| PART 1 Expression of long non-coding RNA ENST00000606790.1 in Papillary thyroid carcinomaObjective: To investigate the expression of Lnc RNA ENST00000606790.1 in papillary thyroid carcinoma and paracancerous thyroid tissue and its correlation with clinicopathological characteristics.Methods: From October 2015 to March 2016,sixty-two thyroid cancer and paracancerous thyroid tissue specimens were collected from the second affiliated hospital of Nanjing Medical University,including 17 males and 40 female patients,aged 17 to 68 years,of which,35 had lymph node metastasis,37 had tumor stage I/II,and 20 had tumor stage III/IV.All the patients had been newly diagnosed through pathological diagnosis and had undergone an operation for the first time;no relevant treatment(including radiotherapy or chemotherapy)had been given to them before.All thyroid cancer tissues were stored in the laboratory of endocrinology.Real-time PCR was used to detect six randomly selected long-chain non-coding RNAs with differential expression in order to verify the results of expression spectrum microarray.Informed consent of the patients was obtained and this experiment was approved by the hospital ethics committee of the second affiliated hospital of Nanjing Medical University.Results:1.Gene spectrum chip results showed that long non-coding RNA expression was high in the thyroid cancer tissues.According to the statistical analysis of the chip data of lnc RNA expression in thyroid cancer tissues compared with para-thyroid cancer tissues,there were 1,976 long non-coding RNAs with expression differences of more than two times,among which,480 were up-regulated and 1,496 were down-regulated.We selected six of the differentially expressed lnc RNAs as experimental candidates(Shown in table 1).2.The expression levels of six lncRNAs in papillary thyroid carcinoma and paracancerous thyroid tissues were consistent with the results of the microarray.3.The expression level of lncRNA ENST00000606790.1 in papillary thyroid carcinoma was significantly higher than that in paracancerous thyroid tissue(P <0.05).Among 62 patients,the expression level of lnc RNA ENST00000606790.1 in the papillary thyroid carcinoma tissues of 41 patients was more than two times higher than that in paracancerous thyroid tissues.4.The relationship between the expression of lncRNA ENST00000606790.1 in papillary thyroid carcinoma tissues and basic clinical data was analyzed and compared systematically.It was found that the up-regulated of lnc RNA ENST00000606790.1 was correlated with lymph node metastasis;however,it was not significantly correlated with other factors(such as gender,age,and polycentricity.et al).PART 2 Investigation of the effect of long non-coding RNA ENST00000606790.1 expression on human Papillary thyroid carcinoma cellsObjective: To downregulate the expression of lncRNA ENST00000606790.1 in IHH4 cells and analyze its effect on proliferation,invasion,vitality,and related proliferative protein expression in IHH4 cells.Methods: Small interfering RNA(Si RNA)targeting human LncRNA ENST00000606790.1 were designed,synthesized,and transfected into papillary thyroid carcinoma IHH4 cells,which were divided into two groups: NC group(untransfected IHH4 cells)and Si RNA treated group(IHH4 cells transfected with ENST00000606790.1-Si RNA).The expression level of Lnc RNA ENST00000606790.1 in each group was detected through Real-Time PCR.First,we used the plate clone formation assay after transfecting papillary thyroid carcinoma IHH4 cells with ENST00000606790.1-Si RNA to analyze the effect of the Lnc RNA on proliferation ability.Second,a Transwell invasion assay was used to observe changes in the invasion ability of cells after transfection.Then,a CCK8 assay was used to analyze the activity of IHH4 cells after transfection.Finally,we analyzed the expression of relevant proliferative proteins in the transfected cells through western Blot analysis to further study the effect of Lnc RNA ENST00000606790.1 on the signal pathway in papillary thyroid carcinoma IHH4 cells.Results:1.RT-PCR was used to detect the expression of Lnc RNA ENST00000606790.1 in normal thyroid cells,PTC and IHH4 cell lines.The results indicated that the expression level of Lnc RNA ENST00000606790.1 in PTC and IHH4 cells was significantly higher than that of normal thyroid cells,and the difference was statistically significant.(P < 0.01).2.The results of plate clone formation assay showed that 10 days after transfection of IHH4 cells,the cell number formation rate of the si RNA group transfected with ENST000006790.1-Si RNA was 48.4 ± 0.8%,while that of NC group was 91.9 ±1%.In Si RNA group,the number formation rate of clone cells in Si RNA+740-Y-P group was 71.9 ± 0.6% after the addition of PI3 K activator 740-Y-P.The results showed that the proliferation of IHH4 cells transfected with ENST000006790.1-Si RNA was significantly inhibited.The number of cloned cells in Si RNA+740-Y-P group was significantly higher than that in Si RNA group.The difference was statistically significant(P < 0.01).3.Transwell invasion assay showed that the number of IHH4 cells was(361.4 ±4.750)in NC group and(202.4 ± 3.757)in Si RNA group and(286.4 ± 3.541)in si RNA+740-Y-P group,Compared with NC group,the number of IHH4 cells in Si RNA group was significantly lower than that in NC group,however,the number of IHH4 cells in Si RNA +740-Y-P group was significantly higher than that in Si RNA group,The difference was statistically significant.(P<0.01).3.CCK8 experiment results showed that compared with the blank group(NC group),IHH4 cells transfected with ENST000006790.1-specific Si RNA significantly decreased the proliferation activity of tumor cells after interference,and increased the viability of IHH4 cells after adding PI3 K activator 740-Y-P.The difference was statistically significant(P<0.01).4.To further investigate the molecular mechanism of Lnc RNA ENST00000606790.1 on the biological behavior of thyroid cancer cells,the expression levels of AKT and CHK1 were detected by Western Blot between the control group and ENST00000606790.1 Si RNA interference group.The results showed that,compared with the control group,the expression levels of protein AKT、p AKT 、PI3K、p-PI3 K and protein CDC25 c in IHH4 cells were significantly decreased after ENST00000606790.1-Si RNA interference was down-regulated,while the expression levels of downstream protein CHK1 was significantly increased,suggesting that the effect of ENST00000606790.1 on the biological behavior of IHH4 cells may be achieved by regulating the PI3K/AKT signaling pathway.Conclusion:The expression level of LncRNA ENST00000606790.1 in Papillary thyroid carcinoma was significantly increased,and it was related to lymph node metastasis(P <0.05).Lnc RNA ENST00000606790.1 may,therefore,be a new target for the treatment of Papillary thyroid carcinoma. |
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