Enhancing Sorafenib Sensitization In HCC Cells Through Suppressing SPRY4-IT1/miR-101-3p/EZH2 Pathway

Background and objective:Hepatocellular carcinoma(HCC)is a malignant tumor that threatens human life and health severely.The symptom of HCC is dormant,and most of the patients have lost the optimal period for surgical treatment.Therefore,chemotherapy has become the preferred treatment method for the middle and advanced HCC.However,HCC patients are prone to develop chemotherapy resistance clinically.The insensitivity of tumor cells to chemotherapeutic drugs leads to poor efficacy.Therefore,it is particularly important to explore the molecular mechanism of HCC resisting to chemotherapy and the corresponding measures to increase chemotherapy sensitization.Sorafenib is an FDA-approved molecular targeting drug for HCC and is considered a first-line chemotherapy drug due to its efficacy and safety.However,there are still some patients who have no obvious curative effect on sorafenib treatment,which instead increases the side effects and economic burden of treatment brought by sorafenib.Therefore,exploring the molecular mechanism of HCC resisting to sorafenib therapy and developing feasible sensitization measures has become a research hotspot in recent years.Long non-coding RNAs are a class of RNAs that are more than 200 nt but don’t have protein-coding function.Most lncRNAs act as ce RNAs which competitively binding with miRNAs,reducing or inhibiting the binding ability of miRNAs to the 3’-UTR region of it’s target m RNAs,thereby up-regulating the expression of downstream target genes of miRNAs,and ultimately participating in the regulation of various physiological functions of cells.Micro RNAs are small RNAs with a length of about 22 nt,and miRNAs can inhibit the expression of target genes by specific complementary base pairing with the 3’-UTR of target m RNA.SPRY4-IT1 is an intron lncRNA which containing 708 nt.Studies have reported that SPRY4-IT1 is significantly overexpressed in a variety of tumors and promotes the development of tumors.As a miRNA with a length of 21 nt,miR-101-3p presents a low expression level in various tumor tissues and cell lines,and promotes the proliferation and migration of tumor cells by regulating the expression level of its downstream target m RNAs.EZH2 is a methyltransferase that methylates lysine 27(K27)on histone H3.Studies have reported that EZH2 can enhance the proliferation of tumor cells and thus promote the development of tumor.Up to date,whether the SPRY4-IT1/miR-101-3p/EZH2 pathway exists in HCC cells and whether it plays an important role in sorafenib sensitivity regulation in HCC cells are unknown.Therefore,it is important to identify the SPRY4-IT1/miR-101-3p/EZH2 pathway and investigate its role in sorafenib sensitivity regulation in HCC cells.Which will contribute to the effective clinical application of sorafenib.Contents and methods:Part Ⅰ Suppression of the SPRY4-IT1 expression increased sorafenib sensitivity in HCC cells1.Total transcriptome sequencing was performed to screen the lncRNAs with significant change in expression level in sorafenib-treated HCC cells.2.Real-time PCR was used to test the top 10 lncRNAs that could be stably upregulated by sorafenib in HCC cells.3.CCK-8 assay was used to detect whether knockdown of SPRY4-IT1 could enhance sorafenib sensitivity in HCC cells.4.Clonal formation assay and Brd U flow cytometry were used to check whether knockdown of SPRY4-IT1 can reinforce the interruptive effect of sorafenib on the proliferation of HCC cells.Part Ⅱ SPRY4-IT1 reduced sorafenib sensitivity in HCC cells by down-regulating the expression of miR-101-3p1.Bioinformatics predicted miRNAs that might bind to SPRY4-IT1 and their binding sites.2.Real-time PCR was used to screen the miRNAs that might bind to SPRY4-IT1 and could be stably down-regulated by sorafenib in HCC cells.3.CCK-8 assay was used to detect whether overexpression of miR-101-3p could increase sorafenib sensitivity in HCC cells.4.Clonal formation assay and Brd U flow cytometry were used to determine whether the up-regulation of miR-101-3p could promote the proliferation inhibition effect of sorafenib on HCC cells.5.Real-time PCR was used to detect whether over-expression or down-regulation of SPRY4-IT1 could regulate the expression of miR-101-3p.6.CCK-8 assay was used to determine whether up-regulation of miR-101-3p could attenuate the reduced sorafenib sensitivity induced by SPRY4-IT1 overexpression in HCC cells.Part Ⅲ miR-101-3p increased sorafenib sensitivity in HCC cells by downregulating EZH2 expression1.Bioinformatics predicted whether miR-101-3p could directly bind to the 3’-UTR region of EZH2 and its binding sites.2.Real-time PCR and Western blot were used to detect the expression of EZH2 in sorafenib-treated HCC cells.3.CCK-8 assay was used to determine whether EZH2 inhibitors could increase sorafenib sensitivity in HCC cells.4.Clonal formation assay and Brd U flow cytometry were used to determine whether inhibition of EZH2 could promote the proliferation inhibition effect of sorafenib on HCC cells.5.Real-time PCR and Western blot were used to detect the effect of over-expression or silencing of miR-101-3p on EZH2 expression.6.The DLR assays were used to detect whether there was direct binding between miR-101-3p and the 3’-UTR region of EZH2 m RNA.Part Ⅳ SPRY4-IT1 regulated sorafenib sensitivity in HCC cells by modulating the miR-101-3p/EZH2 pathway1.Real-time PCR and Western blot were used to test the effect of up-level of SPRY4-IT1 on EZH2 expression.2.Real-time PCR and Western blot were used to examine the effect of silencing SPRY4-IT1 on EZH2 level.3.Real-time PCR and Western blot were used to determine whether silencing miR-101-3p could block the down-regulation of EZH2 caused by SPRY4-IT1 inhibition.4.Real-time PCR and Western blot were used to detect whether the over-expression of miR-101-3p could block the up-regulation of EZH2 caused by SPRY4-IT1 overexpession.Results:PartⅠ Suppression of the SPRY4-IT1 expression increased sorafenib sensitivity in HCC cells1.Ten lncRNAs with significant changes at expression level were screened by complete transcriptome sequencing in sorafenib-treated HCC cells.2.SPRY4-IT1 was stably up-regulated in sorafenib-treated HCC cells.3.Knokdown of SPRY4-IT1 significantly enhanced sorafenib sensitivity in HCC cells.4.SPRY4-IT1 knockdown promoted the proliferation inhibition effect of sorafenib on HCC cells.Part Ⅱ SPRY4-IT1 reduced sorafenib sensitivity in HCC cells by down-regulating the expression of miR-101-3p1.Bioinformatics prediction showed that multiple miRNAs can combine with SPRY4-IT1.2.The gene level of miR-101-3p was declined in sorafenib-treated HCC cells.3.Overexpression of miR-101-3p significantly enhanced sorafenib sensitivity in HCC cells.4.The up-regulation of miR-101-3p promoted the proliferation inhibition effect of sorafenib on HCC cells.5.Overexpression or silencing of SPRY4-IT1 could down-regulate or up-regulate miR-101-3p expression in HCC cells.6.Upregulation of miR-101-3p attenuated the reduced sorafenib sensitivity induced by SPRY4-IT1 overexpression in HCC cells.Part Ⅲ miR-101-3p increased sorafenib sensitivity in HCC cells by downregulating EZH2 expression1.Bioinformatics prediction showed that miR-101-3p directly bound to the 3’-UTR region of EZH2 m RNA.2.The expression level of EZH2 was stably up-regulated sorafenib-treated HCC cells.3.EZH2 inhibitors significantly enhanced sorafenib sensitivity in HCC cells.4.Inhibition of EZH2 promoted the proliferation inhibition effect of sorafenib on HCC cells.5.Overexpression of miR-101-3p down-regulated the expression of EZH2 in HCC cells.6.Silencing of miR-101-3p up-regulated the expression of EZH2 in HCC cells.7.miR-101-3p directly bound with the 3’-UTR region of EZH2 m RNA.Part Ⅳ SPRY4-IT1 regulated sorafenib sensitivity in HCC cells by modulating the miR-101-3p/EZH2 pathway1.Overexpression of SPRY4-IT1 significantly up-regulated the level of EZH2 in HCC cells.2.Silencing SPRY4-IT1 significantly down-regulated the level of EZH2 in HCC cells.3.Silencing miR-101-3p blocked the down-regulation of EZH2 caused by SPRY4-IT1 inhibition in HCC cells.4.Over-expression of miR-101-3p blocked the up-regulation of EZH2 caused by SPRY4-IT1 overexpession in HCC cells.ConclusionIn HCC cells,lncRNA SPRY4-IT1 can be significantly up-regulated by sorafenib,and knockdown of SPRY4-IT1 significantly enhances sorafenib sensitivity in HCC cells.Through down-regulating the expression of miR-101-3p,SPRY4-IT1 decreases the interaction between miR-101-3p and the 3’-UTR of EZH2 m RNA,resulting in the up-regulation of EZH2,and ultimately reduced sorafenib sensitivity in HCC cells.The SPRY4-IT1 si RNA or EZH2 inhibitors can effectively enhance sorafenib sensitivity in HCC cells.