Effect Of Long Non-Coding RNA PVT1 On Proliferation,Invasion And Migration Of Papillary Thyroid Carcinoma Cells Via MiR-195-5p/TERT Signaling Pathway

Thyroid cancer is one of the most common malignant tumors of the endocrine organs,which has been steadily increasing in morbidity and mortality over the years.There are three classifications for thyroid cancer based on the pathological characteristics,including papillary thyroid carcinoma(PTC),follicular thyroid carcinoma(FTC),and anaplastic thyroid carcinoma(ATC).Among the three kinds of thyroid cancers,PTC is the main form of thyroid cancer,whose morbidity is the highest and accounting for approximately 80 %,while ATC is the most aggressive solid and its 5-year survival rate is 3 to 5 months after diagnosis.Previous studies refer that radiation,chemotherapy,and surgery are the common treatment methods for thyroid cancer,but they all produce poor satisfactory.In addition to genetic and environmental factors,increasing papers have shown that epigenetic alteration may play pivotal roles in the development and progression of various kinds of tumors including thyroid cancer.Although being not well understood in human cancers,long noncoding RNAs(lnc RNAs,>200 nt)have been demonstrated to play crucial roles in tumorigenesis in recent years.For example,lnc RNA HOTAIR is significantly upregulated in hepatocellular cancer,and lnc RNA H19 has been proved to be associated with tumorigenesis of bladder cancer and gastric cancer.Generally,the mechanism of lnc RNA regulating gene expressions could be transcriptional or post-transcriptional level.To date,only several lnc RNAs including lnc RNA PTCSC3,lnc RNA NAMA,and lnc RNA BANCR have been demonstrated as the key lnc RNAs in the development and progression of thyroid cancer.Hence,there is a long way to discover the roles of lnc RNAs in thyroid cancer.The lnc RNA PVT1 is encoded by a gene that has been known since it resides in the well-known cancer risk region chromosome 8q24.Increasing evidence has shown that lnc RNA PVT1 plays pivotal roles in a variety of diseases,such as ovarian cancer development,pancreatic cancer prognosis,and non-small cell lung cancer tumorigenesis.Although lnc RNA PVT1 has been reported to play roles in various cancers,few have reported the possible roles and mechanism of lnc RNA PVT1 in regulating thyroid cancer cell proliferation.mi RNA is a small noncoding RNA molecule that plays a key role in posttranscriptional regulation of gene and RNA silencing and involved in the development of diverse tumors.The circulating mi RNAs have been identifed as specifc biomarkers for the diagnosis of various cancers,such as lung cancer,kidney diseases,breast cancer,hematologic cancer,prostate cancer,and thyroid cancer.Recent studies reveal that exogenous mi R-195 transfection can inhibit the proliferation of colorectal cancer cells and hepatocellular carcinoma cells.Rescuing the expression of mi R-195 in breast cancer cells signifcantly inhibits cell survival and promotes cellular apoptosis.Although mi R-195 has been extensively studied in recent years,its role in the development of thyroid cancer and its molecular mechanisms of upstream and downstream regulation are still poorly understood.Telomerase reverse transcriptase(TERT)is the most important component of telomerase because it acts as the major limiting agent for telomerase activity.The primary function of TERT is the elongation of telomeres,but recent studies have revealed additional functions,such as increasing cell proliferation and invasion resistance to apoptosis.We previously reported that the expression of h TERT was positively correlated with many clinicopathological variables,including tumor size,depth of invasion,histological grade,lymph node metastasis,TNM stage and distance of metastasis.TERT cannot be detected in non-immortalized cells,but is highly expressed in most cancer cells,including cancer stem cells.Due to its crucial function in carcinogenesis and universal,specific expression in cancer cells,TERT is an ideal anti-cancer target.Thus,it is important to understand the mechanisms regulating TERT.Many studies have focused on TERT regulation at the transcriptional level,while regulation at the post-transcriptional level,such as modulation by micro RNAs,has not been thoroughly explored.The research includes the following two parts.The first part is the effects of lnc RNA PVT1 and mi R-195-5p on proliferation,invasion and migration of papillary thyroid cancer cells.The second part is the effects of PVT1/mi R-195-5p axist on the proliferation,invasion and migration of papillary thyroid cancer cells through regulating TERT.In order to reveal the effect of long non-coding RNA PVT1 on proliferation,invasion and migration of papilla thyroid cancer cells through mi R-195-5p/TERT signaling pathway.Methods:Part One The effects of lnc RNA PVT1 and mi R-195-5p on proliferation,invasion and migration of papillary thyroid cancer cells.1.The expression level of PVT1 and mi R-195-5p in 32 pairs of papillary thyroid carcinoma(PTC)clinical samples and normal human thyroid cells Nthyori 3-1 and 4 papillary thyroid carcinoma(PTC)cells was measured by q RT-PCR and the correlation between PVT1 and mi R-195-5p expression were analyzed by using Pearson correlation.2.The targeting relationship between PVT1 and mi R-195-5p was measured by bioinformatics analysis.Wild and mutant PVT1 reporter plasmids were constructed,and the targeted binding relationship between PVT1 and mi R-195-5p was verified by dual luciferase reporter assay.The expression of mi R-195-5p after silencing PVT1 was detected by q RT-PCR.3.The effect of silencing PVT1 and combined inhibition of mi R-195-5p on K1 proliferation in papillary thyroid carcinoma(PTC)cells was examined by MTT assay.4.Transwell assay was used to detect the effect of silencing PVT1 and combined inhibition of mi R-195-5p on K1 invasion and migration in papillary thyroid carcinoma(PTC)cells.Part Two The effects of PVT1/mi R-195-5p axist on the proliferation,invasion and migration of papillary thyroid cancer cells through regulating TERT.1.The potential target gene of mi R-195-5p was predicted by bioinformatics analysis.The wild and mutant TERT 3’-UTR reporter plasmid was constructed,and the dual luciferase reporter assay verified that TERT is the target gene of mi R-195-5p.QRT-PCR and Western blot were used to detect the effect of mi R-195-5p on the expression of TERT in papillary thyroid carcinoma(PTC)K1 cells.2.The effect of overexpression of mi R-195-5p and combined with exogenous TERT on cell proliferation of papillary thyroid carcinoma(PTC)K1 cells was examined by MTT assay.3.The effect of overexpression of mi R-195-5p and combined with exogenous TERT on cell invasion and migration of papillary thyroid carcinoma(PTC)K1 cells was detected by Transwell assay.4.The expression of TERT in 32 pairs of papillary thyroid carcinoma(PTC)clinical samples and normal human thyroid cells Nthyori 3-1 and 4 papillary thyroid carcinoma(PTC)cells was measured by q RT-PCR.The correlation between TERT and mi R-195-5p expression was analyzed by Pearson correlation.5.The effect of silencing PVT1 on the expression of TERT in papillary thyroid carcinoma(PTC)K1 cells was detected by q RT-PCR and Western blot.6.The effect of silencing PVT1 and combined with exogenous TERT on cell proliferation of papillary thyroid carcinoma(PTC)K1 cells was examined by MTT assay.7.Transwell assay was used to detect the effect of silencing PVT1 and combined with exogenous TERT on cell invasion and migration of papillary thyroid carcinoma(PTC)K1 cells.8.Correlation between the expression of TERT and mi R-195-5p in 32 pairs of papillary thyroid carcinoma(PTC)clinical samples and normal human thyroid cells Nthyori 3-1 and 4 papillary thyroid carcinoma(PTC)cells was measured by Pearson correlation analysis.Results:Part One The effects of lnc RNA PVT1 and mi R-195-5p on proliferation,invasion and migration of papillary thyroid cancer cells.1.High expression of PVT1 in papillary thyroid carcinoma(PTC)was significantly negatively correlated with low expression of mi R-195-5p.2.Bioinformatics predictions show that PVT1 can bind to mi R-195-5p.The dual fluorescent reporter gene assay and q RT-PCR demonstrated the targeted regulatory relationship between PVT1 and mi R-195-5p.3.Silencing PVT1 significantly inhibited the proliferation of papillary thyroid carcinoma(PTC)K1 cells,while inhibition of mi R-195-5p reversed the inhibitory effect of silencing PVT1 on cell proliferation.4.Silencing PVT1 significantly inhibited the invasion and migration of papillary thyroid carcinoma(PTC)K1 cells,while inhibition of mi R-195-5p reversed the inhibitory effect of silencing PVT1 on cell invasion and migration.Part Two The effects of PVT1/mi R-195-5p axist on the proliferation,invasion and migration of papillary thyroid cancer cells through regulating TERT.1.Bioinformatics predicts that TERT is a potential target gene for mi R-195-5p.The dual fluorescent reporter gene assay and q RT-PCR and Western blot experiments confirmed that mi R-195-5p can specifically inhibit the expression of TERT m RNA and protein in papillary thyroid carcinoma(PTC)K1 cells,and TERT is the target of mi R-195-5p.2.Overexpression of mi R-195-5p significantly inhibited cell proliferation of papillary thyroid carcinoma(PTC)K1 cells,whereas exogenous TERT reversed the inhibitory effect of overexpression of mi R-195-5p on cell proliferation.3.Overexpression of mi R-195-5p significantly inhibited the invasion and migration of papillary thyroid carcinoma(PTC)K1 cells,while exogenous TERT reversed the inhibitory effect of overexpression of mi R-195-5p on cell invasion and migration.4.High expression of TERT in papillary thyroid carcinoma(PTC)was significantly negatively correlated with low expression of mi R-195-5p.5.Silencing PVT1 significantly inhibited the expression of TERT in papillary thyroid carcinoma(PTC)K1 cells.6.Silencing PVT1 significantly inhibited cell proliferation of papillary thyroid carcinoma(PTC)K1 cells,whereas exogenous TERT reversed the inhibitory effect of silencing PVT1 on cell proliferation.7.Silencing PVT1 significantly inhibited the invasion and migration of papillary thyroid carcinoma(PTC)K1 cells,while exogenous complementary TERT reversed the inhibitory effect of silencing PVT1 on cell invasion and migration.8.High expression of PVT1 in papillary thyroid carcinoma(PTC)showed a significantly positive correlation with high expression of TERT.Conclusion:The high expression of PVT1 in papillary thyroid carcinoma(PTC)was significantly negatively correlated with the low expression of mi R-195-5p,and was significantly positively correlated with the high expression of TERT.PVT1 can up-regulate the expression of TERT by sponging mi R-195-5p,thereby promoting the proliferation,invasion and migration of papillary thyroid carcinoma K1 cells.