Clinical Significance Of BCAR1Expression In NSCLC And Its Effect On Biological Behavior Of A549Cells

Background and objectivesEvery year, there are1.35million new lung cancer cases in the world. Worldwide, lungcancer is the leading cause of cancer-related death. Due to the intricate biological functions,prognosis of lung cancer remains very poor. Hence, it is vital to unveil the biologicalfunctions of the disease for the sake of improving therapeutic efficacy.Breast cancer anti-estrogen resistance1(BCAR1), also entitled p130cas was one ofthe CAS protein (crk-associated substrate) family members, and involved in proliferation,apoptosis, motility, invasion and differentiation. BCAR1overexpression has beeninvestigated in breast, prostate, ovarian carcinoma and glioblastoma. In breast and ovarian,there was the negative relation between BCAR1expression and prognosis. For instance,Dorssers et al, using a specific enzyme-linked immunosobent assay (ELISA), investigatedand found that the abundance was inversely correlated with relapse-free survival and oversurvival time. In addition, Alpa et al suggested that increase BCAR1was associated withpoor clinical prognosis and silencing BCAR1expression decreased tumor growth throughstimulation of apoptotic cell death. A study revealed a role for BCAR1in enhancingmesenchymal invasive movement in glioblastoma cells via expression of the non-tyrosinekinase vascular endothelial growth factor (VEGF) receptor neuropilin1(NRP1).In conclusion, BCAR1play a vital role for tumorigenesis. To our knowledge, there isnot still been published any article about relationship between BCAR1expression andNSCLC. Herein, this study, firstly, will detect BCAR1expression in serum and tissue ofNSCLC, and construct RNAi lentiviral vector for BCAR1in order to infect A549cells; thestudy, secondly, will examined effect of silencing BCAR1on proliferation, motility,invasion and cell cycle of A549cells. Aim of this study will clarify that there is the relationbetween BCAR1expression and NSCLC. Finally we presume BCAR1may be a potentialtherapeutic target for NSCLC. Methods1. BCAR1serum levels were examined in65cases with NSCLC,28cases with benignpulmonary tumor,30cases with tuberculosis and40healthy persons using ELISA forinvestigating the relationship between BCAR1and clinicopathological features of NSCLC.2. BCAR1expression was inspected in70cases with NSCLC tissues and adjacentnormal tissues and28cases with benign pulmonary tumor by immunohistochemistry (IHC)and tissue microarray (TMA); P-P38and P38were also examined by the same method inorder to analyze association between BCAR1and both.3. Apoptotic index (AI) of70cases with NSCLC tissues was inspected by Tunel, andrelation between BCAR1and AI were analyzed.4. Designing and synthesizing four groups (PLVT350,PLVT351,PLVT352,PLVT353)of small interfering RNA (siRNA) target for human BCAR1, they were respectivelyinserted to the pMagic4.1with endonuclease. They were transformed into DH5α competentcells and positive clones were screened by PCR amplification and DNA sequencing. Afterthe positive clones were amplified and extracted, the positive clones and adjuvant plasmidssimultaneously infected293T cells in order to product, concentrate and calculate lentiviralparticles. Then, A549lung cancer cells were infected by the lentiviral particles; transfectionand interfering efficiency of the lentiviral particles was respectively assessed usingfluorescent microscope and Real-time PCR; BCAR1proteins were detected by Westernblot.5. Proliferation, motility and invasion of normal, control and inference group cellswere respectively detected by clone-forming assay, cell wounding assay and Transwellinvasion assay; flow cytometry (FCM) was used to detected cell cycle distribution ofaforesaid three groups; P-P38and P38expression of aforesaid three groups were examinedby Western blot.Results1. Serum BCAR1levels were significantly higher in NSCLC group than in benignpulmonary tumor group and in healthy controls(P=0.000). However, there was noappreciable difference of the levels between NSCLC group and pulmonary tuberculosisgroup(P>0.05). Serum BCAR1levels did not correlate with age, gender, lymph nodemetastasis (P>0.05). In spite of no significant difference of serum BCAR1levels between adenocarcinoma and squamous carcinoma (P>0.05), the level of bronchioloalveolarcarcinoma was significantly lower compared with other subtypes of NSCLC (P=0.02).Serum BCAR1levels gradually increased with the progression of tumor staging anddecreased after removal of the tumors. Serum BCAR1levels were obvious higher inpulmonary tuberculosis group than in either benign pulmonary tumor group or healthyperson group(P=0.000), positively and significantly correlated with the diameter of thetuberculosis lesion(rs=0.92, P=0.000), and decreased after removal of the tuberculosislesions. Serum BCAR1levels of cavitary pulmonary tuberculosis were higher incomparison with tuberculoma(P=0.000).There was no appreciable difference of serumBCAR1levels between benign pulmonary tumor group and healthy controls (P>0.05). Byreceiver operating characteristic (ROC) test, NSCLC and pulmonary tuberculosis can bedistinguished by using serum BCAR1level combining with tumor markers(P=0.000)insteadof sole use of serum BCAR1level alone(P>0.05).2. The positive expression rate of BCAR1protein was98.6%,4.2%respectively inNSCLC and adjacent normal tissues. However, there was negative expression in benignpulmonary tumor. There was obvious difference in BCAR1protein positive expressionamong aforesaid three groups (χ2=143.93,P=0.000). BCAR1protein positive expressionwas not correlated with age, gender, lymph node metastasis, TNM stage and pathologicaltype(P＞0.05), was negatively correlated with the degree of neoplastic differentiation(χ2=13.639,P=0.001).BCAR1low expression were in favor of survival in comparison withBCAR1high expression(P=0.014).3. Four lentiviral vectors were successfully constructed; value of their viral titer:PLVT350for3.45×108TU/ml, PLVT351for2.13×108TU/ml, PLVT352for3.01×108TU/mland PLVT353for2.47×108TU/ml; the transfection efficiency of the lentiviral vectors ofA549lung cancer cells was more than90%; except PLVT350, other3the lentiviral vectorswere efficient targets (interfering rate of PLVT351, PLVT353and PLVT352wasrespectively82%,82%and73%). BCAR1expression of PLVT351-A549cell line wassignificantly low in comparison with the normal A549cells by Western blot(P<0.001).4. Compared with normal and control group, the clone-forming rate, healing rate andnumber of invading cells of interference group significantly decreased, but proportion of G1phase substantially increased; P-P38expression was lower in interference than other two groups rather than P38.Conclusions1. Serum BCAR1levels were significantly high in NSCLC than benign and controlgroup; its levels gradually increased with the progression of tumor staging and decreasedafter removal of the tumors. Serum BCAR1may be seen as new marker for diagnosis,assessing progression and therapeutic efficacy of NSCLC and pulmonary tuberculosis;Specificity of serum BCAR1for NSCLC can be increased by combining tumor markers.2. BCAR1expression was significantly high in NSCLC than adjacent normal tissues;there was the negative relation between BCAR1expression and differentiation andprognosis, but no significant correlation between BCAR1and age, gender, lymph nodemetastasis. In addition BCAR1expression was positively related to P-P38expression,however, negatively related to apoptotic index. BCAR1protein expression in NSCLC wassignificantly higher than adjacent normal tissues and benign pulmonary tumor,was alsonegatively associated with the degree of neoplastic differentiation and prognosis. Therefore,BCAR1protein might be used as new marker of NSCLC for diagnosis and prognosis.3. To Construct RNAi lentiviral vector for human breast cancer anti-estrogenresistance1(BCAR1) gene and detect its stable expression in A549lung cell line. RNAilentiviral vector for human BCAR1gene and its stable expression in A549lung cell line hasbeen successfully constructed and established, in order to facilitate further study on thedevelopment and progression of lung cancer.4. Proliferation, motility and invasion of A549cells significantly decreased bysilencing BCAR1gene. In addition, silencing BCAR1gene may result in increase of G1phrase and down-regulation of P-P38expression.BCAR1is a potential therapeutic target ofNSCLC...