The Molecular Pathway Of The Interaction Between MALAT-1,CCR7 And Correlated Genes In Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma（OSCC）is one of the most common malignant tumors in oral cavity which often have an early occurrence of lymph node metastasis.The incidence of oral squamous cell carcinoma is increasing every year.Although modern medicine has made great progress in the treatment of malignant tumors,the 5 year survival rate of OSCC is still hovering at around 50%.Although the causes of cancer are complex,but in the past two decadesscholars have been committed to study the reasons of malignant tumor formation mechanism over environmental,genetic and other aspects to find a better diagnosis,as well as methods of treating malignant tumors.This article focuses on the molecular pathway of lymph node metastasis in oral squamous cell carcinoma,the cell signaling pathway and the key link to block,to find a new target for the prevention and treatment of oral squamous cell carcinoma.The cell carcinogenesis and metastasis is a complex process involving multiple factors,multiple genes and multiple correlations.In recent years,longnon-coding RNA（lncRNAs）has been shown to play an important role in the development and progression of cell development and metabolism,as well as the development of tumor cells.In some of the cases tumors even do not exist the protein-coding gene variation,and only have lncRNAs expression abnormalities,several lncRNAs can make the transcription process changes.Metastasis-associated lung adenocarcinoma transcript 1（MALAT-1）is the first lncRNAs to be found that associated with tumor metastasis.MALAT-1 was synthesized by RNA polymerase Ⅱ,and its expression product was localized in nuclear speckle,while serine / arginine riched protein（SR protein,SR protein Family）is also located in the nuclear speckle,based on the co-localization of the two elements,some scholars through a series of studies found that MALAT-1 and SR protein phosphorylation is directly related.The localization of MALAT-1 in nuclear speckle is the structural basis for the regulation,l o c a l i z a t i o n a n d a c t i v a t i o n o f S R p r o t e i n f a m i l y m e m b e r s.It is been confirmed that there is an interaction between MALAT-1 and miRNA-320 s,MALAT-1 can regulate the expression of hsa-miR-320a（a member of the hsa-miRNA-320 s family）.Y-box binding protein 1（YB-1）is encoded by the coding protein gene YB-1,which is a multifunctional cold shock protein with the function of binding nucleic acid,and considered to be a tumor protein involved in all aspects of tumor development.Studies have found that YB-1 can bind to miRNA-320 s after the regulation of miRNA expression and regulation.Analysis of the data showed the biological information in metagenomics that one of the target genes of chemokine receptors CCR7 may be hsa-miR-320 a,there had been no empirical research support for now.The process of tumor cell migration to specific tissue is similar to that of leukocyte migration to the site of infection.Chemokines and their receptors are closely related to the occurrence,development,metastasis and prognosis of tumor.Chemokines and their receptors can accurately and effectively regulate the transfer of tumor cells to specific tissues and organs is now recognized theory.The focus of this study is based on the idea that chemokines and their receptors mediate the transfer of tumor cells in the same way as chemokines regulate the movement of leukocytes during inflammation.And MALAT-1 regulates the expression of downstream hsa-miR-320 a,however,one of the targets of the hsa-miR-320 a is the chemokine receptor CCR7.Theoretically,it forms a complete cell signaling pathway.In conclusion,MALAT-1 regulates the expression of chemokine receptor CCR7 in oral squamous cell carcinoma by activating SR protein-SRSF1 to regulate the processing of chemokine receptor mRNA precursor miRNA-320 s,which may affect the lymph node metastasis of oral squamous cell carcinoma.The research is divided into three parts: 1、 real-time quantitative detection of oral squamous cell carcinoma,qualitative observation of MALAT-1,miRNA-320 s,SRSF1,YB-1 and CCR7 molecule expression;2、 at the cellular level with tongue cancer cell lines SCC-9,SCC-25,by Co-immunoprecipitation and Western blotting test related RNA（MALAT-1,miRNA-320s）associated with SRSF1 protein or YB-1;3、by transfection of miRNA gene regulation tec hnology inhibitor silencing miRNA-320 d after Co-immunoprecipitation and Western blotting and real-time fquantitative detection of the expression of chemokine receptor CCR7,analysis the possible correlation.Innovation: The innovation of this topic is to demonstrate for the first time that the positive correlation between lnc RNA MALAT-1 and chemokine receptor CCR7 in the OSCC cell lines.MALAT-1,SRSF1 and miRNA-320 d could bind to each other,and miRNA-320 d could affect the expression of CCR7.The possible molecular pathways of MALAT-1 affecting CCR7 expression have not been reported in the literature.[Material and Methods]Part 1: RT-Qpcr detect the expression of MALAT1,SRSF1,miRNA-320 s,YB-1 and CCR7 in oral squamous cell carcinoma tissues and the adjacent tissues 1.cutting 、preserving specimensThe specimens were divided into 6 groups: oral squamous cell carcinoma tissues and adjacent normal tissues cut from operation.freezing tube,-80 ℃ refrigerator.2.the differential expression of MALAT1,SRSF1,miRNA-320 s,YB-1 and CCR7 in oral squamous cell carcinoma and adjacent normal tissues were detected by real-timequantitative PCR.2.1 total RNA extraction.2.2 RNA reverse transcription.2.3 Real-time qPCR.3.statistical analysisSPSS19.0 and Graphpad prism 7was used for statistical data analysis,the measurement data between the two groups using paired t test,on the basis of P<0.05 as a statistically significant difference.Part 2: Correlation of RNA（MALAT-1,miRNA-320s）with SRSF1 protein or YB-1 in tongue carcinoma cell lines 1.Cell cultureHuman tongue squamous cell carcinoma cell line（SCC-9、SCC-25）,DMEM high glucosewith 10% fetal bovine serum culture,subculture medium at 37 ℃,95% saturated humidity,5%CO₂,passage to the sixth generation cells for the experimental use.2.RNA Co-immunoprecipitation（RIP）2.1 Grouping:Negative control group: negative control antibody rabbit IgG provide from the Sigma company;The experimental group 1:MALAT-1-anti-SF2 antibody compound;The experimental group 2: MALAT-1-anti-YB-1 antibody compound;2.2 The method:Using Sigma’s Imprint RNA Co-immunoprecipitation Kit to perform RIP test,Western Blot to detect whether there were interactions between MALAT-1 and SRSF1,RT-qPCR to detect the output of the compound.3.Western blot assay was used to detect the expression of cell lysates and the expression of RIP complex.The cleavage efficiency of cell lysate was detected by Western blot assay.The expression of the target molecule and the expression of the complex was detected by WB.4.Real time qPCR4.1 detect the purity and concentration of RIP complex after transfection;4.2 RNA reverse transcription for cDNA;4.3 Real-time qPCR.5.Statistical analysis:Graphpad prism 7 is applied to Real time quantitative PCR data for statistical analysis,measurement data between the two groups using paired t test,on the basis of P<0.05 as a statistically significant difference.Part 3: Gene regulation technique was used to observe the expression of CCR7 by silencing miRNA-320 d According to the results of the second part of the experiment: hsa-miRNA-320d（one of the hsa-miRNA-320 s family members,screened a,b,d,e）has a correlation with MALAT-1.1.Cell miRNA inhibitor transfection1.1 grouping（1）negative control group: miRNA Inhibitor Control #1（Negative）（4464058）;（2）interference group: miRNA-320 d inhibitor（4464066）;1.2 methodUse Lipofectamine’s RNAiMAX Transfection Kit to transfect miRNA-320 s Inhibitor to OSCC cell line to silence the expression of intracellular miRNA-320 d.2.Western blot assay was used to detect the expression of cell lysates and the expression of RIP complex.3.Real time quantitative PCR3.1 Detect the purity and concentration of RIP complex after transfection;3.2 RNA reverse transcription to cDNA;3.3 Real-time qPCR.4.Statistical analysis:Graphpad prism 7 is applied to Real-time qPCR data for statistical analysis,measurement data between the two groups using paired t test,on the basis of P<0.05 as a statistically significant difference.[Results] Part 1: RT-Qpcr detect the expression of MALAT1,SRSF1,miRNA-320 s,YB-1 andCCR7 in oral squamous cell carcinoma tissues and the adjacent tissues1 The expression of MALAT-1,SRSF1,miRNA-320 d and YB-1 with lymph node metastasis in oral squamous cell carcinoma was higher than that in without lymph node metastasis oral squamous cell carcinoma tissue and adjacent tissues.（P < 0.01）2 The expression of miRNA-320 d with lymph node metastasis in oral squamous cell carcinoma was lower than that in without lymph node metastasis oral squamous cell carcinoma tissue.（P <0.05）Part 2: Correlation of RNA（MALAT-1,miRNA-320s）with SRSF1 protein or YB-1in tongue carcinoma cell lines1 MALAT-1 can be combined with SRSF1.（see WB band and Real-time qPCR statistical analysis）2 miRNA-320d（one of the miRNA-320 s family）and SRSF1 can be combined with each other.（see WB band and Real-time qPCR statistical analysis）3 MALAT-1,miRNA-320 d and YB-1 did not combine with each other.（see WB band）The results show that: MALAT-1,miRNA-320 d have a combination through SRSF1.Part 3: Gene regulation technique was used to observe the expression of CCR7 by silencing miRNA-320dThe expression of CCR7 was enriched by silencing of miRNA-320 d.It was proved that there was an interaction between miRNA-320 d and CCR7.[Conclusion]1 By real-time qPCR detection,the results showed that MALAT-1,SRSF1 and ccr7 in lymph node metastasis group were significantly higher than those without lymph node metastasis group;2 By Co-immunoprecipitation and Western blot analysis,it was found that RNA（MALAT-1,miRNA-320d）could bind with SRSF1 protein;3 Real-time qPCR quantitative RNA-protein complex MALAT-1,miRNA-320 s content,MALAT-1,miRNA-320 d through SRSF1 have a combination;4 Gene regulation technology silencing miRNA-320 d,Co-immunoprecipitation and Western blot and real-time qPCR experiments showed enhanced expression of chemokine receptor CCR7,inferred that interactions exist between MALAT-1,SRSF1,miRNA-320 d,CCR7,revealedMALAT-1 effect of chemokine receptor CCR7 on the molecular level,to provide a theoretical basis for further research using MALAT-1 as a molecular target for oral squamous cell carcinoma lymph node metastasis related to the foundation and application.