The Expression Of Long Non-coding RNA HOTAIR And EMT Related Factors In Esophageal Squamous Cell Carcinoma And Their Relationship With Radiosensitivity

Objective:1) To analyse the expression differences and relevance of HOTAIR andEMT relatived factors (Snail、E-cadherin、β-catenin), and to detect the significance androles of their expressions in esophageal squamous cell carcinoma(ESCC).2) To detectexpression level differences of HOTAIR and EMT related factors and radiosensitivitydifferences in different ESCC cell lines, to analyse the relationship between HOTAIRexpression level and cellular radiosensitivity.3)We established a radioresistant Eca109cell mode (Eca109R60/2) by fractional radiation to detect the radioresistance differenceof Eca109and Eca109R60/2cell lines, and to analyse the possible role of HOTAIR andEMT related factors in radioresistance.Method:1) HOTAIRmRNA expression level, EMT-related factors mRNA andprotein expression levels were detected in96ESCC and paracarcinoma tissues and102pre-radiotherapy tissues using qRT-PCR and Western blot.2) Four ESCC cell lines(K150、K450、TE-1、Eca109) were used in this experiment. mRNA expression levels ofHOTAIR and EMT related factors were detected using qRT-PCR, also the protein levelsof EMT related factors were detected by Western blot; radiosensitivity differences weremeasured by Colony formation assay.3) We exposed the parental Eca109cell line torepeated2Gy/fraction radiation of a total dose of60Gy. Then we examined cell cycle andapoptosis of radiated and non-radiated cell lines with FCM; MTT was used to detectproliferation state of the two cell lines at different times; Colony formation assay wasused to compare radiation biology parameters of the two cell lines. qRT-PCR was used todetect the mRNA expression of HOTAIR and EMT related factors, western blot assay was further used to detect the expression levels of EMT related protein.Results:1) HOTAIRmRNA[13.78(0.79,25.94) vs1.00(1.00,1.00)], Snail mRNAand protein[mRNA:3.50(1.18,6.12) vs1.00(1.00,1.00), protein:1.43(1.04,2.09) vs0.76(0.40,1.19)], β-catenin mRNA and protein[mRNA:1.09(0.48,2.04) vs1.00(1.00,1.00), protein:1.40(1.02,1.89) vs0.73(0.32,1.09)]expression levels in ESCC weresignificantly higher than that in paracarcinoma tissues, with statistical significance, P＜0.01. While the expression level of E-cadherin mRNA and protein[mRNA:1.09(0.48,2.04) vs1.00(1.00,1.00), protein:0.20(0.04,0.47) vs0.49(0.22,0.90)] in ESCC wassignificantly lower than that in paracarcinoma tissues, with statistical significance, P＜0.01.The HOTAIRmRNA, SnailmRNA and protein, β-catenin mRNA and proteinexpression levels showed significantly increased trends from well-differentiated cancer topoorly-differentiated caner, also the more advanced TNM stage the higher the HOTAIRmRNA, Snail and β-cateninmRNA and protein, and had significant Rank correction, P＜0.01; While E-cadherin mRNA and protein expression level showed a decreased trendfrom well-differented cancer to poorly-differented caner, and had significant Rankcorrelation, P＜0.01, also the more advanced TNM stage the lower the expression ofE-cadherin expression, with significant Rank correction, P＜0.01. HOTAIRmRNA, SnailmRNA and protein, β-catenin protein expression levels in the group with limphnode andorgan metastasis were significantly higher than that in the group without metastasis, P＜0.01; While, the expression of E-cadherin in the group with limph node and organmetastasis were significantly lower than that in the group without metastasis, P＜0.01. Inthe96esophageal squamous cancer tissues, the mRNA expression of HOTAIR and Snailwas positively correlated, rs=0.4173; the expression of HOTAIR and E-cadherin wasnegatively correlated, rs=-0.3270; the expression of HOTAIR and β-catenin was alsopotively correlated, rs=0.3049. And the protein expression level of Snail in theHOTAIRmRNA high group was significantly higher than the HOTAIRmRNA low group,while the protein expression of E-cadherin showed the reverse. In the102radiotherapypatients，the expression of HOTAIR, Snail, β-catenin in radiotherapy valid group wassignificantly lower than that in invalid group，while the E-cadherin showed the opposite.Muvariated logistic analysis showed the lymph node metastasis, HOTAIR, Snail andE-cadherin were the factors that impact radiotherapy result and2years’ survival.2) ThemRNA expression of HOTAIR (1.00±0.00) in Eca109cell line was significantly lowerthan that in K450and TE-1cell lines, with statistical significance. The expression ofSnail mRNA and β-catenin mRNA in Eca109cell line was significantly lower compared with that in K450and TE-1(1.66±0.10,1.62±0.10, P＜0.01) cell lines, with statisticalsignificance. β-catenin mRNA expression in Eca109cell line was also sigficantly lowerthan that in K150cell line (1.51±0.16, P＜0.01). The expression of E-cadherin mRNA(1.00±0.00) in Eca109cell line was significantly lower compared with that in K450(0.53±0.03, P＜0.01) and TE-1(0.69±0.06, P＜0.01) cell lines. The expression of Snailprotein and β-catenin protein in Eca109(0.11±0.01,0.05±0.01) and K150(0.13±0.02,0.08±0.01) cell line was significantly lower compared with that in K450(0.21±0.01,0.12±0.02, P＜0.01) and TE-1(0.19±0.01,0.12±0.02, P＜0.01) cell lines. Theexpression of E-cadherin protein in Eca109(0.07±0.01) cell line was significantly highercompared with that in K150(0.05±0.06, P＜0.05), K450(0.04±0.01, P＜0.01) and TE-1(0.02±0.01, P＜0.01) cell lines, with statistical significance. From the cell survival curve,we found that Eca109cell line was more sensitive to radiation than other cell lines. Theradiosensitivity parameters also showed that D0(3.31±0.26) and Dq(1.30±0.02) ofEca109were significantly lower than K150(D0:5.78±0.35; Dq:0.17±0.011), K450(D0:3.99±0.15; Dq:0.25±0.01) and TE-1(D0:4.90±0.51; Dq:0.20±0.02), which furthersuggested that Eca109was the most sensitive cell line in comparison with the other threecell lines. Cell survival fraction (SF2) can reflect cell intrinsic radioresistance. The resultof pearson analysis showed that HOTAIR mRNA and Snail protein expression wassignificantly correlated with intrinsic radioresistance, respectively(r=0.6426and0.6580,P＜0.05), which indicated that radioresistant cells had higher production of HOTAIRmRNA and Snail protein than radiosensitive cells.3) Compared to parental Eca109cellline, Eca109R60/2cell line showed a statistically significant decrease in G2phase(6.22±1.94vs32.83±5.63) and increase in S phase (54.95±3.60vs30.27±1.72), P＜0.01.The apoptosis rate of Eca109R60/2was statistically significantly lower compared withthat of Eca109(1.97±0.45vs7.33±0.45), P＜0.0001. Then, by using the MTT method,the growth curve showed that in the initial4days the growth rate of Eca109R60Gy/2cellline was almost comparrel with Eca109cell line, But after5days, Eca109R60Gy/2cellline grew much faster than Eca109cells and displayed significantly stronger growthability. After that, clone formation assay showed that colony-forming rate ofEca109R60Gy/2cell line was significantly higher than that of the Eca109cells. Theradiobiological parameters, such as D0(5.455±0.2789Gy vs3.439±0.1904Gy), Dq(2.485±0.5878gy vs1.135±0.2301Gy) and SF2(0.767±0.014vs0.646±0.012), theirvalues in Eca109R60/2were statistically significant higher than the parent cell line, P＜0.05. The mRNA expression of HOTAIR (P=0.0306), Snail(P＜0.0016) and β-catenin (P=0.0005) in Eca109R60/2cell line were statistically significant higher than that inparental cell line, and the expression level of E-cadherin in Eca109R60/2cell line wasalso statistically significant higher than that in Eca109(P=0.0061). The protein expressionof Snail (P＜0.0001) and β-catenin (P=0.0005) in Eca109R60/2cell line werestatistically significant higher than that in parental cell line, and the expression level ofE-cadherin (P=0.0020) in Eca109R60/2cell line was statistically significant higher thanthat in Eca109, P＜0.01.Conclusions:1) HOTAIR,Snail and β-catenin were overexpressed in ESCCtissues,while E-cadherin was depressed compared with para-cancerous tissues,The abovefactors may play a key role in the progression of ESCC and was expected to become anew therapeutic target in ESCC.2) Different ESCC cells shows different mRNA andprotein levels of HOTAIR and EMT related factors. These result indicated that there wasa significant correlation between HOTAIR and EMT related factors expressions and cellintrinsic radiosensitivity. The above factors may be potential targets in the radiotherapyof ESCC.3) By exposing the parental Eca109cells to repeated fractions of a total dose of60Gy, we acquired a radioresistant cell. The results suggested that HOTAIR with itspotential targets of EMT related factors had some positive influence to theradioresistance in some way.