Study On The Mechanism Of Long Non-coding RNA HCP5 And Its Coding Protein Promoting Malignant Progression Of Triple Negative Breast Cancer

Research Background and Objectives:The latest cancer statistics in 2020 showed that breast cancer has become the highest incidence of malignant tumor in the world.Triple negative breast cancer（TNBC）was an aggressive type of breast cancer with high rates of cancer recurrence and metastasis.The curative effect of TNBC is poor due to lack of effective therapeutic targets.Long non-coding RNAs（lnc RNAs）are traditionally considered to be a class of RNA transcripts more than 200 nucleotides and without protein-coding ability.However,recent studies have shown that some lnc RNAs have coding functions.At present,a variety of lnc RNAs have been found to be involved in the occurrence and development of TNBC,which are expected to be new targets for the treatment of TNBC.Lnc RNA HCP5 is a complex of human histocompatibility leukocyte antigen HLA and P5,which is mainly expressed in immune cells.Studies have shown that HCP5 is abnormally expressed in hepatocellular carcinoma,lung adenocarcinoma and ovarian cancer.This study aims to explore the role and regulatory mechanism of lnc RNA HCP5 and its encoding protein in TNBC,so as to provide new theoretical support for the diagnosis and treatment of TNBC.Research Methods:（1）The expression level of lnc RNA HCP5 in normal breast epithelial cells and breast cancer cells was analyzed by real-time PCR.（2）The expression of HCP5 in adjacent control breast tissues and breast cancer tissues was analyzed by RNAscope technique.（3）CCK-8 assay and cell survival/death assay were used to analyze the effects of HCP5 knockdown on the proliferation and viability of TNBC cells.（4）The genes differentially expressed after HCP5 knockdown were analyzed by RNA sequencing technology,and verified by antibody microarray and Western blot assay.（5）The effect of HCP5 knockdown on TNBC tumorigenesis ability was analyzed in vivo in nude mice.（6）Bioinformatics method was used to predict the coding ability and coding protein of HCP5.（7）Immunohistochemical assay was used to analyze the expression of HCP5 encoded protein in adjacent control breast tissue and breast cancer tissue,and the correlation analysis of clinicopathological features was conducted.（8）The expression of HCP5 encoding protein in breast cancer cells was analyzed by Western blot.The subcellular localization of HCP5 encoded protein was performed by nucleo-cytoplasmic separation technique.（9）The effects of HCP5 encoding protein knockdown and overexpression on proliferation,migration,apoptosis and cycle of TNBC cells were observed by flow cytometry.（10）RNA sequencing technology was used to detect the genes differentially expressed after HCP5 encoding protein knockdown and followed by pathway enrichment analysis.（11）Flow cytometry and laser confocal experiments were performed to observe the changes of lipid ROS content after knockdown of HCP5 encoded protein and addition of ferroptosis stimulant and inhibitor.（12）The changes of mitochondrial morphology in cells with ferroptosis stimulator were observed by electron microscopy after the changes of HCP5 protein expression.（13）Iron detection assay was used to observe the changes of intracellular Fe²⁺concentration after knockdown of HCP5 encoding protein by adding ferroptosis stimulants and inhibitors.（14）The expression of ferroptosis pathway related proteins were analyzed by Western blot.（15）The effect of ferroptosis stimulators on tumorigenesis ability of TNBC after knockdown of HCP5 encoding protein in nude mice was observed in vivo.Results:（1）Lnc RNA HCP5 was highly expressed in both TNBC cell lines and cancer tissues.（2）Knocking down HCP5 could inhibit the proliferation of TNBC and the growth of allograft tumor.（3）HCP5 knockdown promoted the apoptosis of TNBC cells,resulting in the decreased expression of apoptosis inhibitor BIRC3 and increased expression of Caspase-3.（4）Lnc RNA HCP5 has the encoding ability and can encode a protein with a length of 132 amino acids,which was named HCP5-132aa.（5）HCP5-132aa was highly expressed in TNBC tissues.（6）HCP5-132aa knockdown inhibited the proliferation and migration but promoted apoptosis and ferroptosis of TNBC cells.（7）In vivo experiments in nude mice confirmed that knockdown of HCP5-132aa could inhibit tumor growth in synergism with ferroptosis stimulant.Conclusion:Lnc RNA HCP5 promoted the malignant progression of TNBC through apoptosis pathway,and Lnc RNA HCP5 could encode the protein HCP5-132aa,which promoted the malignant progression of TNBC by regulating cell apoptosis and ferroptosis.This study revealed the role and regulatory mechanism of lnc RNA HCP5and its encoding protein in TNBC,provided a research basis for the clinical search for new TNBC targets.