| Backgroud:Lung cancer is one of the most lethal cancers,in which NSCLC accounts for 85% of lung cancer cases.The incidence rate of lung cancer has reached a peak in some countries,but in many areas,especially in China,the incidence rate is increasing year by year.At present,the cause of non-small cell lung cancer is not completely clear.Some studies have shown that smoking is the most important factor leading to lung cancer.However,with the decline of smoking rate in most western countries,NSCLC still plays a leading role in patients,and its inducement may be the result of the joint action of environmental,emotional,genetic and other factors.The treatment of NSCLC includes early operation,concurrent radiotherapy and chemotherapy for local advanced tumor,targeted therapy and palliative chemotherapy for metastatic disease.One of the important findings of tumor therapeutics is somatic mutation of EGFR TK domain in NSCLC and its correlation with EGFR inhibitor response.EGFR gene amplification is more common in the Oriental population,and HER2 gene amplification,which is closely related to it,is more common in East Asian patients,which may also have an impact on the treatment of NSCLC.The introduction of antiangiogenic drugs and EGFR protein TK inhibitors improved the response rate of NSCLC patients.However,with the prolongation of treatment time,patients will be resistant to some drugs,especially EGFR-TKI.Therefore,it is urgent to explore the mechanism of drug resistance in patients with non-small cell lung cancer,which is conducive to new treatment methods to improve the quality of life of patients.Non coding RNA(lncrna)and micro RNA(micro RNA)play a regulatory role in the occurrence,development and acquired drug resistance of lung cancer.Therefore,we start with lncrna to explore the new type of lncrna rhpn1-as1 targeted micro RNA,and further study the role and related mechanism of lncrna in acquired drug resistance of non-small cell lung cancer gefitinib.Aim:1.To investigate the expression of rhpn1-as1 in non-small cell lung cancer cells resistant to gefitinib2.To investigate how rhpn1-as1 regulates the resistance of NSCLC to gefitinibMethods:1.Tumor tissue specimens from patients with gefitinib resistance have been tested for expression levels in tissues.The expression of RHPN1-AS1 in the gefitinib-resistant group is significantly lower than that in the gefitinib-sensitive group.The expression of gefitinib was increased and the resistance of gefitinib was down-regulated.IC50 representing gefitinib was negatively correlated with the amount of pc DNARHPN1-AS1.2.After the expression of RHPN1-AS1 was inhibited by transfection with small interfering RNA,the number of early apoptotic cells in gefitinib-treated cells was reduced,resulting in increased cell mobility under gefitinib stimulation.Cell lung cancer resistance to gefitinib requires the presence of RHPN1-AS1.3.RHPN1-AS1 over-expressing cell line was constructed and the over-expression efficiency of RHPN1-AS1 was detected by quantitative PCR in real time.The results showed that drug-resistant NSCLC cells that over-expressed RHPN1-AS1 shifted left to IC50 of gefitinib,indicating overexpression RHPN1-AS1 confers sensitivity to gefitinib in non-small cell lung cancer cell resistant strains.4.The biotin avidin pull-down experiment was used to verify the recognition of RHPN1-AS1 by MiR-299-3p.Using double luciferase reporter gene assay,we found that only RHPN1-AS1-WT can bind to MiR-299-3p.5.Target prediction using Targetscan.The prediction results showed that TNFSF12 may be the target of MiR-299-3p,and then different methods were used to confirm the authenticity of the prediction results.6.Detection of apoptosis and migration by transfection,flow cytometry and Transwell showed that RHPN1-AS1 regulates the resistance mechanism of gefitinib-resistant strains by targeting the MiR-299-3p / TNFSF12 pathway.Results1.The expression of rhpn1-as1 in gefitinib resistant group was significantly lower than that in gefitinib sensitive group.With the increase of rhpn1-as1 expression,the drug resistance of gefitinib decreased.IC50 representing gefitinib was negatively correlated with pcdnarhpn1-as1.2.After inhibition of rhpn1-as1 expression by si RNA transfection,the number of early apoptotic cells in gefitinib treated cells was decreased,which led to increased cell mobility stimulated by gefitinib.The presence of rhpn1-as1 was necessary for the resistance of non-small cell lung cancer to gefitini.3.Rhpn1-as1 overexpression cell line was constructed and the overexpression efficiency of rhpn1-as1 was detected by real-time quantitative PCR.The results showed that IC50 of drug-resistant NSCLC cells overexpressing rhpn1-as1 was shifted to the left,indicating that overexpression of rhpn1-as1 endows drug-resistant NSCLC cells with sensitivity to gefitinib4.The identification of rhpn1-as1 by MiR-299-3p was verified by biotin avidin pull-down test.By double luciferase reporter gene assay,we found that only rhpn1-as1-wt could bind to MiR-299-3p.5.Target scan is used for target prediction and the results show that TNFSF12 may be the target of MiR-299-3p.Then different methods are used to verify the authenticity of the prediction results.6.Apoptosis and migration were detected by transfection,flow cytometry and Transwell,which showed that rhpn1-as1 regulated the mechanism of gefitinib resistance by targeting MiR-299-3p / TNFSF12 pathway.Conclusion In this study,we first used real-time quantitative PCR to detect the expression level of rhpn1-as1 in 44 gefitinib sensitive and 40 gefitinib resistant tumor tissue samples.The results showed that the expression of rhpn1-as1 in gefitinib resistant group was significantly lower than that in gefitinib sensitive group.In order to further confirm,we also transfected pcdna-rhpn1-as1 in different amounts on p C9 cell line.The results showed that with the increase of rhpn1-as1 expression,the drug resistance of gefitinib decreased.Next,a small interfering RNA was transfected to inhibit the expression of rhpn1-as1,and then the drug resistance of the two cells after rhpn1-as1 silencing was detected.Compared with the control group,down regulating rhpn1-as1 expression can improve the drug resistance of gefitinib sensitive strains.These data indicate that NSCLC cells need the presence of rhpn1-as1 for gefitinib resistance.We constructed rhpn1-as1 overexpression cell line in gefitinib resistant NSCLC cells,and tested the resistance of rhpn1-as1 overexpression cell line to gefitinib.The results showed that the overexpression of rhpn1-as1 gave NSCLC cell line sensitivity to gefitinib.We used Starbase v.2.0 to predict the potential mi RNA for direct interaction with rhpn1-as1,and found that MiR-299-3p and MiR-7-5p were the most likely targets.In order to further verify the binding of MiR-299-3p to rhpn1-as1,we mutated the sequence of RHPN1-as1 and detected the double luciferase reporter gene.It was found that only rhpn1-as1-wt could bind to MiR-299-3p.QRT PCR was used to detect the expression of MiR-299-3p in NSCLC patients.Compared with sensitive gefitinib NSCLC patients,the expression of MiR-299-3p was up-regulated in gefitinib resistant NSCLC patients.In conclusion,rhpn1-as1 can inhibit the expression of rhpn1-as1,and act directly as a sponge on rhpn1-as1.We measured the expression of TNFSF12 in NSCLC tissue and cell line in real time.The results showed that the expression of TNFSF12 in gefitinib resistant patients was significantly lower than that in gefitinib sensitive patients.Then,luciferase reporter gene was detected,suggesting that TNFSF12 is the direct target of MiR-299-3p.Subsequent experiments showed that rhpn1-as1 regulated the resistance mechanism of gefitinib resistant strains by targeting MiR-299-3p / TNFSF12 pathway. |
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