Studies On The Upregulation Expression Of SSBP1 In Colorectal Cancer Cells Affecting The Viability Of Tumor Cells By Regulating Mitochondrial Mass

Colorectal cancer(CRC)is one of the most common cancers in the world.Its morbidity and mortality are in the forefront of many carcinomas.Despite the progress and application of many treatment approaches,it is still difficult to be cured thoroughly and be satisfactory with a 5-year survival rate.Although great efforts have already been made in precise medicine and gene associated study in recent years,the mechanism of tumor growth and development is still not fully clear.As we all know,the energy supply way of cancer cells is mainly tend to anaerobic respiration,which is not a common way in normal colonic epithelium cell.However,the molecular mechanism that mediates this process,especially the process and mechanism of molecular interaction within,upon,and surrounding mitochondria,has not been totally known.At present,the mechanism and importance of mitochondrial volume(mass)and oxidative phosphorylation(potential)for colorectal cancer cell proliferation and drug resistance are not clear.So far,many literatures have reported the pathways of tumor drug resistance.However,the role of mitochondria in tumor drug resistance and cell oxidative stress is rarely described in details.Although mitochondrial damage is common after chemotherapy,there is no consensus on whether it is related to the ability of tumor proliferation and division and the sensitivity to chemotherapy.In this report,we have generated a representative proteomic map of three pathological stages(stage?,stage?,stage?)and adjacent normal intestinal tissues of CRC.In each stage,we have collected the proteomic analysis results of multiple patients,in order to find possible common differences in a larger cohort.From these maps,we canrecognize the significant changes of mitochondrial protein expression pattern,especially the key regulatory factor SSBP1.The absence of SSBP1 in colon cancer cell lines can trigger the decline of mitochondrial quality and the increase of tumor cell death,and this effect is further strengthened in the presence of cisplatin.These results suggest that mitochondrial biosynthesis and maintenance may play an important role in the survival of tumor cells,and that SSBP1 may be the next treatment target in the case of drug resistance.Main experimental steps:1.The colon cancer tissues and adjacent normal intestinal tissues were collected.The patients were grouped according to the pathological stage.2.Proteomic analysis was carried out according to groups.According to the results,gene expression thermogram was drawn and Pearson correlation method was used to compare the pathological stages.The analysis of the main components of the samples confirmed the correlation between the tumor group with different pathological stages and the normal sample group.Meanwhile,the mucinous adenocarcinoma tumor samples were also separated from other samples.In addition,the volcano map was used to show the differentially expressed genes between normal and tumor samples.3.According to the data of cell morphology in proteomic results,the network analysis of the up-regulated proteins in tumor samples was carried out in the database Reactome,and several highly related proteins were identified.4.Gene set enrichment analysis of KEGG pathway was used to identify the differential expression of multiple cell pathways between normal tissues and tumor samples.5.The thermogram of mitochondrial gene expression was drawn to show the correlation between the expression profile of each gene and the expression level of all mitochondrial proteins.The volcanic map shows the most unstable genes expressed in all tumor and health samples,including high and low expression genes.6.According to the complete list of mitochondrial protein expression,network analysis was carried out by using a database called Reactome to establish the relationship between various proteins.7.TCGA data were collected and matched with single cell RNA sequence.The proteomic data we observed were compared with TCGA data.The expression level between normal and primary tumor samples was calculated by violin map,and the t-SNE visualization map of single cell sequencing data set of colorectal cancer samples was drawn.Carry out secondary verification.8.According to the above results,SSBP1 gene was identified as the main research object.9.Two colon cancer cell lines SW480 and HT29 were selected.SSBP1 sirna was customized.SSBP1 si RNA was transfected into SW480 and HT29 cell lines by liposome and SSBP1 was silenced.10.The validity of ssbp1-sh was confirmed by Western blotting and RT-PCR.11.SW480 colon cancer cell lines were divided into ssbp1-nc group,ssbp1-sh group,ssbp1-NC-cis group and ssbp1-sh-cis group.ATP concentration and mitochondrial ROS were measured respectively.12.HT29 colon cancer cells were divided into four groups according to the previous step.The concentration of adenosine triphosphate(ATP)and mitochondrial related reactive oxygen species(ROS)were detected.13.In HT29 colon cancer cell line,the expression of Bax and bcl-2 protein in the above four groups were detected.14.In SW480 and HT29 cell lines,flow cytometry was performed by 7-AAD staining.SSBP1 was depleted by targeting si RNA or scramb,and cell death was measured.The mitochondrial mass and potential of the two cell lines were evaluated by flow cytometry based on mitotic tracker dye.15.In order to observe the growth of cells,the scratch experiments of control group and si RNA group were carried out.16.The control group and si RNA group were added with cisplatin and 5-FU respectively for 24-hour cell culture.17.The tumor formation experiment was carried out in mice.The different responses of SW480 and HT29 cells to cisplatin and 5FU(tumor growth rate)in SSBP1 silenced and non-silenced colon cancer cell lines were observed.Main experimental results:1.In colon cancer,the type and quantity of protein expression are correlated with pathological stage.2.In colon cancer cells,there are some genes related to cell replication and synthesis,such as PCNA,EIF3 b and so on.In addition,there are significant increases in SERPINH1,TNC,S100A11,CEACAM5,HSPH1,FN1,NSUN2,LARs,PSME3,IPO5,PRKDC,STAT1,DDB1,RANGAP1 and so on.3.In colon cancer cells,there are some genes related to the surface protein of epithelial cells,such as col14a1,MUC2,DCN and so on.In addition,there are significantly decreased CA1,OGN,IGHA2,CLCA1,FCGBP,CA2,ACADM,DPT,FUCA1,RPL8,CTSZ,CTSS,APOBR,GALM and so on.4.In colon cancer cells,the expression of related genes in some pathways increased significantly.The first three pathways were aminoacryi tRNA synthesis,splice some and proteasome.At the same time,the expression of related genes in some pathways decreased significantly.The first three pathways were OXPHOS,BCAA degradation and lysosome.5.Compared with the normal tissues,the expression of SSBP1,TRP1,PYCR1 and TOMM22 were up-regulated,ATP5C1,ACADS,ACADM,MAOA,CKB,VAT,HMGCS2,NMES1 and CASP1 were down regulated.6.In colorectal cancer cells,the expression of SSBP1 is directly involved in the regulation of mitochondrial quality and tumor cell activity.7.There was no significant correlation between the expression of SSBP1 and mitochondrial potential in colon cancer cells.8.When 1 Mm cisplatin was added to colon cancer cell line,the regulation effect of SSBP1 was greater,that is to say,after SSBP1 was knocked down,the death rate of tumor cells was higher.9.When 1uM/ml 5-Fu was added to colon cancer cell line,the regulation effect of SSBP1 was not obvious.10.After SSBP1 was silenced,the growth rate of tumor cells was slowed down.11.Silencing SSBP1 in colon cancer cells can lead to mitochondrial dysfunction(decrease of ATP production and increase of ROS content),and the effect is more obvious after adding cisplatin.12.SSBP1 is involved in the proliferation and apoptosis of colon cancer cells.Specifically,SSBP1 silencing can slow down the growth of tumor cells and increase apoptosis.The regulation effect was more significant after adding cisplatin.Conclusion:As an important mitochondrial gene,the expression of SSBP1 in colon cancer cells is significantly higher than that in normal tissues,and it is involved in the regulation of colon cancer cells growth.When chemotherapeutic drugs resistant occurs,especially cisplatin,SSBP1 may be an another treatment target plot to control the growth of tumor cells.