| According to the latest global cancer statistics,lung cancer ranks first in the incidence and mortality of cancer in the world.In recent decades,although the mechanisms of lung cancer research have been numerous,and designed to produce a number of new targeted drugs.However,due to the complexity of the pathogenesis of lung cancer,the patient's five-year survival rate is still less than 15%.So new effective treatment measures are urgently needed.miRNA(microRNA)is a nucleotide with a length of 18-25 bp.Recent studies have shown that miRNAs play an important role in the development of many human cancers.The research on the mechanism of miRNA in cancer is a hot topic in cancer research.At present,there is still controversy about the function of miRNA-24-3p in lung cancer.miRNA-24-3p can play as an oncogene or tumor suppressor in lung cancer.The high expression of miRNA-21-5p in lung cancer can be used as one of the biomarkers for the diagnosis of lung cancer,and it also plays an important role in suppressing the chemosensitivity of lung cancer cells.However,the specific mechanism of miRNA-21-5p in lung cancer is seldom studied.In this study,we investigated how two important cancer-related miRNAs(miR-24-3p and miR-21-5p)in lung cancer affect the progression of lung cancer by regulating their respective target genes(SOX7 and TGFBI),This study is divided into two parts.The first part is to study the function and mechanism of miR-24-3p in lung cancer.We first detected the expression of miR-24-3p in A549,H1299 and a kind of normal lung epithelial cells(HLF).The results showed that the expression of miR-24-3p in A549 was significantly higher than that in normal lung epithelial cells(P<0.001).So we choose A549 to do cell function experiments.We transfected miR-24-3p mimic and inhibitor into A549 cells.The effects of miR-24-3p on the biological functions of lung cancer cells were studied by cell invasion and proliferation experiments.The results showed that the relative cell number of A549 increased by nearly 20%(P<0.05)and the number of invasive cells increased by nearly 260%(P<0.001)after overexpression of miR-24-3p compared with the control group.And the relative cell number of A549 decreased by 22%(P<0.01)and the number of invasive cells decreased by 50%(P<0.01)compared with the blank control group after inhibiting the expression of miR-24-3p.Then we predicted that SOX7 might be the target gene of miR-24-3p by using three bioinformatics softwares(targetscan,miRanda and PicTc).Through the TCGA database retrieval,we found that the expression of SOX7 in lung cancer was significantly down-regulated compared with normal tissues.To better explore the possible regulatory mechanism of SOX7 down-regulation,we focused on the important regulatory factor of cancer,miRNAs.In order to verify the regulatory relationship between the miR-24-3p and SOX7,we first carried out a double luciferase reporter gene experiment,the results showed that:miR-24-3p can bind to the 3'-UTR region of the SOX7 gene.Then,we transfected miR-24-3p mimic and miR-24-3p inhibitor into two lung cancer cell lines(A549,H1299)and detected the expression level of SOX7 by applying Western blot technique.The results showed that the expression of SOX7 protein in the two lung cancer cell lines decreased by nearly 30%(P<0.05)after overexpression of miR-24-3p compared with the blank control group,while the expression of SOX7 protein in the two lung cancer cell lines increased by nearly 25%(P<0.05)after inhibition of the expression of miR-24-3p.At the same time,we also detected the expression of SOX7 gene mRNA through qRT-PCR assay.The results showed that there was no change in the expression level of SOX7 mRNA after overexpression and interfering with miR-24-3p,suggesting that miR-24-3p regulate the expression of SOX7 gene at post-transcriptional level,which also accords with the mechanism of miRNAs regulating gene expression.These results indicate that miR-24-3p can regulate SOX7 gene expression at post transcriptional level in LC cells.In order to study the effect of miR-24-3p on the expression of SOX7 gene in lung cancer cells,we interfered with the expression of SOX7 gene in A549.However,the relative cell number of A549 decreased by 40%(P<0.001)and the number of invasive cells decreased by 60%(P<0.01)compared with the blank control group after overexpressed the expression of SOX7.The relative cell number increased by 36%(P<0.01)and the number of invasive cells increased by 300%(P<0.01)after inhibiting the expression of SOX7.These results indicate that miR-24-3p and SOX7 have opposite regulatory effects on proliferation and invasion of LC cells.In order to further clarify the effect of miR-24-3p on the proliferation and invasion of LC cells by regulating the expression of SOX7 gene,we carried out the rescue experiments.The results showed that when SOX7 protein was overexpressed,the effect of miR-24-3p on the proliferation and migration of lung cancer cells was reduced,which indicated that miR-24-3p could promote the proliferation and migration of lung cancer cells by inhibiting the expression of SOX7 gene.Finally,we used lentiviral plasmids to construct an over-expressed cell line of miR-24-3p,and validated the effect of miR-24-3p on tumor formation in LC by transplantation tumor model.Transplanted tumor model in vivo showed that the growth of tumor was promoted after overexpression of miR-24-3p,while the growth of tumor was inhibited after overexpression of SOX7 protein.Overexpression of SOX7 and miR-24-3p at the same time slowed the growth of tumor significantly than that of miR-24-3p alone,and the expression of SOX7 protein was between the control group and miR-24-3p alone,suggesting that SOX7 replicated the effect of miR-24-3p on tumor growth.The second part of the study is the regulation of TGFBI by miR-21-5p.Firstly,we retrieved the TCGA database and found that the expression of miR-21-5p in LC was significantly higher than that in normal tissues(P<0.001).In order to better explore the effect of changes in the expression of miR-21-5p on the function of LC cells,we carried out cell proliferation experiments.The results showed that the relative cell number of LC cells increased by nearly 20%(P<0.01)after overexpression of miR-21-5p,but decreased by nearly 30%(P<0.05)after inhibiting the expression of miR-21-5p.These results indicate that miR-21-5p promotes the proliferation of LC cells.We predict that miR-21-5p may regulate the expression of TGFBI genes through bioinformatics software(targetscan,microRNAanda,PicTc).In order to verify the regulatory relationship between the two,we first conducted a double luciferase reporter gene experiment,the results showed that:miR-21-5p can bind to the 3'-UTR region of TGFBI gene;at the same time,we interfered with the expression of miR-21-5p in two lung cancer cell lines(A549,H1299),and detected the expression of TGFBI protein by Western blot.Compared with the control group,the expression of TGFBI protein in A549 cells decreased by nearly 30%(P<0.05)and the expression of TGFBI protein in H1299 cells decreased by nearly 50%(P<0.05)after overexpression of miR-21-5p,while the expression of TGFBI protein in A549 cells increased by nearly 30%(P<0.05),and the expression of TGFBI protein in H1299 cells increased by nearly 60%(P<0.01)after inhibiting the expression of miR-21-5p.These results indicate that miR-21-5p can regulate the expression of TGFBI gene in LC cells.To study the effect of miR-21-5p on the proliferation of LC cells by regulating the expression of TGFBI gene,we used cell transfection technique to interfere with the expression of TGFBI gene in lung cancer cells.The results showed that the relative cell number of LC cells decreased by nearly 30%(P<0.05)after overexpression of TGFBI gene,and increased by nearly 60%(P<0.05)after inhibiting the expression of TGFBI protein.In order to further clarify the effect of miR-21-5p on the proliferation of LC cells by regulating the expression of TGFBI gene,we carried out rescue experiments.The results showed that when TGFBI protein was overexpressed,the effect of miR-21-5p on the proliferation of lung cancer cells could be reduced,suggesting that miR-21-5p could promote the proliferation of lung cancer cells by inhibiting the expression of TGFBI gene in vitro.In summary,this study found that miRNA-24-3p promoted the proliferation and invasion of lung cancer cells,and for the first time found that miRNA-24-3p exerted their carcinogenic miRNA by targeting SOX7 gene.This study provides new evidence for the role of miRNA-24-3p in lung cancer and first reported that SOX7 gene may be the target genes of miRNA-24-3p.This study also revealed for the first time that miRNA-21-5p promoted the proliferation of lung cancer cells by targeting TGFBI gene,and revealed a new mechanism of miRNA-21-5p in lung cancer.In this study,we found miR-24-3p and miR-21-5p influence lung cancer progression by targeting SOX7 or TGFBI,respectively.But whether they have superimposed effects on the progression of lung cancer remains to be further studied. |
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