MiR-193a-3p Promotes Radio-resistance Of Nasopharyngeal Cancer By Targeting SRSF2 Gene And The Hypoxia Sipgnaling Pathway

Background:Tumor is characterized by uncontrolled cell proliferation.Human beings lack effective means to prevent,diagnose and treat it.With the extension of human life span and the change of environment and living habits brought about by the process of social industrialization,the incidence of cancer is on the rise."Cancer incidence and mortality in China in 2013:an analysis based on urbanization level" written by Academician Hejie of the National Cancer Center in 2017 shows,In China,the incidence and mortality of cancer has been rising in recent years,and has become the main cause of disease death.Nasopharyngeal cancer(NPC)is a malignant tumor occurring in the top and lateral wall of nasopharyngeal cavity.It has strong local metastasis and early distant metastasis.It is one of the most common malignant tumors in China.The incidence of NPC is the first one among the malignant tumors of otorhinolaryngology.The common clinical symptoms are nasal obstruction,bloody nasal mucus,ear stuffy feeling,hearing loss,diplopia and headache.Nasopharyngeal carcinoma is mostly sensitive to radiotherapy,so radiotherapy is the first choice for nasopharyngeal carcinoma.Radiotherapy is the radiation-induced ionizing radiation that causes DNA damage in irradiated cells in various forms,including a large number of base damage,single-strand breaks and double-strand breaks.However,as the treatment progresses,the surviving cancer cells are often radiotolerant and eventually lead to tumor recurrence after the sensitive cells are killed by radiation.Because somatic cells have DNA damage induction mechanism,but the stress regulation mechanism of cytotoxicity is very complex,the mechanism of radiotherapy tolerance is still not very clear.About 15%of new cases of nasopharyngeal carcinoma occur in China every year.Therefore,it is urgent to understand the pathogenesis of nasopharyngeal carcinoma and the mechanism of radiotherapy tolerance as soon as possible.MicroRNA is a non-coding RNA molecule with a length of 21 to 23 nucleotides widely existing in eukaryotic cells.It usually pairs with the RNA sequence in the 3'-UTR region of the protein-coding RNA,then inhibits the translation of the protein or causes the degradation of the protein-coding RNA,resulting in the down-regulation of the expression level of these protein-coding genes.MicroRNAs are involved in several key signal transduction pathways,such as DNA damage,Notch,NFkappa B,which alter the chemotherapeutic tolerance of cancer cells,and are considered as promising molecular markers for predicting chemotherapeutic tolerance.MicroRNA also plays a role in the pathogenesis of nasopharyngeal carcinoma.The screening of related microRNAs in radiotherapy-sensitive cell lines and Radiotherapy-resistant cell lines and the study of the mechanism of downstream coding genes regulated by microRNAs can provide new insights and evidence for understanding the pathogenesis of nasopharyngeal carcinoma and the mechanism of radiotherapy tolerance.The human microRNA-193a gene is located in a CpG island(about 1.3KB)on the long arm of chromosome 17(NCBI Gene ID:406968).Since there are no other genes in the range from 10 kB upstream to 4 kB downstream,the microRNA-193a gene is an excellent model for studying the regulation of microRNA transcription.In 2016,Cai Shanbao's team discussed the correlation between 193A and osteosarcoma metastasis.It was found that microRNA-193a-3p could inhibit the metastasis of osteosarcoma.This inhibition may be through its involvement in TGFbeta,Myc/Max signaling pathway to stimulate the downstream gene Rab27B.At the same time,it was found that microRNA-193a-5p could also inhibit the metastasis of osteosarcoma.This inhibition may be through its involvement in ATF2./ATF3/ATF4 signaling pathway stimulates the role of downstream gene SRR,and further proves the association between microRNA-193a-3p and microRNA-193a-5p.Protein splicing factors(SRSFs)rich in serine(S)/arginine(R)are involved in regulating the transcription,processing,output,stabilization and translation of RNA.At present,nine classical SR proteins have been found,named SRSF1,SRSF2,SRSF3,SRSF4,SRSF5,SRSF6,SRSF7,SRSF8 and SRSF9 respectively.Over-expression of SR protein family members plays an important role in the occurrence and development of various tumors.The human SRSF2 gene is located on chromosome 17q25.1 and contains five exons with a total length of about 3317 bp.There were 179 amino acid residues,of which Isoleucine was the most abundant.Stickeler et al.in 1999 showed that SRSF,SRSF2 and SRSF6 genes of SRSF family were highly expressed in breast cancer cells.Fischer et al.showed that SRSF,SRSF2 and SRSF3 genes of SRSF family were highly expressed in malignant ovarian cancer tissues in 2004,and were correlated with the progression of ovarian cancer.Mole et al.showed that SRSF-1,SRSF-2 and SRSF-3 genes of SRSF family were highly expressed in cervical squamous cell carcinoma,while SRSF-2 gene could also promote the development of HPV 16-positive cervical squamous cell carcinoma.Silencing SRSF-2 gene could reduce the number of clones and increase the apoptosis of cancer cells,suggesting that SRSF-2 gene could play an important role in cervical squamous cell carcinoma.It's important.In 20 1 7,Xie Shuqin et al.showed that the high expression rate of SRSF2 in cervical squamous cell carcinoma and HSIL was significantly higher than that in LSIL and chronic cervicitis.Hypoxia signaling pathway,hypoxia is a pathophysiological condition caused by imbalance of cell oxygen consumption and vascular perfusion.Hypoxia is a common feature of solid tumors,which is closely related to the increasing radiation tolerance and poor prognosis of patients.Metazoans have evolved a physiological mechanism to adapt to hypoxia,which is mediated by the transcription factor family HIF,which consists of an oxygen regulatory subunit and a structural expression subunit.In fully oxidized cells,oxygen-regulated subunits are hydroxylated by oxygen-dependent prolyl-4-hydroxylase(PHD)on proline residues.In addition,in the absence of hypoxia,oxygen-regulated subunits are hydroxylated by asparagine acyl,thus preventing the binding of oxygen-regulated subunits to co-activator protein p300/CBP.In the case of hypoxia,the activity of PHD and FIH is limited by substrates,resulting in rapid aggregation of oxygen-regulated subunits,nuclear translocation and dimerization with structural expression subunits.Transactivation occurs when oxygen-regulated subunits bind to common DNA sequences in promoters of target genes.Oxygen regulatory subunits promote the expression of hundreds of genes involved in cellular autonomous and involuntary adaptation to hypoxia.Hypoxia is closely related to the occurrence of tumors.At the same time,hypoxia can be regarded as one of the independent prognostic factors of tumors.It can improve the hypoxic environment of tumors as a goal and open up a new way for the effective treatment of tumors.Chapter 1:The expression level of SRSF2 in sensitive and tolerant nasopharyngeal carcinoma cells was negative correlation to that of miR-193a-3p,and is the direct target gene of miR-193a-3pObjective:To investigate the role of SRSF2 in nasopharyngeal carcinoma sensitive cell line(CNE-2)and tolerant cell line(CNE-1).The correlation between expression level and miR-193a-3p and whether SRSF2 is a direct target genes of miR,193a-3p.Methods:1.The expression of miR-193a-3p in nasopharyngeal carcinoma radiosensitivity(CNE-2)and tolerance cell line(CNE-1)was detected by qRT-PCR.2.Western and qRT-PCR techniques were used to detect the expression of SRSF2 gene in nasopharyngeal carcinoma radiosensitive(CNE-2)and tolerant cell lines(CNE-1).3.Cell transfection technology was used to detect the expression of SRSF2 in nasopharyngeal carcinoma radiosensitivity(CNE-2)and tolerance cell line(CNE-1),respectively.Western and qRT-PCR techniques were used to detect the expression of SRSF2,and qRT-PCR techniques were used to detect the expression of miR-193a-3p in nasopharyngeal carcinoma radiosensitivity(CNE-2)and tolerance cell line(CNE-1).4.Luciferase reporter assay was used to detect SRSF2 a target gene of miR-193a-3p in nasopharyngeal carcinoma radiosensitive(CNE-2)and tolerant cell lines(CNE-1).Result:1.The expression level of miR-193a-3p gene in nasopharyngeal carcinoma radiosensitive cell lines was lower than that in radiotherapy tolerant cell lines(CNE-1).2.The expression level of SRSF2 gene in nasopharyngeal carcinoma radiosensitive cell lines was higher than that in radiotherapy tolerant cell lines(CNE-1).3.The expression of miR-193a-3p was increased and the expression of SRSF2 was decreased in nasopharyngeal carcinoma radiosensitive cell line(CNE-2)transfected with mimic of miR-193a-3p,while the expression of miR-193a-3p was decreased in tolerant cell line(CNE-1)after antagomir,while the expression of SRSF2 was increased.4.Luciferase reporter assay indicates that SRSF2 is the target gene of miR-193a-3p in nasopharyngeal carcinoma cell line.Conclusion:Detection of qRT-PCR,western and Luciferase reporter technology showed that SRSF2 was the target gene of miR-193a-3p.Chapter 2:MiR-193a-3p promotes radiotherapy tolerance of nasopharyngeal carcinoma cells,which is negatively correlated with SRSF2Objective:To investigate the effects of miR-193a-3p and SRSF2 on radiotherapy tolerance of nasopharyngeal carcinoma cell lines.Method:1.Cell transfection technology transfects mimic of miR-193a-3p in nasopharyngeal carcinoma radiosensitive cells(CNE-2).The accelerator energy is 6MV X-ray photon beam irradiation.The dose rate is 300 cGy/min,and the absorbed dose is 2,4 and 6Gy,respectively.The clone formation experiment detects the number of clones.2.Cell transfection technique was used to transfect si-SRSF2 into nasopharyngeal carcinoma radiosensitive cells(CNE-2).The expression of SRSF2 gene in nasopharyngeal carcinoma radiosensitive cells(CNE-2)was detected by qRT-PCR and Western technique.3.Cell transfection technique was used to transfect si-SRSF2 into nasopharyngeal carcinoma radiosensitive cells(CNE-2).The accelerator energy was 6MV X-ray photon beam irradiated vertically.The dose rate was 300 cGy/min,and the absorbed doses were 2,4 and 6 Gy,respectively.The number of clones was detected by clone formation experiment.4.Cell transfection technology transfects antagomir of microRNA-193a-3p into nasopharyngeal carcinoma radiotherapy tolerant cells(CNE-1).The accelerator energy is 6MV X-ray photon beam irradiation.The dose rate is 300 cGy/min,and the absorbed doses are 2,4 and 6 Gy,respectively.The clone formation experiment detects the number of clones.Result:1.Mimic transfected with miR-193a-3p in nasopharyngeal carcinoma radiosensitive cells(CNE-2)was found to be more cloned than control cells(NC)after X-ray irradiation.2.The expression level of SRSF2 gene in nasopharyngeal carcinoma radiosensitive cells(CNE-2)transfected with si-SRSF2,qRT-PCR and western technique was lower than that in control cells(NC).3.Silicon-SRSF2 was transfected into nasopharyngeal carcinoma radiosensitive cells(CNE-2).After X-ray irradiation,clone formation experiments showed that the number of clones increased compared with control(NC).4.Anagomir of miR-193a-3p was transfected into nasopharyngeal carcinoma radiotherapy tolerant cells(CNE-1)by cell transfection technology.The clone formation experiment after X-ray irradiation showed that the number of clones was less than that of control cells(NC).Conclusion:Clone formation experiments showed that miR-193a-3p could promote radiotherapy tolerance of nasopharyngeal carcinoma cells,whereas SRSF2 could decrease radiotherapy tolerance of nasopharyngeal carcinoma cells.Chapter 3:The effect of miR-193a-3p/SRSF2 on radiotherapy tolerance of nasopharyngeal carcinoma cells in vitroObjective:To investigate the effects of miR-193a-3p and SRSF2 on invasion,metastasis and apoptosis of nasopharyngeal carcinoma sensitive cells(CNE-2)and tolerant cells(CNE-1).To investigate the effects of miR-193a-3p and SRSF2 on cancer-related signaling pathways in nasopharyngeal carcinoma sensitive cells(CNE-2)and tolerant cells(CNE-1).Method:1.Cells wound-healing assay was used to detect the metastasis ability of nasopharyngeal carcinoma(NPC)cells transfected with mimic of miR-193a-3p in radiosensitive cells(CNE-2).2.Cells wound-healing assay was used to detect the metastasis ability of nasopharyngeal carcinoma cells transfected with si-SRSF2 in radiosensitive cells(CNE-2).3.Cell invasion ability of nasopharyngeal carcinoma(NPC)cells transfected with mimic of miR-193a-3p in radiosensitive cells(CNE-2)was detected by cell chamber assay.4.The change of invasive ability of nasopharyngeal carcinoma cells transfected with si-SRSF2 in radiosensitive cells(CNE-2)was detected by small cell lab assay.5.Apoptosis assay was used to detect the changes of apoptosis in nasopharyngeal carcinoma radiosensitive cells(CNE-2)transfected with mimic of miR-193a-3p.6.The apoptosis of nasopharyngeal carcinoma cells transfected with si-SRSF2 was detected by fine cell apoptosis assay.7.Cell transfection technology transfected 18 common cancer-related signal transduction pathway reporter gene systems(Qiagen CignalTM reporter assay)in nasopharyngeal carcinoma radiotherapy sensitive(CNE-2)and tolerant cell lines(CNE-1),respectively,to establish the difference spectrum of signal transduction pathway activity between cells.8.Mimic and antagomir of miR-193a-3p were transfected into nasopharyngeal carcinoma radiosensitive(CNE-2)and tolerant cell lines(CNE-1)by cell transfection technology,respectively.Signal transduction pathway reporter gene system was used to identify the activity changes of three different signal transduction pathways.9.Cell transfection technology transfects si-SRSF2 in nasopharyngeal carcinoma radiosensitivity(CNE-2).Signal transduction pathway reporter gene system identifies three different activity changes of intercellular signal transduction pathway.Result:1.Cells wound-healing assay showed that mimics transfected with miR-193a-3p in nasopharyngeal carcinoma radiosensitive cells(CNE-2)enhanced cell metastasis.2.Cells wound-healing assay showed that transfection of si-SRSF2 into nasopharyngeal carcinoma radiosensitive cells(CNE-2)enhanced cell metastasis.3.Cell chamber assay showed that the invasive ability of nasopharyngeal carcinoma cells transfected with mimic of miR-193a-3p in radiosensitive cells(CNE-2)was enhanced.4.Cell chamber assay showed that the invasive ability of nasopharyngeal carcinoma cells transfected with si-SRSF2 in radiosensitive cells(CNE-2)was enhanced.5.Apoptosis assay showed that the apoptosis of nasopharyngeal carcinoma cells transfected with mimic of miR-193a-3p in radiosensitive cells(CNE-2)decreased.6.Cell apoptosis test showed that the apoptosis of nasopharyngeal carcinoma cells transfected with si-SRSF2 decreased.7.Notch,Hypoxia and MEF2 are the three most common cancer-related signal transduction pathway reporter gene systems in nasopharyngeal carcinoma radiosensitive(CNE-2)and tolerant cell lines(CNE-1).8.After transfection of mimic and antagomir of miR-193a-3p into nasopharyngeal carcinoma radiosensitive(CNE-2)and tolerant cell lines(CNE-1),the activity of signal transduction pathways of Hypoxia and MEF2 accorded with the change rule.9.Cell transfection technology transfected si-SRSF2 in nasopharyngeal carcinoma radiosensitivity(CNE-2).The activity of signal transduction pathway of Hypoxia accorded with the change rule.Conclusion:MiR-193a-3p and SRSF2 can promote the invasion and metastasis of nasopharyngeal carcinoma cells and reduce the apoptotic ability of nasopharyngeal carcinoma cells.MiR-193a-3p promotes radiotherapy tolerance of nasopharyngeal carcinoma through hypoxia signaling pathway.