Objective:To investigate the effects and molecular mechanisms of Wilms tumor 1-associated proteins(WTAP)on the cell biological properties of esophageal squamous cell carcinoma(ESCC)cells.Methods:31 pairs of ESCC tissues and their paired paracancerous tissues that were surgically resected at the Second Clinical Medical College of Chuanbei Medical College between September 2019 and April 2021 were collected.The esophageal cancer cells KYSE30,KYSE410,KYSE150,KYSE510,TE-1,and normal human esophageal epithelial cells HET-1A were routinely cultured.Transfection reagents were used to transfect si-NC,si-WTAP#1 and si-WTAP#2 nucleic acids into KYSE150 and KYSE510 cells.The cells were divided into si-NC,si-WTAP#1 and si-WTAP#2 groups.The expressions of WTAP and MAP3K9 mRNA were detected in the cells of each group by qPCR assay.CCK-8 assay,clone formation assay,and scratch healing assay,Transwell assay were employed to detect the effects of knockdown of WTAP expression on ESCC cell proliferation,migration,invasion and apoptosis.WB assay was used to detect the expressions of WTAP,MAP3K9,EMT and MAPK pathway-related proteins in ESCC cells of each group knocked down of WTAP;immunohistochemistry to detect the expression of WTAP proteins in ESCC tissues,immunoprecipitation of methylated RNA(MeRIP)-qPCR assay to detect the level of MAP3K9 m6A in ESCC cells,actinomycin D assay to detect the stability of mRNA of MAP3K9,and database data to analyze the expression,target genes,functional enrichment,and interacting RNA of WTAP.Results:WTAP was highly expressed in ESCC tissues and cells(P<0.05 or P<0.01 or P<0.001)and correlated with the degree of differentiation(P<0.01);the expression of WTAP mRNA and its protein were successfully knocked down in KYSE150 and KYSE510 cells(P<0.01 or P<0.001);the knockdown of WTAP significantly inhibited the proliferation,migration and invasion of KYSE150 and KYSE510 cells(P<0.05 or P<0.01 or P<0.001),and promoted the apoptosis of KYSE150 and KYSE510 cells(P<0.05 or P<0.01).Knockdown of WTAP resulted in a significant decrease in the m6A level of MAP3K9(P<0.05),and its mRNA expression level and mRNA stability were both significantly reduced(P<0.05).Database data analysis showed that WTAP target genes clustered in the MAPK signaling pathway;the expression levels of MAP3K9,p-ERK,N-cadherin,and MMP9 were significantly reduced(P<0.05 or P<0.01),and the expression level of E-cadherin was significantly elevated(P<0.05 or P<0.01)in the KYSE150 and KYSE510 cells after knockdown of WTAP.Conclusions:WTAP is highly expressed in ESCC tissues and cells and correlates with their differentiation.It promotes the stability of MAP3K9 mRNA through m6A modification,activates the MAPK pathway and thus promotes the malignant biological behaviors of ESCC cells.

Objective:To investigate the effects and molecular mechanisms of Wilms tumor 1-associated proteins(WTAP)on the cell biological properties of esophageal squamous cell carcinoma(ESCC)cells.Methods:31 pairs of ESCC tissues and their paired paracancerous tissues that were surgically resected at the Second Clinical Medical College of Chuanbei Medical College between September 2019 and April 2021 were collected.The esophageal cancer cells KYSE30,KYSE410,KYSE150,KYSE510,TE-1,and normal human esophageal epithelial cells HET-1A were routinely cultured.Transfection reagents were used to transfect si-NC,si-WTAP#1 and si-WTAP#2 nucleic acids into KYSE150 and KYSE510 cells.The cells were divided into si-NC,si-WTAP#1 and si-WTAP#2 groups.The expressions of WTAP and MAP3K9 mRNA were detected in the cells of each group by qPCR assay.CCK-8 assay,clone formation assay,and scratch healing assay,Transwell assay were employed to detect the effects of knockdown of WTAP expression on ESCC cell proliferation,migration,invasion and apoptosis.WB assay was used to detect the expressions of WTAP,MAP3K9,EMT and MAPK pathway-related proteins in ESCC cells of each group knocked down of WTAP;immunohistochemistry to detect the expression of WTAP proteins in ESCC tissues,immunoprecipitation of methylated RNA(MeRIP)-qPCR assay to detect the level of MAP3K9 m6A in ESCC cells,actinomycin D assay to detect the stability of mRNA of MAP3K9,and database data to analyze the expression,target genes,functional enrichment,and interacting RNA of WTAP.Results:WTAP was highly expressed in ESCC tissues and cells(P<0.05 or P<0.01 or P<0.001)and correlated with the degree of differentiation(P<0.01);the expression of WTAP mRNA and its protein were successfully knocked down in KYSE150 and KYSE510 cells(P<0.01 or P<0.001);the knockdown of WTAP significantly inhibited the proliferation,migration and invasion of KYSE150 and KYSE510 cells(P<0.05 or P<0.01 or P<0.001),and promoted the apoptosis of KYSE150 and KYSE510 cells(P<0.05 or P<0.01).Knockdown of WTAP resulted in a significant decrease in the m6A level of MAP3K9(P<0.05),and its mRNA expression level and mRNA stability were both significantly reduced(P<0.05).Database data analysis showed that WTAP target genes clustered in the MAPK signaling pathway;the expression levels of MAP3K9,p-ERK,N-cadherin,and MMP9 were significantly reduced(P<0.05 or P<0.01),and the expression level of E-cadherin was significantly elevated(P<0.05 or P<0.01)in the KYSE150 and KYSE510 cells after knockdown of WTAP.Conclusions:WTAP is highly expressed in ESCC tissues and cells and correlates with their differentiation.It promotes the stability of MAP3K9 mRNA through m6A modification,activates the MAPK pathway and thus promotes the malignant biological behaviors of ESCC cells.

Objective To confirm the interference effect of heparin lithium on turbidimetry method for detecting total protein , and to conduct performance evaluation of it .Methods Eight different concentrations of total protein urine specimen with and with ‐out heparin lithium were detected respectively ,to confirm the interference of heparin lithium on the experiment .Adding different concentration of heparin lithium solution to the prepared total protein samples with concentration of 0 .172 ,0 .427 g/L(a series of interference experiment samples ,with heparin lithium concentrations of 0 .000 ,0 .025 ,0 .030 ,0 .035 ,0 .040 ,0 .045 ,0 .050 mg/L ) , repeat four times detection ,and then conduct the dose‐effect experiment of interference .Results The biggest bias of the testing re‐sults in the two paired samples was 159 .37% ,the mini‐bias was 11 .37% ,the difference of the results in two groups was statistical significant(t= 17 .24 ,P< 0 .05) .95% confidence interval of the interference effect in the total protein samples with concentration of 0 .172 g/L was 0 .034 ~ 0 .062 ,while in the total protein samples with concentration of 0 .427 g/L was 0 .043 - 0 .053 .When the heparin lithium were 0 .025 mg/mL and 0 .050 mg/mL respectively ,the results of bias of the total protein samples with concentra‐tion of 0 .172 g/L were 27 .33% ,58 .14% respectively .But in the 0 .427 g/L total protein samples ,the results of bias were 11 .24% 、18 .74% respectively .Dose effect of interference both showed a linear relationship in the series concentration of the hepa ‐rin lithium and interference of sample of 0 .172 g/L and 0 .427 g/L ,and the linear equations were Y = 1 .974X - 0 .001 03 ,Y = 1 .599 X - 0 .000 5 respectively .Conclusion Heparin lithium is the exogenous interfering substance to the detection of total protein in tur ‐bidimetry method ,it is the positive interference to the determination results of total proteins ,especially for the low level of total pro‐tein detection ,and as heparin lithium concentration increases ,the stronger the interference effect .