| Objective: Lung cancer is a global malignancy with leading incidence and extremely high mortality rates worldwide.The prevalence of lung cancer in China is the highest among all malignant tumors,which seriously threatens the healthy life of our residents.Since lung cancer has a long latency period and is often difficult to be detected,according to clinical observation,30.5% of patients are already in advanced stage when detected,which seriously hinders the effectiveness of treatment.The specificity of traditional methods such as surgery,radiotherapy and chemotherapy are not strong,and they can cause greater toxic side effects to patients while playing a therapeutic role,and the actual conditions of use are complicated,so the therapeutic effect is limited to some extent.Although emerging approaches such as targeted therapy,genomic therapy and immunological therapy have been widely used in recent years,the five-year survival rate of patients with advanced non-small cell lung cancer(NSCLC)is still less than 5%.Therefore,studying the mechanism of lung carcinogenesis by discovering new early diagnostic and prognostic markers and finding important signaling targets is of great importance for the diagnosis and treatment of patients with NSCLC in early stages,which can bring better outcomes and effectively improve patients’ quality of life and survival time.Transmembrane protein 125(TMEM125)is a member of the transmembrane protein family.Transmembrane proteins(TMEM),also known as integral membrane proteins,are important components of membrane proteins.Membrane proteins classified as TMEMs must be embedded in or span at least one compartment of the biological membrane.Due to their structural and distribution characteristics,TMEMs exhibit unique biological functions.An increasing number of studies had shown that abnormal TMEM expression is closely related to tumorigenesis,progression,metastasis and immune regulation.TMEM125 protein consists of 219 amino acids(aa)and the gene is localized at 1p34.2.TMEM125 has four transmembrane helices at positions 34-56 aa,66-83 aa,114-136 aa and 146-168.The role of TMEM125 in tumors and its mechanism is not yet clear.By transfecting TMEM125 overexpression plasmid and siRNA in NSCLC cell lines to regulate TMEM125 expression in both directions,we found for the first time that TMEM125 has a role in inhibiting the proliferation and migration of NSCLC cells,however,the specific mechanism by which TMEM125 exerts this role is unclear.The PI3K/AKT pathway is a widespread intracellular signaling mechanism that promotes cell growth and inhibits necrosis.The abnormally activated PI3K/ATK pathway plays an irreplaceable role in the development and progression of NSCLC,which can regulate tumor cell proliferation,invasion and metastasis,thus affecting patient prognosis.We performed transcriptome mRNA sequencing and bioinformatics analysis of lung cancer cell lines overexpressing TMEM125,and found that TMEM125 is closely related to PI3K/AKT pathway,and TMEM125 may play a role in suppressing NSCLC lung cancer cell lines by negatively regulating PI3K/ATK pathway activity.To validate therefore we proposed to detect TMEM125 expression in lung cancer paraffin specimens and count its relationship with clinicopathological factors,and to verify the expression of TMEM125 and the activity of PI3K/AKT pathway in NSCLC cell lines by applying TMEM125 overexpression vectors,siRNA and inhibitors of PI3K/AKT pathway to regulate TMEM125 expression in both directions.Whether TMEM125 plays a role in inhibiting the proliferation and migration of NSCLC cells through PI3K/AKT pathway.This study will enrich the molecular mechanism of NSCLC development and provide a theoretical basis and experimental basis for TMEM125 to become a new target for clinical treatment of NSCLC in the future.Methods: 1.Immunohistochemical staining was applied to determine TMEM125 protein expression in paraffin section specimens of 113 cases of non-small cell lung cancer,and statistical methods such as chi-square test were used to correlate them with clinicopathological factors.2.The TCGA-based UALCAN database was applied to analyze the expression level of TMEM125 mRNA in lung cancer.Kaplan-Meier survival curve was applied for online analysis of the relationship between TMEM125 expression level and clinical prognosis of NSCLC patients.3.The proliferation ability of NSCLC cells was detected by colony formation assay and MTT assay,and the migration ability of NSCLC cells was detected by Transwell migration assay and scratch assay,after the expression of TMEM125 gene was bidirectionally regulated by transfection of overexpression pEGFP-N1-Homo TMEM125 plasmid vector and specific siRNA.4.Transcriptome sequencing was performed on TMEM125 overexpressing cells,and KEGG enrichment analysis was performed on the sequencing results to find the potential targets and pathways of TMEM125 action.5.The changes in expression levels of EMT-related proteins,key proteins in the PI3K/AKT pathway and their downstream proliferation-related proteins Cyclin D1 and p21 and p27 were detected by Western Blot assay after TMEM125 gene disruption or overexpression.6.PI3K/AKT pathway and its downstream protein expression,as well as the proliferation and migration of NSCLC cells were detected after applying PI3 K pathway inhibitor PKI-402 after interfering with TMEM125 and inhibiting the activity of PI3K/AKT pathway.Results: 1.Immunohistochemical staining showed that TMEM125 was lowly expressed in NSCLC tissues,and its low expression level was correlated with tumor lesion size,low tumor differentiation,lymph node metastasis,and high TNM stage.Low expression of TMEM125 was correlated with poor prognosis of lung cancer patients.2.Low expression of TMEM125 was found to be associated with poor prognosis in lung cancer patients by Kaplan-Meier analysis.3.After overexpression of TMEM125 in NSCLC cell lines,MTT and clone formation assays showed that the proliferation ability of NSCLC cells was downregulated,while siRNA interference with TMEM125 expression could significantly upregulated the proliferation ability of NSCLC.Transwell migration assay and scratch assay results showed that overexpression of TMEM125 could significantly inhibit migration of NSCLC cells,while siRNA interference with TMEM125 expression promoted the migration of NSCLC cells.4.Westernblot assay results showed that overexpression of TMEM125 significantly downregulated cell proliferation and EMT-related proteins,as well as key molecules of the PI3K/AKT pathway,while interference with TMEM125 expression upregulated proliferation and EMT-related proteins,as well as key molecules of the PI3K/AKT pathway.5.After applying siRNA to knockdown TMEM125 expression in NSCLC cells,the activity of PI3K/AKT pathway could be effectively inhibited by PI3K/AKT pathway inhibitor,and the up-regulation trend of NSCLC proliferation and migration after interfering with TMEM125 could be reversed,i.e.,the proliferation and migration of NSCLC were effectively inhibited.Conclusions: 1.TMEM125 as an oncogene is lowly expressed in NSCLC,and its expression level is negatively correlated with tumor size,differentiation degree,lymph node metastasis and TNM stage.2.Low expression of TMEM125 is positively correlated with poor prognosis of patients.3.TMEM125 can negatively regulate PI3K/AKT pathway thereby inhibiting the proliferation and migration ability. |
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