Study On The Effect Of FAM111B On The Biological Behavior And Paclitaxel Resistance Of Triple Negative Breast Cancer And Its Mechanism

Objective: Breast cancer(BC)is a malignant tumor occurring in the mammary epithelium or ductal epithelium.The incidence of Breast cancer is increasing year by year.Among them,triple-negative breast cancer mostly occurs in young patients.Compared with other subtypes of breast cancer,the prognosis is worse.Even if surgery is performed as early as possible after early detection,the recurrence and metastasis rate is still high.Although the prognosis of breast cancer has been improved in recent years due to the continuous development of chemotherapy drugs,drug resistance in the process of chemotherapy has brought a great threat to breast cancer patients.More and more studies have suggested that chemotherapy resistance of breast cancer may be due to the relatively single target.Therefore,it is crucial to clarify the underlying molecular mechanism in the occurrence and development of BC and develop new therapeutic targets for improving the prognosis of breast cancer patients.FAM111 trypsin-like peptidase B(FAM111B,NM＿198947)is located on human chromosome 11Q12.1 and encodes a protein with a trypsin-like cysteine/serine peptidase domain at the C-terminus.Studies have shown that FAM111 B may be an oncogene,and relevant literature has shown that FAM111 B plays a cancer-promoting role in lung cancer,cervical cancer,and prostate cancer.However,so far,the role of FAM111 B in malignant tumors,especially in triple-negative breast cancer,is still unclear.Therefore,this project will study the expression of FAM111 B in triple-negative breast cancer,explore the correlation between the expression level of FAM111 B and the clinical prognosis of triple-negative breast cancer,and clarify the molecular mechanism of FAM111 B in promoting cancer in triple-negative breast cancer and its influence on paclitaxel chemotherapy resistance.To clarify the clinical application value of FAM111 B in the treatment of triple negative breast cancer.Methods: Methods: Part one: Analysis of the expression pattern of FAM111 B in breast cancer and its correlation with the prognosis of breast cancer.1.TGGA database was used to analyze the expression level of FAM1111 B in human pan-cancer tissues;Correlation between FAM1111 B expression level and breast cancer stage;2.Kaplan-meier Plotter database was used to analyze the relationship between FAM111 B expression and overall survival of breast cancer.3.Immunohistochemistry was used to detect the difference of FAM111 B expression between triple-negative breast cancer and normal breast tissues.Chi-square test was used to analyze the correlation between FAM111 B expression and clinicopathological parameters in triple-negative breast cancer tissues.Kaplan-meier method was used to draw the survival curve,and the relationship between FAM111 B expression level and overall survival was analyzed.Part Ⅱ: The effect and mechanism of FAM111 B on the biological behavior and paclitaxel resistance of triple-negative breast cancer in vivo and in vitro.1.The expression level of FAM111 B in human triple-negative breast cancer cell lines MDA-MB-231,MDA-MB-468,HCC1806,HCC1937 and human normal mammary epithelial cells Mc F-10 a was detected by Western blot.One cell line with higher FAM111 B expression(H)and one cell line with lower FAM111 B expression(L)were selected for subsequent experiments.2.The expression level and localization of FAM111 B in high and low expression cells were detected by immunofluorescence.3.FAM111 B overexpression plasmid(FAM111B-OE)and its control(Vector)were transfected into FAM111 B low expression cells.At the same time,two FAM111 B interference fragments(FAM111B si RNA1,FAM111 B si RNA2)and their control(NC si RNA)were transfected into FAM111 B high expression cells.The expression level of FAM111 B was detected by real-time q PCR and Western blot.Cell proliferation was verified by CCK-8 at 0 h,24 h,48 h,72 h and 96 h after transfection.48 h after transfection,the cell cycle distribution was detected by flow cytometry.48 h after transfection,the expressions of cyclin D1,cyclin E,PCNA and P16 were detected by Western blot.At 48 h after transfection,cell apoptosis was detected by flow cytometry(Annexin V/PI staining).At 48 h after transfection,Hoechst 33258 staining was used to detect cell apoptosis.48 h after transfection,the expression of Bax,Bcl-2,cleaved caspase-3,cleaved caspase-9,cleaved-PARP1 and PARP1 were detected by Western blot.4.FAM111 B low expression cells were treated with different concentrations(2,5,10,20 μmol/L)of paclitaxel(PTX)for 24 h,and the cell viability was detected by CCK-8.FAM111 B low expression cells were transfected into FAM111B-OE or Vector for24 h,and the cells were treated with the corresponding 1/2 concentration of PTX for 24 h,and the cell viability was detected by CCK-8.Cell apoptosis was detected by flow cytometry(Annexin V/PI staining).The expression of cleaved caspase-3,cleaved caspase-9,cleaved-PARP1 and PARP1 in cells was detected by Western blot,and the effect of FAM111 B on the sensitivity of breast cancer cells to paclitaxel chemotherapy was analyzed.5.To explore the effect of FAM111 B on the proliferation of triple-negative breast cancer cells in vivo by subcutaneous tumor implantation experiment in nude mice.Part Ⅲ: Transcription factor HOXC8 regulates FAM111 B expression.1.The binding site between HOXC8 and FAM111 B promoter was predicted online by the bioinformatics website JASPAR,suggesting that FAM111 B may be transcriptional regulated by HOXC8.NCBI was used to find the 2000 bp before transcription initiation of human FAM111 B as the promoter region.The expression vector of HOXC8 was constructed and transfected into cells.Double luciferase assay was performed to verify the transcriptional regulation of FAM111 B by HOXC8.2.Immunochromatin co-precipitation(CHIP)assay was used to verify the targeting relationship between HOXC8 and FAM111 B.3.HOXC8 triple-negative breast cancer cell line was constructed by sh RNA,and the knockdown efficiency was verified by real-time PCR and Western blot,and the expression changes of FAM111 B in m RNA and protein levels were observed.Results: Part Ⅰ: The expression pattern of FAM111 B in breast cancer and its correlation with the prognosis of breast cancer.1.Compared with non-tumor tissues,TGGA database analysis found that FAM111 B expression was significantly up-regulated in the following types of cancers.BLCA(bladder urothelial carcinoma),BRCA(breast invasive carcinoma),COAD(colon cancer),ESCA(esophageal cancer),LUAD(lung adenocarcinoma),LUSC(lung squamous cell carcinoma),PCPG(pheochromocytoma and paraganglioma),STAD(gastric cancer)and UCEC(endometrial cancer),KIRC(renal clear cell carcinoma),KIRP(papillary cell carcinoma of the kidney).In addition,the expression levels of FAM111 B in READ(rectal adenocarcinoma),THYM(thymic carcinoma)and SKCM(cutaneous melanoma)were significantly decreased compared with normal tissues.Based on the TCGA database,the expression of FAM111 B in breast cancer was significantly increased compared with adjacent breast cancer tissues.2.According to the database,the expression level of FAM111 B was also significantly increased in stage I-IV breast cancer compared with adjacent breast cancer tissues,with statistical significance between each group.FAM111 B was positively correlated with the stage of breast cancer3.Compared with breast cancer patients with low FAM111 B expression level,the overall survival of breast cancer patients with high FAM111 B expression level was significantly shortened.4.FAM111 B is positively expressed in triple-negative breast cancer tissues and cells,mainly expressed in the nucleus and also distributed in the cytoplasm.FAM111 B is negative or weakly expressed in normal breast tissues,while FAM111 B is positive in triple negative breast cancer tissues,and the expression level is higher than that in normal breast tissues.5.Through statistical analysis of clinicopathological data of patients with triple-negative breast cancer,the high expression level of FAM111 B in tumor tissues was correlated with the clinical stage and lymph node metastasis of patients.According to the survival follow-up data of 48 triple-negative breast cancer patients,the overall survival of patients with high FAM111 B expression was shorter than that of patients with low FAM111 B expression.Part Ⅱ: The effect and mechanism of FAM111 B on the biological behavior and paclitaxel resistance of triple-negative breast cancer in vivo and in vitro.1.The expression level of FAM111 B was the highest in triple-negative breast MDA-MB-468 cells and the lowest in triple-negative breast cancer HCC1806 cells by Western blot analysis.2.Immunofluorescence detection showed that FAM111 B was highly expressed in breast cancer cell lines,mainly located in the nucleus.3.After FAM111 B overexpression plasmid was transfected into low expression cells and FAM111 B was transfected into high expression cells,the expression level of FAM111 B was detected by real-time q PCR and Western blot,and the transfection efficiency was good.In HCC1806 cells,the cell activity of FAM111 B overexpression vector transfection group was significantly increased compared with that of empty vector transfection group.In MDA-MB-468 cells,the cell activity of FAM111B1/2 interference vector transfection group was significantly decreased compared with that of control transfection group.4.In HCC1806 cells,compared with the empty vector transfection group,the proportion of cells in G1 phase decreased,the proportion of cells in S phase increased,and the proportion of cells in G2 phase decreased.In MDA-MB-468 cells,the proportion of cells in G1 phase was increased,the proportion of cells in S phase was decreased and the proportion of cells in G2 phase was increased in the FAM111B1/2 interference vector transfection group compared with the control transfection group.In HCC1806 cells,the protein expression levels of cyclin D1,Cyclin E and PCNA in FAM111 B overexpression vector transfection group were increased compared with that in empty vector transfection group,while the protein expression level of P16 was decreased.In MDA-MB-468 cells,the protein expression levels of cyclin D1,Cyclin E and PCNA in FAM111B1/2interference vector transfection group were decreased compared with the control transfection group,while the protein expression level of P16 was increased.5.In MDA-MB-468 cells,the apoptosis rate of FAM111B1/2 interference vector transfection group was significantly increased compared with the control transfection group.The cell nuclei showed bright blue fluorescence.Compared with the control transfection group,the apoptosis rate of FAM111B1/2 interference vector transfection group was significantly increased.Compared with the control transfection group,the protein expression levels of Bax,cleaved caspase-3,cleaved caspase-9 and cleaved PARP1 were increased,and the protein expression level of Bcl-2 was decreased in the FAM111B1/2 interference vector transfection group.6.In HCC1806 cells,when the concentration of paclitaxel was 0.01 nmol/L,the cell inhibition rate of HCC1806 cells was not significantly different from that of the blank group.When the concentration of paclitaxel was 0.1,1,10,100,1000 nmol/L,the cell inhibition rate was significantly increased compared with the blank group.After paclitaxel treatment,the cell activity of FAM111 B overexpression vector transfected group was significantly increased compared with that of empty vector transfected group.After paclitaxel treatment,the apoptosis rate of FAM111 B overexpression vector transfected group was significantly reduced compared with the empty vector transfected group.After paclitaxel treatment,the protein expression levels of cleaved caspase-3,cleaved caspase-9,and cleaved PARP1 were decreased in FAM111 B overexpression vector transfection group compared with empty vector transfection group.7.The results of subcutaneous implant tumor model in nude mice showed that the tumor volume and mass of FAM111 B high expression group(0.78±0.21cm3,0.44±0.11g)were significantly higher than those of the control group.Part Ⅲ: Transcription factor HOXC8 regulates FAM111 B expression1.Double luciferase experiment proved that HOXC8 regulated FAM111 B transcription.2.CHIP experiment proves that HOXC8 can communicate with FAM111 B upstream sites 705-712: ACAATTAT sequence binding.3.After the expression of HOXC8 was inhibited by sh RNA,HOXC8 and FAM111 B were significantly decreased in RNA and protein.Conclusions:1.The expression level of FAM111 B in triple-negative breast cancer was significantly higher than that in normal breast tissue;FAM111B protein expression level is significantly correlated with clinical stage and lymph node metastasis of triple-negative breast cancer.The expression level of FAM111 B is negatively correlated with overall survival of triple negative breast cancer patients.2.FAM111 B is highly expressed in triple negative breast cancer cell lines,mainly expressed in the nucleus and also distributed in the cytoplasm.3.FAM111 B promoted the proliferation of triple-negative breast cancer cells,promoted the process of cell division cycle,and inhibited the apoptosis of tumor cells;High expression of FAM111 B increased the viability of triple-negative breast cancer cells under paclitaxel-induced treatment,inhibited cell apoptosis,and increased the sensitivity of breast cancer cells to paclitaxel chemotherapy.4.Transcription factor HOXC8 can bind to the upstream promoter region of FAM111 B to enhance transcription and increase FAM111 B expression,which promotes the development of triple-negative breast cancer.