| Objetives:Thyroid cancer is the most common malignant tumor of the endocrine system;its morbidity has increased significantly in recent years.Although having good overal prognosis,thyroid cancer still affects the life quality of quite a number of patients,even threaten their life and health because of recurrence and metastasis.ANRIL is highly expressed in many malignant tumors and there are number of studies have shown that ANRIL can promote the proliferation,invasion and metastasis of various cancers,as well as inhibit the apoptosis of cancer cells.However,studies concerning mechanism of ANRIL promoting the development of cancer are limited,and no research has been made on ANRIL in the field of thyroid carcinoma.The purpose of this experiment is to study the role of LncRNA-ANRIL in the invasion and metastasis of thyroid carcinoma and its mechanism.Methods:Totally 105 patients with thyroid cancer in our hospital of recent years were enrolled.Thyroid cancer tissues and thyroid tissues adjacent to carcinoma were collected.At the same time,three kinds of thyroid cancer cell lines K1,TPC-1,SW579 and human thyroid cell line Nthy-ori 3-1 were slelcted to do cell experiments.Quantitative real-time PCR was used to detect the ANRIL expression in thyroid cancer tissues,adjacent normal tissues and each cell line.Meanwhile,immunohistochemical method was employed to detect the protein expression of TGF-β1.siRNA ANRIL and siRNA TGF-β1 were constructed and used for the transfection of TPC-1 and SW579 cell line;the experiment was divided into si-ANRIL group,si-TGF-β1 group,si-ANRIL + si-TGF-β1 group,negative control group and blank control group.MTT method,Transwell assay and tail vein injection of nude mice were conducted to determine the effects of silencing ANRIL and TGF-β on cell proliferation,invasion and metastasis of thyroid cancer cell.The qRT-PCR was applied to detect the regulatory role of ANRIL in tumor suppressor genes p15INK4 b,p14ARF and p16INK4 a in TPC-1 and SW579 cells.TGF-β1 and p-Smad2/3 expressions in TGF-β/Smad signaling pathway were detected by western blot.Results:1.ANRIL expression level was significantly higher in thyroid cancer tissues(10.28±1.21)than in thyroid tissues adjacent to carcinoma(3.47±0.54),with statistical significances(P < 0.001).There was no significant difference in ANRIL expression among different genders,ages,pathological types,tumor sizes,metastasis status,whether tumor is multiple and operation way(P > 0.05),while obvious difference was observed in the expression of ANRIL in different extracapsular invasion status and different lymph node metastasis(P < 0.01).2.The positive rate of TGF-β1 expression was significantly lower in thyroid tissues adjacent to carcinoma than in thyroid cancer tissues(28.57% vs.71.43 %,P < 0.001).Thyroid cancer tissue specimens with lymph node metastasis,extracapsular invasion status had higher positive rate of TGF-β1(all P < 0.01).The positive rate of TGF-β1 expression is independent of patients’ age,gender,pathological type,tumor size,metastasis status,whether tumor is multiple and the operation way(P > 0.05).3.The ANRIL expressions were significantly higher in K1(4.07±0.17),TPC-1(9.69±0.28),and SW579(5.90±0.18)of thyroid cancer cells than in Nthy-ori 3-1(3.02 + 0.14)of normal thyroid cells(P < 0.001).4.The cell counting and MTT experiment showed that cell proliferation was significantly inhibited in si-ANRIL group(P < 0.05).The OD values were significantly higher in si-TGF-β1 group and si-ANRIL+si-TGF-β1 group than in the blank control group and the negative control group at the 24 h and 48 h(P <0.05).Si-ANRIL can obviously inhibit the cell proliferation of TPC-1 and SW579 cells;silencing TGF-β1 can promote the cell proliferation of TPC-1 and SW579;while si-TGF-β1 can reverse si-ANRIL’s inhibition on cell proliferation of TPC-1 and SW579.5.Traswell assay found that inhibiting ANRIL expression can obviously inhibit the invading of TPC-1 and SW579 cells(P <0.05),and inhibiting TGF-β1 can promote the invading of TPC-1 and SW579 cells(P <0.05).Si-TGF-β1 can reverse the inhibition of si-ANRIL on TPC-1 and SW579 cell’s invading.6.Nude mouse experiment found that pulmonary metastasis nodules were obviously lower in TPC-1 and SW579 cells after si-ANRIL transfection through tail intravenous injection than in the blank control group and the negative control group(P <0.01).The pulmonary metastasis nodules were obviously higher in TPC-1 and SW579 cells after si-TGF-β1 transfection through tail intravenous injection than in the blank control group and the negative control group(P <0.05).The pulmonary metastasis nodules were obviously higher in si-ANRIL+si-TGF-β1 group than in si-ANRIL group(P <0.05).Silencing ANRIL inhibited the visceral metastasis of TPC-1 and SW579 cells,while silencing TGF-β1 promoted the visceral metastasis of TPC-1 and SW579 cells.si-TGF-β1 can reverse the inhibited visceral metastasis induced by si-ANRIL.7.The mRNA expression of p15INK4 b,p14ARF and p16INK4 a were significantly higher in si-ANRIL group than in the blank control group and the negative control group(P < 0.05).The mRNA expression of p15INK4 b were significantly lower in si-TGF-β1group and si-ANRIL+si-TGF-β1 group than in the blank control group and the negative control group(P < 0.05).There was no significant difference in the mRNA expression of p14 ARF and p16INK4 a between si-TGF-β1group,si-ANRIL+si-TGF-β1 group and the blank control group and the negative control group(P > 0.05).Si-TGF-β1 can only block the promoting effect on the p15INK4 b expression after silencing ANRIL.8.Expressions of TGF-β1 and p-Smad2/3 were significantly higher in si-ANRIL group than in the blank control group and the negative control group at 24 h after the transfection(P < 0.05).Expressions of TGF-β1 and p-Smad2/3 were significantly higher in than the blank control group and the negative control group than in si-TGF-β1 group and si-ANRIL+siTGF-β1 group(P < 0.05).Conclusion:ANRIL expresses highly in thyroid cancer tissues and thyroid cancer cell lines;it can promote the proliferation,invasion and metastasis of thyroid cancer cell.ANRIL may decrease the expression of p15INK4 b through TGFβ/Smad signaling pathway,thereby promoting the cell invasion and metastasis of thyroid cancer.While silencing ANRIL can inhibit the invasion and metastasis of thyroid cancer cells. |
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