| Objective: At present,the occurrence and development of lung cancer is still the leading cause of cancer death.The results of clinicopathological diagnosis show that about 80～85%of patients are non-small cell lung cancer(NSCLC),and most of the patients have reached stage IIIB and IV at the time of diagnosis,losing the best time for treatment.The latest research suggests that genetic mutations can occur years before cancer is diagnosed.In recent years,with the development of genomics,clinicians have used targeted drugs according to the patient’s own gene mutation to select the best treatment plan to improve the treatment effect and quality of life of tumor patients.Therefore,early diagnosis and treatment are still the focus of lung cancer research.By studying gene expression in lung cancer patients and exploring the molecular mechanism of gene mutation,it can provide better and favorable theoretical evidence for targeted therapy of clinical patients.In recent years,with the rise of molecular biology,non-coding RNAs have attracted more and more attention as potential therapeutic targets for lung cancer.According to reports from domestic and foreign researchers,non-coding RNA is closely related to the occurrence and development of non-small cell lung cancer.Aspartyl-t RNA synthetase antisense 1(DARS-AS1)lnc RNA is located on human chromosome 2q21.3(chr2q21.3).Previous studies have shown that DARS-AS1 is highly expressed in thyroid cancer,renal clear cell carcinoma,prostate cancer and cervical cancer,and is positively correlated with tumor proliferation,migration and invasion.In addition,DARS-AS1 is involved in the occurrence and development of acute myeloid leukemia and myeloma cells.The above findings suggest that DARS-AS1 is involved in tumor carcinogenesis;however,its expression and mechanism of action in NSCLC remain unclear.Studies have shown that miRNAs are single-stranded non-coding RNAs of about 22 nucleotides,and lnc RNAs may act as molecular sponges that regulate micro RNAs(miRNAs)or competing endogenous RNAs(ce RNAs).According to literature reports,miR-302a-3p can inhibit the malignant biological behavior of liver cancer,colon cancer and endometrial cancer cells,enhance the sensitivity of glioma cells to tyrosinase inhibitors,and participate in the regulation of 5-FU chemotherapy and autophagy in rectal cancer.In addition,it was significantly associated with tumor size,histological grade,and vascular invasiveness in pancreatic cancer patients.However,the relationship between miR-302a-3p and NSCLC remains unclear,and the molecular mechanism underlying the association of DARS-AS1 with miR-302a-3p in NSCLC has not been investigated.Increased expression of ACAT1 has been reported in different human cancer cell lines.ACAT1 can be used as a prognostic marker in prostate cancer,and inhibition of ACAT1 expression can also reduce the growth of triple-negative breast cancer cells.Furthermore,ACAT1 is involved in the induction of doxorubicin resistance in endometrial carcinomas.These observations suggest that ACAT1 is a potential novel anticancer target.However,the current study shows that the relationship between ACAT1 and NSCLC has not been reported yet.In addition,it is not clear whether miR-302a-3p affects the malignant behavior of NSCLC by regulating ACAT1.In view of the above research background,this study aims to investigate the expression and biological behavior of DARS-AS1,miR-302a-3p and ACAT1 in non-small cell lung cancer,to prove their mechanism of action in the development of lung cancer,and to be a target for lung cancer patients.Provide a rationale for treatment.Methods: 1.In this experiment,86 clinical tissue samples and 35 pairs of cancer and adjacent tissue,human non-small cell lung cancer cell lines(H661,A549,H1299,H460,H292 and LK2)and human normal bronchial epithelial cells HBE.2.The m RNA expression levels of DARS-AS1,miR-302a-3p and ACAT1 were detected by Real-time PCR experiment,and the endogenous expression of ACAT1 and the expression and changes of AKT/ERK pathway-related proteins were detected by western blot experiment.3.In A549 and H1299 cells,cells were transfected with DARS-AS1-smart silencer and the corresponding negative control to conduct DARS-AS1 knockdown experiments.Transiently transfected with mimics and inhibitors of miR-302a-3p,transiently transfected or interfered with the expression of ACAT1,and obtained cells with overexpression and silencing of miR-302a-3p and ACAT1.4.In situ hybridization experiments were used to detect the subcellular localization of DARS-AS1.5.Dualluciferase reporter gene assay to verify the binding site of DARS-AS1 and miR-302a-3p,and the binding site of miR-302a-3p and ACAT1.6.RNA immunoprecipitation experiments to verify the targeted binding relationship between DARS-AS1 and miR-302a-3p.7.MTS assay is used to detect cell proliferation.8.Transwell assay is used to detect cell migration and invasion capabilities.9.The effect of DARS-AS1 knockout on the growth of non-small cell lung cancer and the expression of ACAT1 in BALB/c nude mice xenograft model.Results: 1.Through GEPIA online database analysis,DARS-AS1 is highly expressed in lung cancer tissues.The expression level of DARS-AS1 in lung cancer clinical tissue samples was significantly higher than that in adjacent tissues.The results of clinicopathological analysis showed that the high expression of DARS-AS1 was positively correlated with tumor size,lymph node metastasis and TNM stage.Using the KaplanMeier database,it was found that the high expression of DARS-AS1 was negatively correlated with the overall survival of patients.2.The results of q RT-PCR experiments showed that the expression levels of DARS-AS1 in non-small cell lung cancer cell lines H661,A549,H1299,H460,H292 and LK2 were higher than those in normal bronchial epithelial cells HBE cells.Knock down the expression of DARS-AS1 inhibits the biological behavior of proliferation,migration and invasion of non-small cell lung cancer cells.3.In situ hybridization experiments confirmed that DARS-AS1 is mainly located in the cytoplasm,and the online biological website analysis DARS-AS1 contains the binding site of miR-302a-3p.q RT-PCR experiments demonstrated that DARS-AS1 negatively regulates the expression of miR-302a-3p.The dual luciferase reporter gene verified the binding site of DARS-AS1 and miR-302a-3p.RNA co-immunoprecipitation experiments further verified the targeted binding of DARS-AS1 to miR-302a-3p to act as a miRNAs sponge.4.The results of q RT-PCR assay showed that the expression levels of miR-302a-3p in non-small cell lung cancer cell lines H661,A549,H1299,H460,H292 and LK2 were lower than those in normal bronchial epithelial cells HBE cells.Overexpression of miR-302a-3p inhibits the biological behavior of proliferation,migration and invasion of nonsmall cell lung cancer cells.Inhibiting the expression of miR-302a-3p could reverse the inhibitory effect of DARS-AS1 knockdown on cells.5.Use the dual-luciferase reporter gene to verify the binding site between the 3’UTR region of ACAT1 m RNA and miR-302a-3p analyzed by the online database.Western Blot method detected the high expression of ACAT1 in non-small cell lung cancer cells,and promoted the biological behavior of cell proliferation,migration,and invasion.6.Through q RT-PCR experiments and Western Blot detection,knockdown of DARS-AS1 can inhibit the m RNA and protein expression of ACAT1,and miR-302a-3p negatively regulates the m RNA and protein expression levels of ACAT1.Inhibitors of miR-302a-3p could reverse the inhibitory effect of DARS-AS1 silencing on ACAT1 expression.7.By Western Blot assay,the mimics of miR-302a-3p can inhibit the AKT/ERK signaling pathway,ACAT1 can activate the AKT/ERK signaling pathway,and the overexpression of ACAT1 reversed the inhibitory effect of the miR-302a-3p mimics on the pathway.8.In the xenograft tumor experiment in nude mice,the injection of H1299 cells stably knocking out DARS-AS1 inhibited the volume and weight of tumor formation in nude mice compared with the control group.Immunohistochemical experiments demonstrated that the injection of H1299 cells stably knocked out DARS-AS1 reduced the positive expression of tumor ACAT1 compared with the control group.Conclusion: 1.The high expression of DARS-AS1 in lung cancer tissue is associated with poor prognosis of patients,indicating that DARS-AS1 can be used as a marker for prognosis.2.Knockdown of DARS-AS1 expression inhibited the biological behavior of NSCLC cell proliferation,migration and invasion.3.miR-302a-3p plays the role of tumor suppressor gene in NSCLC.4.miR-302a-3p targets DARS-AS1 and mediates the inhibitory effect of DARS-AS1 knockdown on NSCLC cells.5.ACAT1 is the target protein of miR-302a-3p plays an oncogene role in NSCLC.6.DARS-AS1 negatively regulates miR-302a-3p and then affects the expression level of ACAT1.7.ACAT1 can activate AKT/ERK signaling pathway.8.Knocking out the expression of DARS-AS1 can inhibit the growth of xenograft tumors in nude mice,which is consistent with the results of in vitro experiments. |
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