data was analyzed by Chi-Square test,and the count data was expressed as an integer.There was a statistical difference at P<0.05.[Result]1.miR-141-3p is highly expressed in XWLC-05,YTMLC,A549 cells;low expression in H157 cells.2.Knockdown of miR-141-3p can inhibit the proliferation,invasion,wound healing and cloning ability of XWLC-05 cells;knockdown of miR-141-3p can inhibit the invasion and wound healing of A549 cells,but the ability to proliferate and clone No significant effect;overexpression of miR-141-3p promoted proliferation,invasion,wound healing and clonal ability of H157 cells3.At the mRNA level,knockdown of miR-141-3p in XWLC-05 cells increased the expression of PTEN and UBAP1.The knockdown of miR-141-3p in A549 cells resulted in an increase in PTEN expression and no significant change in UBAP1 expression.The expression level of PTEN was decreased after overexpression of miR-141-3p in H157 cells,and the expression level of UBAP1 was slightly increased,but there was no significant change.4.At the level of protein expression,knockdown of miR-141-3p in XWLC-05 and A549 cells increased the expression of PTEN and UBAP1;overexpression of miR-141-3p in H157 cells decreased the expression of PTEN and UB AP1.The expression level of miR-141-3p in lung adenocarcinoma tissues was higher than that in normal tissues(P=0.0138).The expression levels of PTEN in lung adenocarcinoma tissues and normal tissues were different and statistically significant.Normal tissues were higher than normal tissues.Cancer tissues(P=0.0260);there was no significant difference in the expression of UBAP1 between lung adenocarcinoma and normal tissues(P=0.2887).The expression level of miR-141-3p in lung adenocarcinoma was negatively correlated with PTEN expression level(r=-0.3190,P=0.0212);the expression level of miR-141-3p in non-Xuanwei area tissues and PTEN and UBAP1 There was a negative correlation between expression levels(r=-0.2225,P=0.1128).There was no significant correlation between the expression level of miR-141-3p and the age,sex,region,clinical stage,primary tumor size and lymph node metastasis(p>0.05).The expression level of PTEN was related to the patient's age,gender and region.There was no significant correlation between stage,primary lesion size and lymph node metastasis(P>0.05).[Conclusion]1.At the cellular level,miR-141-3p has a tumor-promoting gene function in lung adenocarcinoma,which can promote the proliferation,invasion,wound healing and cloning ability of lung adenocarcinoma cells.2.At the molecular level,miR-141-3p can promote the malignant biological behavior of lung adenocarcinoma cells by inhibiting PTEN expression;miR-141-3p only regulates UBAP1 at the protein level.3.At the tissue level,miR-141-3p is expressed in lung adenocarcinoma tissues at a higher level than normal tissues.There was no significant correlation between the expression levels of miR-141-3p and PTEN and clinical pathological parameters in lung adenocarcinoma.