| Thyroid carcinoma is the most common endocrine malignancy.The incidence ofthyroid carcinoma is increasing these years all over the world. In the United States, theincidence with associated APC （%）(annual percentage change （APC） for thyroid cancerwas2.4in1980-1997compared6.6in1997-2009（SEER.cancer.gov）. BRAFV600E mutation is the most prevalent genetic marker in human thyroid cancer. BRAFV600E mutation is associated with lymph node metastasis, extrathyroidal invasion and advancedclinical stages in thyroid papillary carcinoma which is a poor predictor of prognosis.Thyroid cancer patients with BRAFV600E mutation need more advanced thyroidectomy andradioiodine therapy.Radioiodine treatment which can ablate the residual thyroid tissues afterthyroidectomy is very important for thyroid cancer patients. Because evenwell-differentiated thyroid cancer maybe multi-centric with lesions recurring in theremaining tissue. Sodium iodide symporter （NIS） gene is a passive transport across theapical plasma membrane and the first rate-limiting step for iodine uptake. However, inthyroid carcinomas with BRAFV600E mutation NIS gene expression is silenced. As a resultmany patients lost the opportunity to take radioiodine treatment which may cause laterrecurrence. However, the mechanism why NIS gene expression is decreased in thyroidcarcinomas with BRAFV600E mutation is unclear.Our study is to investigate if histone acetylation can play a role in adjusting the NISgene expression in thyroid carcinomas with BRAFV600E mutation. Histone acetylation is akind of post-transcriptional modification that can induce chromatin structure remodelingand make DNA more open to transcription factors which actives gene expression. Histonemodifcations are conserved and stabled in different species like yeast, mouse, rat andhuman. As a result, in our study we can use rat thyroid cell as a model to investigate.PCCL3/BRAF and PCCL3cells were used to express BRAFV600E mutation.PCCL3/BRAF cells, we used1ug/ml DOX to induce BRAFV600E expression. PCCL3cells we did transient expression using empty vector, wt BRAF and BRAFV600E plasmids.Rat NIS promoter area was divided into seven parts: EXON （-20/104）, P1（-297/-107）, P2（-477/-277）, P3（-678/-452）, P4（-1124/-950）, P5（-1874/-1713）, NUE （-2627/-2342）. Weseleceted human cancer cell line BCPAP which harbored BRAFV600E mutation to checkthe mechanism of its’ role to affect NIS promoter histone acetyaltion change. MAPKinhibitor AZD4244, BRAF inhibitor PLX40321um treated to BCPAP cells for48h. Human NIS promoter area was divided into five parts: EXON （42/399）, P1（-692/-370）, P2（-1147/-762）, P3（-1511/-1216）, NUE （-9525/-9287）.One side we lysed for total protein to check total histone acetylation change whenBRAFV600E mutation using WesternBlotting. The other side, we used CHIP assay tocheck NIS promoter area specific histone acetylation sites H3K9/14, H3K18, total H4,H4K16histone acetylation change. Our results showed that BRAFV600E mutation decreasedrat NIS mRNA expression50%to60%in48h in rat thyroid cells. Total histone acetyaltionincreased when BRAFV600E mutation. Rat NIS promoter area: H3K9/14acetylationdecreased in P1, P2and P3parts in PCCL3/BRAF cells induced with DOX, however,EXON and P1parts of NIS promoter area H3K9/14acetylation dereased in PCCL3cellstransient with BRAFV600E. SAHA can increase P1, P3, P4and NUE parts of NIS promoterH3K9/14acetylation in PCCL3/BRAF cells. H3K18and total H4acetylation only affectedP1and P2parts in the NIS promoter area. In PCCL3/BRAF cells, inducibleBRAFV600E decreased NIS promoter area P1and P2parts’ H4K16acetylation and increasedNUE part H4K16acetylation. P3and P5parts H4K16acetylation decreased in PCCL3cellstransient with BRAFV600E. In human thyroid cancer cell line BCPAP, H3K9/14and H4K16acetylation changed in P1part of human NIS promoter. SAHA increased P1, P2partsH3K9/14acetylation and total human NIS promoter area H4K16acetylation.Histone acetylation change mainly occurred in P1, P2and P3parts of NIS promoter.There are more dramatic change of H3K9/14and H4K16acetylation in NIS promoterwhen BRAFV600E mutation.Generally, the results shown H3K9/14, H3K18, total H4,H4K16acetylation decreased in the prominent parts of NIS promoter when inducedBRAFV600E mutation. H3K9/14, H4K16acetylation may play greater role in modulatingNIS promoter activity. BRAFV600E mutation maybe play role in NIS promoter histoneacetylation change across MAPK pathway.Our study describes a novel mechanism of modulating NIS gene expression andprovides evidence of BRAFV600E mutation represses NIS gene expression in thyroid cancer.It is promossing for target specific drug using in thyroid cancer with BRAFV600E mutationthat causing NIS gene silenced. |
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