| Background and Objective:Thyroid cancer?TC?is the most common malignant tumor of the endocrine system,and its incidence ranks first among head and neck malignant tumors.In the past few decades,the incidence has been rising steadily.The vast majority of thyroid cancer are papillary thyroid cancer?PTC?.Most patients with PTC have a good prognosis.However,some patients still have recurrence,insensitivity to I131 treatment and even death.The incidence of anaplastic thyroid cancer?ATC?is very low,but the mortality is very high.The five-year survival rates of patients with ATC are less than10%.Therefore,it is necessary to find new therapeutic targets.Long-chain non-coding RNA?lncRNA?is a kind of RNA with more than 200 bp of nucleotides and can not be encoded to proteins.In recent years,many studies have suggested that it has the function analogous to oncogene or anti-oncogene.LNC RNA LINC00982 was found to be low expressed in a variety of malignant tumors,and studies have demonstrated that itcan affect the proliferation and invasiveness of cancer cells.Genome-wide studies have shown that LINC00982 is down-regulated in thyroid cancer,but its role and mechanism in the development of thyroid cancer are unknown.The purpose of this study was to reveal the expression of LINC00982 in papillary thyroid cancer and its relationship between clinicopathology,to explore its biological role in the proliferation and invasion of papillary thyroid cancer,and to elucidate its mechanism.Methods:1.According to the inclusion and exclusion criteria,148 cases of papillarythyroid cancer were reflected in the present study.Clinical and pathological data were collected,and papillarythyroid cancer tissue and adjacent tissues of the 148cases were collected.The expression of LINC00982 in cancer tissues and adjacent tissues was detected by qRT-PCR,and the difference of LINC00982 expression between cancer tissues and adjacent tissueswas analyzed by paired T test.The difference of LINC00982 expression in cancer tissues between different age,gender and clinicopathological characteristics of patients was detected by T test.2.Two thyroid cancer cell lines BHT101 and B-CPAP were selected for cell transfection experiment.According to the different transfection fragments,they were divided into the blank control group,LINC00982 group?pcDNA4.0-LINC00982transfection?,pcDNA4.0 group?pcDNA4.0 transfection?,siLINC00982 group?siLINC00982 transfection?and siCTRL group?siCTRL transfection?.Through CCK8 experiment,EDU experiment and colony formation experiment,the proliferation of thyroid cancer cells in each group was detected;the cell cycle and apoptosis of thyroid cancer cells were detected by flow cytometry;the migration of thyroid cancer cells was detected by Transwell cell experiment.3.Transfected BHT101 cell lines were subcutaneously injected into nude mice through the left axilla to construct the subcutaneous tumor-bearing model of thyroid cancer nude mice.According to the different injected cells,they were divided into blank control group,LINC00982 group,pcDNA4.0 group,siLINC00982 group and siCTRL group.The volume of the tumors were measured in 3,7,14,21 and 28 days after inoculation,and the experimental data were recorded.Twenty-eight days after inoculation,the nude mice were killed by vertebral dislocation.Subcutaneous tumors were cut off and preserved as required.Then the expression of LINC00982 was detected by qRT-PCR and the expression of Ki67 was detected by immunohistochemistry.4.Proteins were extracted from transfected thyroid cancer cells and nude mice tumors respectively.The expression and activation of PI3K and AKT were detected by Western blot,and the expression of downstream proteins Bcl-2,Cyclin D1,Bax and P21 were also detected.5.In the medium of BHT101 and B-CPAP cell lines stably transfect with LINC00982 fragment,the cells were divided into control group,LINC00982 group and LINC00982+IGF-1 group according to the transfection and intervention.Western blot was used to detect the expression and activation of PI3K and AKT,and the expression of Bcl-2,Cyclin D1,Bax and P21 downstream proteins.CCK8 test,EDU tests and colony formation test was used to detect the proliferation ability of thyroid cancer cells;flow cytometry was used to detect the cell cycle and apoptosis of thyroid cancer cells;and Transwell chamber test was used to detect the migration ability of thyroid cancer cells.Results:1.The expression of LINC00982 in papillary thyroid cancer tissue samples?0.87+0.58?was significantly lower than that in paracancerous tissues?1.5+1.0?,and the difference was statistically significant?P<0.01?.There was no significant difference in LINC00982 expression between different groups of gender,age and multiple tumors?P>0.05?.The expression of LINC00982 was significantly different in different groups of lymph node metastasis,tumor size and clinical stages?P<0.05?.2.The results of CCK8 test,EDU tests and colony formation experiment showed that the proliferation ability of BHT101 and B-CPAP cell lines in LINC00982 group decreased,and that of siLINC00982 group increased.Flow cytometry results showed that BHT101 and B-CPAP cell lines in G0/G1 phase increased,S phase cells decreased and cell apoptosis rate increased,and siLINC00982 group was in G0/G1phase.The results of Transwell cell lab showed that the number of BHT101 and B-CPAP cells in linc00982 group decreased,while that in silinc00982 group increased.3.In the nude mice,28 days after inoculation with BHT101 cells,the expression of LINC00982 in tumors of LINC00982 group increased significantly,the volume of tumors decreased significantly compared with that of normal group,and the positive rate of Ki67 in tumors was lower;the expression of LINC00982 in tumors of siLINC00982 group decreased,the volume of tumors increased significantly,and the positive rate of Ki67 in tumors was higher.4.The results of Western blot assay showed that PI3K expression decreased,AKT activation decreased,Bcl-2,Cyclin D1 protein expression decreased,Bax and P21 protein expression increased in BHT101 and B-CPAP cell lines;PI3K expression increased,AKT activation increased,Bcl-2,Cyclin D1 protein expression increased,while Bax and P21 protein expression decreased in siLINC00982 cell lines.5.Proteins were extracted from nude mice tumors by western blot assay.The results ofwestern blot assayshowed that in LINC00982 group PI3K expression decreased,AKT activation decreased,Bcl-2 and Cyclin D1 protein expression decreased,Bax and P21 protein expression increased;while in siLINC00982 group PI3K expression increased,AKT activation increased,Bcl-2 and Cyclin D1 protein expression increased,Bax and P21 protein expression decreased.6.Western blot assays showed that PI3K expression and AKT activation were returned after the intervention of IGF-1 in BHT101 and B-CPAP cell lines.The expression of Bcl-2 and Cyclin D1 protein increased,while the expression of Bax and P21 protein decreased.7.In BHT101 and B-CPAP cell lines,after the intervention of IGF-1,the results of CCK8 experiment,EDU staining experiment and colony formation experiment showed that the proliferation ability of LINC00982+IGF-1 group was restored;flow cytometry results showed that the cells in G0/G1 phase decreased,S phase cells increased and cell apoptosis rate decreased in LINC00982+IGF-1 group;Transwell chamber experiment results showed that LINC00982+IGF-1 group had a decrease in G0/G1 phase cells.The number of migrating cells increased in the IGF-1 group.Conclusion:Long-chain non-coding RNA LINC00982 is low expressed in thyroid cancer tissues,and is relatively low in patients with late clinical stage,lymphe node metastasis or distal metastasis.Over expression of LINC00982 in vitro can inhibit the proliferation of thyroid cancer cells,change cell cycle,induce apoptosis and reduce cell migration.Over expression of LINC00982 in vivo can inhibit the growth of thyroid cancer cells.We found that LINC00982 can inhibit the activation of AKT by reducing the expression of PI3K,Thus reducing the expression of downstream target proteins of the PI3K/Akt signaling pathway and inhibiting the activity of the PI3K/Akt signaling pathway. |
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