| PART1 Long noncoding RNA expression profiles of papillary thyroid carcinoma by microarray analysisObjective: Long noncoding RNA have emerged recently as key regulatory molecules at almost every level of the regulation of gene expression.The aim of the present study was to determine the long noncoding RNA expression profile in papillary thyroid carcinoma and its potential clinical value.Methods: The global long noncoding RNA expression profile in papillary thyroid carcinoma was measured by human long noncoding RNA microarray. The papillary thyroid carcinoma samples and corresponding noncancerous tissue samples were prospectively collected from 36 patients of Tangshan Gongren Hospital from April 2014 to November 2014. All these samples were snap-frozen in liquid nitrogen immediately after resection. All the cases were confirmed by surgical pathology. All patients were the first operation, all patients were not accepted any treatment before operation. Of these samples, 3 pairs were used for long noncoding RNA microarray analysis and the remaining samples were used for further validation.The scanning picture of chip request signal uniform, clear, no marginal effect or scrape, the results of positive scanning point and negative control area are obvious. Fold Change cut-off is 2.0, P value calculated from paired t-test and cut-off is 0.05. GO and KEGG pathway analyses were performed to discover the function and associated pathways of differentially expressed m RNAs. Classification and subgroup analysis were performed to analysis of the differentially expressed long noncoding RNAs, then using conjoint analysis to analysis long noncoding RNAs and corresponding m RNAs.Quantitative real-time polymerase chain reaction was performed to confirmthe expression of long noncoding RNAs by microarray analysis. the expression of each long noncoding RNA was represented as fold change using2-ΔΔCtmethods.Results:1 Microarray analysis showed 675 long noncoding RNAs and 751 m RNAs to be significantly differentially expressed in papillary thyroid carcinoma samples compared with their adjacent noncancerous tissue samples（Fold Change≥2.0 or Fold Change ≤ 0.5, P < 0.05）. Among these long noncoding RNAs, 312 were upregulated and 363 were downregulated. Among these m RNAs, 499 were upregulated and 252 were downregulated.2 GO analysis was derived from Gene Ontology（http://www.geneontology.org）, which covers three domains: biological process, cellular component and molecular function. Corresponding to the downregulated m RNAs, the total number of DE genes included 170 genes involved in molecular functions, 159 genes involved in biological processes and 187 genes involved in cellular components. The highest enriched GOs targeted by downregulated transcripts were oxygen transporter activity（molecular function） negative regulation of transporter activity（biological process） and myofibril（cellular component）. Corresponding to the upregulated m RNAs, the total number of DE genes included 359 genes involved in molecular functions,362 genes involved in biological processes and 376 genes involved in cellular components. We found that the highest enriched GOs targeted by the upregulated transcripts were response to protein binding（molecular function）,response to stimulus（biological process） and plasma membrane part（cellular component）.3 KEGG pathway analysis indicated that 7 downregulated pathways were identified, and the most enriched network was ‘‘Fc epsilon RI signaling pathway-Homo sapiens（human）’’. Furthermore, this analysis showed that 29 upregulated pathways were identified, the most enriched network was ‘‘p53signaling pathway-Homo sapiens（human）’’.（the recommend P-value cut-off is 0.05）.4 Long noncoding RNAs classification and subgroup analysis indicated that 7 pairs of enhancer-like lnc RNA-m RNA, 9 pairs of antisense lnc RNA-m RNA and 45 pairs of linc RNA-m RNA were differentially expressed between papillary thyroid carcinoma and their paired samples.5 To verify the microarray data, eight differentially expressed long noncoding RNAs（ENST00000503723, ENST00000423539, uc003 tab.3,NR073085, ENST00000515275, ENST00000570022, uc003 qef.1 and ENST00000427243） including four upregulated long noncoding RNAs and four downregulated ones were randomly selected for Real-time PCR in 33 sets of papillary thyroid carcinoma tissues. The Real-time PCR results and microarray data were consistent.PART2 Expression of long noncoding RNA ENST00000423539 in papillary thyroid carcinomaObjective: To detect the expression level of long noncoding RNA ENST00000423539 in papillary thyroid carcinoma, and explore the relationship between long noncoding RNA ENST00000423539 and the clinical signification of papillary thyroid carcinoma.Methods: The samples of papillary thyroid carcinoma and corresponding noncancerous were collected from 57 clinical patients in Tangshan Gongren Hospital from April 2014 to November 2014. There were 17 men and 40 women between 17 and 68 years old. The tissues were snap-frozen in liquid nitrogen immediately after resection. All patients were the first operation. No patients had received preoperative chemotherapy, radiation therapy or other biological therapy. The relative level of long noncoding RNA ENST00000423539 expression in papillary thyroid carcinoma and the paracarcinoma tissues were detected using quantitative real-time polymerase chain reaction. the expression of each long noncoding RNA was represented as fold change using 2-ΔΔCtmethods. All data statistics were processed by SPSS13.0 software, comparison between two groups was done with paired t test analysis. Chi-square tests were used to determine the association between the expression level of long noncoding RNA ENST00000423539 in papillarythyroid carcinoma and clinical features. The clinical factors of patients,including age, gender, tumor stage, tumor size, lymph node involment,multifocal. P value cut-off is 0.05.Results:1 The expression level of long noncoding RNA ENST00000423539 in papillary thyroid carcinoma is significantly higher than in paracarcinoma tissues（P<0.05）. In 36 out of 57 samples, the expression level of long noncoding RNA ENST00000423539 in papillary thyroid carcinoma tissues was at least twice as high as it in the paracarcinoma tissues.2 The association between the expression level of long noncoding RNA ENST00000423539 in papillary thyroid carcinoma and clinical features showed long noncoding RNA ENST00000423539 expression level increased depend on the degree of lymphatic metastasis（P<0.05）, but had nothing related to age, gender, tumor stage, tumor size, and multifocal.PART3 Influence of suppressed long noncoding RNA ENST00000423539 expression on human papillary thyroid carcinoma K1 cells in vitroObjective: To seek out the specific short interference RNA（si RNA） for long noncoding RNA, and to explore the role of long noncoding RNA ENST00000423539 in the proliferation, migration and invasion of papillary thyroid carcinoma K1 cells.Methods: We designed and combined 2 different pairs of si RNAs specific for silencing long noncoding RNA ENST00000423539 and negative control si RNA, which were seperately transfected into papillary thyroid carcinoma K1 cells. The papillary thyroid carcinoma K1 cells were divided into 4 groups: control group（ papillary thyroid carcinoma K1 cells without the transfection）, negative control group（papillary thyroid carcinoma K1 cells with negative control si RNA transfection）, EXP1 group（papillary thyroid carcinoma K1 cells with ENST00000423539-si RNA-302 transfection）, EXP2group（papillary thyroid carcinoma K1 cells with ENST00000423539-si RNA-344 transfection）. Quantitative real-time polymerase chain reactionwas used to detect the interference efficiency of transfection and to selecte the best silencing effect of si RNA. Transfect the specific si RNA for long noncoding RNA ENST00000423539 into papillary thyroid carcinoma K1 cells,divided into 3 groups: control group（papillary thyroid carcinoma K1 cells without the transfection）, negative control group（papillary thyroid carcinoma K1 cells with negative control si RNA transfection）, EXP group（papillary thyroid carcinoma K1 cells with specific ENST00000423539-si RNA transfection）. After 2 weeks after transfecting, the cell proliferation was observed in vitro by colony formation assay, through transwell migration assay at 72 h after transfecting, invasion ability of cells were determined. All data statistics were processed by SPSS13.0 software, Comparison among groups was done with variance analysis. P value cut-off is 0.05.Results:1 ENST00000423539-si RNA-344 of the designed 2 pairs of si RNAs silenced the expression of long noncoding RNA ENST00000423539 effectively, the disruption rates were greater than 70%, which were obviously higher than the aother si RNA（P<0.05）.2 Using colony formation assay, at 2 weeks, the cloning efficiency of papillary thyroid carcinoma K1 cells with ENST00000423539-si RNA-344 transfection group was 55±5%, the cloning efficiency was lower than the other two groups, the difference was statistically significant（P<0.05）. The cloning efficiency of papillary thyroid carcinoma K1 cells without the transfection group and with negative control si RNA transfection group were 96±4%,95±4%, there were no significant difference between the two groups（P>0.05）.3 In Transwell invasion assay, the cell numbers of control group, negative control group and experimental group respectively were（57±3.4）,（55±4.5）and（27±4.3） per view, compared with negative control group, the numbers of papillary thyroid carcinoma K1 cells passed through extracellular cell matrix of experimental group was significantly lower（P<0.05）.Conclusions:1 In the present study, we investigated the long noncoding RNAexpression profiles of papillary thyroid carcinoma using microarray analysis and found that the long noncoding RNA expression levels were altered compared to adjacent noncancerous tissues, this study identified 675 papillary thyroid carcinoma associated long noncoding RNAs, 312 were upregulated and 363 were downregulated.2 The microarray based profiling identified that the m RNA expression levels of papillary thyroid carcinoma were altered compared to adjacent noncancerous tissues, classification analysis showed 61 pairs of lnc RNAm RNA were differentially expressed between papillary thyroid carcinoma and their paired samples.3 Real-time PCR was performed to confirm the expression of long noncoding RNAs by microarray analysis. The Real-time PCR results and microarray data were consistent.4 Expression of long noncoding RNA ENST00000423539 is high in papillary thyroid carcinoma, and closely related to the lymphatic metastasis,that prompt long noncoding RNA ENST00000423539 participate in the metastasis of tumors.5 si RNA ENST00000423539-si RNA-344 can be used to silence the expression of long noncoding RNA ENST00000423539 of human papillary thyroid carcinoma K1 cells, long noncoding RNA ENST00000423539 involved in the regulation of papillary thyroid carcinoma K1 cells proliferation,migration and invasion. The strategy targeted to long noncoding RNA ENST00000423539 gene providing a reliable theory for papillary thyroid carcinoma gene therapy. |
PDF Full Text Request