Mechanism Of Cisplatin Resistance Of LncRNA UCA1/miR-143/Wnt-? Catenin Pathway In Non-small Cell Lung Cancer Cells

Background and purpose:Lung cancer is the most dangerous cancer in the world.Its morbidity and mortality rate rank the first among all cancers,among which non-small cell lung cancer accounts for 80% of all types of lung cancer.The prognosis of early lung cancer is better,but the majority of patients are already advanced when lung cancer is diagnosed,resulting in poor prognosis of lung cancer.According to the step of primary lung cancer diagnosis and treatment guidelines in 2018,for unresectable ? A period,? B NSCLC,basic treatment strategy is still double medicine platinum-based chemotherapy,For no-driver genes ? period lung cancer : squamous cell carcinomas and non-small cell lung cancer basic strategy is also double medicine platinum-based chemotherapy,so the cisplatin in the treatment of advanced lung cancer is still important.In recent years,great progress has been made in the targeted treatment of NSCLC lung cancer,but unfortunately,drug resistance of both targeted drugs and chemotherapy drugs has become a major obstacle to the treatment of NSCLC,and the prognosis of NSCLC is still not optimistic.Therefore,the study of chemotherapy of NSCLC and the drug resistance mechanism of targeted drugs has become a hot issue in tumor treatment nowadays.In recent years,the study of epigenetics and chemical drug sensitivity has gradually become a hot spot.Long non-codingrna(LncRNA)is a non-codingRNA molecule with a length of more than 200 nucleotides,which plays an important role in transcriptional silencing,transcriptional activation,chromosome modification and nuclear transport.It is closely related to the development of various diseases,and its abnormal expression is related to the process of tumor formation,invasion and metastasis.Urothelial carcinoma associated 1(UCA1)was found to be highly expressed in bladder cancer cells for the first time a LncRNA.Later,it was found in many kinds of tumors,such as breast cancer,colon cancer,stomach cancer,hepatocellular carcinoma,tongue squamous cell carcinoma,esophageal cancer,ovarian cancer,melanoma andnon-small cell carcinoma.Studies have found that UCA1 is involved in the occurrence and development of tumors by regulating EMT and cell apoptosis,and is closely related to drug resistance of various tumors.However,the specific molecular mechanism of LncRNA UCA1 resistance to cisplatin in non-small cell lung cancer is not clear.This subject includes the following three parts: part one: the expression of Lnc RNA UCA1 in A549/ DDP cells and its effect on migration and invasion;part two:UCA1 plays a role in regulating A549/ DDP cells by targeting miR-143;part three:Effect of UCA1/ miR-143/ Wnt-? catenin on migration and invasion of A549/ DDP cells and IC50.Part One the expression of Lnc RNA UCA1 in A549/DDP cells and its effect on migration and invasion.Methods:1.The inhibition rate and IC50 of cisplatin-resistant non-small cell lung cancer cells(A549/DDP)established by MTT assay.The expression levels of UCA1 in non-small cell lung cancer cells(A549)and cisplatin-resistant non-small cell lung cancer cells(A549/DDP)were detected by Real-time PCR.3.Establishment of cisplatin-resistant non-small cell lung cancer cells(A549/DDP)stably knocking down UCA1 by lentiviral-mediated method.4.Transwell assay to knock down the effect of UCA1 on migration and invasion of cisplatin-resistant non-small cell lung cancer cells(A549/DDP).5.The effect of knockdown of UCA1 on the expression of EMT-related protein in cisplatin-resistant non-small cell lung cancer cells(A549/DDP)was detected by western blot.Result:1.The inhibition rate of cisplatin-resistant non-small cell lung cancer cells(A549/ DDP)was concentration-dependent,and the IC50 value of cisplatin was 67.58±5.69 ?mol/L.2.The abnormal expression level of UCA1 in A549/ DDP cells is elevated.3.A549/ DDP cells stably knocking down UCA1 by lentivirus were successfully established.4.Knockdown of UCA1 inhibits migration and invasion of A549/ DDP cells.5.Knockdown of UCA1 inhibits EMT of A549/ DDP cells.Part Two UCA1 regulates the function of A549/ DDP cells by targeting miR-143Methods:1.Estimate the target gene of UCA1 by bioinformatics analysis.2.Construction of a recombinant plasmid of wild and mutant UCA1,and verification of the target gene of UCA1 by a dual luciferase reporter assay.3.Western blot and Real-time PCR were used to detect the expression of miR-143 after UCA1 overexpression and knockdown.4.The effects of miR-143 overexpression on migration,invasion and EMT of A549/DDP cells were detected by Real-time PCR and Western blot.Result:1.Bioinformatics predictions show that miR-143 is a target gene for UCA1.2.Dual fluorescent reporter gene experiments verified that UCA1 targets miR-143.3.UCA1 negatively regulates the expression of miR-143.4.Overexpression of miR-143 inhibited migration,invasion and EMT of A549/DDP cells.Part Three Effect of UCA1/ miR-143/ Wnt-?catenin on migration and invasion of A549/ DDP cells and IC50Methods:The effect of miR-143 overexpression on the expression of WNT,?-catenin and CD44 in Wnt/ ?-catenin signaling pathway of A549/ DDP cells was detected by western blot.2.Transwell method was used to detect the effect of Wnt/ ?-catenin signaling pathway inhibitor on migration and invasion of A549/ DDP cells.3.The effect of UCA1/ miR-143/ Wnt-? catenin signaling pathway on IC50 of A549/ DDP cells was detected by MTT assay.Result:Overexpression of miR-143 down-regulated the expression of WNT,?-catenin and CD44 in Wnt/ ?-catenin signaling pathway in A549/ DDP cells.2.Inhibition of Wnt/ ?-catenin signaling pathway inhibits migration and invasion of A549/ DDP cells.3.Knockdown of UCA1,overexpression of miR-143 and inhibition of Wnt-?catenin signaling pathway can reverse the resistance of cisplatin to A549/DDP cells.Conclusion1.The expression level of UCA1 in cisplatin-resistant non-small cell lung cancer cells(A549/ DDP)was significantly increased,the expression level of miR-143 was significantly decreased,the Wnt/ ?-catenin signaling pathway was abnormally activated,and both could promote A549/ DDP.Migration and invasion of cells and EMT.2.UCA1 targets and regulates the expression level of miR-143,and overexpression of miR-143 can inhibit migration,invasion and EMT of A549/ DDP cells.3.miR-143 can inactivate Wnt/ ?-catenin signaling pathway,which inhibits the migration,invasion and EMT of A549/ DDP cells.4.UCA1/miR-143/Wnt-? catenin pathway can reverse the drug resistance of A549/DDP cells.