The Expression Of RASAL2 In Esophageal Squamous Cancer And Its Effects On Cell Proliferation,Migration And Invasion

ObjectivesTo study the expression of Ras protein activator like 2(RASAL2)in esophageal squamous cell carcinoma(ESCC)and its biological function,which concludes cell proliferation,migration and invasion,on ESCC cells.This study may provide a theoretical basis for the development of new anti-tumor drugs for the molecular targets of esophageal cancer.Methods1.Immunohistochemistry was used to detect the expression of RASAL2 in ESCC tissues,and RT-PCR and Western blot methods were used to further detect the expression of RASAL2 in ESCC cells.2.CRISPR/Cas9 gene editing technology was applied to establish stable RASAL2-knockdown ESCC cells.The m RNA and protein expression levels of RASAL2 were detected by real-time quantitative PCR and Western blot for checking RASAL2-knockdown ESCC cells.3.CCK-8 and colony assay were put to detect the proliferation and colony formation capacity in the knockdown group cells.4.Cell scratch assay and transwell assay were conducted to observe the changes of cell migration and invasion ability in each group.5.Western blot was used to detect proteins related to cell migration and invasion in each group.Results1.Immunohistochemical results showed that the expression of RASAL2 in ESCC tissues was lower than that in adjacent tissues(P < 0.05).In ESCC cells,the expression of RASAL2 in KYSE30 and TE-1 cells was higher as detected by RT-PCR and Western blot.2.KYSE30 and TE-1 cells with RASAL2 stable knockout were constructed using CRISPR/Cas9 gene editing technique,and each cell was divided into control group(NC)and knockout group(KD1 and KD2).The m RNA expression levels of RASAL2 in the knockout group of the two types of cells were detected by real-time fluorescence quantitative PCR,which decreased to 75.4%(KYSE30-KD1),28.3%(KYSE30-KD2),83.4%(TE-1-KD1)and 0.6%(TE-1-KD2),respectively,compared with the control group(P < 0.05).Western blot method was used to detect the protein expression level of RASAL2 in the knockout group of the two types of cells,which decreased to 71.46%(KYSE30-KD1),15.17%(KYSE30-KD2),72.81%(TE-1-KD1)and 31.32%(TE-1-KD2),respectively,compared with the control group(P < 0.05).It was confirmed that ESCC cell lines with stable knockout of RASAL2 were obtained.3.The results of CCK-8 experiment showed that OD values in KYSE30 and TE-1 cells were significantly higher in the knockout group than in the control group(P < 0.05).Cell colony formation experiments showed that the colony formation Numbers of cells in the knockout group were 123(KYSE30-KD1),183(KYSE30-KD2),81(TE-1-KD1)and 132(TE-1-KD2),which were significantly higher than those in the control group(49(KYSE30-NC)and 64(TE-1-NC)(P < 0.01),indicating that RASAL2 knockout could promote cell proliferation and colony formation.4.The results of cell scratch test showed that the migration rates of the knockout group were 59.67 %(KYSE30-KD1),86.25 %(KYSE30-KD2),78.20 %(TE-1-KD1)and 81.24 %(TE-1-KD2),respectively,which were significantly higher than those of the control group 20.14 %(KYSE30-NC)and10.56 %(TE-1-NC)(P < 0.001).Transwell test results showed that the number of invasive cells in the knockout group was 1610.00 ± 0.86(KYSE30-KD1),2310.00 ± 1.33(kyse30-kd2),1880.66 ± 2.88(TE-1-KD1)and 2180.00 ± 2.66(TE-1-KD2),respectively,which was significantly higher than that in the control group(50.33 ±2.89(KYSE30-NC)and 33.66 ±1.11(TE-1-NC)(P < 0.001).This suggests that RASAL2 knockout enhances the invasion ability of cells.5.The deficient of RASAL2 promoteed cell proliferation by enhancing the RAS/RAF/ERK signaling pathway.Meanwhile,lack of RASAL2 effectively enhanced EMT and metastasis of ESCC cells via activating ERK thereby down-regulating the expression of E-cadherin and up-regulating the expression of N-cadherin,Snail-1 and ZO-1 proteins.ConclusionsRASAL2 is poorly expressed in esophageal squamous cell carcinoma and may play an anti-tumor effect in esophageal squamous cancer cells.RASAL2 may inhibit EMT by regulating RAS/RAF/ERK signaling pathway and thus suppress the migration and invasion of esophageal squamous cancer cells.