CAFs Promote The Development And Chemoresistance Of Oral Squamous Cell Cancer Through LncRNA LOC100506114

In recent years,the incidence of oral cancer has been increasing year by year.Over 90%of the patients were diagnosed as oral squamous cell carcinoma(OSCC).OSCC has progressed in a multi-step way,from the initial manifestation of precancerous lesions to the final development of invasive cancer.Its prominent features are heterogeneous morphological features and regional anatomical invasion.As one of the sixth most common lethal tumors in the world,most of the deaths are caused by local recurrence of the primary site and local recurrence of peripheral lymph node metastasis.Despite the emergence of new technologies and methods for cancer treatment,especially immunotherapy,only 50%of OSCC patients received such treatment were cured,while platinum-based therapy for patients with metastasis and recurrence was limited and the median survival period was only 6-9 months.Surgery plus radiotherapy and chemotherapy are still the main means of cancer treatment at present.The drug resistance of tumors in radiotherapy and chemotherapy is the most headache problem in cancer treatment,which is also the main reason for the failure of clinical treatment of tumors.Therefore,exploring the causes of drug tolerance and metastasis and recurrence of cancer cells in cancer treatment and elucidating the molecular mechanisms of drug resistance and metastasis and recurrence are particularly important for clinical treatment of tumors including oral cancer.For a long time,tumors have been regarded as the autonomous process of cells independent of their surroundings.Therefore,the research on tumors has focused on the tumors themselves.There is a lot of evidence that the occurrence and development of tumors are closely related to the changes of their microenvironment.The information exchange between tumor cells and tumor microenvironment composed of tumor stromal cells and extracellular matrix promotes the progress of cancer,drug tolerance and invasiveness,and also changes the composition of tumor microenvironment.Tumor-associated fibroblasts(CAFs),also known as activated fibroblasts or myofibroblasts,have been identified as the main cells in tumor matrix.More and more evidences show that the presence of CAFs in various types of tumors is related to the clinical prognosis of cancer patients,and CAFs can promote tumorigenesis,growth,recurrence,metastasis and drug tolerance through various ways.Recent studies have shown that CAFs promote the development and progress of tumors by secreting growth factors,cytokines and other substances acting on tumors and immune cells in an autocrine or paracrine manner.Therefore,the mechanism of CAFs in biological behavior,such as tumorigenesis,development,recurrence,metastasis and drug tolerance,as well as the exploration of CAFs as a new therapeutic scheme for tumors have attracted much attention.Long non-coding RNA(lncRNA)is a kind of RNA that transcribes more than 200 nucleotides and has almost no protein-coding function.Numerous non-coding RNAs(ncRNAs)were initially considered as nonfunctional transcriptional "noise",but a large number of studies have shown that abnormal expression of lncRNAs is associated with diseases such as tumors,as gene transcription regulators,and plays a role in epigenetics.LncRNA regulates gene transcription,translation and expression,chromatin modification,and transport between nuclear and cytoplasm by interacting with DNA,chromatin,protein and RNA as signal molecules,baits,guides and molecular scaffolds.Recent studies have demonstrated the importance of IncRNA in regulating many major biological processes affecting development,differentiation and metabolism,and have put these neglected molecular participants at the forefront.So far,the relationship between the functional changes of lncRNA and CAFs and the role of IncRNA in the development,invasion and drug tolerance of oral cancer are not clear.The purpose of this study was to screen differentially expressed IncRNA LOC100506114 by bioinformatics analysis of paired mesenchymal fibroblasts extracted from normal tissue and tumor tissue samples from patients with oral squamous cell carcinoma(OSCC),and to explore the relationship between the expression of IncRNA LOC100506114 and the prognosis of OSCC.In vivo and in vitro experiments were carried out to further explore the role of the expression of lncRNA LOC100506114 in mesenchymal fibroblasts in promoting invasive growth and drug tolerance of oral squamous cell carcinoma cells,and to reveal the role of lncRNA LOC100506114 in the transformation of normal fibroblasts to tumor-related fibroblasts.Through this study,we have a better understanding of the transformation process of mesenchymal fibroblasts in cancer tissues,improved our understanding of the occurrence and development of oral cancer and drug tolerance process,and provided a new strategy and approach for clinical treatment of oral cancer and other tumors.1.IncRNA LOC100506114 affects the expression of CFAs-related phenotypic markers in mesenchymal fibroblasts and promotes the migration and proliferation of oral cancer cells:As a major member of tumor microenvironment formation,mesenchymal fibroblasts,including tumor-associated fibroblasts,play an important role in tumor development,invasion and metastasis,and drug tolerance.To investigate the role of IncRNA in the transition of mesenchymal fibroblasts from resting state to over-activated state and the effect of tumor-related fibroblasts derived from resting fibroblasts on the migration and proliferation of oral squamous cell carcinoma cells,we first investigated the effect of tumor-related fibroblasts derived from different oral cancer patients and their matched normal tissues,namely tumor-related fibroblasts.Vitamin cells(CAFs)and normal fibroblasts(NFs)were isolated and cultured,and their morphology and expression of recognized phenotypic markers were compared and analyzed.In addition,transcriptome sequencing of 5 paired NFs and CAPs was performed to screen out the differentially expressed IncRNA.The results showed that there were significant differences in the expression of morphological and phenotypic markers between NFs and CAFs,and the expression of CAFs at the level of IncRNA was the most different from that of NFs.The distribution and expression of IncRNA LOC100506114 at tissue and cell levels were verified and evaluated by Q-PCR,immunofluorescence and RNA-FISH techniques.The results were consistent with those of transcriptome sequencing.The expression of IncRNA LOC100506114 in CAFs was significantly higher than that in NFs.Compared with oral squamous cell carcinoma cell lines,IncRNA LOC100506114 was expressed in CAFs,mainly in the nucleus,and in the matrix of tumor tissue,it also showed a high level of IncRNA LOC100506114.We next investigated the effects of over-expression or knockdown of IncRNA LOC100506114 in NFs and CAFs on phenotypic markers of tumor-associated fibroblasts.The results showed that IncRNA LOC100506114 significantly promoted the expression of phenotypic markers of CAFs.To further explore the effect of CAFs expressing IncRNA LOC100506114 on the migration and proliferation of oral squamous cell carcinoma cells,we overexpressed plasmids and small RNA interference fragments(siRNA)by cell transfection and knocked down IncRNA LOC100506114.The results showed that overexpression of IncRNA LOC100506114 and knockdown of IncRNA LOC100506114 significantly promoted or inhibited the migration of oral cancer cells.In addition,a mouse model of subcutaneous transplantation of oral squamous cell carcinoma was established by stably interfering with IncRNA LOC100506114 constructed by lentiviral vector and co-injection with oral squamous cell carcinoma cells to explore the effect of the expression of IncRNA LOC100506114 on the development of oral squamous cell carcinoma in vivo.The results showed that the knockdown of IncRNA LOC100506114 significantly inhibited the effect of CAFs on the growth of oral squamous cell carcinoma,and the peripheral blood and spleen of mice were treated with CAFs.The number of MDSCs in tumors decreased accordingly.These results suggest that IncRNA LOC100506114 promotes the expression of phenotypic markers of CAFs and that the expression of CAFs of IncRNA LOC100506114 promotes the migration and proliferation of oral cancer cells.2.CAFs expressing IncRNA LOC100506114 promotes the migration and proliferation of oral cancer cells through the GDF10/TGF?R1 pathway:In order to further explore the role of IncRNA LOC100506114 in the migration and proliferation of oral squamous cell carcinoma cells in CAFs,we analyzed the results of transcriptome sequencing by bioinformatics and screened some genes related to cell invasion and growth with significant changes.We screened the significant changes in GDF10,MEGF10 and WNT2 expression by verifying gene co-expression analysis at the cellular level.The results showed that there were significant differences in the expression of GDF10,MEGF10 and WNT2.Next,we overexpressed or knocked down lncRNA LOC100506114 on NFs and CAFs respectively to explore its regulation on the expression of GDF10,MEGF10 and WNT2.The results showed that GDF10 had the greatest change multiple,and GDF10 was also affected by the expression of IncRNA LOC100506114 at the protein level.In addition,we detected the expression of GDF10 in CAFs,oral squamous cell carcinoma cells and tissues.Interestingly,the expression of GDF10 in CAFs was significantly higher than that in oral squamous cell carcinoma.In addition,at the tissue level,we also detected that the expression of GDF10 in tumors from patients with oral squamous cell carcinoma with high expression of IncRNA LOC100506114 was significantly higher than that of tumors with low expression of IncRNA LOC100506114.The expression of GDF10 was positively correlated with the expression of lncRNA LOC100506114 in both paired NFs and CAFs and tumors.LncRNA can regulate the expression of target genes through cis-regulatory factors.Therefore,we speculate that IncRNA LOC100506114 may regulate the expression of GDF10 by interacting with transcription factors of target genes.Runx2 transcription factor plays an important role in organogenesis and cell differentiation and involves the expression of GDF10 in tumors.We found that the expression of lncRNA LOC100506114 affects the expression of Runx2 and that lncRNA LOC100506114 can bind to Runx2.GDF10 is a member of the TGFbeta superfamily and is highly homologous with BMP3,also known as BMP3B.As a secretory growth factor related to the growth and differentiation of tissues and organs,GDF 10 plays an important regulatory role in cell differentiation,migration and proliferation,and is involved in the occurrence and recurrence and metastasis of tumors.Current studies mainly focus on the expression of GDF 10 in cancer cells and low expression in non-small cell lung cancer and oral squamous cell carcinoma.These evidences show that GDF 10 plays a role in cancer cells from outside,but the study of tumor stromal cells is still blank.In order to investigate the role of GDF 10 in the migration and proliferation of oral squamous cell carcinoma(OSCC)cells by CAFs,we stimulated the co-culture of OSCC cells and CAFs that knocked down the expression of GDF 10 with OSCC cells by exogenous GDF 10.The results showed that exogenous GDF 10 promoted the migration of oral squamous cell carcinoma cells,while co-culture of CAFs with oral squamous cell carcinoma cells which knocked down the expression of GDF 10 inhibited the migration of oral squamous cell carcinoma cells,and played a role through the TGFbeta R1-SMAD2/3-ERK signaling pathway.GDF 10 also showed a mirror rate of oral squamous cell growth.In addition,we found that exogenous GDF 10 promoted the expression of FAP and alpha-SMA in NFs,while knocking down GDF 10 inhibited the expression of FAP and alpha-SMA in CAFs.These results suggest that IncRNA LOC100506114 regulates the expression of GDF10 in CAFs through Rnnx2 interaction,promotes the malignant transformation of NFs,and promotes the migration and proliferation of oral squamous cell carcinoma cells by activating the TGFbeta R1-SMAD2/3-ERK signaling pathway.3.CAFs overexpressing lncRNA LOC100506114 promotes drug resistance of cancer cells by secreting MK to regulate the expression of lncRNA ANRIL:More and more evidences show that CAFs play a key role in the drug tolerance of cancer cells to antineoplastic drugs and hinder the treatment of cancer.Many studies on the mechanism of drug tolerance mainly focus on the factors of cancer cells themselves,while the factors of tumor stromal cells have not received significant attention.Midkine(MK)is a heparin-binding growth factor that promotes canceration and chemotherapy resistance.In order to investigate the role of CAFs in drug tolerance of oral squamous cell carcinoma(OSCC),we collected conditioned media from CAFs,non-small cell lung cancer(NSCLC),ovarian cancer(OEC)and oral squamous cell carcinoma(OSCC)cells and detected the secretion of MK.The results showed that the secretion of MK in CAFs overexpressing IncRNA LOC100506114 was significantly higher than that in cancer cells.We treated cancer cells with MK neutralizing antibody,DDP and CAFs-derived conditioned medium(CAFs-CM)to explore the effect of MK secreted by CAFs on drug resistance of cancer cells.The results showed that MK secreted by CAFs promoted drug resistance of cancer cells.In exploring the mechanism of drug resistance of oral squamous cell carcinoma cells,we found that the expression of IncRNA ANRIL was high in oral squamous cell carcinoma cells and oral squamous cell carcinoma tissue samples.In view of this,we speculate that MK promotes drug resistance of oral squamous cell carcinoma cells through IncRNA ANRIL.Firstly,we detected that MK promotes drug resistance of cancer cells in a time-dependent manner by cell activity assay.We further found that MK promotes the expression of IncRNA ANRIL in oral squamous cell carcinoma and other cancer cells.Knocking down IncRNA ANRIL significantly inhibited the proliferation and drug resistance of oral squamous cell carcinoma cells and promoted the apoptosis of oral squamous cell carcinoma cells.In view of the important role of ATP-binding cassette protein superfamily(ABC transporter superfamily)in the formation of multidrug tolerance in cancer cells,we will explore the regulatory role of IncRNA ANRIL on members of ABC transporter superfamily.The results showed that IncRNA ANRIL promoted the expression of MRP1 and ABC2,members of ABC transporter superfamily.Next,we examined the effects of IncRNA ANRIL on MK resistance in cancer cells and expression of apoptosis-related proteins induced by antineoplastic drugs.The results showed that IncRNA ANRIL enhanced the drug tolerance of oral squamous cell carcinoma cells induced by MK,alleviated the apoptosis induced by cisplatin by up-regulating the expression of anti-apoptotic protein,and inhibited the effect of MK on drug tolerance of cancer cells and promoted the apoptosis induced by cisplatin.These results indicate that the MK secreted by CAFs plays a major role in the formation of drug tolerance in cancer cells,and CAFs regulate the expression of lncRNA ANRIL related to tumorigenesis and development by MK,which affects drug transporters MRP1 and ABCC2,and promotes drug tolerance in cancer cells.In conclusion,this study mainly explored the role of IncRNA in the changes of CAFs and their roles in tumor cell migration,proliferation and drug resistance:(1)We analyzed the role of lncRNA LOC100506114 in the expression of CFAs-related phenotype markers in stromal fibroblasts and the migration,proliferation and drug tolerance of oral cancer cells;(2)We clarified the role of CAFs in the migration,proliferation and drug tolerance of oral cancer cells through lncRNA LOC100506114.The potential molecular mechanism of interaction with Runx2 to activate GDF10/TGF beta R1 signal;(3)The mechanism of CAFs expressing IncRNA LOC100506114 promoting drug tolerance of cancer cells through MK secretion was revealed.These results suggest that inhibiting the combination of CAFs and anti-cancer drugs may be a new strategy for cancer treatment.lncRNA LOC100506114/GDF10 in CAFs may be a potential molecular target for inhibiting the proliferation and migration of tumors and drug tolerance in the future.