| Gastric Carcinoma(GC)is a malignant tumor originating from gastric mucosal epithelium,its mortality rate are the highest in various common malignant tumors in China.As one of the most common occurring malignant cancers,the incidence and mortality of gastric cancer has decreased recently,but it is still very poor for the prognosis of patients and 5-year survival rate is very low.Because of lack of early diagnostic markers in early stage of gastric cancer,the vast majority of patients with gastric cancer already reached the advanced stage and a cancer metastases had occurred when they were diagnosed,so that most patients had missed an efficient treatment in the pre-metastasis stage.In recent years,an ever increasing number of experiments has confirmed that tumor stem cells were resistant to radiotherapy and chemotherapy drugs,and associated with tumor invasion and metastasis.As a newly discovered EMT inducible factor,studies have shown that paired related homebox 1(PRRX1)is able to promotes the development of EMT in breast cancer,pancreatic cancer and colorectal cancer,while EMT is associated with invasion and migration.In this study,we examined the expression of PRRX1 in gastric adenocarcinoma cells and its corresponding sphere cells,and explored the biological effects of down-regulated PRRX1 on gastric adenocarcinoma cells,and provided new evidences for role of PRRX1 in the development of human gastric adenocarcinoma.In our experiments,various differentiated human gastric adenocarcinoma cells and sphere cells were cultured,and their expressions of PRRX1 were detected by qRT-PCR technique at the level of mRNA,and then verified by Western blot at the protein level.The highest expression of PRRX1 cell lines were selected.Secondly,the lentivirus LV3-shRNA-PRRX1 and the control LV3-shRNA-NC vector,which are down-regulated PRRX1,were constructed,then the poorly differentiated human gastric adenocarcinoma cell line MGC803 were transfected by these two vectors respectively to establish both stable PRRX1 down-expression cell line MGC803-shRNA-PRRX1 and its internal control cell line MGC803-shRNA-NC.qRT-PCR and Western blot were used to verify whether the lentiviral vector were stably transcribed and expressed at the transcriptional and protein levels.qRT-PCR and Western blot assay were also used to examine the effects of down-regulation of PRRX1 on gastric stem cell(CSC)markers and epithelial-mesenchymal transition(EMT)markers in gastric adenocarcinoma cells.Furthermore,the immunofluorescence technique was used to verify the effects of EMT markers.Finally,we performed Transwell assay,Sphere formation assay,MTT experiments,soft agar plate cloning experiments,drug resistance experiments and low cytometry experiments to explore the effects of PRRX1 down-regulation on invasion,migration,proliferation,drug resistance and cell cycle of gastric adenocarcinoma cells.Our results are as follow:1.qRT-PCR and Western blot results showed that PRRX1 has the lowest expression in highly differentiated human gastric adenocarcinoma cell lines MKN28 cells and spheres cells,and was highly expressed in the moderately differentiated human gastric adenocarcinoma cell lines SGC7901 cells and its spheres cells,the PRRX1 expression was highest in poorly differentiated human gastric adenocarcinoma cell lines MGC803 cells and its spheres cells.2.Both of the transcriptional level and the protein level results showed that the PRRX1 low expression lentivirus was stably transcribed and expressed in human gastric adenocarcinoma cell line MGC803,the lentiviral MGC803-shRNA-PRRX1 and MGC803-shRNA-NC stable cell lines were successfully constructed.The results of qRT-PCR,Western blot and immunofluorescence indicated that the expression of EMT marker E-Cadherin was significantly decreased after PRRX1 down-regulation,Vimentin expression was significantly increased,and the expression of other EMT transcription factors(N-cadherin,Snail,Twist)was also significantly increased.Therefore,down-regulation of PRRX1 can promote the occurrence of EMT in human gastric adenocarcinoma cells;The expression of tumor stem cell markers(Lgr5,CD44,Nanog,ABCG2,MSI1)were significantly increased,indicating that down-regulation of PRRX1 was closely related to CSCs and is related to the malignancy of tumors.3.Transwell assay,wound healing assay,MTT assay,soft agar plate cloning assay and others in vitro cell biological function experiments showed that PRRX1 down-regulation can promote invasion,metastasis,and proliferation ability in human gastric adenocarcinoma MGC803 cells;Sphere formation assay showed that PRRX1 down-regulation can promote sphere formation in human gastric adenocarcinoma MGC803 cells;The drug resistance assay results indicated that down-regulation of PRRX1 could increase the drug resistance of human gastric adenocarcinoma cell line MGC803 against cancer drugs and reduce the sensitivity of gastric adenocarcinoma cells to drugs;The results of flow cytometry assay showed that down-regulation of PRRX1 disturbs the cell cycle and promoted the progression of gastric adenocarcinoma cells from G0/G1 phase to the S phase and promote cell proliferation.In conclusion,down-regulation of PRRX1 induces the occurrence of EMT and is closely related to CSCs,it is also closely related to the occurrence and malignant development of human gastric adenocarcinoma.Down-regulation of PRRX1 promotes the invasion,metastasis,proliferation,non-adherent proliferation and drug resistance of human gastric adenocarcinoma cells,indicating that down-regulation of PRRX1 plays an important role in gastric cancer metastasis and drug sensitivity.Our results also suggest that PRRX1 may belong to one of the cancer-resistance genes.This study greatly promoted our understanding of molecular mechanism of PRRX1 in gastric adenocarcinoma development and progression,and laid a molecular foundation for targeted therapy of gastric cancer. |
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