The Role And Mechanism Of MiR-155-5p And LncRNA H19 On Radioresistance Of Esophageal Squamous Cell Carcinoma Cells

Esophageal cancer is the most common malignancy in gastrointestinal cancers.It is the ninth most common cancer and the sixth cause of death from cancer.China is a high incidence area of esophageal cancer.More than half of the new cases and deaths of esophageal cancer occur in China every year.It has two main subtypes:esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).These two subtypes are different in epidemiology and biology.Although EAC is more common in the United States and many European countries,ESCC is the main histological subtype of esophageal cancer in the world,accounting for more than 80%of esophageal cancer.More than 90%of esophageal cancer cases in China are classified as squamous cell carcinoma,and the mortality rate is the fourth.Operation,radiotherapy and chemotherapy are three conventional treatments for esophageal cancer.At present,surgery is the exact treatment of early esophageal cancer.However,the early diagnosis and treatment of esophageal cancer is not satisfactory.50%of the patients have already had extrathoracic metastasis.For the patients with advanced/unresectable tumors,neoadjuvant chemotherapy and radiotherapy has become the standard treatment.Even for those early patients,preoperative/postoperative radiotherapy and chemotherapy are widely used in clinical practice.Although some promising results have been observed in the clinical trials of neoadjuvant or adjuvant therapy,the 5-year overall survival rate of the disease is still between 15-25%,and the resistance to radiotherapy and chemotherapy is one of the main reasons.The ionizing radiation causes DNA damage in many forms.DNA double strand break is the main and most lethal form of damage.Chemotherapy drugs selectively kill tumor cells with high proliferation capacity by interfering with active DNA replication,gene transcription,protein translation,modification or folding or cytoskeleton formation during cell proliferation.However,with the killing of sensitive cells,the surviving tumor cells often have the resistance of radiotherapy and chemotherapy and lead to tumor recurrence.Because of the highly conserved DNA damage sensing mechanism in the cells,the stress regulation mechanism of cytotoxic effect is very complex.At present,the mechanism of resistance of radiotherapy and chemotherapy is not very clear.Therefore,the treatment of esophageal squamous cell carcinoma is still a difficult problem.Further understanding of the molecular mechanism that affects the sensitivity of tumor cells to radiotherapy and chemotherapy will help to improve the survival rate of patients with personalized treatment plans in the future.High throughput transcriptome studies show that 70-90%of human genome sequences can be transcribed,but only less than 2%of the genome regions can encode proteins.Most of the transcription comes from the gene regions that transcribe to noncoding RNAs.As the two main types of noncoding RNAs with regulatory function,microRNA(miRNA)and long non-coding RNA(LncRNA)interact and influence with each other,whose abnormal expression and function directly participate in the occurrence and development of various types of tumors,and affect the curative effect and prognosis of patients.MicroRNA is a kind of noncoding RNA molecule with 21 to 23 nucleotides in eukaryotes.It usually binds specifically to the 3’-UTR(untranslated region,UIR)region of mRNAs,then suppresses the translation or causes the degradation of it,leading to the down-regulation of the expression of these protein coding genes.MiRNA is involved in many key signaling pathways and affects the response of tumor cells to chemoradiotherapy.It is considered to be a promising molecular marker of chemoradio-resistance.The abnormal expression of miR-155-5p is a carcinogenic feature in many hematological malignancies and solid tumors,including ESCC.It can promote cell proliferation,inhibit cell apoptosis,induce epithelial stromal transformation,invasion and migration,tumor metastasis and recurrence.However,conflicting reports have been published regarding the role of its dysregulation in radio-and chemo-resistance.For example,interference of miR-155-5p caused resistance to chemotherapy drugs and ionizing radiation in parental human epidermoid carcinoma cells and breast cancer,respectively.But the opposite behaviors have also been observed,indicating a greatly increased sensitivity to chemotherapy drugs and radiation.Another important member of the noncoding RNA family is the long noncoding RNA with a length of more than 200 bp.Long noncoding RNA has a similar structure to mRNA,with a 5’ cap structure and a 3’ polyadenylated tail,which can even be spliced.H19 is one of the earliest lncRNAs.It can regulate the expression of downstream genes via binding with miRNA and proteins.It also can affect the occurrence and development of tumors by regulating Wnt/β-catenin signaling pathway and blood vessel generation signaling pathway.Dysregulation of H19 plays an oncogenic or antitumor role in many types of tumors such as bladder cancer and breast cancer et al.It is reported that H19 is involved in the invasion and metastasis of esophageal cancer cells,plays a role in the chemoradio-resistance of liver cancer cells,leads to poor prognosis and promotes sternness of breast cancer patients.Since little is known about the relationship between miR-155-5p/lncRNA H19 and the radioresistance of ESCC cells,in the present study,we systematically elucidated the function and mechanism of these two types of noncoding RNA and their downstream genes in the radioresistance of ESCC cells at the molecular and cellular levels.Part I:The DNA methylation regulated mir-155-5p affects chemoradiosensitivity of esophageal squamous cell carcinoma by down regulating MAP3K10Purpose1.To study the relationship between the expression level of miR-155-5p and chemoradio-sensitivity in ESCC cells,as well as the proliferation rate,invasion and migration,and to elucidate the mechanism of DNA damage repair mediated by miR-155-5p.2.Determine the DNA methylation status of CpG island upstream of miR-155 gene and clarify the influence of DNA methylation on the regulation of transcription of miR-155.3.Reveal the role of the downstream target gene of mir-155-5p in the regulation of ESCC cell proliferation,invasion,migration and chemoradio-resistance,as well as the activated signal pathway.MethodsThe expression of miR-155-5p in ESCC and its correlation with prognosis were obtained from TCGA.The expression level of miR-155-5p and radiation resistance profiles in six ESCC cell lines were detected by RT-qPCR and clonogenic survival assay.CCK-8 assay was used to detect the proliferation and sensitivity of cells to chemotherapy drugs.Wound-healing and transwell chamber assay were used to detect cell migration and invasion.DNA methylation in CG Island upstream of miR-155-5p gene was determined by bisulphite conversion sequencing(BSP).Western blot was used to detect the expression level of mir-155-5p downstream gene and DNA damage repair related protein.The effect of miR-155-5p and its downstream target gene MAP3K10 on chemosensitivity,invasion,migration and proliferation of ESCC cells were established by miRNA mimic/antagomir and siRNA/over expression vector transfection.The direct targeting effect of miR-155-5p on MAP3K10 and the effect of them on JNK signaling pathway were established by the Dual-Luciferase Reporter Assay system.Result1.TCGA analysis showed that miR-155-5p was highly expressed in esophageal squamous cell carcinoma,and high expression of miR-155-5p in patients with ESCC have a worse prognosis than those with low expression2.MiR-155-5p is differentially expressed in ESCC cells and positively associated with radioresistance.Upregulation of miR-155-5p by mimic transfection,significantly increased cellular survival rate after radiation treatment,compared to the control group;on the contrary,downregulation of miR-155-5p by antagomir significantly decreased the survival rate of the cells after radiation,indicating that the expression of miR-155-5p renders rsistance of ESCC cells to radiation.3.After increasing(or decreasing)the level of mir-155-5p,the protein level of γ-H2AX decreased rapidly(or slowly)with time,and the protein level of Ku80 increased rapidly(or slowly)without affecting the protein level of Rad51.This indicates that miR-155-5p can improve the DSB repair rate by upregulating Ku80 and activation of NHEJ.4.In addition,overexpression of miR-155-5p promoted resistance to traditional chemotherapy drugs,invasion,migration and proliferation.On the contrary,downregulation of it by antagomir inhibited the chemoresistance,invasion,migration and proliferation of cells.5.Examination the DNA methylation status of CG Island upstream of miR-155-5p by BSP,we found that the DNA methylation level was negatively correlated with the expression of miR-155-5p.After treatment of hypermethylated cells with 5-aza-dC,the level of DNA methylation decreased and the expression of mir-155-5p increased,which confirmed that the transcription of miR-155-5p gene was negatively regulated by DNA methylation.6.Through bioinformatics prediction and Array analysis,we screened out that MAP3K10 was the downstream gene of miR-155-5p.Further western blot showed that the expression level of MAP3K10 was negatively correlated with miR-155-5p.And dual-Luciferase Reporter Assay showed that miR-155-5p could directly bind to MAP3K’10 3’-UTR,which confirmed that MAP3K10 was the target gene of miR-155-5p.7.Knockdown of MAP3K10 by siRNA resulted in a phenotype similar to that of mimic transfection,and overexpression of MAP3K10 caused a phenotypezsimilar to that of antagomir.Inhibition of MAP3K10 reduced JNK signal activity.While overexpression of MAP3K10 incerased JNK signal activity.Thus,miR-155-5p affected JNK signal pathway activity by downregulating MAP3K10.ConclusionIn summary,the present study revealed that miR-155-5p,whose expression under the control of DNA methylation confers resistance to radiation and chemotheraputic drugs,enhances proliferation,migration and invasion,and promotes DNA damage repair via repairing the DSB more efficiently.MiR-155-5p contributes a great deal to the positive regulation of ESCC chemoradio-resistance and proliferation via targeting MAP3k10 to inhibit the JNK pathway.The present study provides a new predictor of chemoradiotherapy efficacy for the guided personalized treatment of ESCC.Part Ⅱ:Knockdown of H19 suppressed the radioresistance via upregulation of miR-22-3p and downregulation of WNT1 in esophageal squamous cell carcinoma Purpose1.Determine the proliferation rate,migration ability and sternness in radioresistant ESCC cells.2.Investigate the effect of lncRNA H19 on the radiosensitivity and other cellular behaviors of ESCC cells.3.Reveal the downstream gene of lncRNA H19 and the signal pathway involved in the radiation resistance in ESCC cells.MethodsThe expression levels of H19 in ESCC was analyzed using the online database starBase.The oncomine database was used to further verify the association between H19 expression and patient age,gender,and tumor stage.The overall survival rates of ESCC patients were analyzed using the KM plotter database.Radio-resistant ESCC cells KYSE-150R were established by repeated X-ray irradiation.Clonogenic survival was conducted to identify the value of survival fraction.The optical density values were obtained via MTS assays to determine proliferation rate.Cells migration and sternness were observed through Transwell and sphere formation assays.The expression levels of H19,miR-22-3p and WNT1 were detected using RT-qPCR.H19-siRNA was transfected and the relationships between H19,miR-22 and Wntl were analyzed.The direct targeting effect of miR-22 on WNT1 was established by the Dual-Luciferase Reporter Assay system.Western blot was used to detect the expression level of WNT1 and the downstream protein levels of β-catenin,Cyclin D1 and C-Myc after forced reversal of H19 and miR-22-3p.Results1.We firstly screened the H19 according to the online database starBase,and then the Oncomine database and KM plotter database showed that H19 expression was significantly upregulated in the ESCC tissues and associated with poor prognosis.2.Secondly,an ESCC radioresistant cell line,KYSE-150R was established.Clonogenic survival showed that radiation decreased the value of survival fraction.MTS assays suggested that optical density values in KYSE-150R cells were significantly higher than that in KYSE-150 cells.Transwell and sphere formation assays showed migration capacity and sphere forming ability was enhanced.Also,the expression of sternness-associated genes(OCT4,SOX2 and NANOG)was increased in KYSE-150R cells.3.In addition,RT-qPCR showed that H19 was upregulated in KYSE-150R cells,and clone formation assays showed that knockdown of H19 decreased the survival fraction values.MTS,transwell and sphere formationassays suggested that H19 inhibited proliferation,migration and sternness in radioresistant KYSE-150 cells.4.Inhibition of HI 9 led to upregulation of miR-22-3p,and downregulation of WNT1,β-catenin,Cyclin D1 and C-Myc,and inhibition of Wnt pathway activity in KYSE-150R cells.5.Dual-luciferase reporter assay showed that miR-22-3p could directly bind to WNT1 3’-UTR;upregulation of miR-22-3p by mimic transfection in KYSE-150R cells reduced the mRNA and protein levels of Wntl significantly,and the protein levels of β-catenin,Cyclin D1 and C-Myc,suggesting that overexpression of miR-22-3p repressed WNT1 expression and inhibited the activation of Wnt pathway.ConclusionOur results demonstrate that H19 knockdown downregulates the Wntl expression and inhibited the activation of Wnt pathway via upregulating miR-22-3p expression,which leads to the inhibition of proliferation,migration and sternness in the radioresistant ESCC cells.This study provides a new target to inhibit the radioresistance of ESCC at the molecular level.