The Function And Molecular Mechanism Of LncRNA LOC730101 In Promoting Drug Sensitivity Of Ovarian Cancer By Inhibiting Autophagy Through BECN1

PurposeOvarian cancer(OC)is the most lethal gynecological malignancy,which has the characteristics of complex histopathology,high malignancy and poor survival prognosis.At present,surgery combined with chemotherapy is currently accepted as the standard treatment mode for OC treatment in the worldwide.Platinum-based chemotherapy,PARP inhibitor(PARPi)and angiogenesis inhibitor targeted therapy have achieved certain curative effects,but drug resistance and recurrence are still the bottlenecks of OC treatment.Therefor,it is an urgent challenge for OC to be overcome towards precision therapy by studying the mechanism of OC drug resistance,finding new drug resistance targets and developing new effective treatment strategies.Previously,we compared the tissue samples of platinum-resistant and platinum-sensitive patients with ovarian cancer by transcriptome sequencing and found that the long non-coding RNA(lnc RNA)LOC730101 was significantly overexpressed in the tissues of platinum-sensitive patients,and the high expression of LOC730101 in OC cells promoted the sensitivity to platinum and PARPi.Then,through RNA pull down experiment combined with mass spectrometry analysis,it was found that LOC730101 specifically binds to the autophagy key protein Beclin1(BECN1).Western blotting and immunofluorescence experiments showed that high expression of LOC730101 inhibited autophagy and promoted DNA damage caused by drugs in OC cells.Based on the above research,we put forward the scientific hypothesis that "LOC730101 may prevent the DNA damage-induced repair mechanism by inhibiting autophagy through binding to BECN1 and improve the drug sensitivity of ovarian cancer".This subject systematically explored the association and molecular mechanism between LOC730101 and BECN1 to inhibit the occurrence of autophagy and the relationship between autophagy and DNA damage,which is expected to provide a key and effective experimental basis for improving the sensitivity of OC drugs,the selection of treatment options,and the judgment of prognosis.MethodsTranscriptome high-throughput sequencing was used to analyze differentially expressed genes in the tissues of platinum-sensitive and platinum-resistant ovarian cancer patients,and screened out significantly differentially expressed lnc RNAs.The expression in ovarian cancer tissues and cells was verified by q PCR,and LOC730101 was screened to be highly expressed in platinum-sensitive ovarian cancer tissues and cells.The relationship between LOC730101 and ovarian cancer prognosis was obtained by analyzing the TCGA ovarian cancer database.Cell lines that stably express or stably interfere with the expression of LOC730101 were constructed using lentivirus expression system and lentivirus carrying sh RNA,respectively.The growth,survival and apoptosis of cells treated with cisplatin or PARPi were detected by cell viability,colony formation assay and flow cytometry.Fluorescence in situ hybridization experiments and RNA nucleocytoplasmic isolation experiments confirmed the subcellular localization of RNA.Proteins that specifically bind to LOC730101 were screened by RNA pull down and mass spectrometry analysis.Co-IP analysis was applied to identify the protein-protein interactions.q PCR and Western Blot were used to detect gene expression at RNA and protein levels.The tumorigenic ability of ovarian cancer cells and the antitumor treatment response to cisplatin and PARPi drugs were evaluated in a nude mouse model of subcutaneous xenografts.H&E staining was used to observe the tissue morphology of the transplanted tumor.The expression levels of RNA and protein in paraffin tissue of ovarian cancer were detected by in situ hybridization and immunohistochemistry,respectively.Results1.LOC730101 is highly expressed in platinum-sensitive ovarian cancer cells and ovarian cancer tissues and progression-free survival is longer in ovarian cancer patients with high LOC730101 expression;2.High expression of LOC730101 inhibited ovarian cancer cell survival and proliferation and promoted apoptosis,while knockdown of LOC730101 promoted ovarian cancer cell survival and proliferation and inhibited apoptosis after treatment with cisplatin and niraparib,respectively;3.LOC730101 specifically binds to BECN1,a autophagy key protein,to inhibit cellular autophagy and promotes apoptosis;4.LOC730101 inhibits the formation of autophagosome BECN1-VPS34 by reducing BECN1 phosphorylation;5.The specific binding domains of LOC730101 and BECN1 are the2001-3784 nt sequence of LOC730101 and the 1-134 aa region of BECN1.The binding of LOC730101 and BECN1 blocks the phosphorylation site of BECN1;6.After cisplatin combined with 3-MA or niraparib combined with3-MA,the proliferation inhibition rate and apoptosis rate of ovarian cancer cells increased significantly,and the colony formation rate decreased significantly;7.LOC730101 promotes DNA damage by inhibiting histone H2A ubiquitination;8.The high expression of LOC730101 promotes the accumulation of the autophagic substrate p62,which binds to RNF168 and inhibits the expression and activity of RNF168;9.An immunodeficient mouse subcutaneous tumorigenesis model validates the mechanism of action of LOC730101 in promoting drug sensitivity in ovarian cancer through inhibition of autophagy.Conclusion(1)LOC730101 is highly expressed in platinum-sensitive cells and tissues of ovarian cancer and positively correlates with survival prognosis of ovarian cancer patients;(2)LOC730101 promotes the sensitivity of ovarian cancer cells to cisplatin and PARPi by inhibiting autophagy.The combination of cisplatin and PARPi with autophagy inhibitors contribute to the sensitivity of ovarian cancer cells to the drugs;(3)LOC730101 specifically binds to BECN1 and inhibits autophagosome BECN1/VPS34 formation by reducing BECN1 phosphorylation,thereby suppressing autophagy in ovarian cancer cells after cisplatin and PARPi treatment and promoting drug sensitivity in ovarian cancer;(4)LOC730101 promotes drug sensitivity in ovarian cancer cells by inhibiting the expression and activity of RNF168 through p62 and thereby affecting H2 A ubiquitination-mediated DNA damage repair.