The Role Of GPX7 In Papillary Thyroid Carcinoma

Background:Thyroid cancer is the most common malignant tumor in endocrine organs.The main pathological types are papillary thyroid carcinoma(PTC),follicular carcinoma,undifferentiated carcinoma and myeloid cancer and so on.PTC is the most common type of thyroid cancer.In general,there is slower development,longer course,better prognosis of PTC patients,but because of no clinical symptoms,many patients went to hospital later,were diagnosised PTC accompanied by lymph node metastasis,membranes invasion,etc.,resulting in poor prognosis.It is necessary to early detection,early diagnosis,and early treatment of PTC.Although most studies reported that the pathogenesis of PTC was related to BRAF gene mutation,RET/PTC gene rearrangement and so on,but the pathogenesis of PTC is still not clear,we should study further the pathogenesis of PTC,which has important clinical significance.Glutathione peroxidases(GPXs)in mammalian cells are the most important oxidoreductases in the body and consist of eight GPXs,named GPX1-GPX8,among which GPX1-GPX4 and GPX6 are selenocysteine-containing GPXs,GPX5,GPX7 and GPX8 were non-selenocysteine-containing GPXs.In general,selenocysteine-containing GPXs can reduce H2O2 by catalytic reduction of glutathione(GSH)to reduce the oxidative stress response of the disease.The mechanisms of non-selenocysteine-containing GPXs of peroxidase activity are controversial,because of lack the GSH binding domain.Reactive oxygen species(ROS)are reactive oxygen-containing molecules that include oxygen ions and peroxide.ROS are produced as natural products from multiple intrinsically means.Some scholars had suggested that oxidative stress is related to the occurrence and development of tumor.Oxidative stress is generated when there is an imbalance between systemic manifestation of ROS and the biological detoxification processes.ROS includes O2-,H2O2,HO2-,-OH and other active oxygen molecules.Persistent exposure to oxidative stress leads to systemic abnormalities,such as autoimmunity,carcinogenesis,diabetes mellitus,obesity,neurodegeneration,and aging.The main physiological function of the thyroid is the synthesis of thyroid hormones.H2O2 is a key enzyme in the synthesis of thyroid peroxidase,which is the essential substrate for thyroid hormone synthesis.So H2O2 plays a very important role in the normal thyroid physiological function.When the body is abnormal,H2O2 can be accumulated in the thyroid gland,making the thyroid continue to be exposed to high concentrations of H2O2.The effect induced by ROS environment,is oxidative stress.This effect can make oxidative DNA damage or gene low methylation of thyroid cells.Studies have confirmed that a large number of ROS can promote cell proliferation,invasion,metastasis and angiogenesis,and can escape the occurrence of apoptosis.Therefore,abnormal oxidative stress response may be related to the occurrence and development of PTC.Glutathione peroxidase 7(GPX7)was one of GPXs family,mainly involved in maintaining peroxidase activities.In recent years,the study found that GPX7 abnormal expression was related to the occurrence and development of a variety of tumors(including esophageal adenocarcinoma,breast cancer,hepatocellular carcinoma,etc.),but the role of GPX7 in thyroid cancer has not been reported.It is known that NF-κB is an important nuclear transcription factor that regulates DNA transcription in eukaryotic cells and has multiple transcriptional regulation.It has been confirmed that NF-κB is involved in the development and progression of various malignant tumors.It has been reported that the role of GPX7 in esophageal adenocarcinoma(EAC)may play a role through the NF-κB signaling pathway.Cyclin D1 can regulate the cell cycle,is one of the indicators of cell proliferation activity.It has been reported that CyclinD1 is an important downstream target gene of NF-κB p65.The study of the relationship between GPX7 expression in NF-κB and Cyclin D1 in papillary thyroid carcinoma has not been reported.The aim of this study was to investigate the expression of GPX7 in PTC and the relationship between the expression of GPX7 with the clinicopathological parameters,to investigate the possible mechanism of GPX7 in the development and progression of PTC.Objective:1.To observe the expression of GPX7 and the change of proliferation and apoptosis of PTC cell from PTC tissue and cell level,in order to investigating the role of GPX7 in PTC.2.To investigate the relationship between the expression of GPX7 and NF-κB p65 and Cyclin D1 from tissue and cell level,in order to investigate the possible mechanism on regulation of PTC cell proliferation and apoptosis.3.To study the expression of GPX7 protein in thyroid papillary carcinoma with Hashimoto’s thyroiditis(PTC+HT),in order to exploring the effect of HT inflammatory microenvironment on the expression of GPX7 in papillary thyroid carcinoma.Method:1.The expression of GPX7 in 30 cases of PTC and 14 cases of nodular goiter tissues were detected by immunohistochemistry.The relationship between the expression of GPX7 and clinical clinicopathological features were analyzed.2.To constructe of GPX7 gene interference lentiviral vector,infect into K1 cells to make GPX7 gene silencing.To observe the change of cell proliferation,apoptosis,and clonal formation of K1 cell after GPX7 gene silencing by a variety of cell function experiments(Celigo cell count,flow cytometry detection of apoptosis,Caspase3/7 detection,MTT detection,cell cloning formation experiment).3.The expression of NF-κB p65 and Cyclin D1 protein in 30 cases of PTC and 14 cases of nodular goiter were detected by immunohistochemistry.The relationship between the expression of GPX7 with NF-κB p65 and Cyclin D1 was analyzed.4.The expression of NF-κB p65 and Cyclin D1 in K1 cells before and after GPX7 gene silencing were compared by Western blotting test.5.The expression of GPX7 in 29 cases of PTC+HT,30 cases of PTC,16 cases of HT and 14 cases of nodular goiter tissues were detected by immunohistochemistry.To investigate the effect of inflammatory microenvironment of Hashimoto’s thyroiditis on the expression of GPX7 and its role in papillary thyroid carcinoma.6.Immunohistochemical evaluation criteria: each case were random observation of five high power field.Scoring methods were utilised in the evaluation of Immunohistochemical for both staining intensity and percentage positive of stained tumor cells.Percentage of positive cells: 0,<10% of tumor cells were stained;1,11% to 25% of tumor cells were stained;2,26% to 50% of tumor cells were stained;3,51% to 75% of tumor cells were stained;4,>75% of tumor cells were stained.staining intensity: 0,no staining;1,weak staining;2,moderate staining;3,strong staining.The score of multiply them was the final score: weak positive(+),1~4 scores;moderate positive(++),5~8 scores is divided into;strong positive(+++),9~12 scores.7.Statistical analysis: All data was analyzed with SPSS statistical software version 20.0.Statistical analysis was performed with the ttest.To compare the variables between the groups,χ2 tests was performed for categorical data.Correlations between parameters were done with the Spearman correlation test.The tests were performed at a significance level of 0.05.Results:1.the results about the role of GPX7 on proliferation and apoptosis of PTC(1)immunohistochemical results:(1)GPX7 was expressed in the cytoplasm of thyroid cells.The positive rate of GPX7 expression in the PTC group was 100.0%,mainly medium-strong positive,and the positive rate of GPX7 expression in the nodular goiter group was 30.0%,mainly weakly positive.The expression of GPX7 in PTC group was significantly higher than that in the nodular goiter group(P < 0.05).(2)The expression of GPX7 was positively correlated with the maximal diameter of tumor(R = 0.601,P <0.05),and the maximal diameter of GPX7 high expression group(1.56 ± 0.56cm)was significantly higher than that of GPX7 low expression group(0.56 ± 0.13cm)(P <0.05).(2)cell test results:(1)Two kinds of human thyroid papillary carcinoma cell lines TPC-1 and K1 were selected.The results of QT-PCR showed that GPX7 gene had high expression in TPC-1 and K1 cells.the expression in K1 cell is higher,so we selected K1 cells for the subsequent experimental study.(2)After preparation of GPX7 gene interference lentiviral vector,with Western blot exogenous verification method,to find that the expression of GPX7 after gene knockdown was significantly reduced,indicating that the target of GPX7 gene knockdown is success.(3)After GPX7 gene interference with lentiviral vector infected with K1 cells,we observed that the cell infection efficiency was more of 80% by ordinary microscopy and fluorescence microscopy,and the cell status is normal.The expression of GPX7 gene in the K1 cells was inhibited by qPCR method,and the knockdown efficiency was 81.5%,which indicated that K1 cells after had good effect and could be followed by experimental observation.(4)Celigo cell count showed that the number of cell proliferation was significantly decreased in GPX7 gene silencing group.The proliferation fold was(5.00 ± 0.32)in GPX7 gene silencing group,lower than(8.44 ± 0.26)in control group.(5)The percentage of apoptotic cells by flow cytometry in GPX7 gene silencing group(24.08 ± 0.28)% was significantly higher than that in control group(4.33 ± 0.15)%.(6)Caspase 3/7 activity in the GPX7 gene silencing group was(257.82 ± 21.28),which was significantly higher than that in the control group(100.00 ± 5.38).(7)By MTT method,the OD490 value fold of GPX7 gene silencing group was(3.081 ± 0.160)lower than(4.897 ± 0.091)times in the control group.(8)Cell clone formation test showed that GPX7 gene silencing group clone number(283 ± 6),compared with the control group(710 ± 10),is significantly reduced.2.the results about the possible mechanism of GPX7 in thyroid papillary carcinoma cells(1)immunohistochemical results:(1)NF-κB p65 was expressed in the thyroid cell cytoplasm and / or nucleus.The positive rate of NF-κB p65 expression in PTC group was higher than that in nodular goiter group.The positive expression rate of NF-κB p65 in the PTC group was 83.3%,mainly medium positive.(2)Cyclin D1 was expressed in the thyroid nucleus.The positive rate of Cyclin D1 expression in PTC group was higher than that in nodular goiter group.PTC group was significantly higher than that of group A.The positive expression rate of Cyclin D1 in the PTC group was96.7%,mainly medium positive.(3)GPX7,NF-κB p65 and Cyclin D1 were significantly positive correlated(r=0.553,0.522,0.674,all of P <0.05).(4)The positive rate of GPX7 combined with NF-κB p65 in the PTC group was 83.3%.The positive rate of GPX7 combined with Cyclin D1 in the PTC group was 100.0%.The positive rate of GPX7 combined with NF-κB p65 and Cyclin D1 in the PTC group was 83.3%.(2)cell test results:(1)After GPX7 gene interference with lentiviral vector infected with K1 cells,the cell infection efficiency was higher.And qPCR method confirmed that the GPX7 gene was effectively knocked down.(2)After GPX7 gene knocked down with lentiviral vector infected with K1 cells,we observed that the expression of NF-κB p65 protein in the papillary thyroid carcinoma cell line(K1 cell line)after GPX7 gene silencing was not significantly changed,and the expression of Cyclin D1 protein was significantly higher than that before knockdown(P <0.05).3.the results about the effect of inflammatory microenvironment on the expression of GPX7(1)Immunohistochemical results showed that the expression of GPX7 in PTC+HT group(6.21±2.29)was significantly lower than that in the PTC group(8.52±2.41)(P < 0.05).(2)Univariate analysis showed that GPX7 was associated with HT,TG-Ab,TPO-Ab,tumor site and capsule invasion,and GPX7 was negatively correlated with HT,TG-Ab and TPO-Ab levels,positively correlated with capsule invasion and Tumor site.(3)Multivariate linear regression analysis showed that the factors affected GPX7 expression was HT inflammatory microenvironment.(4)Clinicopathological features analysis showed that that PTC tumors with HT inflammatory environment were confined to unilateral,and the incidence of invasion was lower.Conclusion:1.Immunohistochemistry and cell test results suggest that PTC cells have high expression of GPX7.2.Immunohistochemistry and cell test results suggest that GPX7 has the effect of promoting the proliferation and reducing the apoptosis of PTC cells.3.The expression of GPX7,NF-κB p65 and Cyclin D1 protein in papillary thyroid carcinoma was positively correlated,suggesting that they maybe have relationship on the development and development of papillary thyroid carcinoma.4.The expression of NF-κB p65 protein after GPX7 gene silencing did not change,suggesting that the effect of GPX7 on PTC was not related to NF-κB signaling pathway or by regulating its protein expression.5.The expression of Cyclin D1 protein after GPX7 gene silencing was increased.The mechanism of this phenomenon is unclear.6.By single factor and multivariate linear regression analysis,it was found that the combination of Hashimoto’s thyroiditis was the factor on affect the expression of GPX7 in papillary thyroid carcinoma tissues,and they were negative correlationship,suggesting that the inflammatory environment of Hashimoto’s thyroiditis could inhibit expression of GPX7 in PTC.