The Mechanism Of Long Non-coding RNA NORAD Involving In The CDDP Resistance Of Esophageal Squamous Cell Carcinoma Through Upregulating MTDH

China is a country with a high incidence of esophageal cancer,and squamous cell carcinoma is the dominant pathological subtype.Most patients with esophageal squamous cell carcinoma（ESCC）were in advanced or locally advanced stage at the time of presentation and lost the opportunity for surgery.cis-diamminedichloro-platinum（CDDP）-based chemotherapy regimen is one of the most important parts of the comprehensive treatment system of ESCC,which is not only the first choice of postoperative adjuvant therapy,but also the first-line treatment for unresectable or recurrent ESCC.However,most patients are faced with the clinical problem of drug resistance,which seriously affects the clinical efficacy.At present,little is known about the molecular mechanism of cisplatin resistance in ESCC,which restricts the establishment of new treatment strategies.Part One The biological significance of lnc RNA NORAD in CDDP resistance of ESCC cellsObjective:This part aimed at establishing the ESCC cells which were resistant to CDDP,and screening out the key long non-coding RNA（lnc RNA）between CDDP-resistant ESCC cells and parental cells and thereby revealing the epigenetic mechanisms promoting CDDP resistance in ESCC.Methods:1.Increasing drug concentration method was used to establish the CDDP-resistant strains of KYSE30 and TE1 cells,which was termed as KYSE30/CDDP-R and TE1/CDDP-R cells.2.Cell counting kit-8（CCK-8）assay was conducted to reveal the half maximal inhibitory concentration(IC₅₀)value of CDDP in treating CDDP-resistant ESCC cells and matched parental cells.The resistance index of KYSE30/CDDP-R and TE1/CDDP-R cell was calculated.3.CCK-8,wound healing and transwell assay were performed to detect the proliferation,migration and invasion of CDDP-resistant ESCC cells and matched parental cells.4.Bioinformatics analysis was performed to screen the differential lnc RNAs between CDDP-resistant and CDDP-sensitive ESCC tissues in the GSE45670 dataset.The upregulated lnc RNAs in CDDP-resistant ESCC tissues were used as candidate lnc RNAs in subsequent study.5.Quantitative real-time polymerase chain reaction（q RT-PCR）was conducted to detect the expressions of candidate lnc RNAs in established CDDP-resistant ESCC cells and matched parental cells.6.Short-hairpin RNA（sh RNA）was used to establish the KYSE30/CDDP-R and TE1/CDDP-R cells with non-coding RNA activated by DNA damage（NORAD）-knockdown,and lentivirus transfection was used to establish the KYSE30 and TE1 cells with NORAD-overexpression.After exposing to different concentrations of CDDP,the cells were detected by CCK-8 to reveal the effect of NORAD on the IC₅₀ value of CDDP.Colony formation assay was performed to evaluate the sensitivity of ESCC cells to CDDP after altering the expression of NORAD.Western blotting was conducted to detect the expressions of DNA damage markerγH2AX and apoptosis marker caspase-3and cleaved caspase-3 in ESCC cells treated with CDDP.Flow cytometry was performed to evaluate the cell cycle of ESCC cells treated with CDDP.7.The KYSE30/CDDP-R cells with NORAD-knockdown and the KYSE30 cells with NORAD-overexpression were used to establish the xenograft tumor-bearing mouse model.The cells transfected with sh-NC and empty vector（EV）was used as corresponding control,respectively.The tumor-bearing mice were given CDDP or PBS.The growth of tumor was observed.The tumor tissues were harvested and detected by immunohistochemistry（IHC）to assay the expression of the markers of DNA damage markerγH2AX and apoptosis marker caspase-3 and cleaved caspase-3.Results:1.The CDDP-resistant strains of ESCC cells were successfully established.The resistance index of KYSE30/CDDP-R and TE1/CDDP-R cells to CDDP was 2.09 and 2.08,respectively.2.Compared to KYSE30 and TE1 cells,KYSE30/CDDP-R and TE1/CDDP-R cells exhibited significantly enhanced migration and invasion capacity（P<0.05）,while the proliferation capacity showed no difference.3.The bioinformatics analysis showed that,in the GSE45670 dataset,there were 5 upregulated lnc RNAs in the CDDP-resistant ESCC tissues,compared to the CDDP-sensitive ESCC tissues.These lnc RNAs were NORAD,HOXA-AS2,linc00996,TENM3-AS1 and CEP83-DT.4.q RT-PCR showed that,NORAD,HOXA-AS2,linc00996 and TENM3-AS1 was upregulated in KYSE30/CDDP-R cells（P<0.05）,and NORAD、HOXA-AS2、linc00996 and CEP83-DT was upregulated in TE1/CDDP-R cells（P<0.01）.Among the candidate lnc RNAs,NORAD exhibited the most significant upregulation in CDDP-resistant ESCC tissues.5.CCK-8 showed that,the IC₅₀value of CDDP for treating the KYSE30/CDDP-R cells transfected with sh-NC,sh-NORAD-1 or sh-NORAD-2 was 5.83,2.93 and 3.10,respectively.The IC₅₀value of CDDP for treating the TE1/CDDP-R cells transfected with sh-NC,sh-NORAD-1 and sh-NORAD-2was 5.86,2.97 and 3.15.The IC₅₀value of CDDP for treating the KYSE30 cells transfected with NORAD-overexpression and EV was 2.93 and 6.26,respectively.The IC₅₀value of CDDP for treating the TE1 cells transfected with NORAD-overexpression and EV was 2.98 and 6.44.6.Colony formation assay showed that,under the interference of CDDP,the colonies of the CDDP-resistant ESCC cells of sh-NORAD group were significantly less than those of the sh-NC group（P<0.001）,while the colonies of the parental ESCC cells of NORAD-overexpression group were significantly more than those of the EV group（P<0.001）.7.Western blotting showed that,under the interference of CDDP,the expression ofγH2AX and cleaved caspase-3/caspase-3 ratio of the CDDP-resistant ESCC cells of sh-NORAD group were significantly higher than those of the sh-NC group（P<0.001）,while the expression ofγH2AX and cleaved caspase-3/caspase-3 ratio of the parental ESCC cells of NORAD-overexpression group were significantly lower than those of the EV group（P<0.001）.8.Flow cytometry showed that,under the interference of CDDP,the G1phase proportions of the CDDP-resistant ESCC cells of sh-NORAD group were significantly more than those of the sh-NC group（P<0.05）,while the G1 phase proportions of the parental ESCC cells of NORAD-overexpression group were significantly more than those of the EV group（P<0.05）.9.The results from xenograft tumor-bearing mouse model showed that,under the interference of CDDP,the NORAD-knockdown KYSE30/CDDP-R cells-formed tumor began to recede from the 3rd week,and the ultimate tumor sizes were significantly lower than the control group（P<0.01）.Meanwhile,under the interference of CDDP,the EV-transfected KYSE30/CDDP-R cells-formed tumor began to recede from the 3rd week,but the NORAD-overexpression KYSE30 cells remain growing continuously.The ultimate tumor sizes of NORAD-overexpression KYSE30 cells were significantly bigger than the EV-transfected KYSE30/CDDP-R cells（P<0.001）.Summary:1.The CDDP-resistant strains of KYSE30 and TE1 cells were successfully established,and was termed as KYSE30/CDDP-R and TE1/CDDP-R.Compared to matched parental cells,migration and invasion of the KYSE30/CDDP-R and TE1/CDDP-R cells were significantly enhanced.2.The expression of NORAD was significantly increased in CDDP-resistant ESCC cells.Knockdown of NORAD increased the sensitivity of CDDP-resistant ESCC cells to CDDP,while overexpression of NORAD decreased the sensitivity of parental ESCC cells to CDDP.Part Two NORAD upregulates MTDH to promote CDDP resistance in ESCCObjective:This part aimed at focusing NORAD to establish a competing endogenous RNA（ce RNA）molecular regulatory network and revealing its effect on CDDP-resistance in ESCC in vivo and in vitro.Methods:1.Fluorescence in situ hybridization（FISH）was conducted to detect the subcellular location of NORAD in the CDDP-resistant ESCC cells and matched parental cells.2.Bioinformatics analysis was performed to screen the differential micro RNAs（mi RNAs）between CDDP-resistant and CDDP-sensitive ESCC cells in the GSE83362 dataset.The downregulated mi RNAs in the CDDP-resistant ESCC cells were used as candidate mi RNAs in subsequent study.3.The q RT-PCR was performed to detect the expressions of candidate mi RNAs in the CDDP-resistant ESCC cells with NORAD knockdown.4.The q RT-PCR was conducted to determine the expression of mi R-224-3p in the CDDP-resistant ESCC cells and matched parental cells.5.FISH and nuclear and cytoplasmic fraction isolation experiment were performed to determine the subcellular location of NORAD and mi R-224-3p in the ESCC cells.6.The bioinformatics database star Base was analyzed to predict the binding site on the transcript of NORAD for interacting with mi R-224-3p.Dual-luciferase reporter assay was conducted to verify the binding of NORAD and mi R-224-3p in the KYSE30/CDDP-R and TE1/CDDP-R cells.RNA immunoprecipitation（RIP）was performed to determine the enrichment of NORAD and mi R-224-3p on Argonaute 2（Ago2）protein.7.The online bioinformatics database mi RWALK,micro T,Target Scan and mi Rmap were analyzed to predict the target downstream gene of mi R-224-3p.The bioinformatics analysis results were overlapped with the results from the GSE45670 dataset to screen out the potential target gene of mi R-224-3p in the ESCC cells.8.Western blotting was performed to the expression of metadherin（MTDH）in KYSE30/CDDP-R,TE1/CDDP-R cells and matched parental ESCC cells.9.The binding between mi R-224-3p and the 3’-UTR of MTDH m RNA was confirmed by using dual-luciferase reporter assay.mi RNA rescue experiment was performed to verify the NORAD/mi R-224-3p/MTDH axis in the KYSE30/CDDP-R and TE1/CDDP-R cells.10.IHC was performed to determine the expression of MTDH in the tumor tissues of ESCC xenograft tumor-bearing mouse model.11.With mi RNA rescue experiment,western blotting and flow cytometry was conducted to determine the effect of NORAD/mi R-224-3p/MTDH axis on the sensitivity of ESCC cells to CDDP.Results:1.In the GSE83362 dataset,there were 3 downregulated mi RNAs in the CDDP-resistant ESCC cells,compared to the CDDP-sensitive ESCC cells.These mi RNAs were mi R-28-5p,mi R-224-3p and mi R-7-5p.2.q RT-PCR results showed that,the expression of mi R-224-3p was significantly upregulated in the KYSE30/CDDP-R and TE1/CDDP-R cells after knockdown of NORAD,compared with the control group（P<0.001）,while the expressions of mi R-28-5p and mi R-7-5p exhibited no significant differences（P>0.05）.3.q RT-PCR showed that the expression of mi R-224-3p in KYSE30/CDDP-R and TE1/CDDP-R cells was significantly lower than that in matched parental ESCC cells（P<0.001）.4.FISH showed that the dominant subcellular location of NORAD and mi R-224-3p in the ESCC cells was cytoplasm.5.Dual-luciferase reporter assay showed that mi R-224-3p directly bound on the binding site#1 on the transcript of NORAD.NORAD and mi R-224-3p occupied the same Ago2 protein to form an RNA-induced silencing complex（RISC）.6.The results from bioinformatics database showed that there were 405downstream m RNAs which showed potential to be regulated by mi R-224-3p.By overlapping the results with the TOP 20 upregulated m RNAs from the GSE45670 dataset,MTDH was found to be the potential target gene of mi R-224-3p in the CDDP resistance process in ESCC.7.Western blotting showed that the expression of MTDH in KYSE30/CDDP-R and TE1/CDDP-R cells was significantly higher than that in matched parental ESCC cells（P<0.001）.8.Luciferase reporter assay showed that mi R-224-3p directly bound on the binding site#1 on the 3’-UTR of MTDH m RNA.The NORAD/mi R-224-3p/MTDH axis in the ESCC cells was confirmed by conducting the mi RNA rescue experiment.9.The IHC staining of MTDH located in the cytoplasm of tumor tissues of ESCC xenograft tumor-bearing mice.The expression of MTDH in the tumors formed by KYSE30/CDDP-R cells with NORAD-knockdown was significantly lower than the control group（P<0.001）,and the expression of MTDH in the tumors formed by KYSE30 cells with NORAD-overexpression was higher than the EV-transfected group（P<0.001）.10.Colony formation assay showed that,knockdown of NORAD significantly increased the colonies of the KYSE30/CDDP-R and TE1/CDDP-R cells under intervention of CDDP（P<0.001）,while this phenomenon was rescued by mi R-224-3p inhibitor（P<0.001）.Likewise,overexpression of NORAD significantly decreased the colonies of the KYSE30 and TE1 cells under the intervention of CDDP（P<0.001）,and this phenomenon was rescued by mi R-224-3p inhibitor（P<0.001）.11.Flow cytometry showed that,knockdown of NORAD significantly increased the G1 phase proportion of KYSE30/CDDP-R and TE1/CDDP-R cells under the intervention of CDDP（P<0.001）,while this phenomenon was rescued by mi R-224-3p inhibitor（P<0.001;P=0.001）.Likewise,overexpression of NORAD significantly decreased the G1 phase proportion of KYSE30 and TE1 cells under the intervention of CDDP（P<0.001）,and this phenomenon was rescued by mi R-224-3p inhibitor（P<0.001）.Summary:1.The mechanism of NORAD in promoting CDDP resistance of ESCC was acting as a ce RNA to sponge mi R-224-3p and thereby upregulates MTDH.2.Interfering the mi R-224-3p could rescue the regulatory effect of NORAD on MTDH and reverse CDDP resistance in ESCC cells.Part Three NORAD/mi R-224-3p/MTDH axis mediated nuclear accumulation ofβ-catenin to promote CDDP resistance and malignant behaviors in ESCCObjective:This part aimed at determining the downstream signaling pathway of NORAD/mi R-224-3p/MTDH axis in CDDP resistance and discussing its effects the malignant behaviors in ESCC.Methods:1.mi RNA rescue experiment and western blotting were performed to detect the expression and phosphorylation of potential downstream signaling pathway-related molecules in KYSE30/CDDP-R and TE1/CDDP-R cells.2.After knockdown of NORAD,cytoplasmic fraction isolation experiment and western blotting were conducted to detect the expression ofβ-catenin in cytoplasm and nucleus of KYSE30/CDDP-R and TE1/CDDP-R cells.3.mi RNA rescue experiment and immunofluorescence were conducted to determine the effect of NORAD/mi R-224-3p/MTDH axis on the subcellular location ofβ-catenin in KYSE30/CDDP-R and TE1/CDDP-R cells.4.The effect of NORAD/mi R-224-3p/MTDH axis on the epithelial-mesenchymal transition（EMT）-related proteins（i.e.E-cadherin,N-cadherin and MMP9）in KYSE30/CDDP-R,TE1/CDDP-R cells and their parental cells was detected by using mi RNA rescue experiment and western blotting.5.The effect of NORAD/mi R-224-3p/MTDH axis on migration and invasion of KYSE30/CDDP-R,TE1/CDDP-R cells and their parental cells were determined by conducting wound healing and transwell assay.Results:1.Western blotting showed that,in the KYSE30/CDDP-R and TE1/CDDP-R cells,knockdown of NORAD reduced the phosphorylation ofβ-catenin,and mi R-224-3p inhibitor rescued this phenomenon.Knockdown of NORAD and mi R-224-3p inhibitor both had no effect on the expression and phosphorylation of MAPK,Akt and NF-κB.2.Cytoplasmic fraction isolation experiment and western blotting showed that,knockdown of NORAD suppressed nuclear accumulation ofβ-catenin in the KYSE30/CDDP-R and TE1/CDDP-R cells（P<0.01）.3.Immunofluorescence showed that,knockdown of NORAD or MTDH significantly decreased the nuclear expression ofβ-catenin in KYSE30/CDDP-R and TE1/CDDP-R cells,while this phenomenon was rescued by mi R-224-3p inhibitor.4.Western blotting showed that,knockdown of NORAD significantly increased the expression of E-cadherin and decreased the expressions of N-cadherin and MMP9 in the KYSE30/CDDP-R and TE1/CDDP-R cells,while this phenomenon was rescued by mi R-224-3p inhibitor.Likewise,overexpression of NORAD significantly decreased the expression of E-cadherin and increased the expressions of N-cadherin and MMP9 in KYSE30and TE1 cells,and this phenomenon was rescued by mi R-224-3p mimic.5.Wound healing assay showed that,knockdown of NORAD significantly promoted migration of KYSE30/CDDP-R and TE1/CDDP-R cells（P=0.001;P=0.007）,while this phenomenon was rescued by mi R-224-3p inhibitor（P<0.001;P=0.003）.Likewise,overexpression of NORAD significantly suppressed migration of KYSE30 and TE1 cells（P<0.001;P=0.024）,and this phenomenon was rescued by mi R-224-3p mimic（P=0.001;P=0.002）.6..Wound healing assay showed that,knockdown of NORAD significantly promoted invasion of KYSE30/CDDP-R and TE1/CDDP-R cells（P<0.001）,while this phenomenon was rescued by mi R-224-3p inhibitor（P<0.001）.Likewise,overexpression of NORAD significantly suppressed invasion of KYSE30 and TE1 cells（P<0.001）,and this phenomenon was rescued by mi R-224-3p mimic（P<0.001）.Summary:1.β-catenin is the downstream signaling pathway element which was activated by NROAD/mi R-224-3p/MTDH axis in ESCC.NROAD/mi R-224-3p/MTDH axis promotes the nuclear accumulation ofβ-catenin to facilitate its phosphorylation.2.NORAD/mi R-224-3p/MTDH axis promoted EMT and enhance migration and invasion in ESCC.Conclusions:1.NORAD is a crucial lnc RNA promoting CDDP resistance in ESCC.Knockdown of NORAD in CDDP-resistant ESCC cells reversed CDDP resistance,and overexpression of NORAD in parental cells induced CDDP resistance.2.NORAD/mi R-224-3p/MTDH axis promotes phosphorylation ofβ-catenin by mediating its nuclear accumulation.,and subsequently promotes CDDP resistance and induces EMT in ESCC.