The Long Noncoding RNA BCYRN1 Promotes The Tumorigenesis Of NSCLC Through Activating Wnt Pathway

Background and objectiveWith the acceleration of the process of urbanization,industrialization and globalization,the deterioration of the ecological environment,the changes in human lifestyles,and the effects of biological and genetic mutations,the incidence and mortality of malignant tumors in the world are on the rise.According to the World Health Organization,there were about 17.5 million new cases of cancer worldwide and 8.7 million deaths in 2015 which were about 67% of them in developing countries.At the same time,in China,4.3 million new cancer cases were reported that is the highest in the world and the number of cancer-related deaths exceeded 2.8million which accounted for 32 percent of the total in the world.The lung cancer is the leading common incident cancer and the top cause of cancer-related deaths among other types of cancers.Malignant tumor has become a serious threat to the safety of human life and social development of public health problems,so it is very important on prevention and treatment of lung cancer.Lung cancer consists of non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC),which non small cell lung cancer accounts for about 85%.As a result of the contribution of the improvement of operative methods,application of targeted drugs and inhibitors of check point,it prolongs the total survival in recent years.But owing to diagnosis lately and secondary drug resistance,recurrence and progression of tumor which 5-year survival rate is less than 20%.Therefore,exploring the pathogenesis of tumor,finding effective biomarkers for early diagnosis,monitoring drug resistance and evaluating prognosis,have become a hot spot in the research of lung cancer.Most studies have shown that the abnormality of gene expression and regulation is the internal cause and foundation for the occurrence,development and metastasis of lung cancer.Early researches on lung cancer are mostly focused on the mechanism of coding protein gene in the process of lung cancer transformation.However,with the development of gene sequencing technology,it is found that less than 3% of human genome encodes proteins,whereas 87.3% of human genome does not have the protein encoding capability,which would be transcribed into the non-coding RNAs(nc RNAs).Depending on their length,nc RNAs are further classified into small noncoding RNAs(< 200 nt in length)and long noncoding RNAs(lnc RNAs),which are largely polyadenylated and >200 nucleotides in length transcripts.It has been gradually recognized that these non-coding RNAs are involved in the gene regulation of transcription,post-transcription,epigenetic regulation and other pathways which have very important biological functions.In recent years,lnc RNAs not only involve in the development and early metastasis of lung cancer,but also play a vital role in the prognosis of lung cancer patients,chemotherapy and targeted drug resistance.Therefore,studying on the main regulatory mechanism of lnc RNAs in the occurrence and development of NSCLC will provide a new target and new approach for the prevention and treatment of NSCLC.In order to better research the relationship between lnc RNAs and tumors,we had collected the clinical specimens of patients with non-small cell lung cancer and the gene chip technology was used.The differentially expressed lnc RNA BCYRN1(Brain Cytoplasmic RNA1)was selected from lung cancer tissues and cancer-adjacent tissues.By detecting the expression of lnc RNA BCYRN1 in tumor and adjacent tissues of different types of non-small cell lung cancer patients,the clinical features of patients were recorded,and the relationship between lnc RNA BCYRN1 and tumor size,pathological type,clinical stage and other clinical features were observed.By culturing non-small cell lung cancer cells and human normal bronchial epithelial cells,extracting total RNAs,transient transfection of si RNA-BCYRN1,down-regulating the expression of lnc RNA BCYRN1,we observed the proliferation and migration of lung cancer cells after the silencing of target gene,even the effect of apoptosis and the expression of Wnt/?-Catenin signaling pathway targets.Methods1?Collection of the lung cancer tissue samples: During the period from January2016 to January 2017,The tumor tissues and adjacent tissues of primary lung cancer were collected from the patients underwent the radical resection of lung cancer in ××× Hospital.The adjacent tissues must be at least 5 centimeter apart from the edge of the cancer tissues.Meanwhile,the postoperative pathological evidences had been screened out.There were 60 cases of NSCLC with no preoperative history of radiotherapy,chemotherapy,immunization and targeted therapy.All specimens were obtained and operated in accordance with the ethical norms and procedures of clinical trials.2 ? Cell lines and cultivation conditions: Four human NSCLC cell lines(including A549,SPC-A1,H460 and H1650)and a normal human bronchial epithelial cell line(16HBE)were purchased from ATCC Cell Bank(Shanghai,China).Cells were cultured in Dulbecco,s modified Eagle,s medium(DMEM;Thermo Fisher Scientific,Waltham,MA,USA)or RPMI1640(GIBCO-BRL;Thermo Fisher Scientific,Waltham,MA,USA)supplemented with 10% fetal bovine serum(Hyclone,USA)in a humidified atmosphere of 5% CO2 at 37?.3?Total RNA extraction and q RT-PCR analysis: Trizol method was used to extract total RNAs from tissue cells.The expression of lnc RNA BCYRN1 in tumor tissues and adjacent tissues and the expression level of lnc RNA BCYRN1 in all cell lines were detected by real-time fluorescence quantitative PCR.4?si RNA transient transfection: The BCYRN1-specific small interfering RNAs and negative control si RNA were purchased from Life Technology(Invitrogen,Shanghai,China).Lung cancer cells at approximately 60-70% confluence were transfected using Lipo2000 reagents(Invitrogen,CA,USA)according the manufacturer's instructions.48 hours after transfection,cells were harvested for q RT-PCR.5?Cell proliferation assay: The cells were seeded on 96-well plate.The ability of cell proliferation was evaluated by using CCK-8 kit(Cell Counting Kit-8)at 6hrs,12 hrs,24hrs,48 hrs and 72 hrs respectively after transfection process.The absorbance of each group was detected by automatic enzyme marker.The cell viability had been calculated according to the formula of cell proliferation rate.Methods6?Cell invasion assay: After 24 hrs of transfection,the cells were digested with trypsin and made into cell suspensions which were seeded in the 24-well plate upper chamber of the Transwell application.The lower chamber was filled with 600 ?L of RPMI1640 medium supplied with 10% fetal bovine serum.After incubating for48 hrs at 37?,the migrated cells were stained with 0.5% crystal violet solution.The cell numbers were determined by counting the penetrating cells under a microscope at 200× magnification in random fields in each well.7?Cell apoptosis analysis: Appropriate amount of cells were seeded on 6-well plate and were cultured of 24 hrs.Then,the cells were transfected with si RNA.After48 hrs of transfection,the cells were collected,washed,fixed,permeated and incubated with fluorescent SA-FLOUS to stain.The apoptosis of the cells was detected by flow cytometry.8?Detection of Wnt/?-Catenin signaling Pathway-Related proteins: After 48 hrs of transfection,the cells were collected.The content of Wnt signaling pathway related proteins were extracted to detect,such as ?-Catenin,and the expression of target protein c-Myc and Cyclin D1.9?Statistical analysis: SPSS 22 software was used for the statistical analysis.Chi square test was used to compare the enumeration data between the two groups.The independent samples t test was used to compare the measurement data.Values of P<0.05 were considered statistically significant.Results1?The expression of lnc RNA BCYRN1 in tumor and adjacent tissues of 60 patients with NSCLC: The expression of BCYRN1 in NSCLC tumor and its matched adjacent tissues were 11.803 ±2.145 and 2.375 ±0.341,respectively.The difference was statistically significant.2?The relationship between the expression of BCYRN1 and clinic pathological features of lung cancer: According to the median of 2.85 BCYRN1 relative expression level(cancer tissue/adjacent tissue),it was divided into two groups: high expression group(?2.85%)and low expression group(<2.85%).It was found that the high expression of BCYRN1 was not associated with the age of the patients,sex,pathological type,smoking status and tumor size(P>0.05).But was correlated with histological grade(P=0.002),lymph node stage(P=0.013),clinical stage(P=0.025),Vascular invasion(P=0.006).3?The expression of lnc RNA BCYRN1 in different cell lines: Compared with the normal human bronchial epithelial cell lines,the expression levels of BCYRN1 in four lung cancer cell lines were significantly higher than those in the control group(8.56 times,7.84 times,5.79 times and 9.43 times,respectively).The difference was statistically significant(P<0.05).4 ? The expression level of BCYRN1 in each cell lines after transfection of si RNA-BCYRN1: Compared with the untransfected cells,the relative expression level of BCYRN1 in each cell line decreased significantly(P<0.05).5?The impact of lnc RNA BCYRN1 on cell proliferation: The ability of cell proliferation was evaluated by using CCK-8 kit(Cell Counting Kit-8)at 6hrs,12 hrs,24hrs,48 hrs and 72 hrs respectively after transfection process.The results showed that compared with the negative control group transfected with si RNA-NC,the proliferation ability of si RNA-BCYRN1 transfected cell lines decreased obviously(P < 0.05),and the inhibition became more significant after 12 hrs of transfection.6?The effect of lnc RNA BCYRN1 on cell invasion ability: Compared with the negative control group and the blank group,the invasion numbers of cells transfected with specific si RNA-BCYRN1 decreased significantly(P < 0.05)in the four non-small cell lung cancer cells.7?The effect of lnc RNA BCYRN1 on apoptosis: After silencing the BCYRN1,the apoptotic numbers of four cell lines,A549,SPC-A1,H460 and H1650,were significantly increased(P<0.05).8?The impact of lnc RNA BCYRN1 on Wnt/?-Catenin signaling pathway: After down-regulation of BCYRN1 expression,the expression of ?-Catenin,c-Myc and Cyclin D1 in all cell lines decreased significantly,suggesting that BCYRN1 is related to Wnt/?-Catenin signaling pathway.Conclusion1 ? Lnc RNA BCYRN1 is highly expressed in non-small cell lung cancer(NSCLC)and plays a oncogenic role,which is closely related to the occurrence and development of NSCLC.2 ? There was no significant difference between the expression of lnc RNA BCYRN1 and age,sex,smoking,pathological type and tumor diameter,but in low differentiation of tumor tissue,mediastinal lymph node metastasis,clinical stage ?,and vascular invasion,the expression of lnc RNA BCYRN1 was significantly higher(p<0.05).3 ? In A549,SPC-A1,H460 and H1650 cells,Lnc RNA BCYRN1 is highly expressed and regulates tumor transformation by promoting cell proliferation,invasion and inhibition of cell apoptosis.4?Lnc RNA BCYRN1 positively regulates the Wnt/?-Catenin signaling pathway,which increases the expression of Wnt signal pathway related proteins and participates in the occurrence and development of NSCLC.