| Back ground:The incidence rate of secondary kidney diseases such as hypertensive nephropathy and diabetic nephropathy increased year by year[1] and with the progression of primary glomerulonephritis and other primary kidney diseases,eventually the final stage of irreversible renal function decline,we call it End-Stage Renal Disease(ESRD).ESRD patients can only rely on renal replacement therapy such as kidney transplantation,hemodialysis or peritoneal dialysis to maintain their lives.The survival rate and quality of life are seriously reduced,and at the same time,it also increases the burden of family,society and country[2].Moreover,the disease has the characteristics of poor prognosis,high cost and long time-consuming,which seriously endangers human health worldwide.Due to the influence of kidney source,most ESRD patients can only rely on hemodialysis or peritoneal dialysis to maintain their life.Although hemodialysis or peritoneal dialysis can prolong the survival time of ESRD patients,their acute and chronic complications seriously threaten the life of ESRD patients,especially cardiovascular diseases(CVD)complications.Domestic and foreign studies show that over 50% of ESRD patients die from CVD[3],leading to an increase in incidence rate and mortality of CVD,which is Vascular Calcification(VC)[4].VC is very common in ESRD patients.Once ESRD patients enter hemodialysis or peritoneal dialysis treatment,the progress of VC will accelerate,and the presence and degree of VC can be the most powerful independent predictor of cardiovascular events and death[5].For ESRD patients,the initiating factor of VC is the imbalance of calcium and phosphorus metabolism[6],the occurrence of VC is regulated by many factors,and the key factor is the transdifferentiation of VSMCs into osteoblasts[7].One of the risk factors for promoting the transdifferentiation of VMSCs into osteoblasts is high phosphorus[8].Hyperphosphatemia is very common in the chronic complications of ESRD patients,so it is of great clinical significance to study the mechanism of hyperphosphatemia and VC.Some studies have shown that fibroblast growth factor 23(FGF23)is closely related to VC[9],but whether FGF23 is a marker in VC,as well as an inhibitory or promoting factor in VC,has not been determined.In the early stage of non chronic kidney disease(CKD)or CKD,FGF23 increased urinary phosphorus excretion,inhibited the metabolism of vitamin D and parathyroid hormone(PTH),maintained the balance of calcium and phosphorus metabolism,and showed the protective effect on cardiovascular system.However,with the progression of CKD to ESRD stage,the expression of Klotho in kidney decreased with the decrease of glomerular filtration rate(GFR).There is a direct correlation between VC and Klotho deficiency in CKD patients.Klotho is a suppressor of VC.The body of ESRD patients shows a resistance state to FGF23.The regulation of FGF23 on calcium and phosphorus weakens or even disappears,which leads to the disorder of calcium and phosphorus metabolism in the body.In addition,secondary hyperparathyroidism and the adverse reactions of vitamin D deficiency,the metabolism of bone and mineral in the body shows an overall imbalance State,a series of disorders and imbalances promote the occurrence and development of VC.The activation of Wnt/?-Catenin signal promotes the osteogenic differentiation and calcification of VMSCs[10].However,whether the Klotho/FGF23 axis mediates the osteogenic differentiation and calcification of VMSCs by regulating the signal molecules in Wnt/ ?-Catenin signal has not been reported.Therefore,?-GP is proposed to induce the calcification of VMSCs in vitro to explore the effect of Klotho/FGF23 on its calcification and its possible mechanism.Long chain non coding RNA(lncrna)is defined as the RNA molecular family of transcription,which has more than 200 nucleotides,but has no protein coding ability,and is related to the development of biology,cell physiology and various diseases[11].The main working mechanism of lncrna is to interact with a variety of proteins and micro RNAs.It is reported that small nuclear RNA host gene 29(Lnc RNA-SNHG29)is involved in the development of osteosarcoma and colorectal cancer[12].Some studies have shown that Lnc RNA-SNHG29 can reduce the calcification of VMSCs and the expression of osteogenic related factors,which is a negative regulator of vascular calcification.However,information on specific regulatory mechanisms was not provided.Therefore,the role of Lnc RNA-SNHG29 in vascular calcification is not clear.mi R-200b-3p is involved in a variety of regulatory mechanisms in different diseases(such as lung adenocarcinoma,pancreatic cancer,prostate cancer,etc.)[13,14].Recently,Kong confirmed the presence of highly expressed mi R-200b-3p in the arteries of degenerative atherosclerosis[15].However,the role of mi R-200b-3p in VMSCs is not clear.We used the biological information software Starbase to predict and found that Lnc RNA-SNHG29 and mi R-200b-3p have a binding region,and mi R-200b-3p and ?-Klotho also have a binding region,and some studies found that Lnc RNA-SNHG29 was down regulated in calcified vascular smooth muscle cells as a negative regulator of VC.In addition,mir-200 c expression was up-regulated in hyperphosphate induced aortic explant calcification,and mir-200 b expression was up-regulated in aortic stenosis caused by aortic valve calcification.However,the specific molecular mechanism of mir-200 b in vascular smooth muscle calcification is not clear at present,all of which suggest that lncrna plays an important role in VC,but the specific mechanism of Lnc RNA-SNHG29 in VC Unclear.Based on the review of relevant literature and the prediction and analysis of biological information software Starbase,we speculated that Lnc RNA-SNHG29 could play a role in the calcification of VMSCs by targeting down-regulation of mir-200-3p to activate ?-Klotho mediated signaling pathway.The study is intended to provide a basis for Lnc RNA-SNHG29 as a new treatment target for VC related diseases.Part ?: The role of Klotho and FGF23 in the calcification of VMSCs induced by ?-GPObjective:VMSCs were cultured in vitro and induced by 10 m M ?-GP.The calcification model of VMSCs was established to investigate the expression level of Klotho and FGF23 in the calcification process of VMSCs induced by ?-GP and the effect of overexpression of Klotho and FGF23 on the calcification of VMSCs induced by ?-GP.Method:1.VMSCs were isolated from the thoracic aorta of rats for primary cell culture.After identification of VMSCs,natural purification method was used for subculture for experimental study.10 m M ?-GP was used to stimulate VMSCs to induce calcification.The experiment was divided into NP group and HP group to explore the best time for ?-GP to induce VMSCs calcification;alizarin red staining was used to observe the calcification in the cells of NP group and HP group on day 0,5,9 and 12;meanwhile,the intracellular calcium of NP group and HP group on day 0,5,9 and 12 was measured;q RT-PCR was used to detect the expression of Klotho and FGF23 m RNA in NP group and HP group The expression of Klotho and FGF23 in NP group and HP group was detected by blot.2.The recombinant adenoviruses expressing Klotho and FGF23 were constructed by QRT PCR and western The results showed that there were 5 groups:NP group,HP group,HP + Adlaz group,HP + Ad-Klotho group,HP + Ad-FGF23group;alizarin red staining was used to detect the calcification of cells in each group;intracellular calcium content of VMSCs in each experimental group was analyzed and the activity of alkaline phosphatase(ALP)in VMSCs was detected.Result:1.?-GP induced VMSCs to promote calcification in a time-dependent manner.With the extension of time,the calcification of VMSCs was more obvious.The intracellular calcium content gradually increased in a time-dependent manner.On the9 th day of intervention,the intracellular calcium content of VMSCs increased compared with that on the 0th day,and on the 12 th day of intervention,the intracellular calcium content of VMSCs increased significantly compared with that on the 0th day.Considering the needs of cell activity and calcification,we used ?-GP The expression of Klotho and FGF23 m RNA and the expression of Klotho and FGF23 protein were down regulated after ?-GP induced VMSCs.2.After overexpression of Klotho Gene in VMSCs,the expression of klothomrna and Klotho protein in VMSCs was up-regulated.After overexpression of FGF23 gene in VMSCs,the expression of FGF23 m RNA and FGF23 protein inVMSCs was up-regulated.In the process of ?-GP induced VMSCs calcification,after overexpression of Klotho Gene or FGF23 gene,the calcification of VMSCs was alleviated to a certain extent,the calcium deposition was decreased,and ALP was decreased The activity decreased.Conclusion:1.?-GP can induce the transformation of VMSCs into osteoblasts and promote the occurrence of calcification.2.During the calcification of VMSCs induced by ?-GP,the expression of Klotho and FGF23 decreased.3.Overexpression of Klotho and FGF23 can reduce calcium deposition,ALP activity and calcification of VMSCs induced by ?-GP.Part ?: The mechanism of Klotho/FGF23 regulating ?-GP Induced Calcification in VMSCsObjective:To investigate the mechanism of FGF23/Klotho axis regulating ?-GP Induced Calcification of VMSCs.Method:1.The experiment was divided into two groups: NP group and HP group.The intervention time of cell culture was 12 days.The expression of Wnt/ ?-Catenin signal pathway related genes Runx2,BMP9,wnt7 b,?-catenin and ?-SMA m RNA was detected by q RT-PCR in NP group and HP group The expression of Wnt/ ?-Catenin signal pathway related proteins Runx2,BMP9,wnt7 b,?-catenin and ?-SMA,Klotho and FGF23 in NP and HP groups were detected by blot.2.The adenovirus overexpression vectors of Klotho and FGF23 were constructed.The experiment was divided into three groups: HP + Adlaz group,HP +Ad Klotho group and HP + Ad FGF23 group.QRT PCR was used to detect the m RNA expression of Wnt/?-Catenin signal pathway related genes Runx2,BMP9,wnt7 b,?-catenin and ?-SMA in HP + Adlaz group,HP + Ad Klotho group and HP +Ad FGF23 group The expression of Wnt/?-Catenin signal pathway related proteins Runx2,BMP9,wnt7 b,?-catenin and ?-SMA,Klotho and FGF23 in HP + Adlaz group,HP + Ad Klotho group and HP + Ad FGF23 group were detected by blot.3.The gene expression of Klotho and FGF23 was silenced.QRT-PCR and Western blot were used to detect Klotho m RNA,FGF23 m RNA and Klotho and FGF23 protein expression.The experiment was divided into 7 groups: NP group,HP group,HP + DKK1 group,HP + sh Klotho group,HP + sh FGF23 group,HP +sh Klotho + DKK1 group,HP + sh FGF23 + DKK1 group.The intervention time of cell culture was 12 days.The expression of Wnt/?-Catenin signal pathway related genes Runx2,BMP9,wnt7 b,?-catenin and ?-SMA m RNA was detected by q RT-PCR,and the expression of Wnt/ ?-Catenin signal pathway related proteins Runx2,BMP9,wnt7 b,?-catenin and ?-SMA protein was detected by Western blot.Result:1.In the process of ?-GP inducing VMSCs to promote calcification,Wnt/?-Catenin signaling pathway was activated,wnt7 b,?-Catenin m RNA expression and wnt7 b,?-Catenin protein expression were up-regulated,Klotho and FGF23 protein expression were down regulated,VMSCs transformed into osteoblasts,and osteoblast specific markers Runx2 and BMP 9 were down regulated The expression of m RNA and Runx2,BMP9 protein were up-regulated;the expression of ?-SMA,a specific expression marker of VMSCs,was down regulated.2.In the process of ?-GP inducing VMSCs to promote calcification,after overexpression of Klotho Gene or FGF23 gene,wnt7 b,?-catenin,Runx2,BMP9 m RNA expression and wnt7 b,?-catenin,Runx2,BMP9 protein expression were down regulated,?-SMA m RNA and ?-SMA protein expression were up regulated.3.In the process of ?-GP inducing VMSCs to promote calcification,Klotho Gene or FGF23 gene expression was silenced,klothomrna,FGF23 m RNA,Klotho,FGF23 protein expression was down regulated;Wnt/?-Catenin signal pathway was activated,wnt7 b,?-catenin m RNA and wnt7 b,?-catenin protein expression was up regulated,Runx2,BMP9,wnt7 b,?-catenin were up regulated The expression of m RNA and Runx2,BMP9,wnt7 b,?-catenin protein was up-regulated,the expression of ?-SMA m RNA and ?-SMA protein was down regulated;after DKK1 blocked Wnt/?-Catenin,the calcification of VMSCs was alleviated obviously,the expression of Runx2,BMP9,wnt7 b,?-catenin m RNA and Runx2,BMP9,wnt7 b,?-catenin protein decreased,the expression of ?-SMA m RNA and ?-SMA protein was up regulated.Conclusion:Klotho/FGF23 regulates the calcification of VMSCs induced by ?-GP through Wnt/?-Catenin signaling pathway.Part ?: The mechanism of Lnc RNA-SNHG29 down regulates mir R-200b-3p involved in Klotho/FGF23 mediated vascular calcificationObjective:The previous two parts showed that Klotho and FGF23 could protect the calcification of VMSCs induced by ?-GP,and Klotho/FGF23 regulated the calcification through Wnt/?-Catenin signaling pathway.In order to further study the regulatory mechanism of Klotho/FGF23,we used the biological information software Starbase to predict and found that Lnc RNA-SNHG29 and mi R-200b-3p have binding regions,and mi R-200b-3p and ?-Klotho also have binding regions,and some studies found that Lnc RNA-SNHG29 was down regulated in calcified vascular smooth muscle cells as a negative regulator of VC.In addition,mir-200 c expression was up-regulated in hyperphosphate induced aortic explant calcification,and mir-200 b expression was up-regulated in aortic stenosis caused by aortic valve calcification.However,the specific molecular mechanism of mir-200 b in vascular smooth muscle calcification is not clear at present,all of which suggest that lncrna plays an important role in VC,but the specific mechanism of Lnc RNA-SNHG29 in VC Unclear.Based on the results of previous studies and the prediction of Starbase software,we hypothesized that Lnc RNA-SNHG29 targeted mi R-200b-3p and mi R-200b-3p targeted ?-Klotho to regulate the calcification of VMSCs.This part of the experiment will clarify the relationship between Lnc RNA-SNHG29 and mi R-200b-3p,the relationship between mi R-200b-3p and ?-Klotho,and its regulatory role in the calcification of VMSCs,providing a new strategy for the treatment of VC.Method:1.10 m Mol / L ?-GP was used to stimulate human aortic smooth muscle cells(HASMC)to construct calcification model in vitro,and the activity of HASMC was measured by MTT on the 0,3,6,9,12,15 d after intervention;the calcification of HASMC was observed by alizarin red staining on the 0,3,6,6,9,12,15 d after intervention The expression of Runx2,BMP2 and ?-SMA protein in HASMC was detected by Western blot on day 0,3,6,9,12 and 15,and SNHG29 and mi R-200b-3p were detected by q RT-PCR on day 0,3,6,9,12 and 15;2.Using 10 m M ?-GP to stimulate HASMC to induce calcification,pcdna3.1-SNHG29 plasmid and SNHG29 empty plasmid were constructed.The expression of SNHG29 m RNA in HASMC was detected by q RT-PCR.The experiment was divided into four groups: NP group,HP group,HP + pc DNA3.1group,HP + pc DNA3.1 group-In SNHG29 group,alizarin red staining was used to observe the calcification of HASMC in each group,and the intracellular calcium content and ALP activity of HASMC in each group were detected;Western blot was used to detect the expression of Runx2,BMP2 and ?-SMA protein in HASMC in each group.3.The biological information software Starbase was used to predict whether mi R-200b-3p was a potential target of SNHG29.The relationship between SNHG29 and mi R-200b-3p was detected by double luciferase reporter gene.The experiment was divided into four groups: NP group,HP group,HP + inhibitor group,HP +mi R-200b-3p inhibitor group.Alizarin red staining was used to observe the calcification of HASMC in each group,and the intracellular calcium content and ALP activity of HASMC in each group were detected;Western blot was used to detect the expression of Runx2,BMP2 and ?-SMA protein in HASMC in each group.4.Starbase was used to predict whether ?-Klotho was a potential target of mi R-200b-3p.The target relationship between ?-Klotho and mi R-200b-3p was detected by double luciferase reporter gene.The experiment was divided into four groups: mimics NC group,mi R-200b-3p mimics group,inhibitor NC group,NP group,mi R-200b-3p inhibitor group.The expression of ?-Klotho,FGFR1 and FGF23 m RNA was detected by QRT PCR,and the expression of ?-Klotho,FGFR1 and FGF23 protein was detected by Western blot.5.The experiment was divided into six groups: HP + pcDNA3.1 group,HP +pc DNA3.1-SNHG29 group,HP + pc DNA3.1-SNHG29 + mimics NC group,HP +pc DNA3.1-SNHG29 + mi R-200b-3p mimics group,HP + pc DNA3.1-SNHG29 + sh NC group,HP + pc DNA3.1-SNHG29 + sh-?-Klotho group.Alizarin red staining was used to observe the calcification of HASMC in each group,and the intracellular calcium content and ALP activity of HASMC in each group were detected;q RT-PCR was used to detect the expression of ?-Klotho,FGFR1,FGF23 m RNA in each group,and Western blot was used to detect the expression of Runx2,BMP 2,?-SMA,?-Klotho,FGFR1,FGF23 protein in each group.Result:1.During the calcification process of HASMC induced by ?-GP in vitro,with the prolongation of calcification time,the activity of cells decreased gradually,the content of calcium increased gradually,the expression of Runx2 and BMP2 increased,the expression of ?-SMA decreased,and HASMC transformed into osteoblasts.Considering the needs of cell activity and calcification,we used ?-GP to treat HASMC for 12 D as the time point of follow-up experiment.The expression of SNHG29 and mi R-200b-3p showed the opposite trend.With the prolonged exposure of HASMC to ?-GP environment,the expression of mi R-200b-3p gradually increased.2.In the process of ?-GP Induced Calcification of HASMC in vitro,overexpression of SNHG29 reduced the calcification of HASMC,decreased the intracellular calcium content,decreased the activity of ALP,decreased the expression of Runx2 and BMP2 protein,and increased the expression of ?-SMA protein,suggesting that overexpression of SNHG29 can partially reverse the calcification of HASMC induced by ?-GP.3.The biological information software Starbase predicted that SNHG29 and mi R-200b-3p could be combined.Luciferase activity analysis showed that the overexpression of mi R-200b-3p significantly reduced the luciferase signal in SNHG29-WT.The expression level of SNHG29 has a direct correlation with mi R-200b-3p,showing a significant negative correlation.Silencing the expression of mi R-200b-3p can reduce the calcification of HASMC induced by ?-GP,decrease the deposition of intracellular calcium,decrease the activity of ALP,down regulate the expression of Runx2 and BMP 2 m RNA,and up regulate the expression of ?-SMA protein.4.The biological information software Starbase predicted that ?-Klotho and mi R-200b-3p could be combined.Luciferase activity analysis showed that the overexpression of mi R-200b-3p significantly reduced the luciferase signal in ?-Klotho WT.The expression level of ?-Klotho has a direct correlation with mi R-200b-3p,showing a significant negative correlation.Over expression of mi R-200b-3p,down-regulation of ?-Klotho,FGFR1 and FGF23 m RNA in HASMC,down-regulation of ?-Klotho,FGFR1 and FGF23 protein in HASMC,down-regulation of mi R-200b-3p,up-regulation of ?-Klotho,FGFR1 and FGF23 m RNA in HASMC,up-regulation of ?-Klotho,FGFR1 and FGF23 protein in HASMC,up-regulation of ?-Klotho,FGFR1 and FGF23 protein in Western blot.5.QRT PCR showed that the transfection efficiency of sh-a-klotho showed that?-Klotho Gene was silenced and ?-Klothom RNA expression was significantly decreased;SNHG29 overexpression significantly inhibited ?-GP Induced Calcification nodule formation,intracellular calcium deposition and ALP activity,but up regulation of mi R-200b-3p and down regulation of ?-Klotho could reverse this effect,and also reverse the over expression in calcification model SNHG29 induced the inhibition of Runx2 and BMP2 and the activation of ?-SMA;the detection of the expression of key regulatory factors involved in each experimental group showed that the increased SNHG29 could promote the expression of ?-Klotho,FGFR1 and FGF23 in ?-GP induced HASMC calcification.However,when mi R-200b-3p-mimics or sh-?-Klotho were used,the trend could be reversed.Conclusion:Lnc RNA-SNHG29 can inhibit the calcification of HASMC induced by ?-GP by down regulating the mi R-200b-3p axis of Klotho/FGF23.Lnc RNA-SNHG29 can be used as a new target for the treatment of VC. |
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