| BACKGROUNDLung cancer accouts for the highest incidence and mortality of malignant tumors.Non-small cell lung cancer?NSCLC?,served as the most common subtype of lung cancer,represents more than 80%of all lung tumors.The progress of NSCLC anti-tumor therapy strategy changes rapidly,but chemotherapy remains regarded as an important cornerstone role.Systemic chemotherapy based on platinum combination therapy is the first choice for most patients with advanced NSCLC.Cisplatin is the most common third generation platinum drug that interferes with the replication of DNA,which affects the transcription and translation of tumor cells.However,cisplatin resistance is responsible for the failure of cancer therapy,meanwhile,it has an important impact on the prognosis of patients.As various factors cross interaction,the mechanism of cisplatin resistance is complex and diverse.In recent years,the research of epigenetics and chemosensitivity has become a hot topic.Long chain noncoding RNAs were reported involved in the regulation of gene expression at the level of epigenetic,transcriptional and post transcriptional levels in the form of RNA,and were closely related to the occurrence of drug resistance in tumor cells.Therefore,it is critical to find out the drug resistance related LncRNAs and study its molecular mechanism for NSCLC patients to improve the therapeutic efficacy ultimately.LincRNA-ROR can induce pluripotent stem cells by targeting the three factors in the promoter region,which plays an important role in DNA damage and stem cell self-renewal.Recent research has shown that LincRNA-ROR was involved in the proliferation,invasion,metastasis and chemotherapy sensitivity of various malignant tumor cells,and affected the prognosis of cancer patients.However,the relationship between LincRNA-ROR and chemotherapy drug resistance in non-small cell lung cancer has not been reported previously.OBJECTIVEThe purpose of our study was to investigate the impact of long chain noncoding RNA-ROR in the cisplatin resistance by regulating PI3K/Akt/mTOR signaling pathway in non-small cell lung cancer.METHODS1.Real-time quantitative polymerase chain reaction?RT-qPCR?was used to detect the expression of LincRNA-ROR mRNA in A549 cell lines and A549/DDP cell line;MTT method was used to compare the effect of DDP concentration on the inhibition of proliferation of A549 cells,to determine the most suitable concentration of DDP.2.The A549/DDP cells were transfected with the plasmid vector,and the expression of LincRNA-ROR was up-regulated and downregulated.Organization of groups in our study was as follows:?1?blank control group?2?,NC group?transfected with non related sequences of shRNA negative control group?,?3?siROR group?with LincRNA-ROR shRNA?,?4?ROR overexpression group?transfected with pCDNA3.1-LincRNA-ROR?,Wortmannin group?5??including Wortmannin inhibitor,WOR,a specific inhibitor of the PI3K,110kD and PI3K catalytic subunit combination,can inhibit the PI3K/Akt/mTOR signal pathway??6?overexpression of ROR + Wortmannin group.3.The mRNA content of LincRNA-ROR,PI3K,Akt,mTOR,Bax and Bcl-2 were detected by RT-qPCR;The protein content of Akt,p-Akt,mTOR,p-mTOR,PI3K,p-PI3K,Bax and Bcl-2 were detected by Western blot;Effect of LincRNA-ROR on proliferation in vitro of A549/DDP resistant cell lines was detected by clone formation test plate.Effect of LincRNA-ROR on migration and invasion in A549/DDP cell lines were detected by cell scratch rate,experiment and cell invasion assay and cell migration assay.Influence of LincRNA-ROR on apoptosis in A549/DDP cell lines was detected by flow cytometry.4.Animal model of A549/DDP tumor cell line transfected with NSCLC in nude mice was established to evaluate the growth rate of the tumor,and to observe the change of expression of LincRNA-ROR in the effect of cisplatin resistance.RESULTS1.Real time fluorescence quantitative PCR method confirmed that expression of mRNA of LincRNA-ROR in A549/DDP cell lines was significantly higher than that in A549 cell lines?A549/DDP vs A549:6.83±0.81 vs 1.37 ± 0.12;t=12.67,P= 0.006??P<0.05?.MTT results showed that with the increase of DDP concentration?0-8 ug/ml?,the inhibition of cell proliferation was stronger.DDP concentrations were 0.5,1,2,4 and 8 ug/ml,inhibition of cell proliferation rates were?2.25 + 0.21?%and?9.77 + 1.32?%and?32.74 + 2.07?%and?62.61 + 2.78?%and?68.08 + 2.95?%in the concentration of DDP was 4 ug/ml and 8 ug/ml,the cell proliferation inhibition rate had no statistically difference.DDP concentration of 4 ug/ml was the most suitable concentration in this study.2.The results of Real time fluorescent quantitative PCR assay revealed that compared with the blank control group,the mRNA expression of LincRNA-ROR in the si-ROR group was decreased?P<0.05?,but the mRNA expression of LincRNA-ROR in the ROR over expression group and ROR +Wortmannin group was significantly increased?P<0.05?.The mRNA expression levels of PI3K,Akt,mTOR in si-ROR group and Wortmannin group were significantly decreased,?P<0.05?,The mRNA expression of PI3K,Akt,mTOR in ROR overexpression group significantly increased?P<0.05?.3.The results of Western blot showed that compared with the control group,the protein expression of p-Akt,p-p-mTOR,p-PI3K and bcl-2 in si-ROR group and Wortmannin group were decreased significantly,while the expression of Bax was significantly increased?P<0.05?.The protein expression of P-Akt,p-mTOR,p-PI3K and bcl-2 in ROR overexpression group were significantly increased,while the protein expression of Bax was significantly decreased?P<0.05?.However,there was no significant difference in the expression of +Wortmannin protein in ROR group?P>0.05?.4.The clone formation test results showed that compared with the control group,the inhibitory effect of DDP on lung adenocarcinoma A549 cell lines was increased in si-ROR group and Wortmannin group,meanwile,the ability of proliferation was reduced in A549/DDP cell lines?P<0.05?.There was no significant difference between the two groups?P>0.05?in the si-ROR group and the Wortmannin group.Compared with the blank control group,the inhibitory effect of DDP on A549 cell lines was decreased in the over expression group of ROR,and the ability of proliferation on A549/DDP cell lines was increased?P<0.05?.5.Cell scratch assay,cell invasion assay and cell migration assay results showed that compared with the blank control group,cell migration and invasion ability of si-ROR group and Wortmannin group was decreased significantly?P<0.05?,the difference between the two groups was not statistically significant?P>0.05?.Compared with the blank control group,the cell migration and invasion ability of ROR over expression group was significantly increased?P<0.05?.6.Flow cytometry showed that compared with the blank control group,the effect of apoptosis on A549/DDP cell lines in si-ROR group and Wortmannin group was significantly increased?P<0.05?.Compared with the blank control group,the effect of apoptosis on A549/DDP cell lines was significantly reduced in the ROR over expression group?P<0.05?.7.The results of subcutaneous implantation model in nude mice showed that compared with the blank control group,the weight of tumor in NC group and si-ROR group were?3.65 ± 0.28?g,?3.55 ± 0.37?g and?1.03 ± 0.15?g.8.Compared with the blank control group,the mRNA expression of Akt,mTOR and PI3K in the siROR group was significantly decreased.?P<0.05?.9.Compared with the blank control group,the mRNA expression of bcl-2 in the siROR group was significantly decreased,while the mRNA expression of bax in the siROR group was significantlyincreased?P<0.05?.CONCLUSIONLincRNA-ROR was highly expressed in human A549/DDP cisplatin resistant lung adenocarcinoma cell lines.LincRNA-ROR promoted tumor proliferation,migration,invasion and inhibited apoptosis of cisplatin resistant cells in non-small cell lung cancer by regulating the PI3K/Akt/mTOR signaling pathway.Our study provided a new sight and theory evidence for the drug resistance mechanism of lung cancer. |
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