| Debilitating lower back pain(LBP)is a common disorder of the back,which affects more than half of the world’s adult population.Intervertebral disc degeneration(IVDD)can result in debilitating pain and is a key factor contributing to LBP.IVDD reduces the life quality and inflicts significant financial burdens on LPB patients.The characteristics of IVDD include increased extracellular matrix breakdown,reduced hydration caused by abnormal matrix synthesis,loss of disc height and decreased ability to absorb load.The adjacent vertebral bodies are linked by intervertebral disc(IVD)which provides flexibility and mechanical stability to the body trunk during axial compression.IVD is composed of specialized connective tissue structures in which there are three morphologically distinct regions including the nucleus pulposus(NP),annulus fibrosis(AF)and cartilaginous endplates.Although the precise mechanisms of IVDD are not well-elucidated,it is believed that multiple factors contribute to the etiology of IVDD.For example,it is demonstrated that aberrant proliferation of NP cells and formation of NP cell clusters play important role in IVDD.Epidermal growth factor receptor(EGFR),which can induce NP cell proliferation after binding to its ligands,is positively associated with the grade of tissue degeneration in IVDD.Erb B receptor feedback inhibitor 1(ERRFI1),also known as mitogen-inducible gene-6(Mig-6),is a negative regulator of EGFR and plays an important role in attenuation of EGFR signaling network.Mechanical stress can induce the release of various cytokines within the IVD region and cause pain,inflammation and tissue damage.Tumor necrosis factor(TNF)-α can decrease the expression of proteoglycans and type II collagen,two major structural components of IVDs.Interleukin(IL)-1β induces proteoglycan breakdown and inhibits biosynthesis of matrix by IVD cells.IL-6 potentiates the catabolic actions of IL-1β and TNF-α on NP cells and decreases the proteoglycan synthesis.These cytokines promote matrix degradation and contribute to the pathology of IVDD.Suppression of these inflammatory cytokines have been utilized as therapeutic approaches for the treatment of IVDD.Micro RNAs(mi RNAs)are a class of endogenous,noncoding small RNAs(22 nucleotides),which pair to the 3’ untranslated region(3’UTR)and regulate gene expression.Mi RNAs have been shown to regulate several cellular functions including cell proliferation,differentiation and development.Mi RNAs were also reported to regulate the process of IVDD.For example,mi R-494 promoted NP cells apoptosis and extracellular matrix degradation.Mir-10 b could promote NP cell proliferation by targeting homeobox D10 in IVDD.Recent study using microarray demonstrated a total of 25 mi RNAs were upregulated,while 26 downregulated,in IVDD,among which mi R-2355-5p was found to be upregulated.Until now,the precise roles of mi R-2355-5p and ERRFI1 in IVDD have not been well investigated.In the current study,we monitored the expression of mi R-2355-5p and ERRFI1 in IVDD and explored their roles in IVDD.Part 1 The expression patterns of mir-2355-5p and ERRFI1 in IVDDObjective: To explore whether mir-2355-5p and ERRFI1 play a role in IVDD.Methods:1.The expression levels of mi R-2355-5p and ERRFI1 in human NP tissue samples from 35 patients with intervertebral disc degeneration(IVDD)and 15 normal NP tissue samples(Normal)were measured by q RT-PCR.2.The expression levels of mi R-2355-5p and ERRFI1 in human NP cells treated with 10 mg/ml LPS for 6,12,24 or 48 h were determined by q RT-PCR.3.The correlation between mir-2355-5p and ERRFI1 m RNA levels in NP tissue samples from 35 patients with IVDD was analyzed by Pearson correlation analysisResults:1.Up-regulated mi R-2355-5p and down-regulated ERRFI1 were associated with IVDDSignificantly decreased ERRFI1 m RNA level was detected in IVDD tissues when compared with normal tissues(P<0.01).In contrast,the mi R-2355-5p expression level in IVDD tissues was significantly higher than that normal tissues(P<0.01).The expression of ERRFI1 was inversely proportional to the expression of mi R-2355-5p in IVDD tissues(R=-0.3765,P<0.05).2.The expression of mir2355-5p and ERRFI1 in NP cells was responsive to LPSLPS treatment decreased the expression of ERRFI1 in a time-dependent manner.6 hours of LPS treatment significantly reduced the m RNA level of ERRFI1.In contrast,LPS treatment promoted mi R-2355-5p expression in a time-dependent manner.We detected significantly increased mi R-2355-5p level in 12 h,24 h and 48 h LPS-treated NP cells.Conclusions: Up-regulated mi R2355-5p and down-regulated ERRFI1 were associated with IVDD.And the expression of mir2355-5p and ERRFI1 in NP cells was responsive to LPS.Part2 The Function of ERRFI1 in NP cellsObjective: To test whether ERRFI1 could inhibit NP cell growth,and explore the potential effects of ERRFI1 on inflammatory cytokines production in LPS-treated NP cells.Methods:1.We transfected NP cells with ERRFI1 overexpression plasmid(ERRFI1-oe)or empty vector(Vec)and monitored the RNA and protein level of ERRFI1 by q RT-PCR and western blot 48 h post transfection.2.CCK-8 was used to detect the cell viability of NP cells transfected with ERRFI1-oe or empty vector(Vec)from day 1 to day 5.3.The RNA and protein level of cyclin D1 and PCNA in NP cells transfected with ERRFI1-oe or empty vector(Vec)were determined by q RT-PCR and western blot.4.We transfected NP cells with ERRFI1-oe or control plasmid and then treated them with 10mg/ml LPS for 48 h.The RNA and protein levels of TNF-α,IL-1β and IL-6 in NP cells transfected with ERRFI1-oe or empty vector(Vec)and then stimulated by LPS were measured by q RT-PCR and ELISA.Results:1.Overexpression of ERRFI1 inhibited NP cell growthThe m RNA and protein expression levels of ERRFI1 in NP cells transfected with ERRFI1-oe showed significant increase compared with NP cells transfected with empty vector(P<0.001),indicating that ERRFI1 was successfully overexpressed in NP cells;NP cells transfected with ERRFI1 plasmid displayed lower cell viability when compared to NP cells transfected with control plasmid and The difference became significant at day 5 post transfection(P<0.001).In addition,overexpression of ERFFI1 significantly inhibited the m RNA and protein levels of cyclin D1 and PCNA(P<0.05),both of which were cellular markers of proliferation.Therefore,our results demonstrated that ERFFI1 prevented NP cell proliferation.2.ERRFI1 suppressed inflammatory cytokines production in LPS-stimulated NP cellsInflammatory mediators have been implicated in the development of IVDD.Previous studies also described that ERRFI1 could negatively regulate inflammatory mediator production.Consistent with previous report,LPS treatment induced significant productions of TNF-α,IL-1β and IL-6.In contrast,NP cells transfected with ERRFI1 produced significantly less TNF-α,IL-1β and IL-6 after LPS treatment,indicating that ERFFI1 suppressed LPS-induced inflammatory cytokine production in NP cells.Conclusions: ERRFI1 inhibited LPS-induced pro-inflammatory cytokine productions in NP cells.Part3: The specific mechanism of mi R2355-5p involved in regulating NP cell related functionsObjective: To investigate whether mi R2355-5p is involved in regulating related functions of NP cells and its regulatory mechanismMethods:1.Use the online software Targetscan to predict the downstream fu nctional targets of mi R-2355-5p.2.Validate whether ERRFI1 was a functional target of mi R-2355-5p by dual-luciferase reporter assay system.3.Perform a pull-down experiment in NP cells with 3 ’biotin-labele d mi R-2355-5p or mi R-NC to further verify the interaction between mi R-2355-5p and ERRFI1 m RNA.4.The effects of mi R-2355-5p overexpression or inhibition on the expression level of ERRFI1 in NP cells were detected by q RT-PCR and western blot.5.Perform co-transformati-on combination experiments(NC-inhibitor+si-NC;NC-inhibitor+si-ERRFI1;mi R-2355-5p-inhibitor+si-NC;mi R-2355-5p-inhibitor + si-ERRFI1)to test whether mi R-2355-5p is involved in r egulating the effects of ERRFI1 on NP cells.Results:1.Mi R-2355-5p targeted 3’UTR of ERRFI1mi R-2355-5p was predicted to bind to ERRFI1 3’UTR.Luciferase assay results showed that expression of mi R-2355-5p significantly decreased the luciferase activity of the reporter gene with wild type but not mutated 3’UTR construct.In addition,the binding of mi R-2355-5p to ERRFI1 m RNA was confirmed by m RNA pull down assay.The q RT-PCR and western blot results showed that mi R-2355-5p overexpression in NP cells resulted in significantly decreased m RNA and protein levels of ERRFI1.In contrast,mi R-2355-5p inhibitor significantly increased m RNA level and protein expression of ERRFI1.Taken together,mi R-2355-5p inhibits the expression of ERRFI1 by directly binding to ERRFI1 3’UTR.2.Mi R-2355-5p Regulated the Effects of ERRFI1 on NP CellsThe q RT-PCR and western blot results showed that the transfection of control inhibitor and ERRFI1 specific si RNA significantly decreased m RNA level of ERRFI1,indicating an efficient knocking down of ERRFI1 by ERRFI1 si RNA.Co-transfection of mi R-2355-5p inhibitor and control si RNA resulted in significantly increased ERRFI1 m RNA,indicating inhibition of mi R-2355-5p enhanced ERRFI1 expression.Co-transfection of mi R-2355-5p inhibitor and ERRFI1 si RNA did not change m RNA level of ERRFI1 compared to control cells.In addition,transfection of ERRFI1 si RNA totally blocked the-upregulation of ERRFI1 m RNA induced by mi R-2355-5p inhibition,indicating that ERRFI1 si RNA knocking down was very efficient.The CCK-8 assay showed that NP cells transfected with control mi RNA and ERRFI1 si RNA displayed increased cell viability when compared to control cells.In contrast,NP cells transfected with mi R-2355-5p inhibitor and control si RNA displayed decreased viability.These data indicated that mi R-2355-5p displayed an opposite function to ERRFI1.Knocking down ERRFI1 in mi R-2355-5p inhibitor transfected-NP cells enhanced the cell viability,indicating the effect of inhibiting mi R-2355-5p on cell growth was dependent on ERRFI1.Similarly,we identified that knocking down ERRFI1 resulted in increased m RNA and protein expressions of cyclin D1 and PCNA Inhibition of mi R-2355-5p significantly decreased the expressions of cyclin D1 and PCNA,and these effects were reverted by knocking down ERRFI1.Finally,we identified that knocking down ERRFI1 enhanced LPS-induced productions of TNF-α,IL-1β and IL-6 at both m RNA and protein levels In contrast,mi R-2355-5p inhibitor suppressed LPS-induced pro-inflammatory cytokine production,while these effects can be reverted by knocking down ERRFI1,indicating the effect of inhibiting mi R-2355-5p on LPS-induced inflammatory cytokine production was also dependent on ERRFI1.Conclusions:Mi R-2355-5p negatively regulated ERFFI1 and prevented the effects of ERRFI1 on inhibiting NP cells proliferation and inflammation. |
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