Mechanism Study Of Long Noncoding RNA UCA1 In Cisplatin Resistance Of Cervical Cancer

Part I The expression verification on the screening of Long Noncoding RNA Chip and genetic screeningObjective: Using microarray technology to observe the long chain encoding RNA(Long noncoding RNA-Lnc,RNA)expression profile in cervical cancer tissue and to analyse the difference of long noncoding RNA expression between in cervical cancer tissues and paracancerous tissues line.Methods: 1 Application of microarray technology to finish the inspection of the long chain non encoding RNA in Cervical cancer tissues and paracancerous tissues expression profile.2 The results on the microarray of expression profile were verified by Real-time and PCR;3 The proper candidate molecules were selected for further studies according to the expression differences.Results: 1.The analysis of long chain noncoding RNA microarray showed that the expression profile of long noncoding RNA gene in Cervical cancer tissues was significantly different compared with paracancerous tissues line.Compared with Cervical cancer tissues there were 47 noncoding RNA with more than4 times differential expression in paracancerous tissues line of cervical cancer,of which 26 were up-regulated and 21 were down regulated.(P<0.05);2 PCR screened significantly different 8 Lnc RNA.For the expression level in Cervical cancer tissues and paracancerous tissues,Real-time PCR analysis results were consistent with the microarray results;3 The detection results showed that Lnc RNA UCA1 in Cervical cancer tissues was highly expressed and UCA1 was chosen as candidate molecules to carry out the follow-up study.Part II The Expression and Significance of Long noncoding RNA UCA1 in Cervical Carcinoma TissuesObjective: To investigate the expression of long chain noncoding RNA UCA1 in cervical carcinoma tissues;Methods: 1.qPCR was used to detect the expression of lnc RNA and UCA1 in cervical cancer tissues.2.The difference of UCA1 expression in cervical tissue among patients with different pathological types,FIGO stage and the depth of tumor invasion was analyzed and compared.Results: 1.The relative expression of lnc RNA and UCA1 in cervical cancer tissues detected by q PCR was28.4±7.8 in the cervical cancer group,10.9±3.8 in the paracancerous group,4.2±2.3 in the non-neoplastic group,and12.2 ± 16.9 in the cervical CIN group.The expression of UCA1 in cervical carcinoma was higher than that of cervical cancer tissues,and the expression is higher than that of non-cervical neoplasia group(p<0.05).2.The expression of UCA1 and tumor pathological type,FIGO staging and the depth of tumor invasion were significantly associated(p<0.01).Part III Investigate the Role and Mechanism of Long noncoding RNA UCA 1 in Cisplatin Resistance of Cervical CancerObjective: The aim of this part of study was to investigate the effect of RNA UCA1 on the cisplatin resistant of cervical cancer and preliminarily analyze the related molecular mechanism.Methods: 1 Increased dose of cisplatin by passive stimulation to establish the cisplatin resistant cell line He La He La/DDP;2.We applied RT-PCR to detect He La cell of cervical cancer and the expression level of Lnc RNA UCA1 in cisplatin resistant He La/DDP.3.The UCA1 overexpression and gene silencing were performed in He La cell of cervical cancer by using pc DNA3.0-UCA1 plasmid and UCA1 si RNA.The cell proliferation was determined by using CCK-8 assay,Edu assay and flow cytometry analysis.4.Western-blotting was employed to determine the expression level of the apoptosis associated protein caspase-3,p21 and survivin and CDK2 in UCA1 regulating He La cell line.Results: 1.The result of PCR showed that the expression of UCA1 in Cisplatin resistant He La/DDP cell of cervical cancer was significantly up-regulated.2.The results of CCK-8 assay,Edu assay and flow cytometry analysis showed that UCA1 overexpression could significantly decrease the cell apoptosis caused by cisplatin in He La cells.3.The results of western-blotting showed that UCA1 overexpression could down-regulate the level of the apoptosis-associated protein caspase-3 and p21 and up-regulate the level of cell cycle related protein survivin and CDK2 at the same time.This further validated the results for the silencing UCA1.Conclusion: 1 The high expression of long chain non encoding RNA UCA1 in Cervical cancer tissues is verified through the chip screening and RT-PCR.2 The high expression of long chain noncoding RNA UCA1 in cervical cancer and cervical CIN tissues;the analysis of the relationship among the expression of UCA1,FIGO in patients with cervical cancer staging and the depth of tumor invasion combined with clinical data;3 Long chain noncoding RNA UCA1 promotes the effect of cisplatin resistance probably through down-regulating level of Caspase-3 and p21,and upregulating the apoptosis-associated protein of survivin and CDK2.