| BackgroundNK/T cell lymphoma(NK/Tcl)is a kind of non-hodgin lymphoma subtype with poor prognosis.Most of which are derivad from mature NK cells,and a few are derived from NK-like T cells,and the cell phenotype is highly heterogeneous and complex.NK/Tcl mainly occurs in upper respiratory tract and digestive tract,with different clinical manifestation,rapid progress and frequent recurrence,but slow progress in diagnosis and treatment.Currently,there is no more effective therapeutic traetment.MicroRNAs(miRNAs)are an important part of non-coding RNAs and target a variety of mRNA to regulate gene expression through RNA degradation and/or inhibition of translation.MiRNA plays an important role in the connection betwween tumor cells and tumor cell microenvironment.By regulating various molecular signaling pathways and releasing tumor cytokines or growth factor,miRNA can regulate the occurrence and development of tumors.Studies have shown that some miRNAs can play the roles of oncogenes ans tumor suppressor genes in specific tumors.MiR-148 a is a number of the miR-148/miR-150 family,which is composed of three highly conserved mature miRNAs,including miR-148 a,miR-148 b and miR-152.MiR-148 a is a novel myogenic miRNA that promotes differentiation of myoblasts and skeletal muscle cells by acting on target protein ROCK1.Meanwhile,miR-148 a can act on different target genes to perform its function in various tumors.Recent studies have shown that miR-148 a is down-regulated in gastric cancer,colorectal cancer,pancreatic cancer,liver cancer,breast cancer and non-small cell lung cancer,while up-regulated in osteosarcoma.Moreover,it was found that miR-148 a could play the role of oncogene or tumeo suppressor gene in different tumor cells.However,currently there are no relevant studies on the role of miR-148a-3p in NK/T cell lymphoma.This paper intends to study the biological function of miR-148a-3p in NK/T cell lymphoma.ObjectiveMiR-148a-3p has not been reported in NK/T cell lymphoma cells.In this study,the expression and function of miR-148a-3p in NK/T cell lymphoma cells were studied in vitro.Further elucidate the function of miR-148a-3p and seek new therapeutic targets for NK/T cell lymphoma.Methods1.NK/T cell lymphoma cell lines NK92 cells and YT cells were donated by the key lymphoma laboratory(Mingzhi Zhang,department of oncology,the first affiliated hospital of Zhengzhou University),and normal NK cells were purchased from the cell bank of the Chinese academy of sciences.The overexpression of LV3-miR-148a-3p mimics mediated by lentivirus and LV3-miR-148a-3p inhibitor interference vector were purchased from Shanghai gemma.2.QRT-PCR was used to detect the expression of miR-148a-3p in NK/T cell lymphoma cells and normal NK cells.3.Lentivirus mediated overexpression vectors(Lv3-miR-148a-3p mimics)and Knockout vectors(Lv3/anti-hsa-miR-148a-3p)were used to infect YT cells and NK92 cells,respectively.After infection,the expression of miR-148a-3p,the proliferation,migration,invasion and apoptosis of YT and NK92 cells were detected.4.SPSS 21.0 software was used for statistical analysis.The relative expression level of miR-148a-3p was compared by one-way ANOVA among the three groups.It's going to be the mean plus or minus the standard deviation.When P <0.05,the difference between sample was considered statistically significant.Repeat all experiments at least 3-5 times.Results1.The relative expression levels of miR-148a-3p in normal NK cells,YT cells and NK92 cells were 8.62±0.35,14.55±0.27 and 24.48±0.44,respectively.The relative expression levels in NK92 cells were significantly higher than those in the other two groups,and the difference was statistically significant(P<0.05).The expression level of miR-148a-3p in NK/T cell lymphoma cells was significantly increased.The expression level of NK92 cells was about 3 times that of the control group,and the expression level of YT cells was about 1.7 times that of the control group.The difference between the two was statistically significant(P<0.05).2.After infecting YT cells of NK/T cell lymphoma with each modified lentivirus.The relative expression levels of the knockout group(LV3/anti-hsa-miR-148a-3p),overexpression group(miR-148a-3p)and negative control group(LV3-nc)were 4.84±0.4,21.55±0.33 and 10.02±0.69,respectively.Compared with the knockout group and negative control group,the expression levels of overexression group were significantly increased in YT cells.The different was statistically significan t(P<0.05).After infecting NK92 cells of NK/T cell lymphoma with each modified lentivirus.The relative expression levels of knockout group(LV3/anti-hsa-miR-148a-3p),overexpression group(miR-148a-3p)and negative control group(LV3-nc)were 8.44±0.3,31.13±0.52 and 11.11±0.5,respectively,in NK92 cells of lentivirus infected NK/T cell lymphoma NK92 cells.The difference was statistically significant(P<0.005).3.CCK-8 was used to detect the effect of miR-148a-3p on the proliferation activity of YT and NK92 cells.Compared with the negative control group(LV3-nc),the proliferation activity of the overexpression group(miR-148a-3p)was significantly increased,while that of the knockout group(LV3/anti-hsa-mir-148a-3p)and the negative control group(LV3-nc)was significantly decreased.4.Transwell assay was conducted to detect the effect of miR-148a-3p on YT and NK92 cell migration.compared with the knockout group(LV3/anti-hsa-miR-148a-3p)and the control group(LV3-nc),the migration ability of miR-148a-3p cells in the overexpression group(miR-148a-3p)was significantly increased.5.Transwell assay was used to test the effect of miR-148a-3p on the invasion ability of YT and NK92 cells.compared with the knockout group(LV3/anti-hsa-miR-148a-3p)and the control group(LV3-nc),the invasion ability of miR-148a-3p cells in the overexpression group(miR-148a-3p)was significantly increased.6.Annexin V-PE/7-AAD double staining was used to detect the effect of miR-148a-3p on apoptosis of NK/T cell lymphoma cells.In YT cells,the apoptosis rates of the knockout group(LV3/anti-hsa-miR-148a-3p),the overexpression group(miR-148a-3p)and the negative control group(LV3-nc)were 14.2±0.84%,2±0.5% and 5.2±0.57%.Respectively,after the LV3/anti-hsa-miR-148a-3p group was infected with YT cells,the apoptosis ratio was significantly increased.In NK92 cells,the apoptosis rates of the knockout group(LV3/anti-hsa-miR-148a-3p),the overexpression group(miR-148a-3p)and the negative control group(LV3-nc)were 54.8±3.96%,43.4±0.55% and 44.1±1.08%.Respectively,after the LV3/ anti-hsa-miR-148a-3p group was infected with NK92 cells,the apoptosis ratio was significantly increased.In NK/T cell lymphoma YT and NK92 cells,the apoptosis ratio of LV3/anti-hsa-miR-148a-3p knockout group was significantly increased,while that of miR-148a-3p overexpression group was significantly dacreased,and the difference was statistically significant(P<0.05).Conclusions1.MiR-148a-3p was significantly increased in NK/T cell lymphoma cells,suggesting taht miR-148a-3p may be involved in the occurrence and progression of NK/T cell lymphoma.2.Inhibition of miR-148a-3p expression in YT cells and NK92 cells can inhibit cell proliferation,migration,invasion and promote apoptosis.suggesting that miR-148a-3p play an oncogene role in NK/T cell lymphoma cells and is expected to be a new treatment target for NK/T cell lymphoma. |
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