The Expression And Clinical Significance Of TXNIP In Breast Cancer

The process of tumor formation is a multi-stage,complicated process and involves the activation of proto-oncogenes and the inactivation of tumor srppressor gene.The proto-oncogene is not carcinogenic,when the body was stimulated by bacteria,viruses,physical and chemical factors,the proto-oncogene was activated and then the tumor formation.There is a kind of tumor suppressor genes in human body which reponsibility of the normal cell division,dysplasia,hyperplasia and cancerous.The funtion of suppressor genes was lost when the suppressor genes was attracted by virus infection and ionizing radiation damage.The changes lead to normal balance of cell division and proliferation is broken, cell division and proliferation is uncontrollable.At last,the abnormal hyperplasia occurs and cell mutates, tumor formation.In recent years, several studies have shown that TXNIP genes take important roles in tumor progression and metastasis. TXNIP genes is a new tumor suppressor genes, which has decreased in several human cancers such as liver cancer, lung cancer. TXNIP is related to tumor metastasis because the proliferation of tumor cells and the inhibition of apoptosis process were cause by TXNIP gene loss.With the completion of the human genome project, a vartety of genetic screening has become hotspot,exploring tumor progression and metastasis-related genes on theprognosis of the tumor have important guiding sifnificance to guide treatment measures and to development new therapeutic measures.BackgroundsBreast cancer is one of the most common malignant tumors in women, accounting for7%-10%of the whole body in all kinds of malignant tumors in our countryr It is reported that breast cancer of women tend to be NO.1in the malignant tumors in our country some big cities.The incidence of breast cancer at the age of20is very low, but rapidly rising after20years old.The incidence of breast cancer is high at45to50years old. The incidence of postmenopausal continues to rise,it’s may be associated with the improvement of the estrone levels of the old ladies.A higher incidence of breast cancer in first menstrual flow at an earlier age or post-menopausal at a later age,and late childbirth (first child after age35) or not feeding after birth.In recent years, along with the improvement of diagnosis and treatment of breast cancer.The5-year survival rate in postoperative patients of breast cancer,and the quality of life of postoperative breast cancer patients are all has greatly improved, but the effects of the treatment of advanced breast cancer remains to be improved.Therefore, in order to early discovery, early treatment and improve the clinical curative effect, study on breast cancer risk factors has become one of the focus of clinical research.Thioredoxin binding protein (thioredoxin-interacting protein and TXNIP), also known as vitamin D3increase protein1(vitamin D3up-regulating proteinl, VDUP1), is one of the members of a-rhodopsin repressor protein family, and is the only one protein in the family can combined with thioredoxin (Trx thioredoxin).TXNIP can inhibite resistance function of the active Trx cysteine residues oxidation through the combination with the active Trx cysteine residues, at the same time involved in regulating the body active oxygen system and process of mitochondrial pathways, inducing cell apoptosis, expression TXNIP in cell mediated role in the process of development. Coding TXNIP genes in chromosome1q21-22, contains8exons,4174bp long. TXNIP genes was found for the first time in human leukemia cell line HL-60which treatment with1,25(2)25-hydroxyitamin D-3. Some studies have shown that TXNIP genes is related to tumorigenesis and metastas by out of control of cell proliferation cycle and NK cell dysplasia.Currently reports on TXNIP expression in breast cancer is rare,and there is no universally accepted conclusion in relationship with clinical pathological characteristics and TXNIP. Now,in the study expression level of TXNIP mRNA was detected by qRT-PCR in16breast cancer tissues and matched paraneoplastic tissues, The expression level of TXNIP protein was detected by Western blot in7breast cancer tissues and matched paraneoplastic tissues, The expression level of TXNIP protein was also detected by immunohistochemistry (IHC) in50breast cancer tissues and matched paraneoplastic tissues. Using SPSS13.0statistical software, respectively discusses breast cancer patients’ age, clinical stage, histologic differentiation,TXNIPmRNA expression levels in breast cancer and matched tissue adjacent to carcinoma, and TXNIP protein of expression level in breast cancer and cancer adjacent tissues and, etc.Methods1. The object of researchIn this study we collected50cases of breast cancer and matched tissue adjacent to carcinoma which saved by Nanfang hospital, Southern Medical University between In2011-2012.All of the patients did not receive chemotherapy or radiotherapy Before the surgery.The pathological diagnosis Infiltrating ductal carcinoma.Among them,there are29cases of patients younger than50years of age and21cases of patients older than50years of age.The average age is44.68years old.There are20cases in Ⅰ～Ⅱ TNM stage and30cases in Ⅲ～Ⅳ TNM stage.There are26cases of The high differentiation breast cancer,24cases of The low differentiation breast cancer,Tissue samples wax block is conventional slice2. The methods of the researchA.The express of TXNIP mRNA which tested by QRT PCR method.a. Sample preparation of total RNA:PBS cleaning tissue samples,cut up,grinding it in the liquid nitrogen.Then,extraction of total RNA in tissue cells by TRIZOLmethod,use ultraviolet spectrophotometer and agarose gel electrophoresis to extract the integrity, purity and content.b.a cDNA synthesis,Before the reverse transcription join in6.5ul directly to RNA Reverse transcription in the system to Synthesis of cDNA.Reaction system is10ul,Among them,PrimeScript RT Enzyme Mix10.5ul、5xPrimeScript Buffer2ul、 Oligo dT Primer(primers)0.5ul。Reverse transcription by The PCR instrument.Reaction conditions is:37℃15min,85℃5s.c.c.qRT-PCR using SYBR Green fluorescent dye method to A real-time quantitative PCR amplification.The PCR reaction system is20ul,among them,2×SYBRpremixEXTaq TM10ul,upstream and downstream primers are both0.4ul (10umol/L)、cDNA2ul, dH2O7.2ul。TXNIP and beta actin PCR reaction Are separatelied by a real-time quantitative PCR.Amplification conditions is:95℃pre modified30s;58℃95℃degeneration10s, annealing15s,72℃extension of10s,45cycle after72℃for10min.At the same time,make a positive control during the reaction process.Use water instead of cDNA to negative control.Calculate theTXNIP relative expression of mRNA by2-ΔCT.ΔCT =CTTXNIP-CTβ-actin。tXNIP primer used in the sequence:the upstream is5’-GACTTCGGAGTACCTGCGCTATG-3’,the downstream is5’-AAGCTCAAAGCCGAACTTGTACTCA-3’, the beta actin primers sequences:the upstream is5’-TGG CAC CCA GCA CAA TGA A-3’,,the downstream is5’-CTAAGT CAT AGT CCG CCT AGAAGC A-3’).B.Use western blot detecte TXNIP protein expressiona.extract the total protein in breast cancer tissue and the corresponding tissue adjacent to carcinoma,cut up,use PBS to clear up three times,grinding it in the liquid nitrogen,at last,mix200ul protein pyrolysis liquid to have Ice bath cracking.Extract Proteins of cells.b.Accorde to the50ug/hole on the sample,electrophoretic separation in10%sds-page gel,transfer protein from Sds-page gel to PVDF membrane by turning wet method,then,Enclosed it for90min at room temperature in TTBS containing5%skimmed milk powder,mix A fight(rabbit anti TXNIP antibodies, dilution degrees of1:200).incubate it for the night,4℃,use TTBS to fully rinse (10min,3times),mix Sheep rabbit IgG, ready-to-use,keep it in37℃role for40min,use TTBS to fully rinse (10min,3times),use the chemical fluorescence (ECL) color, tabletting, developing, fixing and observe the results.C.Immune histochemical methodUse immunohistochemical SP method,make conventional dewaxing hydration of Near the cancer of breast biopsy for breast cancer and matched,mix0.01mol/L citric acid buffer, repair antigen by microwave repair method,Cool to room temperature,use3%of hydrogen peroxide to block endogenous peroxidase,incubate it for15min,use PES to clear up it for three times,3min for once,mix Goat serum closed fluid,incubate25min at room temperature,mix50ul rabbit anti TXNIP monoclonal antibody (1:100),4℃incubate for the night,use PBS to clear up it for three times,every time for5min,Add50ul Sheep rabbit IgQ ready-to-use,incubation for45min at room temperature,use PBS to wash it for5times,every time for5min,DAB chromogenic1～3min, haematin redyeing, conventional dehydration, transparent, neutral rubber sealing piece.At the same time set the Yin, positive controlb. Semi-quantitative result judgment and Immunohistochemical staining,No tan is zero; Light tan is1; Tan is two points; Brown is three points.The same observed under optical lens positive cells number.No positive cell number is0; Positive cells was10%or less is1; Positive cells was11%～50%is two points; Positive cells number>50%is three points.The dyeing depth and number of positive cells percentage score multiplied, the above2points, and is positive.c.Statistical processing,use SPSS13.0software,use2inspection,if the P<0.05,it have statistically significance。Results1.The TXNIP mRNA level of QRT-PCR detectionAfter testing TXNIP mRNA expression levels of16cases of breast cancer tissue and its corresponding tissue adjacent to carcinoma, we can found that TXNIP mRNA levels of average have no Obvious difference (P>0.05) between breast cancer alongside with the corresponding carcinoma tissue.2. The Western blot detection of TXNIP protein expression levelsAt the same time,we Randomly selected7cases of breast cancer and its corresponding samples of breast tissue adjacent to carcinoma,the western blot analysised TXNIP expression level difference between them,we could find tXNIP levels in breast cancer tissueis inferior to levels in normal tissue adjacent to carcinoma in5cases in7cases of samples,and in Other2cases in7cases of samples,tXNIP protein content had not significantly change.(As shown in the figure1)3.The expression level in breast cancer tissue of TXNIP immunohistochemical detectionA.TXNIP expression in the tissue adjacent to breast cancer and breast cancerBoth of them have expressed.The situation which positive dyeing is tan diffuse or granularmainly distributed in the cytoplasm,part of the nucleus is also expressed.The positive expression rate of breast cancer tissue adjacent to carcinoma is72.00%,the positive expression rate of breast cancer is52.00%,there are significant difference,P<0.05.(as shown in the figure2to4).B.The relationship between TXNIP and clinicopathological features of breast cancerI-II TXNIP protein positive expression rate of breast cancer is80.00%.Ⅲ～Ⅳ TXNIP protein positive expression rate of breast cancer is33.33%,there are significant differences (P<0.05) between stage Ⅰ～ⅡI and Ⅲ～Ⅳ TXNIP protein expression level of breast cancer. The tXNIP protein positive expression rate of Ⅰ～Ⅱ phase matching the tissue adjacent to carcinoma of breast cancer is85%,there is no significant difference in the TXNIP protein expression levels of Stage Ⅰ～Ⅱ breast cancer and its matching tissue adjacent to carcinoma. The TXNIP protein positive expression rate of stage Ⅲ～Ⅳ breast cancer matched tissue adjacent to carcinoma is63.33%,the TXNIP protein expression levels of stage Ⅲ～Ⅳ breast cancer and its matching tissue adjacent to carcinoma has significant difference (P<0.05). The Positive expression rate of the TXNIP protein high differentiation of breast cancer is73.07%, the positive expression rate of low differentiated TXNIP protein in breast cancer is29.16%. the tXNIP protein expression levels of the middle differentiation and the low differentiation of breast cancer has significant difference (P<0.05). The positive expression rate of high differentiated tissue adjacent to carcinoma of breast pairing TXNIP protein is73.07%, There was no significant difference in the tXNIP protein expression levels of the high differentiation of breast cancer and its matching tissue adjacent to carcinoma.The TXNIP positive expression rate of low differentiated tissue adjacent to carcinoma of breast pairing is70.83%,the tXNIP protein expression levels of low differentiation of breast cancer and its matching tissue adjacent to carcinoma has significant difference (P<0.05).Conclusions①The expression levels of TXNIP mRNA were not consistent with the expression levels of TXNIP protein in cancerous and matched paraneoplastic tissues.②The positive rates of TXNIP were associated with breast cancer clinical stages and histological differentiation. The earlier stage, the higher the degree of differentiation, the expression level of TXNIP protein was higher. The observations suggested that TXNIP protein might be a valuable tumor suppressor.