The Mechanism Of BICC1 Promotes Pancreatic Tumor Cells Metastasis And Invasion Under Hypoxia Microenvironment

BackgroundBICC1,also known as Bicaudal C(Bicaudal C),was first studied in drosophila melanogaster in 1975 by professor Raden.The mouse BICC1 gene is derived from the fruit fly BICC1 gene.The function of BICC1 in mice is also clear.The mouse BICC1 protein contains three KH domains,and other proteins containing the KH domain and BICC1 studies involving drosophila and xenopod indicate that this domain interacts with RNA.BICC1 is involved in the shear and degradation of RNA and plays an important role in the maintenance of morphology and function of some organs such as kidneys and pancreas.Our previous experimental results showed that BICC1 expression was significantly increased in the surgical specimens of pancreatic adenocarcinoma patients and pancreatic tumor cell lines compared to normal human pancreatic tissue and catheter cells.We in order to further explore BICC1 role in tumor design the experiment,we explored the BICC1 in pancreatic cancer,the effects of high expression of the prognostic significance of survival and BICC1 high expression and the clinical parameters,the relationship between the regulatory mechanism of behind,purpose is including clinical level,cell function,molecular biology and animal experiment levels explore BICC1 function and significance in pancreatic ductal adenocarcinoma.Method1.Paraffin samples of pancreatic adenocarcinoma tissue from the tumor hospital of tianjin were used.First,continuous sections of BICC1 and HIF1α were subjected to spatial co-localization staining.Combined with case survival and prognosis information of paraffin specimens,the expression level of BICC1 protein was comprehensively correlated with all clinical pathological parameters.2.Expression BICC1 and HIF1α adults in the pancreatic tumor cell lines commonly used in this laboratory was detected by using classic molecular biological techniques such as Western Blot and real-time quantitative PCR,and the stable overexpression downexpressed cell lines were constructed according to the expression conditions.Immuno Fluorescence was used to detect the spatial localization of BICC1 in pancreatic tumor cells.Protein-chromatin immunoprecipitation(CHIP)was used to study the correlation between HIF1α dissection and BICC1 expression in pancreatic tumor cells.3.The BICC1 overexpression plasmid and BICC1 short hairpin RNA down-expression plasmid were constructed by the common standard molecular cloning method,and the lentiviral stable transfection system and stable transfection cell lines were constructed.Using Western Blot and real-time quantitative PCR assay,BICC1 and HIF1α were established.The biological functions of BICC1 with overexpression and healthy expression were detected by cell Wound healing,migration and invasion experiments.4.The influence of BICC1 on the movement ability of pancreatic tumor cells in hypoxia environment was studied by using the hyperexpression and health expression BICC1 stable system.The RNA targets of BICC1 for tumor metastasis were screened by transcriptome sequencing rna-sequence.Western Blot and were used for real-time quantitative PCR sequencing.The influence of BICC1 on the movement ability of tumor cells was further determined by block test.5.The molecular mechanism of BICC1 regulatory target was detected by Western blot,rt-pcr and immunofluorescence experiments.6.To construct an in situ tumorigenesis model of pancreatic tissue in NOD-SCID mice;The effect of BICC1 on tumor metastasis of pancreatic cancer was examined in vivo.The results1.The expression intensity of BICC1 in PDAC of pancreatic ductal adenocarcinoma and its corresponding paracancerous normal pancreatic tissue IHC immunohistochemical staining and its significance.We through to the tianjin cancer hospital to collect 120 cases of surgical specimens in BICC1 dyeing and its clinical relevance analysis of paraffin section,and two random from the above cases including 8 cases of surgery paired samples Western Blot,real-time quantitative PCR,found that compared with normal pancreatic tissue adjacent to carcinoma BICC1 expressed in pancreatic tumor volume significantly higher,and the expression of BICC1 levels associated with the survival prognosis of pancreatic cancer,a negative correlation(p = 0.027),We also analyzed The data from TCGA(The Cancer Genome Atlas),and The conclusion was consistent with our immunohistochemical staining results.We further analyzed and compared BICC1 expression level and various common clinical and pathological parameters,and found no statistical difference in BICC1 and pancreatic tumor size,primary lesion infiltration T stage and tumor cell malignancy G stage.In contrast,N staging and UICC-TNM staging were significantly different from lymph node metastasis.Similarly,data from the TCGA database also gave the same answer,suggesting that BICC1 expression is not correlated with tumor T staging,tumor size,and tumor differentiation,and has statistical significance with N staging and TNM staging.The above information suggests that BICC1 may affect the pancreatic cancer metastatic.2.BICC1 plays a role in the progression of pancreatic cancer by regulating the motor capacity of pancreatic tumor cells.Based on BICC1 expression in the commonly used pancreatic cancer cell lines in this laboratory,we successfully constructed two cell stable lines(BxPC-3 and CFPAC-3)with a healthy expression of BICC1,and two cell lines with overexpression of BICC1(BxPC3,ASPC-1).Based on the previous results of immunohistochemistry and TCGA database,we speculated that BICC1 might have an effect on the movement of tumor cells.We will drop of stable expression cell lines for regular training and the control group,the conventional Wound healing experiment on ranging experiment Migration,Invasion and Migration night ever BICC1 express group compared with control subjects both the distance of the movement and Migration of cell count were significantly higher,and low expression ability is poorer.Stable overexpression and in-situ tumor formation in the pancreatic tissue of the control group were used to observe tumor metastasis.It was found that compared with the control group,the number of liver metastases occurred in the over-expression BICC1 group was higher,and the volume of metastatic tumor was larger.3.BICC1 regulates the motor capacity of pancreatic tumor cells through ZEB1 to affect the progression of pancreatic cancer.We performed RNA-seq on BICC1 pancreatic cancer cell line bxpc-3,and the results were analyzed.ZEB1 expression level was significantly reduced,and the two were correlated and positively correlated in the RNA expression level.ZEB1 is currently known to be a strong factor that can affect tumor metastasis.We used Western Blot and real-time quantitative PCR to detect ZEB1 expression in overexpression and down-expression BICC1 stable system,and we found that ZEB1 was indeed correlated with BICC1.This also confirmed that BICC1 affected the movement of tumor cells through ZEB1.4.BICC1 is regulated by HIF1α transcription.Hypoxia is a common microenvironmental feature of all solid tumors,and pancreatic cancer is also a solid tumor,while HIF1α can regulate a variety of genes that affect tumor growth and metastasis against apoptosis.We performed hypoxia treatment on the commonly used pancreatic tumor cell lines in our laboratory,and conducted Western Blot and real-time quantitative PCR experiments,the results indicated that BICC1 expression was significantly increased when the pancreatic cancer cell lines were treated in the hypoxia.In the hypoxic environment of pancreatic tumor,HIF1α was the major contributor.We constructed HIF1α perexpression downregulated and stable overexpression downregulated cell lines.Western Blot and real-time quantitative PCR by referring to the general database,we analyzed and predicted that the structure domain of BICC1 promoter region could be combined with HIF1α.We designed the CHIP and dual-luciferase report,and the results proved that HIF1α primer can indeed bind to BICC1 promoter region and start the transcription of BICC1,increasing BICC1 expression.5.BICC1 expression is correlated with HIF1α substrate expression.By the pancreas tumor surgery patients specimens of paraffin serial section,We done BICC1 and HIF1α immunohistochemical staining,space positioning,and dyeing intensity and area of grading,eventually were classified,and through TCGA database in pancreatic cancer and other common several kinds of tumors at the RNA level correlation analysis,the results found BICC1 expression and HIF1α expression correlation,a positive correlation.6.The effect of BICC1 on the metastasis of pancreatic carcinoma in situ was observed in mice.The over-expression BICC1 cell line and the down-expression ZEB1 cell line of the PanC02 cell line of mouse pancreatic cancer were constructed,and the cell lines were used for in-situ tumor formation of mouse pancreatic tissue.Conventional feeding conditions were used to observe the metastatic tumor.The experimental results showed that the number of liver metastases of the over-expressed BICC1 cell line was significantly higher than that of the control group and the number of liver metastases was decreased after the removal of ZEB1 compared with that of the over-expressed BICC1 group.The experimental results showed that BICC1 promoted tumor metastasis by regulating the expression of ZEB1.Conclusions1.Abnormally high expression of BICC1 in pancreatic cancer tissues and pancreatic tumor cell lines;The expression level of BICC1 in pancreatic tumor tissues was negatively correlated with the prognosis of the patients.2.BICC1 overexpression will be combined with ZEB1 mRNA,resulting in increased ZEB1 expression,thus enhancing the mobility of pancreatic tumor cells.By promoting the movement ability of tumor cells,the ability of tumor metastasis is enhanced.3.HIF1α replication can regulate and increase the transcription of BICC1,increase the level of BICC1 protein,and play a role in promoting the metastasis and movement of pancreatic tumor cells.