| Objective:To explore mi R-19 a targeting regulation Of SOCS3(Suppressors Of Cytokines Signaling3)to activate JAK1-STAT3(Janus Kinase1-Signaling Transducers And Activators Transcription3)signal pathway affect biological characteristic of non-small cell lung cancer cells as the proliferation,migration,invasion and apoptosis.Methods and materials:Thirty patients with primary non-small cell lung cancer(NSCLC)that were surgically resected from January 2019 to June 2019 in the Department of Thoracic Surgery of the Second Affiliated Hospital of the University of South China were selected and matched with normal lung tissues adjacent to the same lung cancer(distance lesions >5cm,no tumor cell invasion was confirmed by pathological sections and immunohistochemistry),and all the samples were pathologically confirmed to be NSCLC.Human nonsmall cell lung cancer cell line A549 was cultured and transfected with logarithmic growth phase cells.The experimental cells were grouped according to different transfection agents: H-mi R-19 a group(A549 cells transfected with mimic-mirna19a);L-mi R-19 a group(inhibitor-mi RNA19 a transfected A549 cells);P-SOCS3 group(A549 cells in L-mi R-19 a group were treated with SOCS3 agonist);The Control group.After the transfection,the cells were cultured again for 48 h.The expression of mi R-19 a and SOCS3 in NSCLC tissues and para-cancerous groups was detected by q RT-PCR to explore the correlation between mi R-19 a and SOCS3 and clinical characteristics of lung cancer,such as clinical stage,tumor classification,and the presence of distant metastasis.In vitro analysis: The transfection success was confirmed by q Rt-PCR to detect the expression of mi R-19 a and SOCS3 in each group.The expression levels of SOCS3,JAK1-STAT3 and Bcl-2\Bax were detected by Western-blot.Next,MTT(tetramethylorniazolium)colorimetry was used to detect cell survival and growth in each group.Cell apoptosis rate in each group was detected by Annexin-V/PI(flow cytometry).Cell migration and invasion in each group were analyzed by scratch and Transwell assay.Results:(1)Compared with the expression of mi R-19 a and SOCS3 in lung cancer tissues detected by q Rt-PCR,the expression of mi R-19 a was higher in non-small cell lung cancer tissues(p<0.05),the difference between the two groups was statistically significant,while SOCS3 expression was higher in the adjacent normal lung tissue,p<0.05 was compared between the two groups.(2)Analysis of clinical characteristics: the expression of SOCS3 mi R-19 a was significantly different in terms of pathological type and clinical TNM stage of lymph node metastasis,etc.,and p<0.05 was compared between the two groups,has statistical significance;The expression of SOCS3 mi R-19 a showed no statistically significant difference in gender and age with or without smoking history(p>0.05)(3)The expression of mi R-19 a and SOCS3 in the transfected cells in vitro was detected by q Rt-PCR,and the expression of mi R-19 a was detected in The H-mi R-19 a group >The Control group > The L-mi R-19 a group >The p-SOCS3 group;So CS3 expression is the opposite;Pair the comparison p<0.05,the difference was statistically significant.The results were consistent with the clinical tissue test results,which verified the successful transfection of cell mi R-19 a plasmid and laid a foundation for further research.(4)WB detection: JAK1 and STAT3 proteins had consistent protein concentrations in A549 cells,of which H-mi R-19 a was the highest,followed by Control and L-mi R-19 a,and P-SOCS3 was the lowest.Pairto-pair comparison of p<0.05.The concentration of SOCS3 protein was the highest at P-SOCS3 level,followed by L-mi R-19 a and Control,and the lowest at H-mi R-19 a.Pair comparison was p <0.05 difference was statistically significant.The expression trend of Bcl-1 protein was consistent with that of JAK1-STAT3.The expression of Bax protein was reversed.(5)MTT was used to detect the survival and growth status of cells.At48 h,the light absorption value of the H-mi R-19 a group at OD570 nm was significantly higher than that of the p-SOCS3 group,followed by the control group and the inhibitor treatment group,and pound-by comparison of p<0.05,the difference was statistically significant.(6)Apoptosis rate of non-small cell lung cancer cells was determined by Annexin-V/PI method.The cell apoptosis rate in each group was the lowest in the H-mi R-19 a group,followed by the Control and L-mi R-19 a group,and the highest in the p-SOCS3 group.Pair the comparison p<0.05,the difference was statistically significant.(7)The results of Transwell and scratch test showed that at 12 h and24h,the number of invaded A549 cells in the lower compartment of Transwell in the PSOCS 3 group was the least,and the scratch healing was the slowest in the scratch test,followed is L-mi R-19 a group.That scratch healing speed in the scratch test and cells of lower compartment of Transwell,H-The mi R-19 a group was larger than the Control group,and the H-mi R19 group had the largest number of invaded cells in the inferior compartment and the fastest rate of scratch healing,compared with other group p<0.05,the difference was statistically significant.Conclusion:(1)Lymph node metastasis,tumor pathological type and clinical TNM stage are important factors affecting the expression of mi R19 a and SOCS3 in NSCLC tissues.(2)miR-19 a regulates SOCS3 in human non-small cell lung cancer to activate JAK1 and STAT3 protein expressions in the JAK1-STAT3 pathway,thereby reducing cell apoptosis and promoting cell proliferation,migration and invasion. |
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