Cytoplasmic Dynein Intermediate Chain 1(DYNC1I1) Promotes Proliferation And Migration Of Gastric Cancer And Its Mechanism

Objective:The function of normal cells is maintained dependent on the fine material transport system within the cell.Compared with normal cells,tumor cells metabolism and signal transduction are more active.Cytoplasmic dynein is an important carrier of intracellular material transport.It is a multi-molecular complex composed of multiple subunits.Its main function is to transport long-distance intracellular substances along the microtubule to the minus end of the microtubule.And nuclear membrane rupture,spindle assembly,chromosome movement,etc.during mitosis.Previous studies have shown that cytoplasmic protein dysfunction can lead to tumorigenesis.During tumor development,cytoplasmic dynein can synergize with certain oncogenic proteins to promote tumor proliferation and metastasis.DYNC1I1 is an important cargo binding subunit of cytoplasmic dynein and its role in tumors is controversial.Early studies have found increased expression in hepatocellular carcinoma,but recent studies have shown that its expression is down-regulated in gliomas,inhibiting tumor progression,and patients with high expression of DYNC1I1 glioma have a better prognosis.At present,the research on the role of DYNC1I1 in tumors is limited,and has not been reported in gastric cancer.Our previous online data analysis showed that DYNC1I1 is a poor prognostic factor for gastric cancer,but its role and mechanism in gastric cancer is not clear.Here,we mainly study the role of DYNC1I1 in gastric cancer and its potential mechanism.Methods: 1.Cell viability was determined by MTT assay.2.ELISA was used to detect the concentration of IL-6 in the cell supernatant.3.Apoptosis was detected using the eBioscienceTM Annexin V-FITC Apoptosis Kit.4.Extract nuclear proteins using the ACTIVE MOTIFTM kit.5.Western Blot was used to detect protein expression.6.Immunofluorescence technique was used to detect the expression and intracellular distribution of P65,P-P65 and other proteins.7.Transient siRNA or plasmid transfection with Lipofectamine 2000 reagent to down-regulate the expression of the target protein.8.Animal experiment established subcutaneous tumor formation and tail vein lung metastasis model of nude mice,and studied the inhibitory effect of silencing DYNC1I1 expression on subcutaneous tumor formation and lung metastasis of gastric cancer.9.RT-qPCR was used to detect the mRNA expression of DYNC1I1,IL-6 and TNPO2.10.Affymetrix sanner 3000 chip detects the level of cellular gene expression profiles.11.Colony formation assay was used to detect the long-term inhibition of gastric cancer cell proliferation after silencing DYNC1I1.12.Cell cycle was detected by flow cytometry with PI(propidium Iodide)staining.13.Analysis of protein complex formation by immunoprecipitation.14.Gene Expression Omnibus(GEO)online database was used to analyze the correlation between DYNC1I1 expression and pathological parameters of gastric cancer,and the relationship between DYNC1I1 expression and prognosis of gastric cancer.15.Statistical analysis: The results of each experiment were repeated 3 times,and the data were expressed as mean ± standard deviation.Statistical analysis were performed using SPSS 17.0 statistical analysis software.The t test was used for comparison between groups,P<0.05 was statistically significant.Results: 1.Gastric cancer patients with high DYNC1I1 expression have a poor prognosis.The gastric cancer dataset GSE62254 was downloaded from NCBI's GEO database(http://www.ncbi.nlm.nih.gov/geo)for the clinical value of DYNC1I1.The data were divided into high DYNC1I1 expression and low DYNC1I1 expression group according to DYNC1I1 gene expression.Chi-square test was used to determine the relationship between DYNC1I1 expression and clinicopathological features of gastric cancer.Analysis showed that DYNC1I1 expression was associated with gastric invasion depth T,lymph node status and clinical(TNM)stage.Univariate analysis and multivariate analysis showed that DYNC1I1 is an independent prognostic risk factor for gastric cancer.The overall survival(OS)of gastric cancer patients was analyzed based on the expression level of DYNC1I1,and gastric cancer patients with high DYNC1I1 expression were found to have shorter survival(P < 0.001).Cox analysis showed that DYNC1I1 was an independent prognostic indicator for gastric cancer(P < 0.05).These data indicate that patients with high DYNC1I1 expression in gastric cancer have a late stage,and patients with high DYNC1I1 expression have poor prognosis.2.DYNC1I1 promotes proliferation and migration of gastric cancer cells in vitro.The expression levels of DYNC1I1 protein in six gastric cancer cell lines and normal gastric cells GES-1 were determined by Western blot.Higher expression was observed in gastric cancer cells compared to normal gastric cell GES-1.In this study,gastric cancer cell lines SGC-7901 and HGC-27 with high expression of DYNC1I1 were selected for further study.MTT and colony formation assays evaluated the effect of DYNC1I1 on cell proliferation.Compared with the control group,knockdown of DYNC1I1 inhibited the proliferation of gastric cancer cells.Colony experiments showed that the long-term proliferation inhibition of gastric cancer cells was more obvious after knocking down DYNC1I1.Transwell results showed that knockdown of DYNC1I1 significantly inhibited the migration of gastric cancer cells,compared with the proliferation inhibition at the same time,the migration inhibition rate was significantly increased,and DYNC1I1 inhibited the migration of gastric cancer cells not due to inhibition of proliferation.These in vitro experiments demonstrated that knockdown of DYNC1I1 inhibited cell proliferation and migration of gastric cancer cells.3.DYNC1I1 promotes the proliferation and metastasis of gastric cancer in vivo.SGC-7901 cells transfected with DYNC1I1 lentivirus(KD-DYNC1I1)and SGC-7901 cells transfected with unwanted sequences(NC-DYNC1I1)were injected subcutaneously into nude mice.Silencing the DYNC1I1 group produced smaller subcutaneous tumors than the control.Immunohistochemistry showed a significant decrease in the level of Ki-67(proliferation marker)in the silenced DYNC1I1 group.The same treatment of SGC-7901 cells was injected into mice to detect the role of DYNC1I1 in tumor metastasis in vivo.Mice were sacrificed 9 weeks after injection of tumor cells into the tail vein to check for the presence of metastatic nodules and metastatic nodules in the lungs.Compared with the control group,lung metastasis nodules were significantly reduced in mice injected with SGC-7901 KD-DYNC1I1 cells.Immunohistochemical staining showed that the expression levels of DYNC1I1 and Ki-67 in the KD-DYNC1I1 tumor group were significantly lower than those in the control group.These data indicate that silencing DYNC1I1 can inhibit tumor proliferation and metastasis in vivo.4.DYNC1I1 functions by up-regulating IL-6.The gastric adenocarcinoma database was downloaded from the cBioPortal for Cancer Genomics website.Genes positively correlated with DYNC1I1 expression and having Spearman coefficient greater than or equal to 0.3 were screened.Then use the DAVID website for pathway analysis.GO analysis showed that the first five of the change folds and the P<0.05 pathway included the IL-6-Jak-STAT signaling pathway,which was significantly associated with cell proliferation and migration.Affymetrix scanner expression analysis was performed using the HGC-27 cell line after knockdown of DYNC1I1.GO analysis of down-regulated genes,the top 5 pathways include "Negative regulation of interleukin-6 secretion." It is suggested that IL-6 may play a leading role in the proliferation and migration of gastric cancer cells mediated by DYNC1I1.The results of ELISA showed that the concentration of IL-6 protein in the supernatant was significantly down-regulated after knockdown of DYNC1I1.Western blot showed that phosphorylated STAT3,which is a classical downstream target of IL-6,was also down-regulated after knockdown of DYNC1I1 expression,but STAT3 did not change significantly.These results indicate that DYNC1I1 may regulate the biological behavior of gastric cancer cells by regulating the level of IL-6,and the IL-6-Jak-STAT pathway may be involved in the regulation of proliferation and migration of gastric cancer cells by DYNC1I1.5.DYNC1I1 promotes the proliferation and migration of gastric cancer cells by up-regulating IL-6.The addition of IL-6 neutralizing antibody to control cells significantly inhibited gastric cancer cell proliferation(~60%).In contrast,the addition of recombinant human IL-6 factor in the cells of the silenced DYNC1I1 group reversed the inhibition of cell proliferation caused by silencing DYNC1I1.The cell migration ability was up-regulated after the addition of recombinant human IL-6 after silencing DYNC1I1.Immunohistochemical staining showed down-regulation of IL-6 expression in tumor tissues of mice silenced with DYNC1I1 in subcutaneous tumors and lung metastatic nodules in mice.Taken together,these results indicate that DYNC1I1 promotes proliferation and migration of gastric cancer cells by up-regulating IL-6.6.DYNC1I1 up-regulates IL-6 expression by promoting NF-? B nuclear transport.RT-qPCR results showed that IL-6 mRNA levels were significantly reduced after knockdown of DYNC1I1.The GeneCard website(GeneCards-Human Genes | Gene Database | Gene Search)was used to predict IL-6 transcriptional regulators,and the top one transcription factor was NF-?B.The results of nucleoplasmic separation showed that TYNC1I1 knockdown resulted in a decrease in the amount of p65 translocated into the nucleus,regardless of P65 and its phosphorylation level.Immunofluorescence experiments further determined the distribution of P65 in gastric cancer cells.Co-immunoprecipitation showed that P65 binds to DYNC1I1.Taken together,these results indicate that DYNC1I1 up-regulates IL-6 expression by promoting nuclear translocation of P65 in gastric cancer cells.In addition,the distribution of DYNC1I1 itself in gastric cancer cells was detected after DYNC1I1 knockdown.The expression of DYNC1I1 in the nucleus did not change significantly,but its expression in the cytoplasm was down-regulated,indicating that knockdown of DYNC1I1 may affect its cytoplasm.Because the role of DYNC1I1 in the cytoplasm is mainly to transport the cargo to the minus end of the microtubule long distance,it is speculated whether it is mainly affected by the function of the transport carrier after knocking down DYNC1I1.Taken together,these results indicate that DYNC1I1 up-regulates IL-6 expression by promoting nuclear translocation of P65 in gastric cancer cells.7.DYNC1I1 up-regulated the expression of TNPO2 in gastric cancer cells.To investigate the specific mechanism of DYNC1I1 in promoting cell proliferation and metastasis in gastric cancer,Affymetrix sanner expression microarray was used to detect the difference in mRNA expression after silencing DYNC1I1 in HGC-27 cells.The results showed that compared with the control group,TNPO2 was the top one gene that down-regulated after knock-down DYNC1I1,which was 29.55-fold.Then,DYNC1I1 was silenced in SGC-7901 and HGC-27 cells,and RT-PCR and Western blot were used to detect the changes of TNPO2 mRNA and protein levels.After silencing DYNC1I1,TNPO2 was down-regulated at both mRNA and protein levels,and all of them were statistically significant.8.Patients with gastric cancer with high TNPO2 expression have a poor prognosis.In order to elucidate the role of TNPO2 in gastric cancer,the expression level of TNPO2 in gastric cancer and adjacent tissues was detected on the GEPIA website,and the expression in gastric cancer was significantly higher than that in normal tissue.The KM-Plotter database performed survival analysis on gastric cancer patients with different TNPO2 expression levels,and found that gastric cancer patients with high expression of TNPO2 had a poor prognosis.9.TNPO2 promotes the proliferation of gastric cancer cells and inhibits the apoptosis of gastric cancer cells.In order to evaluate the biological function of TNPO2 in gastric cancer cells,siRNA was used to transiently silence TNPO2 expression.MTT assay showed that the proliferation of gastric cancer cells was significantly down-regulated after silencing TNPO2.Flow cytometry detected changes in apoptosis levels after silencing TNPO2.The experimental results showed that the level of apoptosis increased after 48 hours of silencing TNPO2 compared to the control group.10.DYNC1I1 down-regulated transcription factor SP1 down-regulated TNPO2 expression.The TNPO2 transcription factor was first predicted by the PROMO website.At the same time,the TNPO2 transcription factor was predicted on the GeneCard website,and the first one was SP1,which was included in the results predicted by the PROMO website.It is speculated that SP1 plays an important role in the regulation of TNPO2 expression by DYNC1I1.RT-qPCR and Western Blot were used to detect the changes of SP1 mRNA and protein levels after silencing DYNC1I1.After silencing DYNC1I1,SP1 mRNA and protein levels were down-regulated.The level of TNPO2 protein was detected after silencing SP1.The results showed that TNPO2 in gastric cancer cells was down-regulated at the protein level after SP1 silencing.These results demonstrate that DYNC1I1 regulates TNPO2 expression by regulating the expression of the TNPO2 transcription factor SP1 in gastric cancer cells.Conclusion: 1.High DYNC1I1 expression is associated with poor prognosis of gastric cancer and is an independent prognostic factor for gastric cancer.2.DYNC1I1 promotes proliferation and migration of gastric cancer cells in vitro and in vivo by promoting the translocation of P65 and up-regulating IL-6 expression.3.DYNC1I1 up-regulates TNPO2 expression by up-regulating transcription factor SP1;4,TNPO2 promotes proliferation of gastric cancer cells and inhibits apoptosis.