Progranulin Regulates The Malignant Biological Behavior And Mechanism Of Papillary Thyroid Carcinoma

Background: Thyroid cancer is one of the most familiar endocrine cancers,and its incidence is steadily increasing worldwide.It is expected that by 2030,thyroid cancer will be the second highest incidence of tumors in women and ninth among men.Generally Thyroid cancer can be classified into six types: papillary thyroid carcinoma(PTC),follicular thyroid cancer(FTC),undifferentiated thyroid carcinoma(ATC),medullary thyroid carcinoma(MTC),poorly differentiated thyroid cancer(PDTC)and Hürtle cell carcinoma.PTC,especially the incidence of low-risk papillary thyroid microcarcinoma(PTMC),is the most common type of thyroid cancer.Most PTCs are relatively indolent with a 10-year survival rate of > 95% after surgery.However,10%of patients diagnosed with advanced stages still have a poor prognosis.It is the followup survey show that the recurrence rate of PTC is also as high as 25%.At present,surgical resolutions is still the mainstay of treatment for PTC,but it is still not ideal for patients with tumors with tumors < 1 cm in diameter or multiple,highly aggressive or metastatic tumors.As its recurrence rate is increasingly rise,the pressure and burden brought to patients are more interesting.Therefore,in-depth study of the formation mechanism of papillary thyroid carcinoma and elucidating the underlying molecular mechanisms leading to the onset and progression of PTC are essential for new technologies to diagnose and treat the disease.Progranulin(PGRN),a protein encoded by the GRN gene located on human chromosome 17,is a secreted protein secreted by epithelial cells.PGRN is secreted by most tissue epithelium cells,and its functions differently from different expression of tissue epithelium cells.The study found that PGRN was expressed at elevated levels in a variety of malignant tumors,involving in the proliferation,invasion,migration and apoptosis of cancer cells.At the same time,the latest study also proves that the serum level of PGRN in patients with papillary thyroid cancer is elevated,with PGRN possibly involved in the invasion and metastasis of papillary thyroid cancer.However,its expression,biological function and molecular mechanism in PTC are still unknown.Methods: Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting were applied to test the mRNA and protein expression of PGRN gene in PTC tissues and paired normal tissues adjacent to tumor which are collected from the hospital.We use statistical results of qRT-PCR and Western Blot experiments to analyze the relationship between abnormal expression of PGRN and clinicopathological features of PTC patients.Thyroid follicular epithelial cell lines(Nthy-ori-3-1,N3)and thyroid papillary cancer cell lines(TPC1,BCPAP,K1)were cultured,and Western blot was used to detect the expression of PGRN in cells.PGRN knockdown cell model and PGRN overexpression model were constructed by transfection of si RNA and lentivirus in PTC cells,respectively.The knockdown and overexpression of PGRN were detected by qRT-PCR and Western blot.The influences of PGRN knockdown and PGRN overexpression in PTC cells on cell proliferation were detected by MTS,EDU,Colony formation and other methods.Scratch experiments examined the effects of knockdown and overexpression of PGRN gene expression on the migration ability of PTC cells.Transwell invasion experiments examined the effects of knockdown and overexpression of PGRN gene expression on the invasion ability of PTC cells.Propidium Iodide(PI)method to detect the effect of knockout and overexpression of PGRN gene expression on PTC cell cycle;PI/FITC method to detect the effect of knockdown and overexpression of PGRN gene expression on apoptosis in PTC.Western blot assay was used to detect the influence of PGRN expression on apoptotic associated protein(BAD、BCL-2、BAX),cell cycle protein Cyclin D1,and epithelialto-mesenchymal transition(EMT)markers.To explore the possible signaling pathways involved in PGRN by the Gene Set Enrichment Analysis(GSEA).Two control group,a knockdown group,a knockdown negative control group and a overexpression group and a overexpression negative group were set up respectively.And the changes in the expression of signaling molecules JAK2,p-JAK2,STAT3,p-STAT3,STAT4,p-STAT4 in each group were examined.In addition,JAK pathway inhibitor JSI-124 was added into the cell culture medium of the above groups,and then the expression levels of signal molecules p-JAK2,p-STAT3,p-STAT4 and other proteins in each group were detected.ResultsWestern blot and qRT-PCR indicated that the relative PGRN mRNA and protein expression in PTC tissues was 23.827±4.450 and 0.958 ± 0.122 respectively,which were significantly higher than that of normal tissues adjacent to cancer(9.276±2.412 and 0.669±0.091)(P <0.05).Immunohistochemical staining showed that the high expression rate of PGRN protein in 70 cases was 84.29%(59/70),which was significantly higher than the high expression rate of 32.86%(23/70)in the adjacent normal group(P < 0.05).The high expression of PGRN was closely related to lymph node metastasis in PTC patients(P <0.05),but not with gender,tumor size,capsular invasion,age,and tumor multifocality.The expression levels of PGRN in the PTC cell line TPC-1 and BCPAP were significantly higher than those in thyroid follicular epithelial cells Nthy-ori-3-1(P <0.05).This work is to study the expression after knockdown and overexpression of PGRN for the effects of malignant biological behavior of PTC cell lines.The results of MTS,EDU and colony formation experiments showed that the cell proliferation ability after PGRN expression knockdown was significantly decreased than that in the control group,and the cell proliferation ability after PGRN overexpression was significantly stronger than that in the control group.Cells by the test results showed that PGRN expression on reduction of apoptosis rate is significantly higher than the control group,and block the cell division phase from G0 /G1 phase to S phase.The apoptosis rate of the PGRN overexpression group was significantly lower than that of the control group,and the cell division transition from G0/G1 phase to S phase was promoted.Transwell experiments and scratch experiments explained that PGRN knockdown significantly decreased the cell invasion and migration ability(P <0.05),while the results of PGRN overexpression group were the opposite.Western blot was used to detect the expression of Cyclin D1,apoptotic associated protein(BAX,BCL-2,BAD),and EMT-related proteins after the knockdown or overexpression of PGRN gene expression.The results showed that the expression of Cyclin D1、BCL-2/BAD and BCL-2/BAX in the two PTC cell lines decreased after the PGRN gene expression knockdown(P <0.05).N-cadherin,MMP2 and Vimentin expression were down-regulated,E-cadherin expression increased(P<0.05);PGRN gene overexpression was completely opposite in the above two cell lines.Enrichment analysis indicated that PGRN and its co-expressed proteins were enriched in the JAK-STAT pathway.Western blot was used to detect the expression of JAK2,pJAK2,p-JAK2,STAT3,STAT3,STAT4,and p-STAT4 proteins in the no-transfected group,knockdown negative control group,knockdown group,overexpression negative control group,and overexpression group.The relative expression levels of p-STAT3 and p-STAT4 proteins decreased significantly(P <0.05),and the relative expression levels of p-JAK2,p-STAT3 and p-STAT4 proteins in the two PTC cell lines after overexpression of PGRN gene increased significantly(P <0.05),while the expression of JAK2,STAT3 and STAT4 proteins did not change significantly.There was no significant difference in protein expression levels of JAK2,p-JAK2,STAT3,p-STAT3,STAT4,and p-STAT4 in the untransfected group of PTC cell lines and negative control groups(P >0.05).After adding JAK2 inhibitor JSI-124 to the overexpression group,Western blot results showed that it partially rescued the effects of PGRN overexpression on apoptosis and EMT-related proteins.Conclusions: The PGRN mRNA and protein expression levels in PTC tissues were significantly higher than those in normal tissues next to cancer,and the abnormal expression of PGRN was closely related to PTC lymph node metastasis.PGRN gene can strengthen the proliferation,migration,and invasion of PTC cells.These abilities are inhibited after knocking down PGRN expression.Meanwhile,knocking down PGRN expression also induces apoptosis and cell cycle arrest.PGRN expression will enhance the expression of Cyclin D1,BCL-2,MMP2,N-cadherin,Vimentin in PTC cells with reducing the expression of E-cadherin,BAD,and BAX.PGRN may affect the biological behavior of PTC cells by regulating the JAK-STAT pathway.So,PGRN may be a potential therapeutic target for papillary thyroid cancer.