Dysregulation Of HRCT1 Promotes Tumor Metastasis And Proliferation In Gastric Cancer

Backgroud:Gastric cancer is one of the most common digestive system malignant tumor in the world,with the poor prognosis and high rate of recurrence and metastasis,seriously impacting peoples health and daily life.The leading causes of death in patients with gastric cancer are invasion and metastasis.The process of invasion and metastasis in gastric cancer is a result of abnormal genes and signaling pathways,which formed a complex network affect the occurrence of tumor metastasis on different levels,and the specific molecular mechanism is not clear.Therefore,how to explore the mechanism of invasion and metastasis in gastric cancer and to find new therapeutic targets have become a international research focus in this field.In our previous study,Microarray assay was used for a genome-wide screen for metastasis-related markers in gastric cancer,more than one thousand of differentially expressed genes were detected in the primary gastric cancer samples with lymph node metastasis(LNM),compared with the gastric cancer samples without LNM.HRCT1 gene were selected for further study finally for its more significant and stable expression between nonmetastatic and metastatic samples.To date,the clinical and expreriment research of HRCT1 in malignancies have no reported so far,especially the exact role and clinical significance of HRCTI in gastric cancer remains largely unknown.Our former study suggest that HRCT1 expression in cancerous tissues with LNM is significantly higher than those in cancerous tissues without LNM,these result provide clues that HRCT1 may play a part in metastasis of gastric caner.In the current study,we firstly detected the expression of HRCT1 in tissue and analyzed the correlation of HRCT1 with clinicpathology parameters and prognosis in gastric cancer.Next,cell invasion and migration assay.EDU and MTS assay,tumor-bearing mice experiment and Microarray assay was used to detect the function and mechanism of HRCT1 promotes invasion,metestasis and proliferation in vivo and vitro.MicroRNAs(miRNAs),a class of small non-coding RNAs,contain 20 to 25 nucleotides,which lead to the suppression of their target genes by binding messenger RNAs at their 3 untranslated region t(3'-UTR),with either inducing mRNA degradation or blocking protein translation.In order to determin wheather or not HRCT1 expression could be regulated by specific miRNAs in gastric cancer,Biology software,Luciferase assay and Western blot were used to predict and verify the potential miRNAs,which may down regulate the expression of HRCT1.Methods:1.Microarray assay was used to screen the diffentially expressed genes between metastatic sample and non-metastatic samples.The primary gastric cancer samples with LNM(5 cases)and without LNM(5 cases)were collected and then the total RNAs were extracted and subjected to mRNA microarray assay.The differentially expressed genes were identified between the above two groups by fold changes and P value.2.The expressions of mRNA were detected by RT-qPCR.The level of HRCT1 mRNA in gastric cancer and gastric cancer cells were quatified by RT-qPCR.3.Immunohistochemistry(IHC)and Westernblot were performed to detected the expression of protein.IHC was carried out to detect the protein expression in 139 cases of formalin-fixed paraffin-embedded(FFPE)tissues of gastric cancers,Westernblot was carried out to investigated the protein expression in BGC823 and SGC7901 cancer cells,which were transfected with PCNA3.1-HRCT1 or si-HRCT1.4.Transwell assay was performed to detect the migration and invasion ability of gastric cancer cells in vitro.After transfected with PCNA3.1-HRCT1 or si-HRCT1,Transwell assay was performed to detecte the migration and invasion ability.5.MTS and EDU assays were used to detect the proliferation ability of gastric cancer cells in vitro.The gastric cancer cells were transfected with plasmids or si-HRCTl,then MTS and EDU assays were performed to examine the proliferation ability of the transfected cells.6.Tumor-bearing mice experiment.The gastric cancer cell were transfected with LV-G416-HRCT1 or Negative control,then were injected into the lateral tail vein or the subcutaneous in axillary fossa of the mice.After five weeks,the mice were dissected for tumor invasion,metastasis and proliferation detection.7.Microarray assay was used to screen the signal pathway regulated by HRCT1.Gene expression microarray profiling was performed in BGC823 cells which transfected with PCDNA3.1-HRCT1 and Negative control.The differentially expressed genes and the signal pathways related by HRCT1 were analyzed,and the result was further validated by qPCR and Westernblot.8.The prediction and validation of the target miRNAs.The miRNAs that potentially suppress HRCT1 expression were firstly predicted by software and were verified by dual-luciferase reporter assay and Westernblot assay.9.Statistical analysis.We used the Student t-test to analyze the difference between two enumeration data,the Chi-square test was used for different analysis between two measurement data.Kaplan-Meier survival analysis and COX regression test were used for patients survival analysis.P<0.05 were taken as statistically significant.Result:1.10 samples of primary gastric cancer,include 5 samples with LNM and 5 samples without LNM,which were collected and subjected to mRNA microarray assay The results showed 1150 differentially expressed genes in metastatic samples,compared with that in non-metastatic samples.HRCT1 gene were selected finally for further study by enhancing the screening criteria and combining with the document report in thirty genes,which were further identified as the differences of their expressions were more significant than the other genes.2.RT-qPCR technique was performed to detect the mRNA expression of HRCT1 in 55 samples of fresh gastric cancer tissue and 27 samples of fresh nontumors gastric mucosa.The result confirmed that HRCT1 transcripts was up-regulated in primary gastric cancer,compared with those in nontumors gastric mucosa,furthermore,the expression of HRCT1 mRNA were significantly higher in 35 metastatic samples than those in 20 non-metastatic samples.In addition,HRCT1 expression were up-regulated in poorly differentiated and metastatic gastric cancer cell lines(BGC823 cells and SGC7901 cells),compared to modestly and well differentiated cell lines(AGS cells and MKN45 cells).3.Immunohistochemistry technique was performed to detect the expression of HRCT1 protein in 139 samples of paraffin-embedded gastric cancer tissue and 51 samples of paraffin-embedded nontumors gastric mucosa The result showed that HRCT1 protein was expressed in cytoplasm,and the positive expression of HRCT1 in primary gastric carcinoma was obviously higher than those in nontumors gastric mucosa.Furthermore,increased expression of HCRT1 was also observed in gastric cancer with distant metastasis,serosal and serosal outside of invasion,poor differentiation,and high clinic stage.In addition,gastric cancer patients with high expression of HRCT1 have poorly prognosis.4.Transwell assay was performed to investigate the migration and invasion ability of gastric cancer cells in vitro.The result suggested that HRCT1 overexpression significantly enhanced migration and invasion of BGC823 cells and SGC7901 cells in vitro,wherears HRCT knockdown dramaticly inhibited migration and invasion.In addition,HRCT1 also promoted the invasion and metastasis ability of gastric caner cells in vivo,overexpression of HRCT 1 increased the ability of BGC823 cells to spread to lung and capsule invasion in LV-416-HRCT1 group.5.MTS and EDU assay suggested that HRCT1 overexpression significantly enhanced proliferation of BGC823 cells and SGC7901 cells in vitro,however,HRCT knockdown dramaticly inhibited growth.In addition,HRCT1 also promoted the proliferation ability of gastric caner cells in vivo.xenograft tumor growth in LV-416-HRCT1 group was significantly faster than those in negative control.6.Microarray assay suggested that overexpression of HRCT1 may up-regulate the activation of HER2-MAPK signal pathway in BGC823 cells.The result was confirmed by RT-qPCR and Westernblot assays.HRCT1 overexpression significantly enhanced activation of HER2-MAPK passway in BGC823 and SGC7901 cells at mRNA and protein level,wherears HRCT1 knockdown dramaticly inhibited activation of HER2-MAPK passway.7.HRCT1 was negatively regulated by miR-124.HRCT1 is the potential target of miR-124,miR-199a-5p,miR-506 and miR-515-3p predicted by software.Dual-luciferase reporter assay showed that the HRCT1 expression was supressed by miR-124,the result was further confirmed by Westernblot.Conclusion:1.HRCT1 expressions were significantly higher in metastatic samples than those in non-metastatic samples,HRCT1 expression was up-regulated in primary gastric cancer,compared with those in nontumors gastric mucosa.In addition,HRCT1 expression were up-regulated in poorly differentiated and metastatic gastric cancer cell lines(BGC823 cells and SGC7901 cells),compared to modestly and well differentiated cell lines(AGS cells and MKN45 cells).2.The result of Immunohistochemistry in large number cases showed that inncreased expression of HCRT1 was correlated with positive lymph node metastasis,distant metastasis,depth degree of invasion,and differentiation.In addition,high expression of HRCT1 was associated with the poor prognosis of gastric cancer patients.3.Cytolgical and zoological experiment showed that HRCT1 promoted the invasion,meatastsis and proliferation ability of gastric caner cells in vitro and in vivo.4.HRCT1 promoted the invasion,meatastsis and proliferation ability of gastric caner cells via activating the HER2-MAPK signal pathway.5.HRCT1 expression could be negatively regulated by miR124 in gastric cancer cells.