The Effect Of FAM135B On Radiosensitivity Of Esophageal Squamous Carcinoma Cells And Its Mechanism

Part one:Effect of FAM135B expression on radiosensitivity of esophageal squamous cell carcinoma cells(ESCC)Objective:To explore the expression status of FAM135B in esophageal squamous cell carcinoma cells,and the relationship between FAM135B expression status and radiosensitivity of ESCC cells.Methods:The expression of FAM135B in SHEE cells and five kinds of esophageal squamous cell carcinoma cells was detected by RT-PCR and Western blot.The effect of FAM135B expression on radiosensitivity of ESCC was evaluated by clone formation assay.The expression of FAM135B in ECA109 and KYSE150 cells was silenced by siRNA,and radiosensitivity of ECA109 and KYSE150 cells was evaluated by colony formation assay.Results:The protein and mRNA levels of FAM135B were assessed using western blot and RT-PCR assays in 6 cells(SHEE,TE-1,TE-10,TE-13,ECA109 and KYSE150)respectively.FAM135B mRNA expression was significantly higher in TE-10,TE-13,ECA109 and KYSE150 cells compared with SHEE cells(P<0.05).The expression of FAM135B mRNA in TE-1,TE-10,TE-13,ECA109 and KYSE150 cells was 2.19,8.98,8.02,7.65 and 12.4 times higher than that in SHEE cells,respectively.FAM135B protein expression was also higher in the 4 cells(TE-10,TE-13,ECA109,and KYSE150)than that in SHEE cells proved by Western blot.The expression of FAM135B protein between TE-1 cells and SHEE cells was no significant difference.The colony formation assay showed that the survival fraction of TE-1,TE-10,TE-13,ECA109 and KYSE150 cells was higher than that of SHEE cells at the corresponding radiation dose.Among the five groups of esophageal cancer cells,the survival fraction of esophageal cancer cells with high expression of FAM135B was higher than that of esophageal cancer cells with low expression FAM135B.The D0 values of SHEE,TE-1,TE-10,TE-13,ECA109 and KYSE150 cells were 1.02,1.36,1.54,1.79,1.65 and 1.69,respectively.The survival fractions at 2 Gy(SF2)were 0.46,0.56,0.60,0.65,0.66 and 0.76,respectively.The SF2 was significant differences between the five groups and SHEE group(P<0.05).Compared with SHEE cells,the radiosensitization ratios of TE-1,TE-10,TE-13,ECA109 and KYSE150 cells were 0.75,0.66,0.57,0.62 and 0.60,respectively.FAM135B knockdown cells(ECA109-FAM135B siRNA and KYSE150-FAM135B siRNA)and control cells(ECA109-NC siRNA and KYSE150-NC siRNA)were successfully constructed by siRNA interference.The results of colony formation assay showed that the survival fraction of ECA109-FAM135B siRNA and KYSE150-FAM135B siRNA cells at 2 Gy irradiation doses were 0.44 and 0.41,respectively,which were significantly different from that in control groups(P<0.05).Radiosensitization ratios(SER)of FAM135B siRNA groups in KYSE150 and ECA109 cells were 1.38 and 1.21 respectively.Conclusion:FAM135B is highly expressed in ESCC cells.High expression of FAM135B was negatively correlated with the radiosensitivity of ESCC cells.The expression of FAM135B in ESCC cells was downregulated by siRNA interference,which increased the radiosensitivity of ESCC cells.Part two:The mechanism of siRNA interference with FAM135B in increasing radiosensitivity of esophageal squamous cell carcinoma cellsObjective:To explore the mechanism of siRNA interference with FAM135B in increasing radiosensitivity of esophageal squamous cell carcinoma cells.Methods:Transcriptome sequencing(RNA-seq)was used to find the signal pathway regulated by FAM135B,and the key proteins of the signal pathway regulated by FAM135B were verified by Western blot.The MTT method was used to detect the cytotoxic effect of signal pathway inhibitor(rapamycin)on esophageal squamous cells,and the 50%inhibitory concentration(IC50)value of the drug was obtained.The cell cycle distribution and apoptosis of KYSE150 cells and KYSE150-FAM135B siRNA cells under non-irradiation and irradiation were analyzed by flow cytometry,and the effect of rapamycin combined on cell cycle distribution was detected.P53,pP53,CDK1,Bcl-2,and Bax protein in KYSE150 cells and KYSE150-FAM135B siRNA cells with or without irradiation were detected by Western blot,and the effect of rapamycin combined on the cell cycle and the protein correlated with apoptosis was analyzed.Clone formation assay was used to analyze the clone formation of KYSE150 cells and KYSE150-FAM135B siRNA cells under irradiation,and the effect of rapamycin combined on cell clone formation was detected.Results:Transcriptome sequencing showed that KYSE150-FAM135B siRNA cells had 327 genes upregulated,520 genes downregulated,and 12203 genes had no significant changes compared with KYSE150 cells.According to the result of KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis,the PI3K/Akt signaling pathway and mTOR signaling pathway maybe the downstream signaling pathway regulated by FAM135B.Western blot analysis showed that down-regulation of FAM135B expression in KYSE150 resulted in the decrease of protein expression of PI3K,p-Akt,mTOR and p-mTOR.The result indicated that the key proteins of PI3K/Akt/mTOR signaling pathway were regulated by FAM135B gene.MTT assay was used to detect the cytotoxic effect of rapamycin on KYSE150 and KYSE150-FAM135B siRNA cells.The results showed that the 50%inhibitory concentration of rapamycin in KYSE150 cells at 24h,48h and 72h was 128.2nm,84.48nm and 72.23nm,respectively.The 50%inhibitory concentration of rapamycin in KYSE150-FAM135B siRNA cells at 24h,48h and 72h were 83.07nm,76.49nm and 67.47nm,respectively.The cell cycle of KYSE150-FAM135B siRNA and KYSE150 cells was analyzed by flow cytometry.The results showed that the proportion of G2/M phase,S phase and G0/G1 phase of KYSE150 cells were 6.47±2.42%,4.83±2.22%and,87.84±3.40%,respectively.The proportion of G0/G1 phase,S phase and G2/M phase of KYSE150-FAM135B siRNA cells were 62.33±8.92%,20.96±8.18%and 13.32±2.88%,respectively.The proportion of G2/M phase cells in KYSE150-FAM135B siRNA cells was significantly higher than that in KYSE150 cells(P<0.05).Western blot analysis showed that the expression of pP53 and CDK1 protein in KYSE150-FAM135B siRNA cells was lower than that in KYSE150 cells,while the expression of P53,Bcl-2 and Bax protein in KYSE150 and KYSE150-FAM135B siRNA cells had no significant change.The results of clone formation assay showed that the number of clones in control group(6Gy RT),rapamycin group(rapamycin+6Gy RT),interference group(FAM135B siRNA+6Gy RT)and combination group(rapamycin+FAM135B siRNA+6Gy RT)were 263.67±11.15,93.67±8.50,26.67± 3.51 and 17.67±4.04,respectively.The number of clone formation in interference group,rapamycin group and combination group was significantly less than that in control group(P<0.05).In addition,the number of cell clones in combination group was less than that in interference group and rapamycin group,and the difference was statistically significant(P<0.05).Flow cytometry assay showed that the proportion of G2/M phase cells in control group,rapamycin group,interference group and combination group was 6.86±0.34%,8.43±0.45%,11.30±1.16%and 18.33±1.24%respectively.The proportion of G2/M phase cells in rapamycin group,combination group and interference group was significantly more than that in control group,and there was a significant difference between combination group,interference group and control group(P<0.01).Combination group had a higher proportion of G2/M phase cells than that of rapamycin group and interference group(P<0.001).The percentage of apoptosis cells in control group,rapamycin group,interference group and combination group was 11.47±2.68%,24.47±1.90%,24.33±2.70%and 45.63±3.05%respectively.The apoptosis ratio was increased in the 3 groups(rapamycin group,interference group and combination group)compared with 6 Gy RT group(P<0.05).In combination group,the apoptosis ratio was higher than that of interference group and rapamycin group(P<0.05).Western blot assay showed that the protein expression of CDK1 and pP53 in combination group,interference group,and rapamycin group was lower than that in control group,and the expression of pP53 in combination group was the lowest.The protein expression of P53 was no change in each group.The expression of Bcl-2 protein in combination group,interference group,and rapamycin group was lower than that in control group,and the expression of Bcl-2 protein in combination group was the lowest.The expression of Bax protein in combination group,interference group,and rapamycin group was higher than that in control group,and the expression of Bax protein in combination group was the highest.Conclusion:FAM135B regulates downstream PI3K/Akt/mTOR signaling pathway.Silencing FAM135B in ESCC cells can inhibit the activation of PI3K/Akt/mTOR signaling pathway,which may be one of the mechanisms to improve the radiosensitivity of ESCC cells.Rapamycin combined with downregulating FAM135B expression have synergistic effects on radiosensitivity,cell cycle regulation and apoptosis induction of ESCC cells.Part three:Effect of RNA interference with FAM135B on radiosensitivity of esophageal squamous cell carcinoma in vivoObjective:To investigate the effect of knockdown FAM135B expression on radiosensitivity of xenografts tumor in nude mice.Methods:The plasmid plenti-u6-fam135bsgrna-sffv-cas9-2a-puro was used to transfect KYSE150 cells.The nude mice were randomly divided into four groups:KYSE150 group(control group),KYSE150+8Gy RT group(irradiation group),KYSE150-FAM135B shRNA group(interference group)and KYSE150-FAM135B shRNA+8Gy RT group(interference irradiation group).The changes of tumor volume and the weight of xenografts tumor were analyzed.The expression of FAM135B,CDK1,Bcl-2 and Bax protein in xenograft tumor tissues was analyzed by immunohistochemistry.Results:KYSE150 cells were successfully transfected by plenti-u6-fam135bsgrna-sffv-cas9-2a-puro plasmid,and KYSE150-FAM135B shRNA cells with low expression of FAM135B protein were obtained.The growth of xenograft tumors in irradiation group,interference group and interference irradiation group slowed down after 6 days of irradiation.After 15 days of irradiation,the weight of xenograft tumors in control group,irradiation group,interference group and interference irradiation group was 1.75±0.16g,1.25±0.18g,1.29±0.14g and 0.74±0.09g,respectively.The weight of xenograft tumors in irradiation group,interference group and interference irradiation group was lighter than that in control group(P<0.05).The weight of xenograft tumors in interference irradiation group was significantly lighter than that in interference group and irradiation group(P<0.05).Immunohistochemistry analysis showed that the expression of Bcl-2,CDK1 and FAM135B protein in interference irradiation group and interference group was lower than that in control group,and the expression of Bax protein was higher than that in control group.Conclusion:Knockdown of FAM 135B can improve the sensitivity of xenograft tumor to radiotherapy.