The Mechanism Of LncRNA H19 Regulating Radiation Sensitivity In Non-small Cell Lung Cancer

Background and purposeRadiotherapy is one of the conventional treatment methods for non-small cell lung cancer(NSCLC),which can be used alone or in combination with other treatment methods such as surgery,chemotherapy,targeted therapy and immunotherapy to enhance the efficacy.The local control rate of tumor is highly correlated with the dose of radiotherapy,however,due to the dose limitation to organ at risk,the dose to the tumor cannot be further improved.Therefore,the method that can improve tumor radiation sensitivity has become one of the main research objectives.Long non coding RNA(lnc RNA)abnormally expressed in tumors can regulate the development of tumors and regulate the radiation sensitivity of many types of tumors.Lnc RNA exert their effects mainly by interacting with micro RNA(miRNA)through competitive endogenous RNA(ce RNA)mechanisms to regulate the biological functions of m RNA.Therefore,the lnc RNA/miRNA/m RNA axis may play an important role in regulating radiation sensitivity in NSCLC.In this study,we investigated the molecular mechanisms related to regulating radiation sensitivity in NSCLC and provided new targets and therapeutic strategies for the combination of precision radiotherapy and RNA drugs in NSCLC.Methods1.Screening lnc RNA potentially regulating radiosensitivity: Whole transcriptome sequencing of A549 and A549-R11(acquired radioresistant cell lines)cells revealed that lnc RNA H19(H19)was highly expressed in radioresistant cells and the corresponding si-H19 sequences were designed.After down-regulated H19,CCK-8and flow cytometry were used to detect the cell proliferation ability and apoptosis rate in A549 and H460 at 24 h,48 h and 72 h after 6 Gy of X-ray irradiation;EdU assay was used to detect the synthesis ability of DNA in the two cells after irradiation.Colorogenic survival assay was performed to examine the radiosensitivity of NSCLC cells to X-rays and carbon ions after down-regulation of H19.2.Bioinformatics screening of miRNA combined with H19 and validation of their functions: The mirbase was used to select candidate miRNA interacting with H19,RTPCR was further screened;Dual-luciferase assay was used to determine the binding of miR-130a-3p to H19.RT-PCR was used to detect the overexpression efficiency of miR-130a-3p minic in A549 and H460 cell lines.After 6 Gy of X-ray irradiation,CCK-8,EdU,flow cytometry and clone survival assay were used to detect the effects of miR-130a-3p overexpression on the proliferation,DNA synthesis,apoptosis and radiation sensitivity of NSCLC cells.Rescue experiments were used to further validate the relationship between miR-130a-3p and H19.3.Screen the target genes of miR-130a-3p and verify the function: Screen the candidate target genes interacting with miR-130a-3p using targetscan database,and further screen the candidate target genes by the correlation with the expression of miR-130a-3p,Dual-luciferase experiments determined the binding of miR-130a-3p to WNK3.Bioinformatics method was performed to analyze the difference of WNK3 expression between lung squamous cell carcinoma and lung adenocarcinoma and normal lung tissues and its impact on survival prognosis.The corresponding siRNA was designed against WNK3 and its silencing efficiency in A549 and H460 cell lines was examined by RT-PCR.CCK-8 and flow cytometry were used to detect the proliferation ability and apoptosis rate of NSCLC cells after 6 Gy of X-ray irradiation;EdU assay was used to detect the synthesis ability of DNA in the two cells after irradiation.Clone survival assay was performed to examine the sensitivity of NSCLC cells to X-ray radiation after down-regulation of WNK3.Western blot was used to detect the expression levels of apoptosis-related molecules,such as cleaved-caspase3,PARP,p-p38,Bax and Bcl-2.4.The function of H19 verified in tumor-bearing nude mice: A549-OEH19 cell line overexpressing H19 was constructed.BALB/c-nu tumor-bearing mouse models were constructed by subcutaneous injection of A549 and A549-OEH19 cells,and then they were divided into six groups,namely,non-irradiated groups(A549 and A549-OEH19),4 Gy X-ray groups(A549 and A549-OEH19),and 4 Gy carbon ion groups(A549 and A549-OEH19).The size of the tumor was measured and recorded every 3days after irradiation.21 days later,the mice were sacrificed and the intact tumor tissue was removed,and the expression level of WNK3 was detected by immunohistochemistry.Results1.After irradiation of A549 and H460 cell lines,compared with NC,the cell proliferation ability after silencing H19 was inhibited;at 48 h and 72 h after irradiation,silencing H19 significantly enhanced the radiation-induced apoptosis rate of A549 cells and H460 cells(p <0.01);Compared with the NC+irradiation group,the DNA synthesis ability of cells the si-H19+irradiation group was significantly reduced(p<0.001);the clone survival results showed that inhibiting the expression of H19 increased the radiation sensitivity of NSCLC cell lines exposed to X-ray and carbon ion radiation.2.Fifty-seven miRNAs that bind to H19 were selected from the database;RT-PCR method was used to further screen miRNA that were negatively correlated with H19 expression and miR-130a-3p expression was increased after silencing H19 in both A549 and H460 cells,indicating that H19 may bind to miR-130a-3p;Dual-luciferase assay confirmed that H19 could bind to miR-130a-3p.Transfection of miR-130a-3p minic in A549 and H460 cell lines increased the expression of miR-130a-3p to 118-fold and 187-fold compared to the NC group.At 48 h and 72 h after irradiation,compared with the NC+irradiation group,the proliferation of NSCLC cells in the miR-130a-3p minic+ irradiation group was significantly inhibited(p<0.01)and the apoptosis rate was significantly enhanced(p < 0.05).At 24 h after irradiation,the DNA synthesis ability of cells in miR-130a-3p minic+ irradiation group was significantly inhibited(p<0.01).Overexpression of miR-130a-3p could increase the sensitivity of NSCLC cell lines to X-rays.Rescue experiments showed that the cell proliferation effect promoted by overexpression of H19 could be inhibited by miR-130a-3p minic.3.Bioinformatics analysis indicated that WNK3 may be the target gene of miR-130a-3p.The expression of WNK3 was down-regulated after overexpression of miR-130a-3p in A549 and H460 cells,indicating that WNK3 may bind to miR-130a-3p.Dual luciferase confirmed direct binding of WNK3 to miR-130a-3p.Bioinformatics analysis revealed that WNK3 expression was increased in lung squamous cell carcinoma and lung adenocarcinoma tissues compared with normal tissues.The expression of WNK3 is negatively correlated with the prognosis of lung cancer patients.After 6Gy irradiation of A549 and H460 cell lines,compared with NC,the cell proliferation ability was inhibited after silencing WNK3.At 48 h and 72 h after irradiation,silencing WNK3 significantly enhanced the apoptosis of radiation-induced cells(p<0.01).Compared with the NC+irradiation group,the DNA synthesis ability of the si-WNK3+irradiation group was significantly decreased(p<0.01).Silencing WNK3up-regulated radiation-induced expression of cleaved caspase3 and PARP,increased the ratio of Bax/Bcl-2,and down-regulated the expression of p-p38.The results of colony survival showed that inhibition of WNK3 expression increased the radiosensitivity of NSCLC cell lines to X-rays.4.The results in vivo study showed that the tumor volume of A549 and A549-OEH19 was reduced after X-ray and carbon ion irradiation compared with the nonirradiated group(p < 0.05).After X-ray or carbon ion irradiation,the tumor volume in the A549 group was smaller than that in the A549-OEH19 group(p < 0.05).The WNK3 immunohistochemical score was lowest after carbon ion irradiation,followed by X-ray irradiation,and highest without irradiation in both the A549 cell group and the A549-OEH19 cell group.Conclusions1.H19/miR-130a-3p/WNK3 axis regulates the radiation sensitivity of NSCLC cell lines by regulating apoptosis and cell proliferation,which is one of the molecular mechanisms related to the radiation sensitivity of NSCLC cells.2.H19 affects the inhibitory effect of X-rays and carbon ions on tumor growth.3.H19 may be a new target affecting the effect of radiotherapy.