| ObjectiveLung cancer(LC)has been the most commonly diagnosed malignancy and the leading cause of cancer-related death in the world.Despite significant developments in the oncological management of lung cancer over recent years,survival remains poor.The condition is especially serious in patients with LUAD that the 5-year mortality rates ranges from 51% to 99% with the advance of clinical stage.Patients with LUAD always have advanced disease at the time of diagnosis(stage III/IV).Besides,they are usually highly resistant to conventional radio-and chemotherapies.As LUAD takes the largest proportion(40%)among all the LC histological subtypes,the aim that increasing the five-year survival rate of LUAD is a critical step to overcome LC.Micro RNAs(mi RNAs)are a class of small(19–25 nt),single-stranded endogenous noncoding RNAs.They exist widely in human cells and body fluids,including blood,urine,cerebrospinal fluid,bronchoalveolar lavage,or breast milk.Mi RNAs cause degradation of m RNA or inhibit translation by binding to partially complementary sequence in 3?-untranslated region(3?-UTR)of the target m RNAs.Normally,micro RNAs typically target multiple functionally related genes to control entire biological processes including cell proliferation,and apoptosis,cell cycle,metastasis,glucose and lipids metabolism,and infection and immune responses.In fact,aberrant mi RNAs also associated with the initiation and progression of certain cancer types.These abnormally expressed mi RNAs function as tumor promoters or suppressors.Our previous study verified that mi R-520c-3p was significantly down-regulated in LUAD tissue compared with the corresponding adjacent normal tissue by testing 31 pairs of samples.Besides,mi R-520c-3p had a modulation on the immune microenvironment of LUAD by directly targeting thymic stromal lymphopoietin(TSLP).TSLP affected DCs in LUAD,then induced CD4+CD25-T cells to differentiate into CD4+CD25+foxp3+ T cells and the migration of CD4+CD25+ T cells,finally forming a inhibitory immune microenvironment which was on behalf of tumor progression,which was an indirectly way of regulating LUAD tumorigenesis and progression.Hsa-mi R-520c-3p is almost undetected in types of cancers,especially in LC.Until now,only a handful of papers show that mi R-520c-3p or mi R-520 c plays an inhibitory role in breast cancer,hepatocellular carcinoma(HCC),Diffuse Large B Cell Lymphoma(DLBCL)and gastric cancer.Therefore,it is full of significance to investigate the role of mi R-520c-3p in LUAD.Purpose1.Look for the characteristics of mi R-520c-3p expression in LUAD as well ad study the biological function of mi R-520c-3p in LUAD.2.Study the molecular mechanism of mi R-520c-3p that regulates tumorigenesis of LUAD and verify whether mi R-520c-3p can directly regulate the biological function of tumor cells through a target gene.Meanwhile,look for signaling pathways involved in that process.MethodsWe collected 165 lung adenocarcinoma tissue samples with corresponding adjacent tissue samples in Tianjin Medical University Cancer Institute and Hospital and tested the expression of mi R-520c-3p in LUAD tissue by real-time PCR.We also collected the prognostic information of patients with the aim of detailed statistical analysis.We studied the biological function of mi R-520c-3p in LUAD cell lines.We over-expressed mi R-520c-3p in LUAD by transient transfected with mi R-520c-3p mimics or negative control to verify whether there were changes in biological behavior of cells.WST-8 was used to test cell proliferation.Cell apoptosis was analysised by Flow cytometry.Cell migration and invasion were measured through transwell assay and wound healing assay.We studied the molecular mechanism of mi R-520c-3p functioning as an anti-oncogene gene on the development of LUAD.Bioinformatics,real-time PCR,western blot,and luciferase reporter assays were applied to verify the target gene of mi R-520c-3p.WST-8,Flow cytometry and transwell assay were used to test the biological behavior changes of the gene.Finally,rescue experiment was made to make sure the mechanism of mi R-520c-3p functioning as tumor inhibitor in LUAD.Western blot was made to identify the signaling pathways taking part in this process.Results1.Compared with the expression of mi R-520c-3p in LUAD,the expression of mi R-520c-3p in the corresponding adjacent tissues was higher.2.Patients with relatively higher mi R-520c-3p expression in early stage of lung adenocarcinoma(TNMI)had a better prognosis than those with relatively lower expression.3.The result turned out that no differences existed in proliferation of LUAD cells between the mi R-520c-3p overexpression group and the negative control group by WST-8.Cells in mi R-520c-3p overexpression group had more apoptosis compared with cells in negative control group via Flow cytometry.Transwell assay and wound healing experiment showed that mi R-520c-3p overexpression group had more cells attached on the lower side of transwell.mi R-520c-3p inhibits migration and invasion of LUAD cells.4.Rab22 a was the direct target of mi R-520c-3p.Mi R-520c-3p promoted cell apoptosis and inhibited the cell migration and invasion of LUAD via Rab22 a.5.When mi R-520c-3p was over-expressed in lung adenocarcinoma,the expression of rac1 and dc42 was decreased,confirming that Rac1/cdc42 signaling pathway was involved in the mi R-520c-3p-Rab22a-biological function of tumor cells in lung adenocarcinoma.Conclusions1.Mi R-520c-3p was lower expressed in LUAD tissues than that in the corresponding adjacent tissues.2.The expression of mi R-520c-3p in patients with early lung adenocarcinoma(TNMI)is related to the prognosis of patients.3.Mi R-520c-3p played a tumor suppressive role in LUAD by promoting tumor cell apoptosis,inhibiting tumor cell migration and invasion ability.4.Rab22 a was a direct target gene of mi R-520c-3p.Mi R-520c-3p inhibited the synthesis of Rab22 a protein,promoting apoptosis and inhibiting the migration and invasion of LUAD cells.5.In lung adenocarcinoma,the Rac1/cdc42 signaling pathway participated in the “mi R-520c-3p-Rab22a-biological function of tumor cells”. |
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