| Gastric cancer(GC),which influences over one million patients per year around the world,is a major health problem in many countries.GC is a heterogeneous disease,stratified by histopathological variation.The Lauren histopathological classification,which consists of "enteric" and "diffuse" types,is the most commonly used approach in GC.Intestinal GC is mainly associated with intestinal metaplasia caused by Helicobacter pylori infection and has tubular or glandular structures,while diffusive GC is usually composed of poorly differentiated tumor cells that lack adhesion and infiltrate the stroma as single cells or small subpopulations.From the perspective of molecular biology,the incidence and advancement of GC are the result of a great number of dangerous factors.Therefore,it is critical to further explore the precise molecular target points and their mechanisms of action in GC.Background:GC is one of the most frequent malignancies,which seriously impair human health,in digestive tract system.GC is featured with large base of population,high proportion of progressive stage,long process of diagnosis and treatment,and poor overall prognosis in China,which has placed an enormous burden on family and society.To prevent the incidence of GC,the exploration of the molecular mechanism of GC occurrence and development is an ideal means.With the popularization of early detection,diagnosis and treatment,the GC outcome has been partially improved.However,in the course of clinical diagnosis and treatment,high recurrence and metastasis is always a problem that needs to be solved.Chemotherapy after surgery is one of the ways to cope with the problem,however,the incidence of chemotherapeutical resistance is very common.Thus,the exploration of the relevant molecular mechanism is very crucial.Multiple genes interact to induce changes in germ and somatic cells in tumor.Many somatic cells are induced by germ cells,and meiosis is the key to germ cell development.For the mechanism of chemotherapy resistance,the restoration of DNA double strand lesion is a significant risk factor in the forming of drug-resistant cell,and several specific molecules in the process of meiosis may be involved in the repair of damage,thus affecting the process of chemotherapy resistance.Meiotic nuclear divisions 1(meiotic nuclear divisions 1,MND1)is the key to the inhibition of meiosis,there have been reports MND1 can regulate the cell cycle related molecules which would be helpful to the progression and tolerance of malignant tumors.To date,the biological functions as well as relevant molecular mechanism of MND1 in GC have never been explored and reported.In this study,we aimed to preliminarily explore the specific functions of MND1 in GC by using clinical samples as well as in vitro and in vivo experiments.Methods:1.The expression level of MND1 in GC was detected by bioinformatics,and the expression level was verified by immunohistochemistry in 159 clinical samples collected.According to the collected clinical information and the expression level of MND1 in clinical samples,the high and low expression groups were divided,and the relationships between MND1 level and clinical stage as well as outcome were verified based on survival information and clinical data.In addition,six paired samples of gastric adenocarcinoma were obtained and MND1 expression was assessed by Western blot.2.Western blot and RT-PCR were adopted to assess MND1 expression in various cell lines of GC,and then the silencing and overexpression of MND1 vectors were constructed,and the stable cell lines were constructed.WB,PCR and immunofluorescence were all performed to verify the silencing and overexpression efficiencies.Subsequently,the cell reproductive capacity between different groups were assessed and compared by using CCK-8,colony cloning,EDU assay and subcutaneous tumorigenesis.Apopmsis,cell cycle,and the changes in the expressions of related proteins in each group were detected by using flow cytometry and WB.Scratch and Transwell assays were performed to further assess the transferred capacities of GC cells in each group.In addition,CCK-8,flow cytometry and subcutaneous tumor formation in nude mice were used to evaluate the changes of the cell proliferation and apoptosis of GC cells with the intervention of MND1 and oxaliplatin respectively,so as to explore its influence on the drug sensitivity of oxaliplatin.3.Bioinformatics searched for transcription factors that had binding sites with MND1 in the database,and WB was performed to evaluate the changes of MND1 after the silencing or overexpression of the screened transcription factors FOXA1.The binding between FOXA1 and MND1 was verified by dual luciferase reporter assay.In addition,the silenced FOXA1 vector was constructed and co-transfected with the silenced MND1 vector,and the proliferation,apopmsis,cell cycle,and transferred capacity of GC cells were assessed and compared between different groups.4.TKT was identified as the interacting protein of MND1 by using CO-IP combined mass spectrometry technology.WB and PCR were performed to evaluate the effects of silencing and overexpression of MND1 on TKT expression.CCK-8,EDU,colony forming,flow cytometry,wound healing,invasion and migration tests were performed to further assess the changes of related biological functions of GC cells in each group.In addition,the effects of the regulation of TKT on the changes of the contents of total lactic acid and glucose were explored.5.The enrichment signal channel of TKT was assessed by using gene set enrichment analysis(GSEA).WB was performed to assess the effects of the regulation of TKT and MND1 on the key proteins of PI3K/AKT signaling pathway.Results:1.Bioinformatics results showed that MND1 was a differentially expressed gene associated with platinum resistance.Statistical analysis of the clinical tissue samples demonstrated that MND1 was highly expressed in GC tissue in comparison with adjacent normal tissue.By the detection of MND1 expression in 6 pairs of fresh GC and adjacent normal tissue samples via western blot as well as in 159 tissue samples of GC patients via immunohistochemical detection,it was indicated that MND1 was highly expressed in GC tissue.The high expression of MND1 was correlated with TNM stage,pathological grade and poor prognosis of GC.2.The results of RT-PCR and WB indicated that,the MND1 expression level was significantly elevated in all GC cells in comparison with that in normal gastric mucosal cell.The combination of RT-PCR and WB further demonstrated that the efficiency of the constructed vector was remarkable,and silencing MND1 inhibited the proliferation,metastasis,influenced apopmsis and enhanced the quantity of arrested cell in G1 phase.In contrast,MND1 overexpression restored the above effects.Moreover,the silence of MND1 improved the sensitivity of GC cells to oxaliplatin.3.FOXA1 could inhibit the transcription of MND1 via binding to its promoter sequence.In addition,the co-transfection of FOXA1 and MND1 can inhibit the proliferation and metastasis,and influence apopmsis and cell cycle of GC cells.4.CO-IP and mass spectrometry results showed that MND1 can bind to TKT,and the results of WB and RT-PCR demonstrated that MND1 promoted the expression of TKT.In addition,TKT can bind to AKT,activates the PI3K/AKT signal pathway,and then promotes cellular proliferation,transferred capacity,and glucose metabolism of GC cells.Conclusions:1.MND1 is overexpressed in GC tissue and is associated with poor outcome.In addition,MND1 promotes proliferation,migration,invasion,apoptosis escape and the progression of cell cycle in GC cells,and silencing of MND1 facilitates the sensitivity of GC cells to oxaliplatin.2.FOXA1 can bind to the promoter of MND1 and suppresses its expression as well as the progression of GC.3.MND1 is co-expressed with TKT,which further promotes GC progression via activating PI3K/AKT signaling pathway. |
PDF Full Text Request