The Mechanistic Studies Of LINP1 In Promoting CSCC Progression And Enhancing Cutaneous Ionizing Radiation Resistance

BackgroundCutaneous squamous cell carcinoma(cSCC)is the second most common cancer in the world,with an annual incidence of more than 1 million.It is highly aggressive and actively metastasizes to lymph nodes,and then spreads to the whole body.It has a high recurrence rate and poor prognosis.The 5-year survival rate is only 22%-56%,and the 1-year survival rate is only about 50%.Therefore,it is imperative to explore the pathogenesis of cSCC and provide new treatment ideas for cSCC.Long non-coding RNAs(LncRNAs)are a group of non-coding RNAs longer than 200 nucleotides with no protein-coding potential.LncRNA plays multiple roles in chromatin modification,post-transcriptional regulation,genomic imprinting,X chromosome inactivation,and miRNA sponge regulation,involving multiple pathophysiological processes,including carcinogenesis with multiple mechanisms.In-depth research in the field of cancer has shown that many LncRNAs are highly expressed in tumors and influence tumor progression as oncogenes or tumor suppressors.In order to explore the important LncRNAs that regulate cutaneous squamous cell carcinoma,we screened the only LncRNA-LINP1 among the significantly highly expressed ncRNAs in cSCC through screening of published data.At present,the research on LINP1 is very limited,and the molecular mechanism of LINP1 is limited to the regulation of NHEJ pathway,so our study aims to explore whether LINP1 regulates the progression of cSCC and its mechanism,and provide new ideas for the development of new drugs and therapies for the treatment of cSCC.LncRNAs are functionally diverse and can be involved in the regulation of a variety of biological processes and diseases,including IR damage.Skin is very sensitive to radiation exposure,skin IR damage is the most common IR damage and is a common complication of radiation oncology,bone marrow transplant pretreatment,and radio-nuclear accidents.Therefore,it is important to explore the key molecular events that affect skin radiation damage and find new ways to reduce damage to the skin when radiotherapy techniques are applied in medicine.Recently,more and more studies have found that LncRNA may play an important role in IR resistance,but the molecular mechanism of its radiation has not been thoroughly studied.In our presequencing,we found that IR can significantly induce the expression of LINP1 in HaCaT cells.Therefore,we hope to explore the role of LINP1 in cutaneous IR resistance and the molecular mechanism and to provide new evidence for enhancing cutaneous IR resistance in oncology radiotherapy and occupational exposure.Method(1)We analyzed the data of cutaneous squamous cell carcinoma in GEO database and screened out the LncRNA LINP1 significantly upregulated compared with normal skin tissue.The higher expression of LINP1 in cutaneous squamous cell carcinoma was verified by qPCR and in situ hybridization(ISH);The role of LINP1 on the proliferation,migration,and invasion of cutaneous squamous cell carcinoma was detected by CCK-8,cell clone formation assay,Transwell migration and invasion assay;Transcriptome sequencing was performed after LINP1 knockout,and KEGG analysis was conducted to explore the key downstream signaling pathways regulated by LINP1;The regulatory effect of LINP1 on the endoplasmic reticulum stress induced apoptosis pathway was verified by Western Blot,qPCR,SYTOX staining,trypan blue staining,flow cytometry and TUNEL staining;The proteins interacting with LINP1 in cSCC cells were collected by RNA pulldown and identified by high performance liquid chromatography combined with mass spectrometry.RNA pulldown,RIP assays and in vitro phosphorylation verified the interaction between LINP1 and eIF2a;ChIP assays and double knockout of LINP1 and DDIT3 were used to prove that the regulation of UPR-induced apoptosis by LINP1 is DDIT3-dependent;Different endoplasmic reticulum stress inducers were performed to induce endoplasmic reticulum stress to detect the effect of LINP1 on endoplasmic reticulum-induced apoptosis by Western Blot and qPCR;Nude mice Xenograft tumor models were established to verify the influence of LINP1 on the growth and biological function of the xenograft tumor in vivo;The expression level of LINP1 in the xenograft tumor was verified by qPCR and ISH,and the expression level of endoplasmic reticulum stress and apoptosis-related proteins were confirmed by Western Blot and qPCR;(2)RNA-seq was conducted to identify the significantly upregulated LncRNAs after IR;The effects of LINP1 on cell viability and apoptosis under IR were determined by CCK-8,clone formation assays and flow cytometry assays;The effects of LINP1 on DNA damage under IR were detected by single-cell gel electrophoresis,γ-H2AX immunofluorescence and Western Blot;The regulation of LINP1 on PCNA and FEN1 was detected by transcriptome sequencing and WB;RNA pulldown and HPLC-MS were used to determine and verify LINP1-interacting proteins in HaCaT cells.Result(1)LINP1 is highly expressed in cSCC tissues and cells,and its expression level is related to the stage of cSCC;LINP1 may play a role of oncogene in cSCC cells,promoting the proliferation,colony formation,migration and invasion of tumor cells;LINP1 significantly regulates endoplasmic reticulum stress-related pathways and apoptosis-related signaling pathways.LINP1 could inhibit ER stress-induced apoptosis-related proteins DDIT3,DR5,ER stress-related markers GRP78,XBP1 and apoptosis-related proteins cleaved-Caspase8,cleaved-Caspase7,cleaved-Caspase3;LINP1 interacts with eIF2α phosphorylation at Ser51 site in cSCC to inhibit the phosphorylation of eIF2α,thereby inhibiting the PERK-eIF2α branch-mediated UPR signaling and subsequent cell apoptosis;LINP1’s effect on cSCC depends on DDIT3、The double knocking of LINP1 and DDIT3 partially restores the effect of LINP1 knockdown;The nude mouse xenograft tumor model proved that LINP1 promotes tumor growth.Consistent with the results of in vitro experiments,Western Blot,qPCR and ISH showed that knockdown of LINP1 promotes endoplasmic reticulum stress and apoptosis-related proteins.(2)LINP1 is highly-expressed in HaCaT keratinocytes after IR.In keratinocytes,LINP1 enhances cell viability and inhibits apoptosis induced by IR and enhanced cutaneous IR resistance;LINP1 inhibits the DNA damage of keratinocytes by IR;LINP1 promotes the expression of PCNA and FEN1 under IR.LINP1 participates in the transcriptional regulation of CDK7 on the DNA damage repair-related protein FEN 1 by interacting with CDK7.Conclusion(1)LINP1 interacts directly with eIF2α to inhibit eIF2α phosphorylation and DDIT3 expression,thereby inhibiting UPR signal-mediated apoptosis,and ultimately promoting cSCC progression.Our findings elucidates the oncogenic and UPR signaling inhibitory effects of LINP1 in cSCC,and highlight a novel regulatory mechanism to regulate UPR signaling and subsequent apoptosis,which may provide a new intervention target for cSCC therapy.(2)LINP1 is up-regulated in keratinocytes after IR.Under IR conditions.LINP1 promotes the transcriptional regulation of CDK7 on FEN1 by interacting with CDK7 to enhance DNA damage repair and cutaneous IR resistance.It provides new ideas for reducing skin damage in tumor treatment.