Study On The Biological Function Of Bit1 And Its Potential Mechanisms In Gastric Cancer

Background:The latest global cancer data of《CA: A Cancer Journal for Clinicians》in 2018 showed that the incidence of gastric cancer ranked fifth and the mortality ranked third among malignant tumors.Predisposition to distal metastasis is the key factor for the poor prognosis of gastric cancer patients.Therefore,seeking novel biomarkers for gastric cancer and further exploring its molecular mechanism are significance for the early and effective diagnosis of gastric cancer and improving the prognosis of patients.The anoikis effector molecule Bit1 is aberrantly expressed in a variety of tumors,which can suppress or promote cancer..Previous studies have preliminarily shown that the expression of Bit1 was low in normal gastric tissues by immunohistochemistry,while the expression of Bit1 was significantly increased in gastric cancer and atypical hyperplasia tissues,suggesting that Bit1 may be involved in the occurrence and development of gastric cancer.But so far,there is no related research on the effect of Bit1 on the biological function of gastric cancer cells and its specific mechanism in gastric cancer.Purpose:The purpose of this study is to investigate the expression and clinical significance of Bit1 and clarify the biological function of Bit1 in gastric cancer.Importantly,it is the first time to reveal the specific mechanism of Bit1 in gastric cancer with Bit1 high expression,which provides novel ideas for clarifying the related research of gastric cancer targeted by Bit1.Methods:The expression and clinical significance of Bit1 in gastric cancer were determined by Western blot,WES,q RT-PCR,immunohistochemistry of tissue chip and Bioinformatics analysis.The gastric cancer cell line BGC-803 with endogenous high expression of Bit1 was screened and identified by Western blot;BGC-803 cells were transfected with lentivirus to interfere with the expression of endogenous Bit1;Wound healing assay and transwell assay were used to analyze the effect of Bit1 on the migration and invasion of gastric cancer cells;TUNEL assay and flow cytometry were used to analyze the effect of different levels of Bit1 expression on gastric cancer cell apoptosis;CCK-8 cell proliferation assay was used to analyze the effect of silencing Bit1 on the proliferation of gastric cancer cells;Transmission electron microscopy was used to detect the effects of Bit1 on the mitochondrial morphology of GC cells.3.Bioinformatics analysis suggested that Bit1 might be associated with genes which were related to EMT in gastric cancer.Immunofluorescence assay and Western blot were performed to detect the effects of different levels of Bit1 expression on gastric cancer cell proliferation,apoptosis-related molecules and EMT marker protein expression,to clarify whether Bit1 can affect the progression of gastric cancer by acting on EMT.4.Bioinformatics analysis suggested that Bit1 might be associated with MMPs and integrin β1 in gastric cancer.Western blot was used to detect the expression of MMP-2,MMP-9 and integrin β1 in gastric cancer cells with different levels of Bit1 expression,suggesting that Bit1 in gastric cancer with Bit1 high expression may enhance the progression of gastric cancer by regulating the expression of MMPs and promoting the degradation of ECM.Results:1.The expression and clinical significance of Bit1 in gastric cancerThe mRNA and protein expressions of Bit1 in gastric cancer tissues and corresponding adjacent tissues were detected by Western blot,WES,RT-q PCR,the results showed that Bit1 expressions in GC tissues were significantly up-regulated compared with tumor adjacent tissues.Immunohistochemical staining showed that the expression level of Bit1 in gastric cancer was closely related to the tumor pathological grade and lymphatic invasion;Bioinformatics analysis showed that PTRH2 was highly expressed in GC and was positively correlated with the tumor stage,lymph node metastasis,gender,Helicobacter pylori infection and prognosis TP53.STRING database analysis found that PTRH2 may be correlated with CDH1,CDH2,VIM,TJP1,MMPs,ITGB1,VEGFC,FIGF,KDR,FLT1,FLT4;KEGG enrichment analysis showed that PTRH2 was negatively correlated with extracellular matrix receptors and positively correlated with cholesterol metabolism.2.The effect of Bit1 expression level on the biological function of gastric cancer cellsBGC-803 cell lines with highly expression of Bit1 was selected for Bit1 sh RNA lentiviral experiments to knock down endogenous Bit1 expression.Wound healing assay showed that wound healing rate of the GC cells treated with Bit1-specific sh RNA was significantly lower than the cells in WT and Vector groups at 24 h and 48 h.Transwell assay showed that the number of cells invading from the upper surface to the lower surface in Bit1 sh RNA1 and Bit1 sh RNA5 groups were significantly reduced,compared with WT and vector groups,indicating that Bit1 down-regulation could significantly inhibit the invasion of gastric cancer cells;CCK-8 assay detected that Bit1 downregulation could significantly inhibit the proliferation ability of gastric cancer cells.Flow cytometry and TUNEL were used to detect the effect of different Bit1 expression levels on apoptosis.The results showed that Bit1 down-regulation could significantly promote the apoptosis of gastric cancer cells;Transmission electron microscopy results showed that transfected with Bit1-specific sh RNA,the GC cells displayed swollen and irregular mitochondria with seriously dilated and disrupted crista.3.Down-regulation of Bit1 can inhibit EMT progression,and thereby hinder the progression of gastric cancerSTRING database analysis found that PTRH2 may be correlated with CDH1、CDH2、VIM、TJP1,which encodes EMT protein.Western blot and immunofluorescence showed that silencing Bit1 could down-regulate the expression of PCNA,Ki67 and Bcl-2,and up-regulate the expression of Bax;Silencing Bit1 could up-regulate the expression of Ecadherin and ZO-1,but inhibit the expression of vimentin and N-cadherin,suggesting that silencing Bit1 might inhibit the progression of gastric cancer by inhibiting EMT.4.Bit1 may inhibit the progression of gastric cancer by affecting the expression of integrin and MMPsThe results of Western blot showed that silencing Bit1 could inhibit the expression of MMP-2,MMP-9 and integrin-β1,and then hinder the progression of gastric cancer.Conclusions:1.Bit1 is highly expressed in gastric cancer tissues,and its expression level is closely related to lymphatic invasion and pathological grade.2.Down-regulation of Bit1 expression can significantly promote the apoptosis of gastric cancer cells inhibit the proliferation,migration and invasion,destroys the mitochondrial morphology in GC cells.3.Down-regulation of Bit1 can inhibit gastric cancer progression by regulating proliferation,apoptosis related molecules by hindering EMT progression.4.Down-regulation of Bit1 can inhibit the expression of MMP-2,MMP-9 and integrin β1,which indicates that Bit1 in gastric cancer with Bit1 high expression may promote the progression of gastric cancer by regulating the expression of MMPs,and then promoting the degradation of ECM.