MiR-335-3p Affects The Sensitivity Of NSCLC Cells To Gefitinib And Cell Proliferation,migration And Invasion

Background and Objective:Almost all patients with EGFR mutant non-small cell lung cancer(NSCLC)will eventually develop acquired resistance after treatment with EGFR-TKIs.The mechanisms of acquired resistance to EGFR-TKIs are still not fully elucidated.Numerous studies have shown that abnormal expression of microRNAs plays an important role in tumorigenesis and tumor progression.An increasing number of studies have confirmed that microRNAs are associated with acquired resistance to EGFR-TKIs.The purpose of this study is to investigate whether miR-335-3p can affect the sensitivity of non-small cell lung cancer cells to gefitinib,whether it can inhibit the proliferation,migration and invasion of tumor cells by negatively regulating the expression of RAP1B,CTHRC1 and SMYD2,and provide experimental evidence for the new direction for the treatment of non-small cell lung cancer patients with acquired resistance to EGFR-TKIs.Methods:(1)HCC827GR cell line was established and the sensitivity of HCC827 and HCC827GR to gefitinib was detected by Cell Counting Kit-8(CCK8)assay.The proliferation,migration and invasion capability of HCC827 and HCC827GR were detected by colony formation assay,wound healing assay,Transwell migration and invasion assay.(2)Real-time PCR was used to detect the expression of miR-335-3p in HCC827 and HCC827GR.(3)After overexpression of miR-335-3p in HCC827GR,the sensitivity of HCC827GR to gefitinib was detected by Cell Counting Kit-8 assay,and the proliferation,migration and invasion capability of HCC827GR were detected by colony formation assay,wound healing assay,Transwell migration and invasion assay.After knocking down miR-335-3p in HCC827,the sensitivity of HCC827 to gefitinib was detected by Cell Counting Kit-8 assay.(4)Refer to TargetScan to predict the target gene of miR-335-3p.Real-time PCR was used to detect the expression of RAP1B,CTHRC1 and SMYD2 in HCC827 and HCC827GR.After overexpression of miR-335-3p in HCC827GR,the expression of RAP1B,CTHRC1 and SMYD2 in HCC827GR was detected by Real-time PCR again.(5)After knocking down RAP1B,CTHRC1 and SMYD2 in HCC827GR,the proliferation,migration and invasion capability of HCC827GR were detected by colony formation assay,wound healing assay,Transwell migration and invasion assay,and the sensitivity of HCC827GR to gefitinib was detected by Cell Counting Kit-8 assay.Results:(1)The 50%inhibitory concentration of HCC827 to gefitinib was 2.39?mol/L,which was more sensitive to gefitinib.While the 50%inhibitory concentration of HCC827GR to gefitinib was 15.97?mol/L,whose sensitivity to gefitinib was significantly decreased.The proliferation,migration and invasion capability of HCC827GR were significantly enhanced than that of HCC827(P<0.001).(2)The expression of miR-335-3p in HCC827GR was significantly lower than that in HCC827(P<0.001).(3)Overexpression of miR-335-3p significantly increased the sensitivity of HCC827GR to gefitinib,and the difference has statistical significance in the concentration of 10?mol/L,20?mol/L and 40?mol/L(P<0.05).Overexpression of miR-335-3p weakened the proliferation,migration and invasion capability of HCC827GR(P<0.01).Knocking down miR-335-3p decreased the sensitivity of HCC827 to gefitinib,and the difference has statistical significance in the concentration of 0.1 ?mol/L,0.5?mol/L and 1?mol/L(P<0.05).(4)TargetScan suggested that the 3'-UTR of RAP1B,CTHRC1 and SMYD2 had potential binding sites of miR-335-3p.The expression of RAP1B,CTHRC1 and SMYD2 in HCC827GR was up-regulated in comparison with HCC827(P<0.01).MiR-335-3p negatively regulated the expression of RAP1B,CTHRC1 and SMYD2.(5)Knocking down RAP1B,CTHRC1 and SMYD2 weakened the proliferation,migration and invasion capability of HCC827GR(P<0.01),but failed to increase the sensitivity of HCC827GR to gefitinib.Conclusion:MiR-335-3p affects the sensitivity of non-small cell lung cancer(NSCLC)cells to gefitinib,and may inhibit the proliferation,migration and invasion capability of tumor cells by negatively regulating the expression of RAP1B,CTHRC1 and SMYD2.This suggests that the overexpression of miR-335-3p may be a new direction for the treatment of non-small cell lung cancer patients with acquired resistance to EGFR-TKIs,but further research and clinical trials are needed.