| Papillary thyroid carcinoma(PTC)originates from thyroid follicular epithelial cells.The incidence of thyroid cancer has been increased in recent years.Thyroid cancer has gradually become the most common endocrine malignant tumor.As the main subtype of thyroid cancer,PTC accounts for about 80% of thyroid cancer and mainly occurs in young women and children.Although the prognosis for patients with early PTC is good,the 5-year survival rate for patients with advanced PTC is only about 59%.Therefore,it urgently needs to find a more effective treatment strategy for PTC.CircRNAs(circRNAs)is a novel class of endogenous non-coding RNAs molecules characterized by covalent closed-loop structure absence of 5’-3’-polarity and a polyadenylated tail.They are abundant,conservative,stable,and show spatio-temporal specificity in different tissues.CircRNAs,as emerging non-coding RNA molecules,some of them are considered as biomarkers and modulators of cancer.Researchers have found that circRNAs may play a role in inhibiting or promoting cancer in a variety of cancers.CircRNA nuclear receptor interaction protein 1(circNRIP1),a new type of circRNA,has been found to be abnormally overexpressed in gastric cancer,and played a role in promoting the progression of gastric cancer.However,the expression of circRNA NRIP1 in PTC has not been reported,and its roles in PTC are unknown.MicroRNAs(miRNAs)are a class of conserved small non-coding RNAs.They cause target gene silencing by degrading mRNA molecules or inhibiting their translation.miRNAs are involved in a variety of biological events,including tumorigenesis and metastasis,suggesting that they play a critical role in the pathogenesis of a variety of human malignancies.Abnormally up-regulated miRNAs,such as miR-146b,miR-222,miR-221,and miR-151,are thought to be related to the development of PTC,especially the metastasis of PTC.In PTC related studies,miR-195 is down-regulated in PTC compared with normal thyroid tissue.However,the role of miR-195 in PTC has not been confirmed,and the molecular mechanism of miR-195 regulation roles in thyroid cancer remains unclear.miR-195-5p is significantly downregulated in many types of human cancers,including PTC,and has been shown to inhibit cancer.In the early stage of this study,Starbase v2 0 and other bioinformatics online analysis showed that there was a targeted complementary sequence between CircRNA NRIP1 and miR-195-5p,suggesting that there may be a regulatory relationship between them.This study aims to detect and analyze CircRNA NRIP1 and miR-195-5p expression in PTC,and circRNA NRIP1 and miR-195-5p in the function of PTC and its possible mechanism,which providing a new potential target for PTC.First,the PTC and normal thyroid tissue adjacent to carcinoma specimens were collected,and HE staining was used to detect their difference in histomorphology,and circRNA NRIP1 and miR-195-5p expression in PTC tumor tissues and the PTC cell line was detected,and the correlation between circRNA NRIP1 and miR-195-5p expression in PTC tissues were analyzed.In addition,the relationship between circRNA NRIP1 and miR-195-5p was predicted and verified;Furthermore,effects of the interference of circRNA NRIP1 on the proliferation,apoptosis,invasion and migration and the P38 MAPK and JAK/STAT signaling pathways and the growth of xenograft tumors were detected in vitro;Finaly,and the effects of circRNA NRIP1/miR-195-5p axis on cell proliferation,invasion,apoptosis,migration and the P38 MAPK and JAK/STAT signaling pathways in TPC1 and IHH-4 cells and the growth of TPC1 xenograft tumor were detected.This study is mainly divided into three parts:Part Ⅰ:Expression of circRNA NRIP1 and miR-195-5p and its significance in PTC;Part Ⅱ:Effect of circRNA NRIP1 on the malignant phenotype of PTC cells and PTC xenograft growth;Part Ⅲ:Study on the mechanism of circRNA NRIP1 affecting malignant phenotype of PTC cells and the PTC xenograft growth.Main content:Part Ⅰ:Expression of circRNA NREP1 and miR-195-5p and its significance in PTCMethods1.Samples of PTC tissues and adjacent normal thyroid tissues were collected,and histomorphological differences were detected by HE assay.2.qRT-PCR was used to detect the expression level of circRNA NRIP1 and miR-195-5p in PTC tumor tissues and control adjacent tissues,PTC tissues at different clinical stages,PTC cell lines including TPC1,B-CPAP,IHH-4,K1,and normal thyroid cells Nthy-ori3-1.3.Analyzing the correlation between circRNA NRIP1 and miR-195-5p expression in PTC tissues was performed.4.The relationship between circRNA NRIP1 and miR-195-5p was predicted and verified by online bioinformatics software and dual luciferase reporter gene assay respectively.Results1.The results of HE staining test showed that there were branched papillae structure in PTC tissues compared with the normal adjacent tissues.2.Compared with control tissues adjacent to carcinoma,in the PTC tumor tissues,circRNA NRIP1 significantly had higher expression,and miR-195-5p expression was decreased obviously.3.Compared with the Nthy-ori3-1 cells in the control group,the expression of circRNA NRIP1 in TPC1,B-CPAP,IHH-4 and K1 was significantly increased,and the expression of miR-195-5p was significantly decreased,especially in TPC1 and IHH-4 cells.4.Compared with the PTC tissues at Ⅰ/Ⅱ stage,the expression level of circRNA NRIP1 was significantly increased in the PTC tissues at Ⅲ/Ⅳ stage,while the expression level of miR-195-5p was significantly decreased.5.CircRNA NRIP1 was negatively correlated with the expression level of miR-195-5p in PTC tissues.6.Online bioinformatics software Starbase V2.0 predicted and dual-luciferase reporter gene experiment verified that circRNA NRIP1 could inhibit miR-195-5p expression by functioning as a molecular sponge.Part Ⅱ:Effect of CircRNA NRIP1 on the malignant phenotype of PTC cells and PTC xenograft growthMethods1.Lentivirus vector with circRNA NRIP1 interference sequence was constructed.Lentivirus packaging and titration were performed in 293T cells.Then,lentivirus infected TPC1 and IHH-4 cells.2.Interference efficiency of circRNA NRIP1 in TPC1 and IHH-4 cells was detected by qRT-PCR.3.MTT assay was performed to measure the effect of circRNA NRIP1 interference on the proliferation of TPC1 and IHH-4 cells.4.The effect of circRNA NRIP1 interference on apoptosis in TPC1 and IHH-4 cells was detected by flow cytometry assay.5.The effect of circRNA NRIP1 interference on TPC1 and IHH-4 cell invasion was determined by Transwell assay.6.The scratch assay detected the effect of circRNA NRIP1 interference on the migration of TPC1 and IHH-4 cells.7.Western blot was used to analyze the effect of circRNA NRIP1 interference on the proliferation,apoptosis,invasion,EMT and the P38 MAPK and JAK/STAT signaling pathways-related proteins expression in TPC1 and IHH-4 cells,including Ki67,Cleaved caspase-7,MMP-1,MMP-7,E-cadherin,Vimentin,α-SMA.8.PTC xenograft models were constructed by injection of TPC1 cells with circRNA NRIP1 interference into mice to detect the effect of circRNA NRIP1 interference on the growth of PTC xenograft growth.Results1.Compared with the control group,circRNA NRIP1 expression in the circRNA NRIP1 interference group was significantly decreased.2.In TPC1 and IHH-4 cells,interference of circRNA NRIP1 expression could significantly inhibit cell proliferation,invasion,migration,EMT and activity of the P38 MAPK and JAK/STAT signaling pathways,and promote apoptosis.3.Compared with the control group,Ki67,MMP-1,MMP-7,Vimentin andα-SMA expression levels were significantly decreased in TPC1 and IHH-4 cells after circRNA NRIP1 interference,while E-cadherin and Cleaved caspase-7 levels were significantly increased.Part Ⅲ:Study on the mechanism of circRNA NRIP1 affecting malignant phenotype of PTC cells and the PTC xenograft growthMethods1.Lentivirus vectors with miR-195-5p overexpression and circRNA NRIP1 overexpression sequences were constructed.After lentivirus packaging and titrated titration,lentivirus infected TPC1 and IHH-4 cells,TPC1 and IHH-4 cells with miR-195-5p overexpression,circRNA NRIP1 overexpression and miR-195-5p overexpression together with circRNA NRIP1 were obtained.2.The effect of circRNA NRIP1 on miR-195-5p expression was detected by qRT-PCR.3.MTT assay was used to detect the effect of circRNA NRIP1-miR-195-5p axis on proliferation of TPC1 and IHH-4 cells.4.Flow cytometry assay was used to detect the effect of circRNA NRIP1-miR-195-5p axis on apoptosis in TPC1 and IHH-4 cells.5.Transwell assay was used to detect the effect of circRNA NRIP1-miR-195-5paxis on invasion of TPC1 and IHH-4 cells.6.The scratch assay was used to detect the effect of circRNA NRIP1-miR-195-5p axis on migration of TPC1 and IHH-4 cells.7.Western blot analyses of the effects of circRNA NRIP1-miR-195-5p axis on the expression of the P38 MAPK and JAK/STAT pathway-related proteins p-P38,P38,p-JAK2,JAK2,p-STAT1 and STAT1 in TPC1 and IHH-4 cells were performed.8.PTC xenograft tumor was constructed by injection of TPC1 cells to analyze the effect of circRNA NRIP1-miR-195-5p axis on the growth of PTC xenograft tumor.Results1.qRT-PCR results showed that circRNA NRIP1 overexpression significantly inhibited the expression of miR-195-5p,while interference of circRNA NRIP1 expression promoted the expression of miR-195-5p.2.In TPC1 and IHH-4 cells,circRNA NRIP1-miR-195-5p axis could promote cell proliferation,invasion and migration and inhibit cell apoptosis,as well as enhance the growth of PTC xenograft tumor by promoting the activity of the P38 MAPK and JAK/STAT signaling pathways,thereby playing a pro-cancer role.Conclusion1.Compared with normal paracancer tissues and normal cells,circRNA NRIP1 was significantly highly expressed in PTC tissues and cells,while miR-195-5p was significantly decreased.The expression of the two molecules was negatively correlated in PTC tissues,and circRNA NRIP1 could sponge and inhibit miR-195-5p.2.In TPC1 and IHH-4 cells,interference of circNRIP1 expression could significantly promote apoptosis and inhibit cell proliferation,invasion,migration and EMT as well as the activity of P38 MAPK and JAK/STAT signaling pathways.3.In TPC1 and IHH-4 cells,circRNA NRIP1 through effecting miR-195-5p expression could inhibit apoptosis and promote cell proliferation,invasion and migration as well as the growth of PTC xenograft tumor by promoting the activity of the P3 8 MAPK and JAK/STAT signaling pathways,so as to,thereby playing a pro-cancer role. |
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