LncRNA UCA1 Increases Multi-drug Resistance Of Gastric Cancer Via Down Regulating MiR-27b

PART I Discussion on the function of LncRNA UCA1 in the Occurrence and Development of Gastric CarcinomaObjective:Discussion on the function of LncRNA UCA1 in the Occurrence and Development of Gastric CarcinomaMethods:Gastrectomy was conducted in the incorporated 56 primary gastric cancer patients who were not treated with chemotherapy or radiotherapy.After the resection,gastric cancer tissues and paracancerous tissues were collected and frozen immediately with liquid nitrogen,and then stored at-80℃ for further application.First,gastric mucosa tissues resected in the surgery were soaked with 4%paraformaldehyde for 48h,followed by paraffin embedding,then collected samples were sliced into 4μm continuous sections and embedded with paraffin.After that.HE staining was performed,subsequently,analysis and evaluation of the slices were made by pathological experts.Furthermore,following immunohistochemical staining,total RNA was extracted from gastric cancer samples.Total RNA of the cells was extracted and subjected to denaturing agarose gel electrophoresis and RNA reverse transcription reaction.After reverse transcription reaction.Inc RNA and mRNA abundance were detected by Real-time PCR.Results:1 I IE and immunohistochemical stainingParaffin specimens of gastric cancer tissues and normal gastric mucosa tissues were stained by HE.and the pathological examination showed that the diagnosis was consistent with the diagnosis before the experiment.Immunohistochemical staining showed that the specimens of gastric cancer were highly expressed,while the normal gastric mucosa was not expressed.2 Significant up-regulation of Inc RNA in gastric cancer tissuesThe expression of Inc RNA UCA1 was detected by Real-time PCR method in pathological tissues(gastric cancer tissues and paracancerous tissues),and a total of 56 pairs of samples were collected.As shown in Figure 1-2.the relative content of Inc RNA UCA1 was significantly lower in paracancerous tissues(1.29±0.26)than in gastric cancer tissues(2.85±0.74).The difference was statistically significant by Wilcoxon matched pairs test(p<0.01),suggesting that Inc RNA UCA1 might be an oncogene in gastric cancer tissues.3 Relationship of the relative content of Inc RNA UCA1 in gastric cancer tissues and paracancerous tissues with the clinical pathological characteristics and prognosis of gastric cancerWe further analyzed the relationship of the C/P ratio expressed by the relative content of Inc RNA in 56 cases of gastric cancer and paracancerous tissues with sex.age.tumor size,stage,degree of invasion,differentiation and lymph node metastasis.The results showed that C/P ratio was significantly correlated with tumor size and tumor differentiation.There was an increasing trend in the expression of low differentiated gastric cancer,and the difference was statistically significant.However.there was no significant correlation of C/P ratio with sex and age,showing none apparent statistical difference,which might be related to the small sample size.In this process,we divided the 56 cases into two groups,namely,mean value of C/P>C/P group and mean value of C/P<C/P group.The postoperative survival time(hour)was statistically analyzed subsequently.Corresponding results indicated that the postoperative survival time in the mean value of C/P<C/P group was significantly longer than that in the mean value of C/P>C/P group,yet the difference was not statistically significant.Possible reason might be that the follow-up time was relatively short and the 5-year survival rate was relatively higher in gastric cancer patients postoperatively.Conclusion:The expression of Inc RNA UCA1 in gastric cancer tissues is significantly higher than that in adjacent normal tissues and is correlated with the size and differentiation of gastric cancer,acting as an oncogene in the progression of gastric cancer.PART ⅡThe search of LncRNA UCA1 enhances gastric cancer multidrug resistance by down-regulating miR-27b expressionObjective:In this study,we aimed to investigate the association between UCA1 and miR-27b in gastric cancer and further studied their involvement in multi-drug resistance(MDR)of gastric cancer.Methods:Human tissue collection28 patients diagnosed with primary gastric cancer,had not received previous chemotherapy or radiotherapy were recruited from Qilu Hospital of Shandong University.This study was approved by the ethics committee of the Qilu Hospital.Informed consent was obtained from the patients before the study.The gastric cancer tissues and adjacent normal tissues were obtained during gastrectomy and were frozen in liquid nitrogen immediately after resection and stored at-80℃ for future use.Cell culture and transfection The human gastric cancer cell line SGC-7901 was obtained from American Type Culture Collection.The SGC-7901 derived adriamycin resistant SGC-7901/ADR.cisplatin resistant SGC-7901/DDP and 5-FU resistant SGC-7901/FU cells were generated by using a conventional stepwise method according to one previous study20,which induces drug resistance by increasing concentrations of the drugs over 8 months.The cancer cells were cultured in RPMI-1640 medium supplemented with 10%FBS,100 μg/mL penicillin.and 100 U/mL streptomycin.Two UCA1 siRNAs were chemically synthesized by Ribobio SGC-7901/ADR,SGC-7901/DDP and SGC-7901/FU cells were transfected with 75 nM UCA1 siRNA or the scramble negative control,while SGC-7901/ADR cells were transfected with 100 nM miR-27b mimics or the scramble negative control using Lipofectamine 2000.48 hours later,cells transfected with siRNA were harvested for qRT-PCR to determine the inhibiting efficiency.The siRNA with higher inhibitive efficacy was used for following studies.The complementary DNA encoding UCA1 was PCR-amplified and inserted into pcDNA3.1 between the BamhI/XhoI sites.SGC-7901 cells were transfected with pcDNA3.1-UCA1 expression vector or 50 nM miR-27b inhibitor(Ribobio)using Lipofectamine 2000 reagent.Bioinformatics analysis.The microarray data of IncRNA profiles in gastric cancer tissues and paired peritumoral tissues were retrieved in NCBI GEO Datasets.The binds sites between UCA1 and miR-27b were predicted using DIANA tools.QRT-PCR analysisTotal RNAs in the tissue and cell samples were extracted using the TRIzol reagent(Invitrogen).Then,reverse transcription was performed to get the first strand cDNA by using the PrimeScript(?)RT reagent kit(TaKaRa,Dalian,Liaoning,China).UCA1 expression was then detected by qRT-PCR.To detect mature miR-27b expression.miRNA specific cDNA was firstly synthesized using the stem-loop primers and the TaqMan MicroRNA Reverse Transcription Kit).Then.qRT-PCR analysis was performed using TaqMan MicroRNA Assay Kit.All qRT-PCR reactions were performed using an ABI Prism 7500.The results were calculated using the 2-AACT methods.The correlation between UCA1 expression and miR-27b expression was assessed using linear regression analysis.RNA fluorescence in situ hybridization(FISH)Biotin-labeled UCA1-Locked Nucleic Acid(LNA)probe.miR-27b-LNA probe and the corresponding control oligo for in situ hybridization were purchased from Exiqon(Vedbaek.Denmark).SGC-7901 and SGC-7901/ADR cells were grown on cover slips and the cells were fixed when reaching 60%-70%confluence.Hybridization was performed according to the methods introduced in one previous study21.The signals were detected using anti-Biotin-Cy3(C5585.Sigma-Aldrich)at 37℃ for 30 min.Nuclei were counterstained with DAPI,then the immunofluorescence were detected under FV1000 fluorescence microscope.IC50 measurement.SGC-7901/ADR cells were transfected with UCA1 siRNA or miR-27b mimics.while SGC-7901 cells were transfected with UCA1 expression vector or miR-27b inhibitors.24 hours after transfection,the cells were seeded in a 96-well plate.24 hours later,the cells were treated with varying concentrations of ADR,DDP or 5-FU for 48 hours.Then,cell viability was measured using a conventional MTT assay.Absorbance was recorded at 490 nm using a microplate reader.IC50 value was determined by creating dose-response curves.Cell apoptosis was detected by using Annexin V-FITC Apoptosis Detection Kit and the apoptosis rates were measured by using a flow cytometer.Western blot analysis,In brief,the samples containing 30 μg of total protein were loaded per lane and then were separated by 10%SDS-PAGE.After that,the protein samples were electrophoretically transferred onto nitrocellulose membranes.The membranes were firstly blocked,washed and then incubated primary antibodies against BCL-2 and cleaved caspase-3 and β-actin(ab8227,Abeam)overnight.After washing,the membranes were then incubated with the HRP conjugated secondary antibodies.Protein bands were visualized by super ECL detection reagent.Statistical analysis was performed using GraphPad Prism 6.0.The difference between groups was evaluated by unpaired,two-tailed Student t test.p<0.05 indicates statistical significance.Results:UCA1 was significantly upregulated in the cancerous tissues and its expression was negatively correlated with miR-27b level.Inhibition of UCA1 significantly restored miR-27b expression in the MDR gastric cancer cells.UCA1 knockdown and miR-27b overexpression reduced IC50 of ADR.DDP and 5-FU in SGC-7901/ADR cells and increased ADR induced cell apoptosis.UCA1 overexpression and miR-27b inhibition increased IC50 of ADR.DDP and 5-FU in SGC-7901 cells and reduced ADR induced cell apoptosis.Following western blot analysis showed that UCA1 knockdown and miR-27b overexpression also decreased anti-apoptotic protein BCL-2 and increased apoptotic protein cleaved caspase-3.Conclusion:UCA1 is negatively correlated with miR-27b expression in gastric cancer.Knockdown of UCA1 restored miR-27b expression in the cancer cells.The UCA1-miR-27b axis is involved in regulation of chemosensitivity of the gastric cancer cells.