The Clinical Relationship Between Obesity And Papillary Thyroid Carcinoma And The Mechanism Of Adiponectin Chemical Analogues

Purposes.1.To clarify the relationship between obesity and aggressive pathological features of papillary thyroid carcinoma.2.To screen potential obesity factors involved in the progress of obesity and papillary thyroid carcinoma with adipokine antibody arrays.3.To verify the therapeutic effect of recombinant adiponectin,constructed from adipose stem cells,on papillary thyroid carcinoma.4.To explore the therapeutic effect and molecular mechanism of AdipoRon in thyroid cancer cells.Method.1.This study retrospectively analyzed 13,995 adult patients with papillary thyroid cancer（PTC）from a single medical center in China.Data stratification was performed to assess the associations of obesity with ultrasound（US）features and aggressive clinicopathological features in different models according to the World Health Organization Body Mass Index（WHO-BMI）and Chinese BMI classification（CN‐BMI）.The odds ratio（OR）of malignant US features and aggressive clinicopathological features were calculated from binary logistic regression models.2.In the previous retrospective study,we found that female obesity affects the TIRADS classification of thyroid nodules,which may interfere with ultrasound evaluation.This may delay the diagnosis and treatment of thyroid nodules and passively promote the progression of thyroid cancer.Thus,we further conducted a prospective study and collected 1,830 cases.Body fat parameters of PTC patients,including full body fat parameters（height,weight,BMI,BSA,body fat percentage）and local body fat parameters（neck circumference,neck length,neck-circumference-to-length ratio,waist circumference,hip circumference,thigh circumference）,Waist-to-body ratio,waist-to-hip ratio,skinfold thickness.Binary logistic regression was used to analyze the predictive significance of different body fat parameters for invasive pathological features of thyroid cancer,and to determine whether obesity interferes with ultrasound evaluation of thyroid nodules.3.Through clinical retrospective and prospective studies,the clinical association between obesity and PTC progression was clarified.Therefore,we applied the adipokine antibody arrays to collect the serum and neck adipose tissue of 80 patients with normal weight and obesity PTC.We selected 40 obesity factors that may be involved in the progression of PTC from the currently known 300 obesity factors and screened potential target factors,namely adiponectin（AdipoQ）.4.In order to verify the accuracy of the results of the adipokine antibody arrays,we detected the serum AdipoQ level of PTC patients with ELISA.The adipose stem cells from the neck adipose tissue were extracted to construct a stable adipose stem cell recombinant AdipoQ cell line.Human-derived recombinant AdipoQ was used to explore the therapeutic effects of AdipoQ in thyroid cancer cells,including cell proliferation and migration.5.The foregoing studies confirmed that AdipoQ is an important obesity factor that inhibits the progression of thyroid cancer.However,due to the specific molecular structure of AdipoQ,the clinical transformation technology of AdipoQ is difficult and the economic cost is high.Therefore,we found a new type of AdipoQ analogue-AdipoRon,as the research object of this topic.First,q-PCR and immunofluorescence were used to determine the presence of adiponectin receptors on the surface of thyroid cancer cells,and further cell function verification was performed,including proliferation ability（CCK-8,plate clone formation and Ed U experiment）,migration ability（scratch experiment And Transwell experiment）,ability metabolism level（key molecules of sugar metabolism and amino acid metabolism）,differentiation level and apoptosis（detection of mitochondrial membrane potential,apoptotic protein caspase 3/7 and AnnexinV fluorescent probe）.6.RNA transcriptome sequencing was used to screen out the differential genes of thyroid cancer cells after AdipoRon treatment.Through bio-information analysis,the signal pathways that may be involved in inhibiting tumor growth（ie,autophagy）are screened out.First,Western-blot,immunofluorescence detection,and LC3B double-labeled fluorescent reporter gene were used to detect the changes of LC3B in thyroid cancer cells K-1 and KTC-1 after AdipoRon treatment.To further clarify whether AdipoRon initiates the autophagy pathway,autophagy inhibitors hydroxychloroquine and3-MA combined with AdipoRon were used to treat K-1 and KTC-1,and Western-blot was used to detect changes in LC3B protein levels.7.To further clarify the specific molecular mechanism of AdipoRon initiating autophagy,Western-blot was used to detect autophagy-related proteins,including mTOR、p-mTOR ^Ser2448、p70S6K、p-p70S6K ^Thr389、ULK1、p-ULK1 ^Ser555、BECN1、ATG3、ATG5、ATG7、ATG12 and ATG16L1.Small interfering RNA-ULK1 was used to knock down ULK1,and AdipoRon was applied again to treat thyroid cancer cells.Western-blot and LC3B double-labeled fluorescent reporter genes were used to detect the expression level of the key autophagy protein LC3B.8.In order to clarify whether AdipoRon plays a role by activating adiponectin receptors,we used small interfering RNA siRNA-AdipoR1 and siRNA-AdipoR2 to knock down adiponectin receptor 1（AdipoR1）and adiponectin receptor 2（AdipoR2）.Western-blot and LC3B double-labeled fluorescent reporter gene were used to detect the expression level of the key autophagy protein LC3B in thyroid cancer cells after AdipoRon treatment.Results.1.Influence of body mass index on the clinicopathological features of 13,995papillary thyroid tumors.a)The BMI,obesity ratio,malignant ultrasound（US）features,and aggressive pathological characteristics of males were significantly higher than those of females.b)The association of obesity with malignant US features and aggressive pathological characteristics was found to be sex-dependent.c)Obesity increased the risk of tumor size(OR_male=1.539 and OR_female=1.521)and multifocality(OR_male=1.659 and OR_female=1.449).However,obesity did not increase the risk of extrathyroidal extension（ETE）or tumor staging in males.d)Obesity was found to have an“interfering effect”on the US assessment of PTC in women.2.Neck-circumference-to-length ratio as a new body fat parameter for predicting the risk of extrathyroidal extension of papillary thyroid carcinoma.a)The neck-circumference-to-length ratio（NCLR）is an independent risk factor for extrathyroidal extension（ETE）of PTC.b)In the normal BMI group,the NCLR can still effectively indicate the risk of ETE.c)NCLR could not interfere with ultrasound evaluation of thyroid nodules.d)NCLR is a good body fat parameter for predicting general obesity（BMI）,central obesity（waist-to-height ratio）,and local neck obesity（neck circumference）.3.Screening of adipokines in neck adipose tissue and blood circulation of thyroid cancer using adipokine antibody arrays.a)There are gender differences in the results of serum adipokine antibody arrays.Compared with the normal weight group,only insulin-like growth factor binding protein-1（IGFBP-1）was down-regulated in the obese men with PTC.In women,AdipoQ,adipsin and RBP4,IGFBP-1,IGFBP2,IL-12p70,IL-12p40,and IL-1rαwere down-regulated.b)In the results of neck adipose adipokine antibody arrays,OPG,IGFBP-2,MSP,Ag RP,IFNg、Pepsinogen II,RANTES and TSP-1 is down-regulated;but in women,PAI-1,resistin,VEGF、Lipocalin-2 and SAA are up-regulated,and only IL-10 is down-regulated4.Therapeutic effect of adipose stem cell recombinant AdipoQ on thyroid cancer cells.a)ELISA experiments verify that the serum AdipoQ level of obese PTC patients is lower than normal weight group;b)Serum AdipoQ levels are negatively correlated with risk of the maximum diameter more than 1cm,ETE and lymph node metastasis of PTC tumors.c)In vitro experiments,we confirmed that the recombinant AdipoQ of adipose stem cells inhibited the proliferation and migration of thyroid cancer cells.5.Therapeutic effect and molecular mechanism of AdipoRon in thyroid cancer cells.a)Taking differentiated thyroid cancer cell lines as the research object,q PCR and WB showed that there are AdipoR1 and AdipoR2 in differentiated thyroid cancer cell lines.Immunofluorescence experiments showed that the two receptors are membrane receptors located on the surface of thyroid cancer cells.b)CCK-8,plate clone formation and Ed U experiment showed that AdipoRon inhibited the proliferation of thyroid cancer cells in a concentration-dependent manner.Scratchand Tranwell experiment showed that AdipoRon inhibits the migration ability of thyroid cancer cells.c)q PCR detected that the key molecules of amino acid metabolism（GLS,SLC1A5 and SLC7A5）and the key molecules of sugar metabolism（GLUT-1,PKM2and LDHA）were down-regulated after AdipoRon treatment.The WST-8 method detected a significant decrease in intracellular glucose 6-phosphate and NADH.Through q PCR detection,it was found that thyroglobulin（Tg）and peroxidase（TPO）RNA levels were up-regulated after AdipoRon treatment.After AdipoRon treatment,the cell mitochondrial membrane potential decreased,and the apoptosis-related proteins Caspase-3/7 and AnnexinV increased.d)Transcriptome sequencing found that after AdipoRon treatment,autophagy-related genes were differentially expressed in thyroid cancer cells.Western-blot detected that LC3BII/I increased significantly after AdipoRon treatment with time-and concentration-dependent.Immunofluorescence detection and mcherry-GFP-LC3B double-labeled fluorescent reporter gene showed that LC3B was up-regulated and autophagy lysosomes increased after AdipoRon treatment.e)Western-blot detects key proteins and their phosphorylation sites in the autophagy pathway.The results show that AdipoRon inhibits p-mTOR^Ser2448and p-p70S6K^Thr389,and simultaneously activates ULK1 and p-ULK1^Ser555.When ULK1was knocked down,its upstream p-p70S6K^Thr389was not affected,but the LC3B II/I ratio no longer increased,indicating that AdipoRon initiates autophagy by activating ULK1.f)The two adiponectin receptors were knocked down and verified by q-PCR and Western-blot in parallel.The results showed that when AdipoR2 was knocked down,we found that ULK1 and LC3B of thyroid cancer cells treated with AdipoRon no longer increased.g)In addition,we found that when ULK1 is knocked down,the mitochondrial membrane potential no longer changes,and the apoptosis-related protein caspase3/7no longer rises.After further knocking down the AdipoR2 receptor,the mitochondrial membrane potential and the apoptosis-related protein caspase3/7 no longer increased.This shows that AdipoRon promotes the apoptosis of thyroid cancer and may act through autophagy.Conclusion.1.Obesity is an independent risk factor for the highly aggressive pathological features of PTC.2.The neck-circumference-to-length ratio can be used as a new body fat parameter for predicting the risk of ETE in PTC.The neck-circumference-to-length ratio will not affect the ultrasound evaluation of thyroid nodules.3.By using adipokine antibody arrays,it was found that the serum AdipoQ was significantly down-regulated in female of PTC combined with obesity.4.Serum AdipoQ level is negatively correlated with tumor maximum diameter>1 cm,invasion and lymph node metastasis.In vitro experiments,we confirmed the adipose stem cells recombinant AdipoQ could inhibit the proliferation and migration of thyroid cancer cells.5.As a chemical analog of AdipoQ,AdipoRon could inhibit the proliferation and migration of thyroid cancer cells and promote apoptosis.6.AdipoRon could inhibit autophagy in thyroid cancer cells by activating AdipoR2/ULK1.