The Study On The Mechanism Of MiRNA-141 Targeting NFIA To Regulate The Radiosensitivity Of Non-small Cell Lung Cancer Cells By Induction Of AKT/ERK Pathway

Objectives: Lung cancer is the leading cause of cancer-related deaths worldwide,accounting for 26% of all cancer-related deaths.Non-small cell lung cancer(NSCLC)constitutes more than 80% of all the lung cancer pathological types.About half of NSCLC has been locally advanced or distant metastasis at the time of diagnosis,thus losing the opportunity of surgery.Therefore,radiotherapy plays an important role in the treatment of NSCLC.However,despite of the continuous improvement of radiation technology,the efficacy of radiotherapy is still unsatisfactory.The intrinsic or acquired radiation resistance is an important factor leading to radiotherapy failure.The occurrence of radioresistance involves many molecular mechanisms,such as the abnormal regulation of signal pathway(such as PI3K/AKT,NF-?B,etc.),the enhancement of DNA damage repair,EMT,the abnormal expression of mi RNA and lnc RNA,and the changes of tumor microenvironment(such as hypoxia).In recent years,a large number of studies have proved that non coding RNA(nc RNA)is involved in the radioresistance,including DNA damage repair,cell cycle regulation,apoptosis and radiation-induced tumor death.So far,the exact molecular mechanisms of intrinsic and acquired radioresistance in NSCLC have not been fully elucidated.Therefore,it is of great clinical significance to explore the molecular mechanisms of radioresistance of NSCLC and find the target of radiosensitization for improving the efficiency of radiotherapy and the survival of NSCLC patients.mi R-141 is one of the members of mi R-200 family,which plays an important role in NSCLC.It has been found that high expression of mi R-141 in human lung cancer tissue is one of the risk factors for poor prognosis.At the same time,mi R-141 was found to be related to the response of lung cancer to treatment,and its high expression can promote cisplatin resistance of NSCLC.NFIA is a member of the transcription factor NFI family.It can regulate cell proliferation and differentiation by transcriptional regulation of target genes.Its dysfunction can participate in the occurrence and progression of tumors.In NSCLC,the down-regulation of NFIA can promote tumor proliferation and migration,and the lack of expression of NFIA can cause radiochemotherapy resistance in oropharyngeal squamous cell carcinoma.In addition,it has been found that NFIA can mediate endocrine resistance in breast cancer.Therefore,based on the results of high-throughput sequencing,the following studies were conducted to evaluate whether mi R-141 and NFIA affect the radiosensitivity of NSCLC and its mechanism.Methods: The first part: The CCK-8 and clone formation assay were used to detect the radiobiological characteristics and the radiosensitivity of four different pathological types of NSCLC cells(adenocarcinoma cell lines A549,H1975,squamous cell carcinoma cell line H226 and large cell carcinoma cell line H460).We established two radioresistant NSCLC cell lines,H226 R and H460 R,by dose-gradient irradiation.The clone formation assay,CCK-8,cell cycle detection and apoptosis detection were then used to determine the radiosensitivity of H226 R and H460 R cells.High-throughput sequencing was used to screen lnc RNA,mi RNA and m RNA which were differentially expressed between H226,H460 cells and H226 R,H460R cells;Gene ontology(GO)and Kyoto Encyclopedia of genes and genomes(KEGG)bioinformatics analysis were used to identify the gene function and signal transduction pathway involved in radioresistance;Quantitative Real-time PCR(q RT-PCR)was used to detect the differentially expressed genes in the two groups to verify the accuracy of sequencing results.The second part: The sequencing results revealed that the up-regulated mi R-141 and down-regulated NFIA may be involved in the radioresistance of NSCLC.Firstly,QRT-PCR was used to detect the expression of mi R-141 in different NSCLC cells after irradiation;Lentivirus was transfected into H226 and A549 cells to construct stable mi R-141 low expression cell line,and CCK-8 was used to detect the cell proliferation activity after mi R-141-inhibition,CCK-8,clone formation assay,cell scratch experiment and apoptosis analysis were used to detect the effect of inhibiting mi R-141 combined with irradiation on cell radiosensitivity;Western blotting was used to detect the expression of Caspase-3,Bax and p53,and the AKT and ERK phosphorylation level after mi R-141-inhibition.Western blotting was used to detect the NFIA expression and AKT and ERK phosphorylation level in radioresistant cell lines and parental cell lines,and to detect the NFIA expression level after mi R-141-inhibition.We overexpressed NFIA in NFIA low expressing cell lines,H226 R and H460 R,and downregulated NFIA in NFIA high expressing cell lines,H226 and H460.Then we detected the expression of NFIA in transfected cells with QRT-PCR and Western blotting.The changes of radiosensitivity in transfected cells were detected by clone formation assay and apoptosis analysis.The number of ?H2AX focus nuclei of transfected cells post-irradiation was detected by immunofluorescence to evaluate the effects of NFIA on DNA damage and repair ability;Western blotting was used to evaluate whether knockdown or overexpression of NFIA led to a change in the activation of AKT and ERK.Results: The first part: 1.NSCLC cells of different pathological subtypes have different radiobiological characteristics,and the radiosensitivity from high to low is H226,H460,H1975,A549;2.Using multiple fractions of IR,we successfully derived the radioresistant cell lines,H226 R and H460 R,from their parental NSCLC cell lines,H226 and H460.Compared with their parental cell lines,the clone forming ability and survival fraction of radioresistant cell lines post-irradiation are higher,indicating the radioresistance of the cells;Further experiments showed that H226 R and H460 R cells increased their radioresistance by inhibiting cell death,changing cell cycle redistribution and reducing radiation-induced apoptosis;3.High throughput sequencing filtered out a total of 47 lnc RNA(23 up-regulated and 24 down-regulated),503 m RNA(193 up-regulated and 310 down-regulated)and 208 mi RNA(120 up-regulated and 88 down-regulated)differentially expressed in H226 R cells,and 122 lnc RNA(46 up-regulated and 76 down-regulated),838 m RNA(508 up-regulated and 330 down-regulated)and 203 mi RNA(95 up-regulated and 107 down-regulated)differentially expressed in H460 R cells;4.Go analysis showed that these differentially expressed transcripts were mainly enriched in the process of response to external stimulation,biological regulation and adhesion,molecular function regulation,transcription factor activity and cell signal transduction,cell proliferation,differentiation and adhesion,cell death and cell movement;KEGG pathway analysis showed that these differentially expressed transcripts were mainly enriched in focal adhesion,PI3K/AKT,ECM receptor and other signaling pathways,and the predicted target genes of differentially expressed mi RNA were mainly enriched in Ras,m TOR,MAPK,TNF,PI3K/AKT signaling pathway,cancer-related signaling pathway and apoptosis signaling pathway;5.QRT-PCR confirmed the differential expression of NEAT1?DDX10?PHLDB2?NFIA?mi R-146a?mi R-205?mi R-141?mi R-200a?mi R-200 b and mi R-200 c in the radioresistant cell lines and the parental cell lines,which were consistent with the sequencing results.The second part: 1.The expression of mi R-141 was up-regulated in different NSCLC cells after receiving ionizing radiation,and the mi R-141 expression reached the peak at 24 hours post-irradiation in A549,H226 and H1975 cells.The expression of mi R-141 in H460 cells gradually increased with the time;2.A549 and H226 cell lines with stable low-expressed of mi R-141 were constructed.It was found that inhibition of mi R-141 reduced tumor proliferation activity.Inhibition of mi R-141 combined with ionizing radiation can increase radiosensitivity by reducing proliferation activity,migration ability and promoting apoptosis induced by ionizing radiation.The activity of AKT and ERK in NSCLC cells decreased after the inhibition of mi R-141;3.NFIA was predicted to be one of the target genes of mi R-141 by Target Scan and DIANA bioinformatics database.The expression of NFIA was significantly down-regulated with the increase of AKT and ERK activity in radioresistant cell lines,H226 R and H460 R.The expression of NFIA was up regulated after the inhibition of mi R-141 in A549 and H226 cells.4.The overexpression of NFIA restored radiosensitivity in H226 R and H460 R cells,whereas the knockdown of NFIA reduced radiosensitivity in H226 and H460 cells.The changes of radiosensitivity was related to the change of radiation-induced apoptosis and DNA damage repair ability.Overexpression of NFIA reduced AKT and ERK phosphorylation,while knockdown of NFIA increased AKT and ERK phosphorylation.Conclusions: 1.The radioresistant cell lines H226 R and H460 R were successfully constructed by dose-gradient irradiation,and the differentially expressed lnc RNA,m RNA and mi RNA were screened by high-throughput sequencing.Further bioinformatics analysis showed that the differentially expressed genes participated in the process of radiation resistance of NSCLC by regulating signal transduction pathway;2.Inhibition of mi R-141 enhanced the radiosensitivity of NSCLC;3.mi R-141 targeting NFIA to regulate the radiosensitivity of NSCLC by induction of AKT/ERK pathway.