| Renal cell carcinoma(RCC)is the deadliest urinary tract tumor.There are about 70,000 new kidney cancer cases in China each year,with about 23,000deaths.The incidence and mortality of RCC are increasing every year.Clear cell RCC(cc RCC)accounts for about 80%of RCC cases and is the most common histological subtype of RCC.Although surgery,inhibitor drugs,and immunotherapy have prolonged the life of patients to a certain extent,the clinical effects of kidney cancer patients in the past decade have not been satisfactory.One reason is that the efficacy of early diagnosis of RCC needs to be improved,and the pathogenic mechanism of RCC is still not fully understood.The occurrence and development of tumors are closely related to Warburg effect.The expression of genes involved in energy metabolism in cc RCC is heterogeneous,suggesting that the Warburg effect may be involved in the development of cc RCC.However,the role and mechanism of the Warburg effect in the progression of cc RCC is largely unknown.Angiogenesis is closely related to tumor growth,invasion,metastasis and prognosis.Microvessel density in renal cell carcinoma is also an important prognostic factor for tumors.Ultrasound medicine is a discipline that com-bines medical,acoustic and electrical engineering techniques.Two-dimen-sional ultrasound(2D-US)can probe some basic characteristics of the tumor,such as the size,shape,boundary,adjacenc,but it cannot image the vascular system within the tumor.Nowadays the rapid development of multimodal imaging systems has improved the early detection and identification of lesions.Microflow Imaging(MFI)technology is a new type of blood flow imaging technology,which applies a new clutter suppression algorithm to separate slow flow signals from tissue motion artifacts.Visualize microvascular and low-velocity blood flow signals in a non-invasive setting using contrast agents,enabling visualization of the microcirculation.Only a few studies have applied MFI techniques to assess tumor vasculature in deep organs so far.As we all know,cc RCC is a tumor with a rich blood supply.Pathological studies have shown that high-density and disordered new blood vessels can be seen in tumor tissue.The number of blood vessels and blood flow grade are signifi-cantly higher than those of benign tumors.Previous studies have shown that increased microvessel density(MVD)is significantly associated with higher pathological grade and shorter patient survival.Therefore,the assessment of microvessels in cc RCC tumor tissue is of great significance for the early diagnosis and prognosis of cc RCC.Real-time shear wave elastography(RT-SWE)technology can non-invasively,rapidly and objectively detect tissue elasticity quantitatively,and provide clinical information on physical para-meters of tissue hardness.In recent years,with the rapid development of new ultrasound technology,multimodal ultrasound has been widely used in the early diagnosis and treatment of various tumor types by providing more comprehensive tissue information from multiple perspectives.Enolase(ENO)is a metalloenzyme,also known as 2-phospho-D-glyce-rate hydrolase In eukaryotes,this protein family includes three distinct isoforms:ENO1 orα-ENO,ENO2 orγ-ENO,and ENO3 orβ-ENO.They are involved in muscle development an regeneration in adult skeletal muscle cells and also in the glycolytic pathway,catalyzing the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate.Numerous studies have shown that ENO,especially ENO1 and ENO2,play an important role in avariety of tumors.Compared to the first two,ENO3 is less studied,but it also plays an important role in tumors.A previous study found that knockdown of ENO3 inhibited the proliferation of STK11-mutant lung cancer cells and showed selective anti-cancer effects.In colorectal cancer,lower ENO2 and ENO3 expression was significantly associated with longer overall survival.These suggest that ENO3 is closely related to tumor progression.The study also found that ENO3 is related to the Warburg effect of cells.However,the expression and function of ENO3 in cc RCC remain unclear.In this study,ultrasonic multimodal imaging was used to collect tumor size,microvessels,elastic modulus and other information,so as to diagnose the tumor and initially predict its progress in combination with cell experi-ments.The results showed that the higher the blood flow level in the larger tumors,the higher the microvascular density,and the Warburg effect was stronger in the cells from the large tumors of cc RCC than in the small tumors.Through further studies,we also found that ENO3 participated in the Warburg effect and increased its expression with the growth of cc RCC tumors,and high levels of ENO3 predicted poor prognosis in patients with cc RCC.This study further elucifies the molecular mechanism by which NSUN5/ENO3 regulates the Warburg effect and the progression of cc RCC.The results showed that the NSUN5/ENO3 axis plays a key role in the progression of cc RCC and may be a potential therapeutic target for cc RCC.Part One Multimodal ultrasound non-invasive detection to assist in the diagnosis of renal malignant tumorsObjective:In order to explore the application of multimodal ultrasound imaging,especially MFI,RT-SWE in renal malignant tumor.Methods:1.A total of 90 patients with renal mass and complete clinical data were selected as the research objects.They underwent 2D-US,CDFI and MFI,RT-SWE examinations,and received surgical treatment.All mass ultrasound images were measured,analyzed,and graded.2.Animal models of human clear cell renal cell carcinoma(cell line 786-O)were established.2D-US,CDFI and MFI were performed to measure tumor size and retain blood flow images.The CEUS examination was then performed,and the dynamic images were retained.Thirty minutes after the end of the angiography,the mice were sacrificed,the whole tumor tissue was taken,fixed in 4%paraformaldehyde.Then paraffin sections were made,and the MVD of the tumor tissue was detected by immunofluorescence.3.The tumor size of cc RCC was measured by 2D-US,grouped as follows:T1,tumor diameter<5 cm;T2,tumor diameter≥5 cm.4.H&E staining was performed on cc RCC tumor of different sizes and normal kidney tissues.5.Primary cc RCC cells were isolated from tumor samples,and glucose consumption,amount of lactate produced,extracellular acid production efficiency,and cell viability were detected in primary cells of tumors of different sizes.Results:1.MFI non-invasive imaging of microvessels has obvious advantages over CDFI(x~2=117.93,P<0.000),and the detection rate of malignant tumors is higher than CDFI.The application of MFI in large tumors showed higher blood flow grading compered to small tumors.2.The pattern of tumor microvessels imaging by MFI has a higher coincidence rate with that imaging by CEUS(87.5%).In terms of imaging tumor blood vessels,the difference between MFI and CDFI was statistically significant(x~2=21.737,P<0.00001).MFI was significantly correlated with CEUS in imaging tumor blood flow(=0.948,P>0.05).More importantly,MFI imaging of tumor microvessels has a good correlation with tumor MVD.3.RT-SWE can be used to evaluate benign and malignant renal tumors,but it cannot distinguish subtypes of RCC.4.Compared with normal renal cells,glucose intake,lactic acid produc-tion,extracellular acid efficiency and cell viability in cc RCC cells increased with the increase of tumor volume,suggesting that cells from large tumors have a stronger Warburg effect.Summary:Multimodal ultrasound imaging of tumor size,microvessels,elasticity value and other information can be used for differential diagnosis of renal malignancies and it combined with the Warburg effect related experi-ments may preliminarily predict tumor progression.Part Two ENO3 promotes cc RCC progression by regulating the Warb-urg effectObjective:To explore the mechanism of regulating Warburg effect to promotes the progression of cc RCC.Methods:1.Expression levels of candidate genes involved in Warburg effect in renal tumors of different sizes were detected by RT-qPCR.2.WB and RT-qPCR to detect the expression level of ENO3 in normal kidney tissue and tumor tissue of different sizes.3.Immunohistochemical staining showed the expression of ENO3 in normal kidney tissue and tumor tissues of different sizes,and made quanti-tative analysis.4.Kaplan-Meier analyzed the survival of cc RCC patients with low(n=130)and high(n=130)ENO3 levels from The Cancer Genome Atlas(TCGA)data.5.WB detection of ENO3 expression in 293A and different cc RCC cell lines(A498,Caki-1,SW839,786-O).6.RT-qPCR detection of ENO3 m RNA expression levels in the above cell lines.7.Two ENO3-targeting sh RNAs and one oe ENO3 vector were used to transfect 786-O and Caki-1 cell lines,respectively.8.The expression of ENO3 m RNA and protein in the transfected cell lines was detected by RT-qPCR and WB.9.Glucose uptake analysis,lactate production analysis,ECAR analysis and detection of cell viability were performed on the transfected cells.Results:1.ENO3 expression in cc RCC tissues increases with tumor growth,leading to poor prognosis.ENO3 expression increased with tumor size.Kaplan-Meier analysis suggested that cc RCC patients with high ENO3 expression had poor prog-nosis.2.Up-regulation of ENO3 can promote cell growth and enhance Warburg effect.Knockdown of ENO3 reduced glucose uptake,lactate production,and ECAR,while overexpression of ENO3 increased these effects.Knockdown of ENO3 significantly decreased the proliferation of 786-O cells,while overex-pression of ENO3 promoted the growth of Caki-1cells.Our experimental data suggest that ENO3 promotes cell proliferation and cc RCC progression by enhancing the Warburg effect.Summary:1.ENO3 expression increases with tumor size.High levels of ENO3suggest poor prognosis in cc RCC patients.2.ENO3 promotes cc RCC progression by enhancing the Warburg effect.Part Three NSUN5-mediated m5C regulates the Warburg effect by sta-bilizing ENO3 m RNAObjective:To explore the mechanism of ENO3 regulating Warburg effect.Methods:1.Total RNA was extracted from normal tissues and RCC tissues,and Me RIP-qPCR was used to detect the enrichment of ENO3 m RNA precipitated by m5C antibody.2.Me RIP-qPCR to detect the level of m5C-modified ENO3 m RNA.3.RT-qPCR detection of m5C methyltransferase candidate genes in 293A,786-O and Caki-1 cell lines.4.Knock out these genes in 786-O cell line,and detect ENO3 m RNA expression by RT-qPCR.5.RT-qPCR and Western blot to detect the expression of NSUN5 m RNA and protein in normal kidney tissue and tumor tissue.6.Kaplan-Meier analysis of survival in cc RCC patients with low(n=130)and high(n=130)NSUN5 levels from The Cancer Genome Atlas(TCGA)data.7.Correlation analysis showed that the expressions of ENO3 and NSUN5were positively correlated in cc RCC tissues.8.786-O cell line was transfected with sh NSUN5-1#,sh NSUN5-2#and control vector(p LKO),and Caki-1 cell line was transfected with oe NSUN5 and p WPI vector.Expression of NSUN5 in the above cell lines was detected by RT-PCR and WB.9.The transfected cell lines were exposed to actinomycin D for 0,2,4 and8 hours.NSUN5 m RNA levels were determined by RT-qPCR.10.Detection of m5C levels by Me RIP-qPCR after NSUN5 knockdown and overexpression.11.786-O cells were simultaneously transfected with sh NSUN5,sh ENO3and two vectors,and Caki-1 cells were simultaneously transfected with sh NSUN5,oe ENO3 and two vectors.Glucose consumption,lactate production,ECAR,cell viability in the above cells were detected.12.The 786-O cells with stably knocking down ENO3,NSUN5 and the above two genes were injected into nude mice to construct a cc RCC xenograft cancer model.13.Tumor volume was measured in each group of mice.The wet weight of the xenograft tumors in each group of mice was analyzed.14.ENO3,NSUN5,and CDK6 protein levels were measured by WB from xenograft tumors.15.Double immunofluorescence staining was used to detect the expre-ssion of CDK6 and ENO3 in xenograft tumor tissue.Results:1.NSUN5-mediated m RNA m5C upregulates ENO3 expression786-O cells with high ENO3 expression had higher levels of m5C,while Caki-1 cells with low ENO3 expression had lower levels of m5C.Candidate m5C methyltransferases were tested in these cell lines and showed that only knockdown of NSUN5 reduced ENO3 expression.Correlation analysis showed that the expression of ENO3 and NSUN5 were positively correlated in cc RCC tissues,and the m5C expression of ENO3 m RNA increased after up-regulation of NSUN5.2.NSUN5 regulates m5C to promote ENO3 expression by stabilizing m RNA786-O cell line transfected with sh NSUN5 vector significantly reduced NSUN5 m RNA and protein expression,while Caki-1 cell line transfected with overexpressing NSUN5 vector increased the expression of NSUN5.Knock-down of NSUN5 significantly reduced ENO3 m RNA levels.Conversely,overexpression of NSUN5 enhanced ENO3 m RNA stability.Me RIP-qPCR confirmed that knockdown of NSUN5 decreased the enrichment of ENO3m RNA m5C,while overexpression of NSUN5 increased m RNA m5C levels,confirming that NSUN5 regulation of m5C promotes ENO3 expression by stabilizing m RNA.3.NSUN5 is involved in the Warburg effect regulated by ENO3sh NSUN5,sh ENO3 and co-transfected 786-O cell line,knockdown of NSUN5 significantly reduced the rate of glucose uptake and lactate production,while knockdown of NSUN5 and ENO3 enhanced the above responses.Conversely,overexpression of NSUN5 in the Caki-1 cell line increased glucose uptake and lactate production rates,whereas simultaneous knockdown of ENO3reversed these effects.The ECAR assay also had similar results.Knockdown of NSUN5 decreased cell viability,while simultaneous knock-down of ENO3further enhanced this effect.In contrast,NSUN5 overex-pression promoted cell viability,while simultaneous knockdown of ENO3 reversed this effect.These results suggest that the NSUN5/ENO3 axis modul-ates the Warburg effect and promotes cc RCC progression.4.Blockade of the NSUN5/ENO3 axis inhibits cc RCC progression in vivoKnockdown of ENO3 or NSUN5 exhibited small tumors of the control vector,and combined knockdown of ENO3 and NSUN5 enhanced these effects.Consistent with this,knockdown of ENO3 or NSUN5 exhibited smaller tumor size or smaller wet weight,while knockdown of both resulted in smaller tumor size or weight.Western blot analysis showed that knockdown of NSUN5inhibited ENO3 and CDK6 expression,while knockdown of NSUN5 and ENO3enhanced this effect.Again,double immunofluorescence staining from tumor tissue confirmed these results.Summary:1.NSUN5-mediated m RNA m5C upregulates the expression of ENO3.2.NSUN5 regulates m5C and promotes ENO3 expression by stabilizing m RNA.3.NSUN5 is involved in the Warburg effect regulated by ENO3.4.Blocking NSUN5/ENO3 axis inhibits cc RCC progression in vivo and this may be a potential therapeutic target for cc RCC.Conclusions:In this study,ultrasonic multimodal imaging was used to collect tumor size,microvessels,elastic modulus and other information,so as to diagnose the tumor and initially predict its progress in combination with cell experiments.The results showed that the higher the blood flow level in the larger tumors,the higher the microvascular density,and the Warburg effect was stronger in the cells from the large tumors of cc RCC than in the small tumors.Through further studies,we also found that ENO3 participated in the Warburg effect and increased its expression with the growth of cc RCC tumors,and high levels of ENO3 predicted poor prognosis in patients with cc RCC.This study further elucifies the molecular mechanism by which NSUN5/ENO3 regulates the Warburg effect and the progression of cc RCC.The results showed that the NSUN5/ENO3 axis plays a key role in the progression of cc RCC and may be a potential therapeutic target for cc RCC. |
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