The Preliminary Research On Function And Mechanism Of SOX6 In Hypoxia-induced Proliferation Of Human Pulmonary Artery Smooth Muscule Cells

Hypoxic pulmonary hypertension（HPH）is a kind of diseases triggerd by hypoxia,and its pathological features mainly focusing on pulmonoary vessels which includes persistent vaso-constriction and structural change,accompanied with high pulmonary artery pressure and right ventricular hypertrophy.In the plains,HPH are often secondary to lung diseases espe-cially accompanied with hypoxic conditions,such as chronic obstructive pulmonary disease.As the important pathogenesis,it may develop into many high altitude diseases.HPH will ag-gravate the heart load,further affect the heart function,and seriously damage the life quality and health of plateau residents.Therefore,clarifying the occurrence and development process of HPH is helpful for us to grasp the pathogenesis and provide important theoretical support for the prevention and treatment.The remodeling of pulmonary vascular induced by hypoxia is one of the main pathological characteristics of HPH,also the basis for high pulmonary ar-tery pressure.Hypoxia-induced proliferation of pulmonary artery smooth muscle cells（PASMCs）is an important pathological change leading to pulmonary vascular structural changes.Hypoxia-induced proliferation of PASMCs has always been the focus of researches for the pathogenesis of HPH,which has been proven to be a complex process co-regulated by multiple factors and mechanisms.Thus,its mechanism has not been clearly illuminated yet.Under hypoxia,changes in ion channels,oxidative stress forming,changes in mi RNA profiles and acid-base imbalance,as well as activation of many signal pathways such as ROCK and HIF,above all can promote the proliferative reaction of PASMCs.More and more studies have shown that the pathogenesis of tumor and the proliferation of PASMCs induced by hy-poxia has many similarities,and it has been proven that a variety of molecular mechanisms regulating tumor cells’proliferation are also applicable to hypoxia-induced proliferation.The expression of SOCS3 changed significantly in the lung tissues of HPH rat,while in-creasing its expression can significantly alleviate the pulmonary vascular remodeling induced by hypoxia.Moreover,SOCS3 has a significant inhibitory effect on the proliferation of vas-cular smooth muscle caused by inflammatory factors and hypoxia,which is closely related to the STAT3 signaling pathway.Recent studies have found that SOCS3 has a characteristic se-quence recognizing SOX6 in the promoter region of SOCS3,and the transcriptional expres-sion of SOCS3 is directly regulated by SOX6.As a member of the Sex Determination Region of Y chromosome（SRY）cluster,SOX6regulates many important growth and development processes such as embryonic development,catilage formation,etc.Recent studies have found that SOX6 can act as a tumor suppressor molecule,which can retain more cells in the G0/G1 phase to inhibit the proliferation of vari-ous tumor cells.However,the role of SOX6 in normal cell proliferation and hypoxic-induced PASMCs proliferation has not been reported yet.In view of the effect of hypoxia on SOCS3 expression,its role and related mechanisms in the proliferation of vascular smooth muscle cells,combined with the function of its up-stream regulatory molecule SOX6 in the regulation of tumor cell proliferation,it is speculated that SOX6 may play an important role in the proliferation of PASMCs induced by hypoxia.The mechanism is closely related to SOCS3 and STAT3 signaling pathways.Therefore,this study intends to explore the role and mechanism of SOX6 in hypoxia-induced proliferation of human pulmonary artery smooth muscle cells through si RNA and lentivirus transfection tech-niques,so as to find a new target for regulating hypoxia-induced proliferation of PASMCs,which can enrich the research on the pathogenesis of HPH,and also find a new direction for the formulation of HPH prevention and treatment measures..Methods1.Human pulmonary artery smooth muscule cells（HPASMCs）were used as the cell model in this study.HPASMCs were treated with 4%O₂ hypoxia to build a hypoxia-induced proliferation model.According to whether under hypoxia or not and the time of hypoxia,they were split into three groups,which include normoxic group cultivated under 21%O₂ condition,hypoxic 24 h group cuitivated under hypoxic condition for 24 h,and hypoxic48 h group cul-tivated under hypoxic condition for 48 h.2.The expression of SOX6 and SOCS3 were interfered by si RNA.Lentivirus transfec-tion was used to increase the expression of SOX6 in HPASMCs,then the cells were treated by4%O₂for 48h.3.The expression of SOX6,SOCS3,PCNA,p-STAT3 was detected by q RT-q PCR and Western blot.CCK-8 test and PCNA protein level were used to detecet the prolifetation level.Results1.The influence of hypoxia on the proliferation of HPASMCs and SOX6 expression:The relative level of CCK-8 and the expression of PCNA protein of HPASMCs in hypoxia were remarkably increased（P<0.05）,while the SOX6 protein was prominently lowered（P<0.01）.2.The role of SOX6 in hypoxia-induced proliferation of HPASMCs:In HPASMCs transfected with si RNA under normal oxygen condition,SOX6 m RNA and SOX6 protein were both obviously lowered（P<0.001）,while CCK-8 relative level and PCNA protein were prominently elevated（P<0.05）.Lentivirus transfection of HPASMCs significantly increased the SOX6 protein in cells（P<0.05）.After treatment with 4%O₂for 48h,the relative level of CCK-8 and PCNA protein in cells were significantly decreased（P<0.05）.3.The mechanism by which SOX6 can regulate the hypoxia-induced proliferation of HPASMCs:Hypoxia and interference of SOX6 expression significantly reduced SOCS3 pro-tein in HPASMCs（P<0.05）,while overexpression of SOX6 significantly increased SOCS3protein in HPASMCs（P<0.01）.Interfering SOCS3 expression with si RNA significantly re-duced SOCS3 m RNA and protein in HPASMCs（P<0.001）,while the relative level of CCK-8and PCNA protein in HPASMCs were significantly increased（P<0.05）.Hypoxia,SOX6knockdown and SOCS3 knockdown could significantly increase p-STAT3 protein in HPASMCs（P<0.01）,while overexpression of SOX6 could significantly decrease p-STAT3protein in HPASMCs（P<0.01）.Conclusion1.Hypoxia significantly reduced the expression of SOX6 and induced the proliferation of HPASMCs.2.SOX6 can regulate the hypoxia-induced proliferation of HPASMCs.3.SOX6 can regulate the hypoxia-induced proliferation of HPASMCs through SOCS3affecting the STAT3 signaling pathway.