MiR-34c Inhibit Malignant Behavior And Reverse The Radioresistance Of Nasopharyngeal Carcinoma

Objective: The aim of this part is to discover the clinical value of miR-34 c in the radioresistance of nasopharyngeal carcinoma,and to explore the effect of miR-34 c in NPC malignant behavior and the radiotherapy resistance in vitro.To explore the downstream targets of miR-34 c for inhibiting malignant behavior and radiotherapy resistance of nasopharyngeal carcinoma,to verify the inhibitory effect of miR-34 c on nasopharyngeal carcinoma and to study its regulation of downstream targets in vivo.To explore whether the exosomes derived from MSCs transfected with miR-34 c can regulate the invasion,migration,proliferation,apoptosis and radiosensitivity of nasopharyngeal carcinoma.Method:1.Using micro RNA sequencing technology to detect micro RNA expression and screen the deferential micro RNA among immortalized nasopharyngeal epithelial cell line NP-69,nasopharyngeal carcinoma cell line CNE-2,and radioresistant nasopharyngeal carcinoma cell line CNE-2R;Analyzing and exploring the difference of miR-34 c expression level between normal nasopharyngeal tissues and nasopharyngeal carcinoma tissues by using bioinformatics technology;exploring the relationship between miR-34 c and the survival time of nasopharyngeal carcinoma patients;Using transwell experiment,colony formation experiment and CCK8 cell proliferation experiment to verify the effect of miR-34 c on invasion,migration and proliferation of nasopharyngeal carcinoma cell lines;Verifying the effect of miR-34 c on the expression of key molecules of invasion,migration and epithelial-mesenchymal transition(EMT)by Western blot;Using flow cytometry technology by Annexin V APC / 7-AAD double staining to detect cell apoptosis,verifying the sensitivity of nasopharyngeal carcinoma cell lines to radiotherapy after overexpression or knocking-down of miR-34c;Radioresistance level of nasopharyngeal carcinoma cell line CNE-2R overexpressing or knocking-down of miR-34 c was detected with different radiation dose,and colony generation experiment and CCK8 cell proliferation experiment were used to verify the impact of miR-34 c on NPC cells.2.Using qRT-PCR to explore the effect of overexpression and knockdown of miR-34 c on m RNA levels of β-catenin;Using Western blot to explore the changes in expression level of β-catenin in nasopharyngeal carcinoma cell lines that overexpressed or knocked down miR-34c;Bioinformatics predicting of the possible binding sites of miR-34 c in the 3’UTR region of β-catenin,and on this basis,constructing the mutant(MUT)and wild type(WT)sequences of the corresponding 3’UTR region.The wild-type sequence contained the potential binding site of miR-34 c,while the mutant sequence would not be recognized and bound by miR-34 c due to base substitution.The mutant or wild-type sequences were inserted into plasmids containing luciferase sequences,respectively,to construct a reporter gene vector.Co-transfecting wild-type or mutant reporter gene vector with miR-34 c into HEK293 T at the same time,and detecting the fluorescence intensity to verify the direct targeting effect of miR-34 c on β-catenin m RNA;Overexpressing miR-34 c and β-catenin at the same time to test the invasion and migration ability of nasopharyngeal carcinoma cells(transwell experiment)and proliferation ability(colony formation assay and cck8 assay),to confirm that β-catenin is targeted by miR-34 c which inhibits the invasion,migration and proliferation of nasopharyngeal carcinoma cells;Rescue experiments were carried out on CNE-2R cell lines overexpressing miR-34 c and β-catenin simultaneously.After different doses of irradiation,colony formation assay was performed to verify the radiosensitivity of CNE-2R cells.Implant CNE-2R cells stably transfected with miR-34 c or miR-Ctrl into BALB /c nude mice and divided the nude mice into 0 Gy(no radiotherapy)group and 8 Gy(radiotherapy)group.For nude mice in the radiotherapy group,a single 8 Gy radiotherapy was performed on the 24 th day of tumor implantation.The tumor volume of nude mice was measured every three days,and the volume growth curve and the tumor growth rate curve after radiotherapy were drawn.On day 36,the nude mice were sacrificed and the tumors were weighed.Immunohistochemical staining was performed.EMT-,proliferation-and apoptosis-related molecules,and β-catenin were stained and statistically analyzed to confirm in vivo that overexpression of miR-34 c can inhibit β-catenin in nasopharyngeal carcinoma tissues.3.Using lentivirus to stably transfect MSCs to increase the expression level of miR-34 c in MSCs,and then extracting their exosomes(MSC-exo-miR-34c).The purity and particle size were confirmed by Western blot and nanosight.Using PCR to verify the expression level of miR-34 c in transfected MSCs and exosomes;Using Transwell assays,CCK8 assays and colony formation assays to verify the effect of exosomes with high expression of miR-34 c on the proliferation and migration ability of nasopharyngeal carcinoma cells;Flow cytometry analysis for Annexin V APC/7-AAD double staining was used to detect the apoptosis and radiosensitivity of nasopharyngeal carcinoma cells after exosome stimulation;Different doses of irradiation were performed after MSC-exo-miR-34 c stimulation for NPC cell line CNE-2R.Colony formation assay was performed and survival curve was drawn accordingly.CCK8 assay was performed to verify the effect of miR-34 c on radioresistance of NPC cells;Implant CNE-2R cells subcutaneously into BALB/c nude mice,and MSC-exo-miR-34 c or MSC-exo-NC or PBS were intravenously injected into nude mice for 14 days from the 7th day of tumor implantation.A single 8 Gy of radiotherapy was given on the 24 th day.The tumor volume of nude mice was measured every three days,and the volume growth curve and the tumor growth rate curve after radiotherapy were drawn.On day 36,the nude mice were sacrificed and the tumors were weighed.Immunohistochemical staining of EMT-,proliferation-and apoptosis-related molecules were performed.Result:1.Deferential expression micro RNA in NP-69,CNE-2 and CNE-2R cell lines detecting by RNA sequencing showed that miR-34 c expression in nasopharyngeal carcinoma cell line CNE-2 was lower than that in immortalized nasopharynx cell line.The expression level of miR-34 c in nasopharyngeal carcinoma cell line CNE-2 was lower than that in immortalized nasopharyngeal epithelial cell line NP-69.In the radioresistant nasopharyngeal carcinoma cell line CNE-2R,the miR-34 c level was lowest among the cell lines;Analysis of the expression of miR-34 c in normal nasopharyngeal tissues and NPC tissues showed that the level of miR-34 c in NPC tissues decreased significantly;Survival analysis for clinical data in GEO database indicated that higher expression of miR-34 c signifcantly shortened the survival time for NPC patients.Overexpression of miR-34 c inhibited the invasion,migration and proliferation ability of NPC cell lines,while inhibiting its expression significantly promoted the above malignant behaviors;Western blot showed that miR-34 c inhibited the procession of EMT and reduced invasion and migration-related proteins in NPC cells;Overexpression of miR-34 c promoted radiotherapy’s(at 8Gy doses)ability to induce apoptosis of NPC cells;while knocking-down of miR-34 c inhibited radiation-induced apoptosis;The results of colony formation assay and CCK8 cell proliferation experiments showed that with the increase of radiotherapy dose,the CNE-2R cell line overexpressing miR-34 c showed more obvious inhibition of colony formation and proliferation ability;while the CNE-2R cell line knocking down miR-34 c increased the level of resistance to radiation significantly.2.Overexpression and knockdown of miR-34 c can down-regulate or up-regulate the expression level of β-catenin m RNA in nasopharyngeal carcinoma cells,respectively;Western blot results showed that the expression ofβ-catenin protein in nasopharyngeal carcinoma cell lines was down-regulated and up-regulated when overexpressed or knocked down miR-34 c,respectively;The luciferase reporter gene experiment results showed that overexpression of miR-34 c could down-regulate the fluorescence of HEK293 T cells transfected with wild-type reporter gene vectors.But for HEK293 T cells transfected with mutant reporter gene vectors,miR-34 c did not have the ability to decrease their fluorescence which indicated that β-catenin is the direct target of miR-34c;Knockdown of β-catenin reduced the colony forming ability of nasopharyngeal carcinoma cells;Overexpression of β-catenin could rescue miR-34c-induced effect on the invasion,migration and proliferation ability of nasopharyngeal carcinoma cells;In CNE-2R cells,the inhibitory role of miR-34 c on radioresiatance could be rescued by β-catenin overexpression.Overexpression of miR-34 c could significantly reduce the volume and weight of subcutaneous tumors in nude mice,and the inhibitory effect is more significant under radiotherapy conditions;analysis of the tumor growth rate showed that after receiving radiotherapy,tumors overexpressing miR-34 c displayed a faster decline in growth rate.Immunohistochemical results showed that overexpression of miR-34 c inhibited the tumor EMT process and tumor proliferation activity,and significantly reduced the expression of β-catenin.In addition,for tumors receiving radiotherapy,overexpression of miR-34 c significantly increased the proportion of apoptotic cells.3.MSCs were transfected successfully and the positive rate was over 85%.PCR showed that the expression of miR-34 c of MSCs were significantly increased after transfection.The exosomes of MSCs were extracted.Western blot experiments showed that TSG101 and CD9 were highly expressed,but calnexin was absent.Nanosight showed that the diameter of the extracted particles was between 50-150 nm,which confirmed that the extracted particles were exosomes.PCR results confirmed that miR-34 c expression level in MSC-exo-miR-34 c was significantly upregulated.Transwell assay showed that MSC-exo-miR-34 c significantly reduced the invasion and migration ability of nasopharyngeal carcinoma cell lines.CCK8 assay and colony formation assay showed that MSC-exo-miR-34 c could significantly inhibit the proliferation of nasopharyngeal carcinoma cells;The results of flow cytometry showed that MSC-exo-miR-34 c treatment could drastically promote the level of apoptosis in NPC receiving radiotherapy;Tumor growth curve and CCK8 results showed that MSC-exo-miR-34 c could strongly improve the radiosensitivity of nasopharyngeal carcinoma;The tumor volume and weight of nude mice in the MSC-exo-miR-34 c group were significantly lower than those in the MSC-exo-NC group.The tumor growth rate curve showed that MSC-exo-miR-34 c also reduced radioresistance while inhibiting tumor growth.Immunohistochemical staining results showed that MSC-exo-miR-34 c could significantly inhibit EMT,reduce cell proliferation and promote apoptosis of NPC.Conclusion: miR-34 c is down-regulated in nasopharyngeal carcinoma,especially in radioresistant nasopharyngeal carcinoma.It has the ability to inhibit malignant behaviors such as invasion,migration and proliferation of nasopharyngeal carcinoma cells,and can induce higher level of radiosensitivity in nasopharyngeal cancer cells.These results help us exploring the key molecules that affect the occurrence and development of nasopharyngeal carcinoma and radiosensitivity,which providing a direction for further exploring about the underlying mechanism.miR-34 c directly inhibits the malignant behavior and radioresistance of nasopharyngeal carcinoma in vivo and in vitro by directly targeting β-catenin.These results help us exploring the specific mechanism of miR-34 c,a key tumor suppressor molecule,in inhibiting malignant behavior and radiation resistance of nasopharyngeal carcinoma.After transfection of MSCs with miR-34 c,the MSCs-exosomes can significantly inhibit the invasion,migration and proliferation of NPC cells,and promote their apoptosis and radiosensitivity.It is confirmed that MSC exosomes can be used as a carrier of the therapeutic molecule miR-34 c to inhibit the malignant behavior of nasopharyngeal carcinoma,which provided a new idea for the treatment of nasopharyngeal carcinoma.