Claims:WE CLAIM:
1. A reliable, liquid biopsy and less invasive method for early diagnosis of oral submucous fibrosis (OSMF) in a subject comprises of analyzing presence or absence of characterized circulating miRNA biomarkers comprising of has-miR-381-3p, has-miR-183-5p and has-miR-486-5p in a sample from the said subject wherein presence of atleast one of the said biomarkers indicates the presence of OSMF in the said subject and absence of all the said three biomarkers indicates absence of OSMF.

2. The method as claimed in claim 1, wherein the said biomarkers are analyzed by quantitative real time polymerase chain reaction (qRT-PCR).

3. The method as claimed in claim 1 wherein the said sample is whole blood.

4. A novel cluster of biomarkers for use in early diagnosis of oral submucous fibrosis (OSMF) in a subject comprises of characterized circulating miRNA biomarkers comprising of has-miR-381-3p, has-miR-183-5p and has-miR-486-5p.

5. The characterized circulating miRNA biomarkers comprising of has-miR-381-3p, has-miR-183-5p and has-miR-486-5p in any of the preceding claims are capable of regulating TGF-ß/ Wnt signaling pathway leading to epithelial mesenchymal transition (EMT) in subjects with OSMF thereby making the said biomarkers potential as biomarkers in OSMF condition

6. The said subject in any of the preceding claims is animals including humans.
Dated this 31st day of July 2018

For UNIVERSITY OF MADRAS
By its Patent Agent

Dr.B.Deepa
, Description:Form 2

THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

“DIAGNOSTIC BIOMARKERS FOR ORAL SUBMUCOUS FIBROSIS (OSMF) AND METHOD OF DETECTION THEREOF”

in the name of UNIVERSITY OF MADRAS an Indian National having address at Dept. Of Biochemistry University of Madras, Guindy Campus, Chennai-600025, Tamilnadu, India

The following specification particularly describes the invention and the manner in which it is to be performed

FIELD OF THE INVENTION:

The present invention generally relates to diagnostic method for detection of fibrosis. More specifically the present invention relates to a novel panel of biomarkers and their use in early diagnosis of oral submucous fibrosis (OSMF).

BACKGROUND OF THE INVENTION:
Oral submucous fibrosis (OSMF) is a premalignant condition of oral squamous cell carcinoma (OSCC) that has high prevalence of malignant transformation. It is a collagen disorder and it exhibits stiffness in the oral cavity and parts of oral mucosa. Blanched appearance of the oral mucosa was observed in the initial stage and there after the disease progresses to the development of palpable fibrous bands in the buccal cavity. As a result, patients experience trismus (restricted mouth opening) and difficulty in the movement of tongue. Latest scientific studies report that, areca nut chewing is the major etiological factor leading to this disease and the incidence rate is very high in Indian population. Since the exact mechanism behind this fibrosis is yet to be identified and there is no proper treatment regime or diagnostic tools available.
The current available diagnostic method used to identify OSMF involves tissue biopsy which is an undesirable invasive technique that involves more time. Thus there exist a need in the state of art to develop a novel reliable, liquid biopsy and less invasive method for early diagnosis of oral submucous fibrosis (OSMF).
OBJECT OF THE INVENTION:
The main object of the present invention is to identify differentially expressing circulating miRNA in oral submucous fibrosis condition and its role in the regulation of TGFß/ Wnt signaling pathway that leads to epithelial mesenchymal transition (EMT) in oral submucous fibrosis (OSMF).
Another object of the present invention is to identify a novel cluster of biomarkers for early diagnosis of oral submucous fibrosis (OSMF).
Another object of the present invention is to identify a novel cluster of biomarkers comprising of has-miR-381-3p, has-miR-183-5p and has-miR-486-5p for early diagnosis of OSMF.
Yet another object of the present invention is to develop methods for early diagnosis of OSFM in a subject comprises of analysing the identified novel cluster of biomarkers in a sample.
Further object of the present invention is to detect OSFM employing the developed method.
BRIEF DESCRIPTION OF DRAWINGS:
Figure 1 illustrates Up- regulated Wnt signaling pathway in OSMF.
From Figure 1 it is inferred that the pathway is activated when a Wnt ligand binds to the seven pass membrane receptor frizzled. The ligand- receptor binding, leads to phosphorylation at the cytoplasmic region of LRP5/6 by GSK-3ß and CK-1 that in turn leads to the recruitment of dishevelled and axin. The net effect of this is the inhibition of phosphorylation of ß- catenin and its degradation. Hence, the accumulated ß- catenin translocates into the nucleus from the cytoplasm, which then binds to the TCF/LEF transcription factors leading to the up regulation of its target genes such as c-myc and cyclin D1. Here, up-regulated protein expression pattern of Wnt5a, Wnt3a, ß- Catenin, cMyc and LEF-1 and down-regulated protein expression pattern of Wnt3a observed in OSMF condition when compared to control individual indicate that Wnt signaling pathway is up-regulated during OSMF condition.
Figure 2 illustrates Up- regulated TGF-ß signaling pathway in OSFM
From Figure 2 it is inferred that an up-regulated protein expression pattern of TGF-ß1, Smad3, pSmad3 and down-regulated protein expression pattern of Smad7 observed in OSMF when compared to control individual indicate that TGF- ß signaling is up-regulated in OSMF condition.
Figure 3 illustrates Up- regulated EMT in OSFM
From Figure 3 it is inferred that Down-regulated protein expression pattern of E- Cadherin, an epithelial marker, and an up-regulated protein expression patterns of mesenchymal markers viz N-Cadherin, a-Sma, Snail, Slug, Twist, Vimentin observed in OSMF condition when compared to control individuals indicate that epithelial mesenchymal transition is taking place during OSMF.

SUMMARY OF THE INVENTION:
The present invention shall disclose a novel cluster of characterized circulating miRNA biomarkers comprising of has-miR-381-3p, has-miR-183-5p and has-miR-486-5p for diagnosis of oral submucous fibrosis (OSMF) in a subject. The present invention also discloses a reliable, liquid biopsy and less invasive method for early diagnosis of oral submucous fibrosis (OSMF) in a subject. The diagnostic method comprises of analyzing presence or absence of characterized circulating miRNA biomarkers comprising of has-miR-381-3p, has-miR-183-5p and has-miR-486-5p in a sample from the subject. If atleast one of the biomarkers is present it indicates the presence of OSMF in the subject and absence of all the three biomarkers indicates absence of OSMF.
DETAILED DESCRIPTION OF THE INVENTION:
The present invention shall disclose a novel panel of biomarkers and their use in early diagnosis of oral submucous fibrosis (OSMF).

Research done over the past decades evidenced the involvement of various signaling pathways in fibrosis condition. For decades, RNA was considered as an intermediate molecule that is responsible for the transfer of genetic information from DNA to proteins. Advances in high through-put technologies made clear the perception of ncRNAs as the functional regulatory molecules that are involved in vital cellular process including chromatin remodeling, transcription, post-transcriptional modifications and signal transduction. Among the ncRNAs, the functional relevance of microRNA (miRNA) and long non- coding RNA have been very much discussed in recent years. MicroRNAs are single stranded endogenous non-coding RNA found in eukaryotes with 20-25 nucleotides in length. Usually, miRNAs are capable of identifying and targeting the mRNA by baseparing after transcription and thus regulating gene expression. A single miRNA can target one or more mRNA.

Based on this concept, we have identified ten circulating miRNA which are differentially expressed during OSMF (Table1 and 2).

We also found that these miRNAs are capable of regulating the TGF-ß/ Wnt signaling pathway leading to epithelial mesenchymal transition (EMT) in patients with OSMF. We have also validated the protein expression level of these signaling molecules indicating their potential as biomarkers in OSMF condition. (Table 3, 4 and 5)
Table 3 Table 4 Table 5
From the results what is inferred that the protein expression of Wnt signaling pathway viz Wnt5a, Wnt3a, GSK-3ß, ß- catenin, cMyc, LEF-1; TGF ß signaling pathway viz TGF-ß1, Smad3, pSmad3, Smad7; epithelial markers viz E- Cadherin and mesenchymal markers viz N-Cadherin, a-Sma, Snail, Slug, Twist and Vimentin studied by western blot analysis revealed the up regulation of Wnt5a, Wnt3a, ß- catenin, cMyc, LEF-1, TGF-ß1, Smad3, pSmad3, N-Cadherin, a-Sma, Snail, Slug, Twist, Vimentin and down regulation of GSK- ß, Smad7 and E- Cadherin, indicate that Wnt/TGF-ß signaling leads to epithelial mesenchymal transition in OSMF condition.

Since we identified circulating miRNA, these are stable in blood and plasma. Thus this could be considered as liquid biopsy, which is less invasive and allows repeated sampling. This could be an advantage over the painful surgical procedure (tissue biopsy) practiced these days in case of OSMF.
In the present study, we have focused on the role of circulating miRNA in the regulation of TGFß/ Wnt signaling pathway that leads to epithelial mesenchymal transition (EMT) in OSMF and its possible role as blood based biomarkers.
1.0 ml of blood was collected from normal healthy individuals and OSMF patients and it was subjected to RBC lysis by using RBC lysis buffer. Then, miRNA was isolated from the WBC obtained, by Trizol+ mirVana miRNA isolation kit with DNase Treatment. The isolated miRNA was subjected to sequencing and it was found that eleven miRNAs are differentially expressed among which three miRNAs are up-regulated. The levels of expression of both the up and down-regulated miRNA were quantitated by quantitative real time polymerase chain reaction (qRT-PCR). (Table.6) .

Then, the role of these identified miRNA in the regulation of TGFß/ Wnt signaling pathway that leads to epithelial mesenchymal transition (EMT) in OSMF was validated by western blot analysis. (Table 3, 4 and 5). Thus we could identify a blood based biomarker in OSMF condition.

Thus the present study invented an up-regulated expression of three circulating miRNA in oral submucous fibrosis condition (OSMF) and their role in TGFß/ Wnt signaling that leads to epithelial mesenchymal transition (EMT) in OSMF. The miRNA could be utilized as blood based biomarkers (liquid biopsy) that involve less invasive methods with more reliability.

In one of the preferred embodiment the present invention shall disclose a reliable, liquid biopsy and less invasive method for early diagnosis of oral submucous fibrosis (OSMF) in a subject. The diagnostic method comprises of analyzing presence or absence of characterized circulating miRNA biomarkers comprising of has-miR-381-3p, has-miR-183-5p and has-miR-486-5p in a sample from the subject. If atleast one of the biomarkers is present it indicates the presence of OSMF in the subject and absence of all the three biomarkers indicates absence of OSMF.

As per the invention, in the diagnostic method, the biomarkers are analyzed by quantitative real time polymerase chain reaction (qRT-PCR).
According to the invention, in the diagnostic method, the sample is whole blood.
In another preferred embodiment the present invention shall disclose a novel cluster of biomarkers for use in early diagnosis of oral submucous fibrosis (OSMF) in a subject which comprises of characterized circulating miRNA biomarkers comprising of has-miR-381-3p, has-miR-183-5p and has-miR-486-5p.

In accordance with the invention, the characterized circulating miRNA biomarkers comprising of has-miR-381-3p, has-miR-183-5p and has-miR-486-5p are capable of regulating TGF-ß/ Wnt signaling pathway leading to epithelial mesenchymal transition (EMT) in subjects with OSMF thereby making the biomarkers potential as biomarkers in OSMF condition
As per the invention the subject is animals including humans.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.