The Influence Of TNF-? And Ang ? On The Proliferation,migration And Invasion Of Hepatocellular Carcinoma Cells Via Regulation Of GRK2 Expression

Hepatocellular carcinoma?HCC?is a common malignancy in China and it occurs mainly in the southeast coastal area.Currently,the global incidence of HCC is showing a rising trend and it is the third leading cause of cancer-related mortality worldwide.According to the"World Cancer Report 2014,"which was published by the World Health Organization on February 3rd,2014,the number of new cancer cases in China ranked No.1 in the world;among these,both the number of new HCC cases and the number of HCC-related deaths were the highest in the world.The causes of HCC have not yet been clarified,and the disease still lacks specific medicines and treatment approaches.As a result,the prognosis for HCC patients is very poor.Therefore,the discussion on the influence of tumor necrosis factor-??TNF-??and angiotensin??Ang??on the proliferation,migration and invasion of HCC cells by regulating the expression of G protein-coupled receptor kinase 2?GRK2?is very significant,which will provide clinicians with new directions of thought toward the prevention and treatment of HCC.TNF-?and Ang?play important biological roles in human physiology,and previous studies have shown that they both participate in the formation and progression of HCC.The biological effects of TNF-?are mainly conducted through two receptors that have different structures:tumor necrosis factor receptor 1?TNFR1?and tumor necrosis factor receptor 2?TNFR2?.TNFR1 is expressed on the surface of almost every histiocyte,and TNFR2 is mainly expressed by cells of the immune system.Therein,the signal transduction of TNFR1 has a dual biological effect of causing inflammation and promoting apoptosis.Ang?is the most important biological effector in the renin-angiotensin-aldosterone system?RAAS?,the main receptors related to the biological effects of Ang?are angiotensin?type-1 receptor?AT1R?and angiotensin?type-2 receptor?AT2R?.Ang?can promote the vasoconstriction,inflammation,fibrosis and cell proliferation in various tissues through AT1R,while the signal transduction through AT2R has the opposite biological effects.G protein-coupled receptors?GPCRs?are seven-transmembrane helical proteins that function as signal transducers and mediate various signaling pathways related to the growth and metastasis of tumor cells.GRK2 belongs to the serine/threonine-protein kinase family and is widely distributed in various tissues.It can specifically phosphorylate and desensitize activated GPCRs,thereby switching the signaling pathways mediated by GPCRs.GRK2 has a variety of different physiological and pathological functions,while its activity and expression can be regulated by many factors.It is reported that GRK2 can obviously reduce the proliferation and invasion of HCC cells,while the expression of GRK2 in HCC tissues significantly decreases with reducing degrees of differentiation.Therefore,GRK2 functions as a negative regulator in the formation and progression of HCC.Previous studies have suggested that TNF-?and Ang?may play roles in the inflammation,proliferation and metastasis of synoviocytes,endotheliocytes and other cells by regulating GRK2 expression.However,there are no reports have investigated the relationship between TNF-?,Ang?and GRK2 in HCC cells.Therefore,this study explores the effects of TNF-?and Ang?on the proliferation,migration and invasion of HCC cells by examining their influence on the expression of GRK2 and relevant receptors TNFR1,AT1R and AT2R,combining with human liver cancer specimens and a mouse HCC model.Objective:This study was carried out to observe the expression level of TNF-?,Ang?,TNFR1,AT1R,AT2R and GRK2 in the serum and liver tissues of HCC and BLT patients,and to research the expression level of TNF-?,Ang?and GRK2 in mice liver tissues at different stages of HCC formation,as well as to determine the related mechanism that TNF-?and Ang?influence the proliferation,migration and invasion of HCC cells via regulation of GRK2 expression.Methods:1.The serum of normal subjects,HCC and BLT patients who were enrolled in our hospital?The First Affiliated Hospital of Anhui Medical University?from 2014 to 2015was collected in this study,and the mean serum level of TNF-?and Ang?in normal subjects,BLT and HCC patients were evaluated by ELISA assay.2.The liver tissues of 32 non-metastatic HCC patients and 32 BLT patients who were treated with hepatectomy in our hospital from 2014 to 2015 were collected in this study,and the expression of TNF-?,Ang?,TNFR1,AT1R,AT2R and GRK2 proteins in HCC,tumor-adjacent and normal liver tissues were evaluated by IHC and Western blot analysis.3.The proliferative activity of HCC cells was tested by CCK-8 assay after incubated with various concentrations of TNF-??2.5,5,10,20,40 ng/ml?and Ang?(1×10^-9,1×10^-8,1×10^-7,1×10^-6,1×10^-5 mol/l)for 12,24 and 48 h,then cells were incubated with TNF-?and Ang?alone or in combination at the specified concentrations for other 12,24 and 48 h.4.The motility of HCC cells was tested by wound healing assay after incubated with TNF-?and Ang?alone or in combination at the specified concentrations for 24 and48 h,while the migratory and invasive ability of HCC cells was tested by Transwell assays after incubated with TNF-?and Ang?alone or in combination at the specified concentrations for 24 h.5.The expression of TNFR1,AT1R and AT2R on the surface of HCC cells were investigated by FCM analysis after incubated with TNF-?and Ang?alone or in combination at the specified concentrations for 48 h.6.The expression of GRK2 in HCC cells was investigated by Western blot analysis after incubated with TNF-?and Ang?alone or in combination at the specified concentrations for 12,24 and 48 h.7.HCC cells were transfected with GRK2 or scrambled siRNAs,and the expression of GRK2 in siRNAs transfected HCC cells was assessed by Western blot analysis,then the proliferative,migratory and invasive ability of siRNAs transfected HCC cells was assessed by CCK-8 and Transwell assays.8.The proliferative activity of GRK2-knockdown HCC cells was assessed by CCK-8assay after incubated with TNF-?and Ang?alone or in combination at the specified concentrations for 12,24 and 48 h,while the migratory and invasive ability of GRK2-knockdown HCC cells was assessed by Transwell assays after incubated with TNF-?and Ang?alone or in combination at the specified concentrations for 24 h.9.C57BL/6J mice of 14-day-old were injected intraperitoneally with DEN to establish the liver cancer model,and the mice were divided into normal control or liver cancer model groups randomly,then the expression of TNF-?,Ang?and GRK2 proteins in mice liver tissues at different stages?0,12,24 and 36wk?of HCC formation were confirmed by Western blot analysis.Results:1.Mean serum level of TNF-?and Ang?in normal subjects,BLT and HCC patientsThe ELISA results confirmed that mean serum level of TNF-?and Ang?in HCC patients were obviously higher than those in BLT patients and normal subjects,and there were no obvious differences in mean serum level of TNF-?and Ang?between BLT patients and normal subjects.2.Expression of TNF-?,Ang?,TNFR1,AT1R,AT2R and GRK2 proteins in HCC,tumor-adjacent and normal liver tissuesThe IHC and Western blot results confirmed that the expression of TNF-?and TNFR1 proteins in tumor-adjacent tissues were obviously increased compared with those in HCC and normal liver tissues.And compared with normal liver tissues,HCC tissues had a higher expression of TNF-?and TNFR1.Meanwhile,the expression of Ang?,AT1R and AT2R proteins in HCC tissues were clearly increased compared with those in normal liver tissues.In addition,compared with normal liver tissues,tumor-adjacent tissues had a higher expression of Ang?and AT1R.Furthermore,the expression of GRK2 protein in HCC tissues was obviously decreased compared with that in tumor-adjacent and normal liver tissues,and there was no obvious difference in GRK2 protein expression between tumor-adjacent and normal liver tissues.3.Effects of TNF-?and Ang?on HCC cells proliferationThe CCK-8 results showed that HepG2 cells were sensitive to 5 ng/ml TNF-?and 1×10^-7 mol/l Ang?,while Bel-7402 cells were sensitive to 10 ng/ml TNF-?and1×10^-7 mol/l Ang?.Then treating HepG2 and Bel-7402 cells with TNF-?and Ang?alone or in combination at the specified concentrations for 12,24 and 48 h,the results showed that the proliferative activity of HCC cells in all three treatment groups was obviously increased compared with the blank control group in a time-dependent manner.Moreover,compared with the Ang?alone and combination groups,the TNF-?alone group had a more evident effect on cell proliferation,and there were no obvious differences between the Ang?alone and combination groups.4.Effects of TNF-?and Ang?on HCC cells migration and invasionThe wound healing and Transwell assays results showed that the migratory and invasive ability of HCC cells in all three treatment groups was significantly enhanced compared with the blank control group.Furthermore,compared with the Ang?alone and combination groups,the TNF-?alone group had a more prominent effect on cell migration and invasion,and there were no obvious differences between the Ang?alone and combination groups.5.Expression of TNFR1,AT1R and AT2R on the surface of HCC cellsThe FCM results verified that TNF-?alone group significantly enhanced the expression of TNFR1 compared with the blank control,Ang?alone and combination groups.In addition,compared with the blank control and Ang?alone groups,the combination group had a higher TNFR1 expression,and there were no obvious differences between the blank control and Ang?alone groups.Meantime,the Ang?alone and combination groups significantly enhanced the expression of AT1R compared with the blank control and TNF-?alone groups,and there were no obvious differences between the blank control and TNF-?alone groups or between the Ang?alone and combination groups.Meanwhile,the combination group significantly enhanced the expression of AT2R compared with the blank control,TNF-?alone and Ang?alone groups.Moreover,compared with the blank control and TNF-?alone groups,the Ang?alone group had a higher AT2R expression,and there were no obvious differences between the blank control and TNF-?alone groups.6.TNF-?and Ang?modulate the expression of GRK2 in HCC cellsThe Western blot results verified that there was no obvious difference in GRK2expression between the blank control,TNF-?alone,Ang?alone and combination groups after treating HCC cells with TNF-?and Ang?alone or in combination at the specified concentrations for 12 h.When the testing time was prolonged to 24 h,the TNF-?alone group significantly inhibited the expression of GRK2 compared with the blank control group,and there were no obvious differences between the blank control and combination groups.After 48 h of treatment,the TNF-?alone,Ang?alone and combination groups significantly inhibited the expression of GRK2 compared with the blank control group.Furthermore,compared with the combination group,the TNF-?alone group had a lower GRK2 expression,and there were no obvious differences between the TNF-?alone and Ang?alone groups.7.Effects of GRK2 on HCC cells proliferation,migration and invasionThe Western blot results demonstrated that the expression of GRK2 protein in GRK2 siRNA transfected HCC cells was obviously decreased compared with that in scrambled siRNA transfected HCC cells.Meantime,the CCK-8 and Transwell results demonstrated that the proliferative,migratory and invasive ability of HCC cells in the GRK2 siRNA transfected group was clearly enhanced compared with the scrambled si RNA transfected group.8.Effects of TNF-?and Ang?on the proliferation,migration and invasion of GRK2-knockdown HCC cellsThe CCK-8 assay results demonstrated that there was no obvious difference in the proliferative activity between the blank control,TNF-?alone,Ang?alone and combination groups after treating GRK2-knockdown HCC cells with TNF-?and Ang?alone or in combination at the specified concentrations for 12 and 24 h.After 48 h of treatment,the TNF-?alone group had a more evident effect on cell proliferation compared with the blank control group,and there were no obvious differences between the TNF-?alone,Ang?alone and combination groups.Meanwhile,the Transwell assays results demonstrated that the migratory and invasive ability in the TNF-?alone group was clearly enhanced compared with the blank control group,and there were no obvious differences between the TNF-?alone,Ang?alone and combination groups.9.Expression of TNF-?,Ang?and GRK2 proteins in DEN-induced mice HCC modelThe Western blot results suggested that there were no significant differences in TNF-?,Ang?and GRK2 proteins expression in normal control group with time passing.However,TNF-?and Ang?proteins expression in liver cancer model group were gradually increased with time passing,while GRK2 protein expression in liver cancer model group was gradually decreased with time passing.Conclusions:1.The expression of TNF-?and Ang?in the serum and liver tissues of HCC patients had increased significantly,and they could promote the proliferation,migration and invasion of HCC cells obviously,which demonstrated that TNF-?and Ang?played key roles in promoting HCC formation and progression.Meantime,TNF-?and Ang?proteins expression in liver tissues of mice HCC model were gradually increased with time passing,which indicated that TNF-?and Ang?played key roles in promoting different stages of HCC formation.2.There are significant differences in the expression of TNFR1,AT1R and AT2R on the surface of HCC cells that induced by TNF-?and Ang?alone or in combination,while TNFR1,AT1R and AT2R proteins expression had obvious differences between HCC,tumor-adjacent and normal liver tissues,which hinted that they both participate in the formation and progression of HCC.Furthermore,over-expression of TNFR1 and AT1R perhaps enhanced the proliferation,migration and invasion of HCC cells,while high AT2R expression had the opposite effect.3.The expression of GRK2 protein in the liver tissues of HCC patients had decreased significantly,and it could suppress the proliferation,migration and invasion of HCC cells obviously,which demonstrated that GRK2 played a key role in suppressing HCC formation and progression.Meanwhile,GRK2 protein expression in liver tissues of mice HCC model was gradually decreased with time passing,which indicated that GRK2 played a key role in suppressing different stages of HCC formation.4.TNF-?and Ang?alone or in combination can significantly suppress the expression of GRK2 in HCC cells in a time-dependent manner.Moreover,the level of inhibition on GRK2 expression in each of the experimental groups was related to their impact on the proliferation,migration and invasion of HCC cells.Meanwhile,downregulation of GRK2 expression in HCC cells can obviously reduce the effects of TNF-?and Ang?alone or in combination on cell proliferation,migration and invasion.Hence,TNF-?and Ang?could promote the formation and progression of HCC by reducing GRK2expression.5.Ang?had a certain degree of inhibition on the effects of TNF-?on HCC cells proliferation,migration and invasion,and the mechanism may be associated with the differential expression of GRK2 and relevant receptors in HCC cells after treatment with the two reagents combined.Additionally,over-expression of GRK2 and AT2R perhaps inhibited the formation and progression of HCC,whereas high TNFR1 and AT1R had the opposite effect.